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Elements of photoreceptor homeostasis: investigating phenotypic manifestations and susceptibility to photoreceptor degeneration in genetic knockout models for retinal disease
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Elements of photoreceptor homeostasis: investigating phenotypic manifestations and susceptibility to photoreceptor degeneration in genetic knockout models for retinal disease
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Content
ELEMENTS OF PHOTORECEPTOR HOMEOSTASIS:
INVESTIGATING PHENOTYPIC MANIFESTATIONS AND
SUSCEPTIBILITY TO PHOTORECEPTOR DEGENERATION IN GENETIC
KNOCKOUT MODELS FOR RETINAL DISEASE
by
Rosanne Marie Yetemian
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR AND CELLULAR BIOLOGY)
August 2010
Copyright 2010 Rosanne Marie Yetemian
ii
DEDICATION
This manuscript is dedicated to my beloved family and friends who have provided
me with the support, encouragement and strength I needed throughout my graduate
career.
iii
ACKNOWLEDGMENTS
It is with sincere gratitude that I thank the following individuals who have helped
me in the completion of my thesis work and allowed me to develop my own sense of
scientific creativity. I would first like to acknowledge the dedication and commitment of
Dr. Cheryl M. Craft, my mentor and friend, who has always had her students’ best
interest at heart. She gave me the confidence and strength I needed to complete this work
and continues to inspire me to achieve great things. She allowed me to think
independently and creatively in the laboratory and supported me, not only with my
scientific research, but throughout all of my endeavors at USC. Where other mentors do
not encourage their student’s interdisciplinary thinking, Dr. Craft allowed me to flourish.
She strongly advocated for me in the completion of a Masters degree in Regulatory
Science and allowed me to maintain an active role in student government. It is only
through her encouragement and support that I was able to accomplish what I have in
graduate school. I am proud to call her my mentor and even more proud to call her friend.
The Mary D. Allen Laboratory for Vision Research would not be without the
generosity of Mrs. Mary D. Allen and her contributions to science by providing us with a
beautiful facility where I’ve spent countless hours conducting experiments, researching,
and writing this dissertation. The success of a team is entirely due to its players.
Therefore, I would also like to thank all members of the Mary D. Allen Laboratory for
their contributions to this work. The dedication of our biochemist and senior research
associate, Bruce Brown, who performed all of the retinal dissections and preparations, is
immensely appreciated. He provided the invaluable guidance and patience that comes
iv
with years of experience. My fellow graduate students, Freddi Isaac Zuniga and Shun-
Ping Huang, I thank you for providing me with your suggestions, guidance, and
friendship these past few years. Our experiences led us to develop a strong relationship
that I know will last for years to come. I would like to also thank Leng-Ying Chen, Dr.
Guey-Shuang Wu, and Teresa Ramirez for their assistance and friendship and Gloria
Arciniega for feeding us and keeping our laboratory looking beautiful. The technical
support of Lawrence Rife, Fernando Gallardo and Ernesto Barron is also sincerely
appreciated in the completion of this work. I thank Dr. Yibu Chen from the
Bioinformatics Service Program at the USC Norris Medical Library for his assistance in
the microarray analyses and Dr. Xuemei Zhu and Kebin Wu for their contributions to the
Nrl
-/-
Grk1
-/-
study. We also thank Drs. Anand Swaroop and Alan Mears for providing the
Nrl
-/-
mice, and Dr. Ching-Kang Jason Chen for the Grk1
-/-
mice, Dr. Jeannie Chen for
Arr1
-/-
mice and Dr. Shlomo Melmed for Pttg1
-/-
mice.
Finally, I would like to thank my dissertation committee, Dr. David Hinton, Dr.
Jeannie Chen, Dr. Alapakkam Sampath, and Dr. Le Ma for their encouragement, support,
and guidance with the progress of my work.
v
TABLE OF CONTENTS
DEDICATION ii
ACKNOWLEDGMENTS iii
LIST OF TABLES viii
LIST OF FIGURES ix
ABSTRACT xii
CHAPTER 1. INTRODUCTION 1
1.1 Retinal Degenerative Disease 1
1.2 Structure and Function of the Mammalian Retina 3
1.3 Photoreceptors: Rods and Cones 6
1.3.1 Basic Photoreceptor Structure 6
1.3.2 Functional Variability 8
1.4 Phototransduction Activation 9
1.5 Phototransduction Recovery 10
1.5.1 G-protein Coupled Receptor Kinase 1 (Grk1) 12
1.5.2 Arrestin 1 (Arr1) 14
1.6 The Neural Retina Leucine Zipper Knockout Mouse (Nrl
-/-
) 16
1.7 Choroidal and Retinal Vasculature 20
1.8 Choroidal and Retinal Neovascularization 22
1.8.1 Choroidal Neovascularization (CNV) 23
1.8.2 Retinal Neovascularization (RNV) 26
1.9 Summary 28
CHAPTER 2. MATERIALS AND EXPERIMENTAL PROCEDURES 29
2.1 Loss of G-protein Coupled Receptor Kinase (Grk1) in Nrl
-/-
Mice 29
2.1.1 Experimental Animals 29
2.1.2 Electroretinography (ERG) 29
2.1.3 Eyecup Embedding and Sectioning 30
2.1.4 Retinal Histology 31
2.1.5 Immunohistochemistry (IHC) 31
2.1.6 TUNEL Analysis of Apoptosis 32
2.1.7 Endothelial Cell Staining 33
2.1.8 Fluorescein Angiography (FA) 34
2.1.9 Affymetrix
TM
GeneChip Microarray and Ingenuity Pathway Analysis 34
2.1.10 Real Time Quantitative Polymerase Chain Reaction (qPCR) 36
2.1.11 Isoelectric Focus (IEF) and Immunoblot Analysis of PGI 37
2.1.12 PGI Activity Assay 38
vi
2.1.13 Molecular Identification and Sequence Analysis of PGI mRNAs 38
2.2 Poly(ADP)Ribose Polymerase-1 (PARP) Activity in Grk1
-/-
Mice 41
2.2.1 Immunoblot Analysis of PARP and PAR 41
2.2.2 PAR Immunohistochemistry in Grk1
-/-
and C57Bl/6J Retinas 42
2.2.3 TUNEL and PAR Colocalization 42
2.2.4 In Situ PARP Activity Assay 43
2.2.5 HT Colorimetric PARP Apoptosis ELISA Assay 43
2.3 Characterization of the Pttg1
-/-
Mouse Retina 45
2.3.1 Pttg1
-/-
Mice 45
2.3.2 Genotyping 45
2.3.3 Electroretinography (ERG) 47
2.3.4 Retinal Histology 47
2.3.5 Immunohistochemistry 47
2.3.6 Fluorescein Angiography (FA) 47
2.4 Background Strain Variability in the Arrestin1 (Arr1
-/-
) Phenotype 49
2.4.1 Arr1(A)
-/-
and Arr1(B)
-/-
Mice 49
2.4.2 TUNEL Analysis of Apoptosis 49
2.4.3 Isoelectric Focusing and Immunoblot Analysis of Prdx6 49
2.4.4 Allele Specific Genotype Analysis of Prdx6 50
2.4.5 Phospholipase A
2
Activity (PLA
2
) in Arr1(A)
-/-
and Arr1(B)
-/-
52
2.4.6 Association Mapping and Genome Wide Array Studies 53
CHAPTER 3. LOSS OF G-PROTEIN COUPLED RECEPTOR KINASE
(GRK1) IN NRL
-/-
MICE LEADS TO NEOVASCULARIZATION,
ENHANCED INFLAMMATORY RESPONSE, AND
LIGHT-INDEPENDENT CONE DYSTROPHY 55
3.1 Introduction 55
3.2 Results 58
3.2.1 Age-Related Light-Independent Cone Dystrophy 58
3.2.2 Apoptosis of Cone Photoreceptors 63
3.2.3 Neovascularization 65
3.2.4 Microarray and Ingenuity Pathway Analysis (IPA) 75
3.2.5 Confirmation of Gene Expression Changes by Quantitative PCR 84
3.2.6 Nrl
-/-
and Nrl
-/-
Grk1
-/-
Mice Have a Single Nucleotide
Polymorphisms (SNP) in Phosphoglucose Isomerase (PGI) 85
3.3 Discussion 88
CHAPTER 4. ACTIVATION OF POLY(ADP)RIBOSE POLYMERASE-1
(PARP) CONTRIBUTES TO LIGHT-MEDIATED
PHOTORECEPTOR DEGENERATION IN GRK1
-/-
MICE 95
4.1 Introduction 95
4.2 Results 100
4.2.1 PARP and PAR Expression in Grk1
-/-
, Arr1
-/-
and C57Bl/6J Mice 100
4.2.2 Correlation of PARP Activity with Cell Death 105
vii
4.2.3 Enhanced PARP Cleavage and Activity Analysis 109
4.3 Discussion 114
CHAPTER 5. CHARACTERIZATION OF THE PITUITARY
TUMOR TRANSFORMING GENE 1 (PTTG1) NULL MOUSE RETINA 118
5.1 Introduction 118
5.2 Results 120
5.2.1 Pttg1
-/-
Mice Lack the DdeI Mutation for Rdle and are Met450Met
for Rpe65 120
5.2.2 Healthy Cone Function and Cone Number in Pttg1
-/-
Mice 121
5.2.3 Pttg1
-/-
Male and Female Mice Have Normal FA Staining Patterns 125
5.3 Discussion 127
CHAPTER 6. MOUSE BACKGROUND STRAIN VARIABILITY
IN THE ARRESTIN 1 NULL (ARR1
-/-
) PHENOTYPE 130
6.1 Introduction 130
6.2 Results 133
6.2.1 Light-Independent Photoreceptor Apoptosis 133
6.2.2 Arr1(A)
-/-
and Arr1(B)
-/-
Mice Harbor Unique Prdx6 Isoforms 134
6.2.3 Prdx6 from Arr1(A)
-/-
and Arr1(B)
-/-
Have Comparable PLA
2
Activity 135
6.2.4 Quantitative Trait Locus (QTL) Mapping and Genome Wide
Association Studies (GWAS) 137
6.3 Discussion 145
CHAPTER 7. CONCLUSION 149
BIBLIOGRAPHY 154
APPENDIX A. A COMPREHENSIVE LIST OF
DIFFERENTIALLY EXPRESSED GENES BETWEEN NRL
-/-
GRK1
-/-
AND
NRL
-/-
RETINAS 168
APPENDIX B. GENE IDENTIFICATION AND CATEGORIZATION
OF REGIONS WITH SIGNIFICANT LOD SCORES IN ARR1(A)
-/-
AND
ARR1(B)
-/-
GENOMIC DNA 182
viii
LIST OF TABLES
Table 3.1: Comparison of Up-Regulated Transcripts Between Nrl
-/-
and 78
Nrl
-/-
Grk1
-/-
Retinas
Table 3.2: Comparison of Down-Regulated Transcripts Between Nrl
-/-
and 79
Nrl
-/-
Grk1
-/-
Retinas
Table 3.3: Biological Pathways Identified as Statistically Significant 81
Table 3.4: Associated Functions of Top Networks. 81
Table 6.1: Chromosomal Locations and LOD Scores for Quantitative 142
Trait Loci (QTL)
Table 6.2: Primer Names and Genetic and Physical Locations for Linkage 143
Analysis Studies
Table 6.3: Microsatellite Markers Identified from the Genome 144
Wide Array and Association Mapping Analysis
ix
LIST OF FIGURES
Figure 1.1: Structure of the Human Retina 5
Figure 1.2: Schematic of Rod and Cone Photoreceptors 7
Figure 1.3: Phototransduction in Vertebrate Rods 10
Figure 1.4: Schematic of Disk Proteins Associated with Phototransduction 11
Recovery
Figure 1.5: Light Microscopy and Ultrastructural Analysis of WT, Nrl
+/-
18
and Nrl
-/-
Mice
Figure 1.6: A Model of Photoreceptor Differentiation in Mice 19
Figure 1.7: The Structure of the Human Eye and Retina 22
Figure 1.8: Schematic Representation of Choroidal and Retinal 23
Neovascularization
Figure 1.9: The Retinal Pigment Epithelium, Bruch’s Membrane and the 24
Choroid
Figure 2.1: PGI mRNA Clone Sequences from Different Published Mouse Strains 40
Figure 2.2: Allele Specific PCR (ASPCR) Primer Design for Prdx6 52
Figure 3.1: Maximum b-wave Amplitude of Dark-adapted ERG Responses 59
in Nrl
-/-
(A) and Nrl
-/-
Grk1
-/-
Mice (B).
Figure 3.2: Age-Dependent Cone Photoreceptor Degeneration in the Nrl
-/-
61
and Nrl
-/-
Grk1
-/-
Mouse Retina
Figure 3.3: Both S-opsin (A-F) and M-opsin (G-L) Expressing Cones 62
Degenerate in the Nrl
-/-
Grk1
-/-
Retina
Figure 3.4: TUNEL Staining is Enhanced in Nrl
-/-
Grk1
-/-
64
Figure 3.5: Retinal Anastomoses in Nrl
-/-
Grk1
-/-
Mice 66
Figure 3.6: Retinal Anastomoses in Nrl
-/-
Grk1
-/-
Mice are Present Beginning 68
at PN21
x
Figure 3.7: VEGF Expression is Enhanced in Nrl
-/-
Grk1
-/-
Retinas Beginning at PN21 71
Figure 3.8: VEGF and IsolectinB4 Staining in a Retinal Blood Vessel of a 73
PN21 Nrl
-/-
Grk1
-/-
Retina
Figure 3.9: Fluorescein Angiography (FA) Staining of Nrl
-/-
and Nrl
-/-
Grk1
-/-
Mice 74
Figure 3.10: Fluorescein Angiography (FA) of C57Bl/6J and Grk1
-/-
Retinas 75
Figure 3.11: Top Canonical Pathways Identified by IPA 82
Figure 3.12: Schematic Representation of the Inflammatory Disease, 83
Inflammatory Response Network
Figure 3.13: Confirmation of Differential mRNA Expression of Klk22 and 84
Pttg1 Using Quantitative RT-PCR (qPCR)
Figure 3.14: Isoelectric Focusing of PGI in Various Retina Tissue Homogenates 87
Figure 3.15: PGI Enzymatic Activity is Not Affected by Grk1 87
Figure 4.1: Structural and Functional Characteristics of PARP-1 98
Figure 4.2: PAR Expression in Light Adapted Grk1
-/-
, Arr1
-/-
and C57Bl/6J Retinas 101
Figure 4.3: PAR Expression is Enhanced at 1 Month in Light Adapted 102
Grk1
-/-
Retinas
Figure 4.4: PARP and PAR Expression in Dark and Light Treated Grk1
-/-
105
and C57Bl/6J Mice
Figure 4.5: PAR IHC and TUNEL Staining 107
Figure 4.6: Quantification of TUNEL and PAR Positive Cells 109
Figure 4.7: PARP Activity ELISA Assay 110
Figure 4.8: In Situ PARP Activity Assay 112
Figure 5.1: Genotyping of the 6 Pttg1
-/-
Parents 121
Figure 5.2: Haemotoxylin and Eosin (H&E) Staining Indicate 122
Healthy Retinal Morphology and Normal ONL Thickness in Pttg1
-/-
xi
Figure 5.3: Maximum b-wave Amplitude of Dark-Adapted ERG 123
Responses in WT and Pttg1
-/-
Mice
Figure 5.4: Arrestin 4 IHC Staining in Pttg1
-/-
Mice is Similar to 124
C57Bl/6J at 1.5 and 9 Months
Figure 5.5: Mouse Arrestin 4 Staining in the Superior Region of 124
Pttg1
-/-
and C57Bl/6J Mice
Figure 5.6: Pttg1
-/-
Male and Female Mice Have Normal Retinal Vasculature 126
Figure 6.1: Quantification of TUNEL Positive Cells from WT, Arr1(A)
-/-
134
and Arr1(B)
-/-
Mice with Increasing Age
Figure 6.2: Isoelectric Focusing and ASPCR SNP Genotype Analysis for Prdx6 135
Figure 6.3: Phospholipase A
2
Activity 136
Figure 6.4: LOD Score Graph of the Linkage Analysis Performed 140
on Arr1(A)
-/-
and Arr1(B)
-/-
Genomic DNA
xii
ABSTRACT
G-protein coupled receptor kinase 1 (Grk1) is essential for light-activated opsin
phosphorylation in phototransduction shutoff, and genetic defects cause Oguchi’s
disease, a form of Retinitis Pigmentosa (RP). To elucidate the recovery function of cone
pigments, we combined Grk1
-/-
murine knockouts with the Neural retina leucine zipper
(Nrl
-/-
), which have an enhanced S-cone phenotype. We observed that with increasing age
and independent of light, the retinas of Nrl
-/-
Grk1
-/-
when compared to Nrl
-/-
developed
progressive cone degeneration and decreased cone protein expression. The degeneration
initially occurs in the central inferior quadrant and spreads with retinal pigment epithelia
(RPE) atrophy. Endothelial cell specific immunohistochemistry and fluorescein
angiography (FA) revealed progressive changes in retinal neovascularization in the Nrl
-/-
Grk1
-/-
at 1 month of age, prior to the onset of significant cone functional deficits and
ONL thinning. Vascular Endothelial Growth Factor (VEGF) expression was also
observed in the inner retina and within blood vessels at post natal (PN) 21 of these mice.
To further delineate the cone degeneration phenotype, we performed microarray analyses,
observed statistically significant changes in retinal transcript levels of >400 genes, and
examined these candidates with Ingenuity Pathway Analysis (IPA). The Oncostatin M
Signaling pathway was the top canonical pathway, and inflammatory disease/response
genes were one of the top networks identified. These data demonstrate that the loss of a
functional Grk1 on the Nrl
-/-
background exacerbates age-related cone dystrophy in a
light-independent manner, mediated partly through the inflammatory response pathway
leading to retinal neovascularization. Our working hypothesis is that Grk1 ablation in
xiii
cones leads to a hypoxic or metabolically compromised environment and subsequently
stimulates increased blood vessel penetration into the retina, leading to increased
inflammation and inevitable cone apoptosis. In addition to being essential for cone
pigment recovery, Grk1 expression maintains a healthy cone environment and provides a
model to examine potential cellular mechanisms of inherited disease associated with
retinal angiogenesis such as Age-related Macular Degeneration (AMD).
1
CHAPTER 1. INTRODUCTION
1.1 Retinal Degenerative Disease
Very few cubic millimeters of tissue in the human body exist that if lost, will lead
to a severe decrement in function or in quality of life. One such tissue is the retina.
Dysfunction or cell death of the neurons that form this highly organized structure lead to
severe disabilities, making scientific investigation into the molecular and biological
characteristics of such diseases crucial for therapeutic intervention and disease
prevention.
Retinal degeneration is the major cause of adult blindness in industrialized
countries and is characterized by the progressive cell death of retinal photoreceptors
(Wright et al., 2010). Photoreceptor degeneration is arguably one of the most genetically
heterogeneous disorders in man, constituting 185 loci, 146 of which have been identified
and seem to account for only half of all the monogenic subtypes (Hartong et al., 2006).
Inherited and multifactorial forms of retinal degeneration exist, and the two main vision
disorders most prevalent are Retinitis Pigmentosa (RP) and Age-related Macular
Degeneration (AMD).
RP is the most common subtype of inherited photoreceptor degeneration, and with
a prevalence of 1 in 3,500, is one of the two main causes of blindness in individuals 20-
64 years old (Buch et al., 2004). Patients with RP present with poor night vision due to
rod dysfunction followed by loss of the mid-peripheral visual field gradually leading to a
small central island of vision due to the preservation of the macula. The most common
form involves primary degeneration of rods followed by cone cell death; however, other
2
forms where cones are more severely affected than rods are also not uncommon (Wright
et al., 2010). Segregation analyses of families with RP were classified as autosomal
dominant, autosomal recessive, X-linked, or due to non-genetic factors, non-Mendelian
genetics, or complex inheritance (Wright et al., 2010). The most common single gene
defects that cause RP are within retinitis pigmentosa GTPase regulator (RPGR),
rhodopsin (RHO) and usherin (USH2A) (Wright et al., 2010). Mutations in the ATP-
binding cassette, subfamily A, member 4 (ABCA4) is the gene most commonly mutated
in inherited macular degenerations, causing autosomal-recessive juvenile macular
degeneration (Stargardt disease), cone-rod degeneration, or RP depending on the severity
(Wright et al., 2010). The genetic architecture of inherited photoreceptor degeneration
such as RP is composed of hundreds of rare alleles in a few hundred genes. The genetic
patterns have thus been analogized to a chain with many links, any of which can be
broken to predispose or cause photoreceptor degeneration.
The overall epidemiological impact of RP is dwarfed by another form of
photoreceptor dystrophy, Age-related Macular Degeneration, or AMD. A multifactorial
cause of photoreceptor degeneration, AMD accounts for more than half of all blindness
and visual impairment in developed countries (Ting et al., 2009). It is the leading cause of
severe vision loss in individuals over 60, and the prevalence rises exponentially with age
(Ting et al., 2009). Patients with AMD are categorized as having early (dry) or late (wet)
stage forms of the disease. Early AMD patients exhibit large, poorly demarcated drusen
and retinal pigment epithelia (RPE) abnormalities. They also have a substantial risk of
developing late AMD, which is characterized by local regions of RPE atrophy and
3
growth of new blood vessels from the choroid that penetrate through Bruch’s membrane
and enter the retina where they can leak and cause damage. Genetic association studies
on AMD patients identified Complement Factor H (CFH) and Age-related Maculopathy
Susceptibility 2 (ARMS) genes as contributors to AMD (Wright et al., 2010), with the
effect sizes of these two susceptibility alleles unusually large by the standard of most
complex traits. AMD is categorized as a complex disease, whereby a slow destruction of
a delicate non-renewing tissue takes places. Current therapeutic strategies for treating
AMD include the use of anti-VEGF medicines, which have been successful in improving
visual acuity, and current research in therapeutics for slowing the rate of disease
progression are currently underway.
Advancement in vision research suggests that the more we learn about the
pathophysiology and genetic basis of the photoreceptor degenerative diseases such as
AMD and RP, the more they will be viewed as disorders of homeostasis. To better
ascertain the characteristics of photoreceptor degeneration, the structure and anatomical
function of the mammalian retina must be well understood.
1.2 Structure and Function of the Mammalian Retina
The gift of sight is a manifestation of the highly regulated anatomic and
physiologic functions of the retina. This sophisticated sensory tissue is not only an
essential component to sight and the most accessible aspect of the brain for neuroscience,
but is truly unique for its ability to encapsulate a great deal of functional activity in a
small volume of neural tissue. The vertebrate retina is responsible for processing all
incoming visual signals and spatial information, orchestrated by a remarkably
4
collaborative effort of six different classes of neurons. A systematic and orderly layering
of photoreceptors, horizontal, bipolar, amacrine, ganglion, and inner plexiform cells
constitute these classes and together, work to transmit an external light stimulus into an
electrical impulse perceived by the brain as sight.
The retina is a component of the central nervous system (CNS) and in essence, is
an outcropping of the brain; made from the same components apart from the light
activated photoreceptors. Morphologically, the retina is inimitable, as the layers of
specialized neurons allow for signal transduction beginning at the outer segments of rods
and cones down to the nerve fiber layer where the optic nerve transmits the electrical
signal to the lateral geniculate nucleus of the brain relaying information to the visual
cortex (Massey, 2006). The layers of the retina are divided appropriately by cell body
and synaptic processes, and the depth within the retina often determines the synaptic
connections (Massey, 2006).
The outer nuclear layer (ONL) of the retina contains rod and cone photoreceptor
nuclei, while the inner nuclear layer (INL) consists of horizontal cells, bipolar cells and
amacrine cells responsible for trophic support, signal processing, and signal transmission
to the ganglion cell layer (Sancho-Pelluz et al., 2008). Radial glial cells (Müller cells) are
also located in the INL. Separating the ONL from the INL is the outer plexiform layer
(OPL) containing cone pedicles and fine dendrites and synapses where photoreceptors
signal to horizontal and bipolar cells. Directly anterior to the INL is the inner plexiform
layer (IPL), which contains the synapses where bipolar cells signal to amacrine and
ganglion cells. Below the IPL is the third nuclear layer, the ganglion cell layer (GCL),
5
housing amacrine and ganglion cells forming the output from the retina to the brain via
the optic nerve (Massey, 2006). The layered morphology of these neurons provides the
structural arrangement critical for adequate circuitry and signaling.
Figure 1.1: Structure of the Human Retina. (A) Schematic representation of the human eye in
which light passes through the pupil, lens and vitreous cavity before reaching the light-sensitive
retina. (B) Human retinal cross section demonstrating its highly structured and layered
morphology. 1) Ganglion cell layer (GCL) 2) Inner nuclear layer (INL) 3) the Outer nuclear or
photoreceptor layer (ONL) 4) Photoreceptor outer segment (OS) 5) Retinal pigment epithelium
monolayer (RPE). Light passes through the nerve fiber layer, inner retinal blood vessels and inner
cell layer before reaching the light-sensitive photoreceptors. (C) Higher resolution schematic of
the RPE and Bruch’s membrane. 1) Photoreceptor OS tips 2) RPE cells 3) Bruch’s membrane 4)
Choroidal capillaries. Reprinted with permission from Wright, A. F. et al., Nature Reviews. 2010.
A B
C
6
1.3 Photoreceptors: Rods and Cones
Within the ONL lie two types of photoreceptor cells, the rods and cones. These
specialized neurons produce graded electrical signals as a response to light stimuli at
unique wavelengths. Within a normal human retina, rods dominate in number,
constituting approximately 95% of the photoreceptors (Baylor, 1996) and there are
approximately 100-120 million in each human eye. Cones make up the remaining
photoreceptors but are just as powerful in function. Arranged like a mosaic, the rods and
cones are tightly packed in a strategic fashion; their location and interactions significantly
influencing their function within the retina.
1.3.1 Basic Photoreceptor Structure
Rods and cones have commonalities in structure that span the INL and IPL layers
of the retina. The outer segment (OS) of rods and cones house their respective light
sensing pigment molecules but have distinct shapes as evidenced by their names: cones
are larger and have a tapering OS, while rods are long and slender with a rod shaped OS.
Whereas the rod OS is made up of a stack of individualized disks unconnected to the
ciliary plasma membrane, the cone OS is a series of invaginations continuously
connected to the cilium plasma membrane (Figure 1.2) (Kevany and Palczewski, 2010).
The cell machinery for protein synthesis including the mitochondria and Golgi apparatus
can be found in the inner segment (IS). Separating the OS from the IS is the connecting
cilium, a short pathway of communication and protein transport (Rosenbaum and
Witman, 2002). The nuclear bodies of rods and cones are located in the ONL, however,
the cone nuclei primarily localize to the top row of the ONL (Massey, 2006). The nucleus
7
contains the primary photoreceptor genome and is responsible for the initiation of genetic
programs in the cell (Chen et al., 2006). Finally, the synaptic terminal of a rod is a small
spherule with only a single ribbon synaptic complex, while cone synapses are larger in
diameter and are characterized as pedicles with multiple ribbon synapses (Chen et al.,
2006). The synapses of both photoreceptors connect to the interneurons, horizontal and
bipolar cells, for further signal propagation.
Figure 1.2: Schematic of Rod and Cone Photoreceptors. Graphical representation of rod and
cone structures accompanied by EM images of rod and cone outer segments that display the
bands and invaginations of these photoreceptors. The OS contain the disks harboring light
sensitive opsin molecules while the energy and protein synthesis machinery are located in the IS.
Whereas rods are long and thin, cones are shorter but larger and their nuclei localize to the upper
portion of the ONL. Mustafi. Prog Retinal Eye Res, 2009 as adapted in Kevany, Physiology,
2010, used with permission.
8
1.3.2 Functional Variability
Even though they harbor similar pathways of light induced activation, rods and
cones are quite distinct in their function, with rods providing the primary source of visual
information at night (scotopic) and cones for high acuity color vision during the day
(photopic). The rod photoreceptors are large in number and extremely efficient at
detecting light; a mechanism initiated by the light sensitive visual pigment rhodopsin, a
G-protein coupled receptor that mediates a complex set of events leading to visual
responses. Most of the expressed rhodopsin is directed into the membrane-bound
lamellae or discs within the OS, the site for photon capture and phototransduction
initiation (Figure 1.3). Rhodopsin molecules are sensitive to blue-green light with peak
sensitivity wavelength at around 500nm. Despite their high level of sensitivity, rods are
not sensitive to color and exhibit relatively low spatial acuity.
The remaining photoreceptors are cones, and though less prevalent, mediate
vision at much higher light intensities than rods and recover sensitivity faster after light
flashes that generate similar membrane currents (Baylor et al., 1979;Perry and
McNaughton, 1991). Cones are not evenly distributed throughout the human retina, with
the greatest density located in the fovea providing maximum visual acuity.
Approximately 5% of photoreceptors in the human retina are cones, while mouse retinas
possess 2-3%. Therefore, in terms of absolute numbers, mouse and human retinas are
similarly rod dominant. Three flavors of human cones exist, each detecting different
wavelengths of the visual spectrum depending on the type of opsin they express: long (L)
(564nm), medium (M) (533nm), or short (S) (437nm) (Baylor, 1996). Mouse cones only
9
express S (359nm) and M (510nm) opsins, and while many cones express both, a greater
level of M opsin is expressed in comparison to S opsin in the mouse retina (Applebury et
al., 2000).
1.4 Phototransduction Activation
Visual perception is a function of highly regulated molecular mechanisms that
originate in rod and cone photoreceptors. Together, rods and cones participate in the
cascade of phototransduction; the process by which light is captured by a visual pigment
molecule to generate an electrical response. Phototransduction begins in the OS, which
are tightly packed with membrane discs full of the visual pigment rhodopsin in rods and
S, M, and L opsin in human cones.
Phototransduction begins when a photon of light excites the rhodopsin molecule
in the form of photo-isomerization of 11-cis-retinal to the activated all-trans-retinal
isomer (Tomita, 1970). The active form of rhodopsin then binds to the G-protein
transducin, through which guanosine diphosphate (GDP) is exchanged for guanosine
triphosphate (GTP) on the guanine-nucleotide binding site of the α subunit of transducin.
The activated transducin molecule then dissociates from the partnering βγ subunits and
binds to the phosphodiesterase (PDE) γ subunit. As shown in Figure 1.3, both transducin
and PDE are peripheral proteins located in the membrane disks. Because there are two
PDEγ subunits per PDE tetramer, two activated transducin molecules are required per
PDEαβ activation. The binding of activated transducin to PDEγ releases the catalytic
PDEαβ subunit, allowing it to hydrolyze cGMP to GMP, thereby closing the cyclic-
nucleotide-gated (CNG) channels that are open during the dark (Yau and Hardie, 2009).
10
Prior to light activation, the concentration of free cGMP is high, and by direct binding,
maintains the cGMP channels in the open state allowing a high concentration of calcium
(Ca
2+
) ions to cause membrane depolarization sustaining the synaptic release of the
glutamate transmitter. Following light activation, a graded decrease in the level of free
cGMP results in the closure of the cGMP gated channels, causing hyperpolarization and
stimulation of an action potential (Shichida and Imai, 1998).
Figure 1.3: Phototransduction in Vertebrate Rods. Light converts rhodopsin into an active
form, Rh*, which activates the heterotrimeric G protein transducin (G
t
) by GTP-GDP exchange.
Active Gta (Gtα*) binds to and activates phosphodiesterase (PDE), which hydrolyzes cyclic GMP
(cGMP), thereby closing the cyclic-nucleotide-gated (CNG) channels that are open in the dark.
Inset: schematic diagram of the ciliary rod photoreceptor, with a light-sensitive outer segment
formed from a highly expanded cilium. Adapted with permission from Yau, K.W. and Hardie,
R.C. Cell. 2009.
1.5 Phototransduction Recovery
Although light exposure is critical for proper photoreceptor function, continuous
light exposure can produce apoptotic cell death by activating the transduction cascade too
strongly and for too long (Fain, 2006). Therefore, in a normal photoreceptor, once the
11
phototransduction cascade is activated by light, it must be turned off. The deactivation of
phototransduction is a complex pathway that is currently under investigation. In order to
reach complete deactivation, each active component must be shut down, including
rhodopsin and the cone opsins. Activated rhodopsin must first be phosphorylated via G-
protein coupled receptor kinase 1 (Grk1), followed by the binding of a member of the
arrestin family, Arrestin 1 (Arr1), which recognizes the phosphorylated form of
rhodopsin. Eventually, the Arr1 is released and rhodopsin is dephosphorylated, most
likely through the activation of a generic phosphatase such as protein phosphatase 2A
(PP2A) (Brown et al., 2002;Palczewski et al., 1989). Next, transducin must be
deactivated through the hydrolysis of GTP to GDP. Finally, PDE activity must be
terminated by reassociation of the inhibitory γ-subunit with the catalytic αβ-subunits
(Figure 1.4) (Hurley and Stryer, 1982;Wensel and Stryer, 1986)
Figure 1.4: Schematic of Disk Proteins Associated with Phototransduction Recovery. The
deactivation reaction of Grk1 phosphorylation of activated rhodopsin followed by Arr1 binding is
shown in the top disk. The bottom disk represents GTP hydrolysis and deactivation of transducin
and PDE. Adapted with permission, Burns, M. and Pugh, E. Physiology, 2010.
12
1.5.1 G-protein Coupled Receptor Kinase 1 (Grk1)
G-protein coupled receptors (GPCRs) are crucial links in the relay of information
from extracellular stimuli to an intracellular response. The superfamily of these seven-
transmembrane spanning receptors contains several hundred members and function in
light and olfactory stimuli, as well as hormone and neurotransmitter signaling (Lohse et
al., 1996). The mechanism by which this crucial signaling mediator is regulated is
through phosphorylation by G-protein coupled receptor kinases (Grks), which are
stimulated by the activated heptahelical receptor and cause their uncoupling from G-
proteins (Shichi and Somers, 1978). The family of Grks consists of serine/threonine
kinases that provide the most specific and rapid mechanisms for desensitizing GPCRs;
and perhaps the most well studied GPCR desensitization signaling is that of rhodopsin
and its kinase, Grk1.
G-protein coupled receptor kinase 1 (Grk1) is referred to as the founding member
of the Grk kinase family, which includes Grk2 (β-adrenergic receptor kinase 1) and Grk3-
7 (Singh et al., 2008). All Grks share a kinase domain similar to those of protein kinase A
(PKA), G, and C (the AGC kinases) and include the C terminal extension characteristic
of this kinase family (Singh et al., 2008). Grk1 is a single subunit polypeptide with a
molecular weight of 63kDa, has been shown to loosely associate with the membrane of
rods and cones, and can be present in the cytosol. Grk1 phosphorylates multiple sites in
the C-terminal tail of rhodopsin but only when rhodopsin is in its active state. This
interaction stimulates kinase activity against peptide substrates up to 160-fold, thereby
13
rapidly turning over activated rhodopsin to the inactive state and allowing signal
transduction to reinitiate.
Grk1 along with the related species specific Grk7, is primarily found in the human
retina and pineal gland and regulates the opsin light receptors (Weiss et al., 2001). Mice
do not express Grk7 in their retinas, therefore Grk1 is solely responsible for the light-
dependent phosphorylation of rhodopsin in rods, and both S and M opsin in mouse cones.
Genetic knockout mice lacking Grk1 expression display profoundly slowed recovery of
cone photoresponses and a light-induced photoreceptor degeneration, suggesting that
Grk1-dependent opsin phosphorylation is essential for the shutoff of cone
photransduction in the mammalian retina (Nikonov et al., 2005;Weiss et al., 1998;Zhu et
al., 2003).
The absence of GRK1 or ARR1 in human populations has been linked to a
recessive genetic disorder, Oguchi disease, a form of RP characterized by congenital
stationary night blindness (Zhao et al., 1998). Interestingly, studies on patient populations
with enhanced S cone syndrome caused by mutations in NR2E3 demonstrate normal
deactivation kinetics in L and M cones without GRK7 and with GRK1 present, but
abnormal response when GRK1 is absent (Cideciyan et al., 2003). This indicates an
alternative function for GRK1 in cones, which may be critical to their survival and
homeostasis. Recent studies on genetic knockout mice for Grk1, Rpe65, and Gnat1
demonstrate a light-independent mechanism for retinal degeneration in the absence of
Grk1, and suggest a second not previously recognized role for this kinase (Fan et al.,
2010). Taken together, current research conducted using the Grk1
-/-
mouse and human
14
population studies on Oguchi disease indicate an important novel functional role for Grk1
in the retina.
1.5.2 Arrestin 1 (Arr1)
The phosphorylation of GPCRs by Grks increases the affinity of the receptor
towards systolic inhibitory proteins known as Arrestins (Arr), leading to the uncoupling
of the receptors and G-proteins and terminating the signaling pathway that triggers the
cellular response. Visual Arrestin, or Arrestin 1 (Arr1) was the first member of the
Arrestin family identified and exhibits significant specificity to light-activated and
phosphorylated rhodopsin thereby inhibiting the activation of transducin (Kuhn and
Wilden, 1987). Genetic knockout mice for Arr1 demonstrate a similar degenerative
phenotype caused by enhanced photoreceptor activation as observed in Grk1 null animals
and are also used as a model to study Oguchi disease (Chen et al., 1999b).
The mammalian Arrestin family is composed of four members: the visual
Arrestins, Arrestin 1 (referred to as rod Arrestin, S-antigen, or the 48kDa protein) and
Arrestin 4 (also known as cone arrestin or X arrestin) and the non visual Arrestins,
Arrestin 2 (or β-Arrestin1) and Arrestin 3 (or β-Arrestin2) (Chen et al., 1999b;Craft and
Whitmore, 1995). Despite the variation in sensory and non-sensory roles, Arrs share a
sequence structural homology; each harboring two receptor-interacting domains, an N-
terminal domain and a C-terminal domain connected by a polar hinge region (Kendall
and Luttrell, 2009). Whereas Arr2 and 3 are ubiquitously expressed and interact with a
plethora of other GPCRs, visual Arr1 and 4 have restricted expression patterns, localizing
mostly to the visual and sensory photoreceptors and pinealocytes (Craft et al., 1994).
15
Arr1 is the only Arr expressed in rods; however Craft and coworkers (Zhu et al., 2005)
discovered a shared Arr4 and Arr1 expression in cone photoreceptors. Recent
characterization of the dual Arr expression in cones is theorized as an evolutionary
specialization of genome duplication events (Nikonov et al., 2008). Arr1 and Arr4 are
therefore both important players in mediating the desensitization mechanism of
phototransduction and maintenance of healthy vision.
Interestingly, the most recent work by Craft’s laboratory group (Brown et al.,
2010) demonstrates a light-independent cone photoreceptor degeneration in Arr1
-/-
mice,
contrary to what has been previously shown in these models when reared in complete
darkness (Chen et al., 1999b). Close inspection of their photoreceptor morphology and
apoptosis staining patterns revealed that Arr1 is important for cone photoreceptor
survival and light adaptation. It is thought that the cone photoreceptor degeneration in
Arr1
-/-
mice is due in part to the calcium overload and oxidative stress, possibly due to an
enhanced metabolic rate and ATP demand (Brown et al., 2010). In light of this recent
discovery, current research is in progress to examine the causative mechanisms behind
the light-independent degeneration in these mice.
Transgenic and knockout animal technologies have become powerful tools for the
understanding of eye disease and the relevance of particular genes with respect to retinal
health. The Arr1
-/-
and Grk1
-/-
mice are thus invaluable tools for the characterization and
functional analyses of these critical phototransduction recovery proteins, both in light
mediated and light independent environments. It is evident that these animal models will
16
continue to be utilized as means for understanding human disease and in the design of
potential therapeutic strategies.
1.6 The Neural Retina Leucine Zipper Knockout Mouse (Nrl
-/-
)
Despite the well characterized physiologic and biochemical differences known
between rod and cone photoreceptors, the complete understanding of cone molecular
determinants and function were once rudimentary. This deficit in knowledge was a
consequence to the fact that all well-studied mammalian retinas are rod dominant, with
only a small percentage of cones decorating the retinal photoreceptor mosaic. With this
in mind, the Neural Retina Leucine Zipper (Nrl) knockout mouse was created as a
potential solution to the problem of cone photoreceptor scarcity. In 2001, Swaroop and
coworkers published work on the Nrl knockout mouse and reported that deletion of the
Nrl transcription factor in mice results in the complete loss of rod function and enhanced
cone function, mediated primarily by S cones (Mears et al., 2001). Morphologic,
electrophysiological, and biochemical studies on Nrl
-/-
mice confirmed that their rod
precursors differentiated as cones instead of rods. The end result was a pure cone retina
currently used extensively in cone function studies.
Despite their beneficial research applications, Nrl
-/-
retinas are characterized to
display morphologic distortion, with whorls and rosettes in their ONL believed to be
secondary to the presence of an abnormally high number of cones (Corbo et al., 2007).
The majority of the cones in these mice express enhanced levels of S opsin and normal
levels of M opsin, a consequence of the transcriptional developmental function of Nrl. By
approximately 8 months, Nrl
-/-
mice were shown to lose the rosette and whorl structures,
17
and exhibit a thinning of the ONL, a degeneration similar to the slow photoreceptor cell
death observed in the rd7 mouse (Akhmedov et al., 2000). Ultrastructural studies on the
Nrl
-/-
retina show considerably shorter and fewer cone outer segments, as well as
abnormal disk morphologies believed to be due to the lack of structural support provided
by rods (Figure 1.5). It is also important to note that the Nrl
-/-
OS discs were often
misaligned and abnormally associated with the retinal pigment epithelium (RPE) (Mears
et al., 2001). Initial characterization of the Nrl
-/-
mouse also identified the transcription
factor to be upstream of Nr2e3 during photoreceptor development (Oh et al., 2008b).
18
Figure 1.5: Light Microscopy and Ultrastructural Analysis of WT, Nrl
+/-
and Nrl
-/-
Mice.
Retinal sections from 5 week old mice were counter stained with toluidine blue (a,b,c). Whereas
Nrl
+/-
retinas appear normal (b), Nrl
-/-
mice have rosettes and an abnormal ONL. Electron
micrographs of WT (d), and Nrl
-/-
retinas (e) (magnification x 1,950). Some cones from Nrl
-/-
mice have normal discs, while others are disorganized (f). Outer segments of WT (g) and Nrl
-/-
(h)
(magnification x25,000). Adapted with permission, Mears, A.J. et a.l, Nature Genetics, 2001.
The Nrl
-/-
phenotype most closely resembles the human condition Enhanced S-
Cone Syndrome (ESCS), a night blindness with an autosomal recessive hereditary
pattern. ESCS is caused by a mutation in the NR2E3 gene and patients have
photoreceptor mosaics dominated by S cones and fewer L and M opsin expressing cones.
In ESCS, the macula of patients are normal and often show cystoid changes and is so
19
named due to the enhanced cone ERG response from short wavelength stimuli and a non-
recordable rod ERG (Nakamura et al., 2002). Despite the morphologic abnormalities
associated with Nrl
-/-
retina, it continues to represent a useful tool in the discovery of
cone genes and protein function studies.
Figure 1.6: A Model of Photoreceptor Differentiation in Mice. Retinal progenitor cells (RPCs)
pass through different stages of competence during development. Post mitotic cells (PMCs)
commit to either a cone or rod fate in response to extrinsic stimuli and transcription factors. In
response to thyroid hormone receptor β2 (Trβ2), some PMCs that commit to cone cell fate
become M cones. Factor X is a yet unidentified molecule that directs gone progenitors down the
S cone pathway. Another pool of PMCs acquire competence to an S cone fate or a rod cell fate.
The Nrl transcription factor drives PMCs to become rods. Nr2e3 is responsible for suppressing
the S cone pathway and Nrl may work in concert with Nr2e3 and Crx to drive rod development.
Adapted with permission, Mears, A.J. et al., Nature Genetics, 2001.
20
1.7 Choroidal and Retinal Vasculature
When normalized for its weight, the retina is the most oxygen consuming tissue in
the body, with a consumption level 50% greater than the brain and kidney (Rattner and
Nathans, 2006). The demands of this heavy metabolic load are met by the blood supply
provided via the choroidal and retinal circulation. An encapsulation of the mammalian
retina, the choroid is a vascularized and pigmented tissue that forms the posterior part of
the uveal tract and consists of four layers: the suprachoroid, stroma, choriocapillaris, and
Bruch’s membrane. Together, these layers form a firm connection to the retinal pigment
epithelia (RPE) and the choroidal vascular system provides nourishment not only to the
choroid itself, but to the RPE and the retina up to the outer aspect of the inner nuclear
layer INL (Ring and Fujino, 1967).
The components of each of the choroidal layers are diverse. The suprachoroid
consists of a plethora of cell types and a variety of fibers, yet despite this, no blood
vessels are present in this layer aside from those that pass through. The stromal layer does
however harbor vascularization along with melanocytes, fibroblasts, collagen and nerve
fibers. Although both arteries and veins are present in this layer, the arteries anastomose
gradually and decrease in size as they divide to form the choriocapillaris (Guyer et al.,
2006). The choriocapillaris layer exhibits a unique structure that is critical for the health
and function of the retina. The oxygen and energy demands of the retina are so high that
over 70% of all the blood in the globe at any one time can be found in the choriocapillaris
(Parver et al., 1980). With large capillaries (40-60μm) and rather thin walls, the blood
vessels composed of endothelial cells allow two to three blood cells to pass through at
21
any given time (Guyer et al., 2006). Separating the choriocapillaris from the RPE layer is
Bruch’s Membrane, a thin (1-4μm) layer of loosely arranged collagen mesh along with
components of the RPE and choriocapillaris (Guyer et al., 2006). Blood vessels that
penetrate through Bruch’s Membrane from the choriocapillaris layer can cause serious
vision complications and defects such as those found in the wet form of Age-related
Macular Degeneration (AMD).
The choroid is not the only source of nutrients and oxygen supply to the retina.
The retinal vascular anatomy provides direct blood supply to the inner two-thirds of the
human retina, a “holangiotic” vascular pattern similarly found in mice, rats, and other
mammals (Guyer et al., 2006). The retinal blood vessels are independent from the
choroidal vasculature and permeate from the internal limiting membrane (ILM) to the
inner nuclear layer (INL) (Campochiaro, 2000). Whereas arteries and veins are found in
the nerve fiber layer, the arterioles and venules extend deeply into the layers of the retina
forming microvascular networks; which include 1) superficial capillaries in the ganglion
cell and nerve fiber layers and 2) deep dense capillaries in the INL (Toussaint et al.,
1961).
22
Figure 1.7: The Structure of the Human Eye and Retina. (A) Cut away view of the eye, with
the cornea at the bottom and the optic nerve at the top. (B) Schematic diagram of the retina and
choroid (CH) showing the major cell types. The choroidal vasculature is at the top and the inner
retina is at the bottom. Vasculature of the inner retina is shown on the lower right. Arrows
indicate the local diffusion of oxygen, nutrients and waste products between choroidal
vasculature and the outer retina (upper arrows), and the retinal vasculature and the inner retina
(lower arrows). Adapted with permission, Rattner, A. and Nathans, J. Nature Reviews, 2006.
1.8 Choroidal and Retinal Neovascularization
Neovascularization is the formation of new blood vessels in previously avascular
tissue by way of both vasculogenesis and angiogenesis (Qazi et al., 2009).
Vasculogenesis is the process by which endothelial cells differentiate from precursor
cells and angioblasts already present in the tissue to form vessels, while angiogenesis
occurs from the sprouting of preexisting blood vessels that invade surrounding tissue
(Campochiaro, 2000). In retinal degenerative disease models, neovascularization is the
process by which the choroid and retina become infiltrated with new blood vessels. The
A B
23
two mechanisms by which this occurs are choroidal neovascularization (CNV) and retinal
neovascularization (RNV).
Figure 1.8: Schematic Representation of Choroidal and Retinal Neovascularization. (A)
Artist’s rendition of CNV. Note the breaks in Bruch’s membrane and penetration into the retina
from the choroidal vasculature. (B) Artists rendition of RNV. Blood vessel penetration occurs
from the ILM and into the retinal layers. Adapted with permission, Campochiaro, Journal of
Cellular Physiology, 2000.
1.8.1 Choroidal Neovascularization (CNV)
Choroidal neovascularization (CNV) is characterized as a non-specific response
to a specific stimulus (Grossniklaus and Green, 2004) whereby new blood vessels
emanate from the choroid into the subretinal pigment epithelium, subretinal space, or
both. This leads to the formation of neovascular membranes, which include vascular
endothelial cells, RPE cells and macrophages (Das and McGuire, 2003). Angiogenesis
and vasculogenesis in the form of CNV are important pathobiologic mechanisms
encountered in a variety of chorioretinal diseases. Many postmortem CNV studies
demonstrate that CNV is a stereotypic nonspecific wound repair response that develops
with chronic inflammation of the RPE, Bruch’s membrane and choriocapillaris (Das and
McGuire, 2003).
24
The pathobiology of CNV begins with a break or defect in Bruch’s membrane,
which can be caused by a trauma, degenerative process, or inflammation (Penfold et al.,
1984). When this occurs, choriocapillary endothelial cells, pericytes, fibrocytes and
inflammatory cells are introduced into the sub-retinal pigment epithelium and/or
subretinal space. Inflammatory, angiogenic and extracellular matrix components exist
together in the microcosm where they compete to induce or suppress CNV. The
combination of endothelial cell growth and coverage by pericytes surrounded by the
ECM is mediated by vascular endothelial growth factor (VEGF) and platelet derived
growth factor (PDGF), which appear essential for CNV formation (Campochiaro, 2000).
Figure 1.9: The Retinal Pigment Epithelium, Bruch’s Membrane and the Choroid. (A)
Electron micrograph of mouse retinal pigment epithelium (RPE) and choroid (CH). The choroidal
vasculature is at the top and photoreceptor outer segments (OS) are at the bottom. The apical face
of the RPE cell is covered with microvilli that contact the outer segments. On the basal face of the
RPE a highly folded plasma membrane faces the choroid. Numerous melanin granules (black) are
seen in the RPE cell; the nucleus is at the far left. (B-F) Schematic illustration of the RPE and
Bruch’s membrane (BM) histopathology associated with various macula degenerative processes.
(B) Normal retina and RPE (C) the TPE with lipofuscin accumulation (D) A druse sandwhiched
between the RPE and Burch’s membrane (E) A druse with adjacent geographic RPE atrophy and
loss of overlying photoreceptors (F) Choroidal neovascularization. IS, inner segment. Adapted
with permission, Rattner, A. and Nathans, J. Nature Reviews, 2006.
Several animal models currently exist for studying CNV, including laser induced,
surgically induced and transgenic knockout mouse models. Experimental laser induced
A B C D E F
25
mice were created using laser spot treatments from a krypton laser to create
photocoagulation injuries to Bruch’s membrane, which have been performed in monkeys,
rats and mice (Ryan, 1979; Tobe et al., 1998). The laser induced model is currently a well
established method used to study CNV pathogenesis and is also used in pre-clinical trials
for the study of anti-angiogenic drugs. Although the physical insult induced by the laser
of Bruch’s membrane differs from the long-term chronic conditions that occur in human
AMD, the laser induced CNV models closely mimic natural cellular responses that occur
in human CNV. Surgically induced forms of CNV are also currently in practice and are
done primarily by the injection of synthetic peptides, viral vectors containing VEGF, and
inert synthetic materials. Many of these models lead to inflammatory destruction of
Bruch’s membrane and subretinal neovascularization.
The most relevant models of CNV for this work are those found in transgenic
knockout mice where CNV is a phenotypic distinction. One such model is the monocyte
chemoattractant protein-1 (Ccl2) or its receptor CC-chemokine receptor-2 (Ccr2)
deficient mouse, which are both current models for AMD (Ambati et al., 2003). These
transgenic mice lacking either Ccl2 or Ccr2 fail to recruit macrophages to the RPE or
Bruch’s membrane, thereby allowing the accumulation of complement factor C5a and
IgG, both of which induce VEGF production (Ambati et al., 2003). Other transgenic
knockout mice that exhibit a CNV phenotype include mice that over express
Apolipoprotein E (ApoE) that were fed a high fat diet and developed AMD like lesions
(Malek et al., 2005). Disruption of ceruloplasmin and hephaestin in mice causes iron
overload and subsequent AMD-like changes as well. These mice develop RPE
26
abnormalities and photoreceptor degeneration (Hahn et al., 2004). Knockout mice for the
very low density lipoprotein receptor gene (Vldlr
TM1Her
) develop new blood vessels in the
OPL of the retina as well as choroidal anastomoses by 3 months (Heckenlively et al.,
2003). Finally, the Cu, Zn superoxide dismutase deficient mouse (Sod
-/-
) has been shown
to exhibit fundus and histological evidence of CNV in approximately 10% of Sod
-/-
mice.
Currently there is no perfect animal model for CNV, however those that are
available to date indicate that VEGF over expression alone is not enough to develop
CNV, and that damage to Bruch’s membrane must also occur. Research on animals using
either laser induced, surgically induced or transgenic models for CNV have both
comparable characteristics to human disease and limitations of the model itself.
Nevertheless, CNV studies are crucial to understanding the mechanism behind diseases
such as AMD and will continue to provide avenues for potential therapeutic targets.
1.8.2 Retinal Neovascularization (RNV)
The architecture of the retinal blood supply renders it possible to have two types
of neovascularization, choroidal and retinal. In RNV, sprouting retinal vessels penetrate
the ILM and grow into the vitreous, and in some cases, grow the other way through the
avascular outer retina into the subretinal space (Campochiaro, 2000). Numerous clinical
and experimental observations indicate that ischemia (or hypoxia) is the driving force
behind RNV (Michaelson and Steedman, 1949). Occlusion of retinal vessels leading to
ischemia is a feature of diseases with RNV, including diabetic retinopathy, retinopathy of
prematurity, central retinal vein occlusion, and branch retinal vein occlusion; diseases
collectively referred to as ischemic retinopathy (Campochiaro, 2000)
27
Animal models for the study of RNV include hypoxia-induced, vascular
occlusion, transgenic mouse models, and intraocular injection of pro-angiogenic
molecules. Hypoxia induced models have been developed in several species and is
performed by exposing mice to hyperoxia, then placing them in normoxic conditions,
causing an ischemic situation which initiates rapid abnormal neovascularization. This
model directly correlates to retinopathy of prematurity (ROP) (Ashton, 1966). Occlusion
of retinal veins by laser photocoagulation or photodynamic therapy has also been used in
RNV studies (Ham et al., 1997). These animal models closely mimic the clinical
characteristics of branch retinal vein occlusion, leading to reduced blood flow, retinal
ischemia and neovascularization.
Transgenic mouse models for RNV provide an adequate resource for the
characterization of this phenomenon. One example is the rho/VEGF transgenic mouse,
which develop retinal and subretinal neovascularization as a consequence of VEGF
expression driven by the rhodopsin promoter (rho/VEGF). The over expression of VEGF
in the retina of these mice leads to new vessel formation originating from the retinal
vasculature and extending into the subretinal space. This model is a close representation
of patients with retinal angiomatous proliferation (RAP) (Miller, 1997). The development
of VEGF
165
over expressing transgenic mice driven by the truncated rhodopsin promoter
developed phenotypes ranging from mild to severe RNV and are also used in RNV
studies (Miller, 1997).
Alongside intraocular injection of pro-angiogenic molecules and inflammatory
responses, the RNV models listed here are well established when compared to CNV
28
models. Ischemia is the main culprit for development and the disease processes
characterized by RNV, such as diabetic retinopathy, ROP, and central retinal vein
occlusion. These models are well characterized and will continue to be used in the study
of such diseases and for the improved understanding of RNV.
1.9 Summary
The retina is a remarkable network of interconnected specialized neurons that
together orchestrate complex yet highly organized electrical signals that are processed by
the brain to achieve visual perception. Experiments on animal models with retinal
dystrophy have made it increasingly apparent that the delicate balance of genetic and
proteomic communication is extremely important in maintaining healthy visual function.
Despite their similarities, the rod and cone photoreceptors are unique in their shape and
function, and the mosaic structure of these photoreceptors is one of the characteristic
dictators for vision differences between animal species. Mice, for example, have different
opsin expressing cones when compared to humans and lack a defined macula, however
they harbor similar morphology and neuronal structures that are extremely comparable to
the human retina and are therefore extensively used in many human disease studies.
Retinitis Pigmentosa (RP) and Age-related Macular Degeneration (AMD) are two of the
leading forms of retinal dystrophy in human populations, and mouse models are
becoming increasingly useful in identifying causative mechanisms and therapeutic targets
to protect against human diseases of the eye.
29
CHAPTER 2. MATERIALS AND EXPERIMENTAL PROCEDURES
2.1 Loss of G-protein Coupled Receptor Kinase (Grk1) in Nrl
-/-
Mice
2.1.1 Experimental Animals
All mice were treated according to the guidelines established by the Institute for
Laboratory Animal Research and the Association for Research in Vision and
Ophthalmology (ARVO) Statement for Use of Animals in Ophthalmic and Vision
Research. The IACUC of the University of Southern California approved all procedures
involved in animal experiments. Control mice used for all experiments were C57Bl/6J
(C57) mice that were born and maintained in 12hr light:12hr dark cyclic light.
Knockout animals Nrl
-/-
(provided by Anand Swaroop) (Mears et al., 2001)and
Grk1
-/-
mice (provided by C.-K. Jason Chen) (Chen et al., 1999a) were created from a
mixture of C57Bl/6J and 129/SvJ founders and are therefore on a similar mixed genetic
background. Nrl
-/-
Grk1
-/-
mice were created by backcrossing Nrl
-/-
mice with Grk1
-/-
mice
as described previously (Zhu et al., 2003). Nrl
-/-
and Nrl
-/-
Grk1
-/-
mice were born and
maintained in either total darkness, ambient light (5 lux white light measured at the cage
level) 12hr light:12hr dark cyclic light, or constant bright light (approximately 8000 lux
white light measured at the cage level).
2.1.2 Electroretinography (ERG)
ERGs were recorded as previously described (Zhu et al., 2002; Zhu et al., 2005;
Zhu et al., 2006). Nrl
-/-
and Nrl
-/-
Grk1
-/-
mice maintained in total darkness, ambient (~5
lux) cyclic light or bright (~8000 lux) constant light were recorded at 1, 3, 5, 7 and 9
months of age. At least 10 mice were recorded for each genotype at each age. The Nrl
-/-
30
and Nrl
-/-
Grk1
-/-
mice were dark-adapted overnight and their eyes were dilated with
topical administration of phenylephrine (2.5%) and tropicamide (0.5%). Mice were
anesthetized via an intraperitoneal injection of ketamine HCl (100 mg/kg body weight)
and xylazine HCl (10 mg/kg body weight), and the cornea anesthetized with 0.5%
tropical tetracaine. Each mouse was placed in an aluminum foil-lined Faraday cage and a
DLT fiber electrode was placed on the right cornea. A platinum reference electrode was
placed on the lower eyelid and another ground electrode on the ipsilateral ear. Photopic
stimuli of 10μs duration of a maximum light intensity (log 2.01 cd s/m2) were delivered
through one arm of a coaxial cable using a Grass PS22 xenon flash. The cable delivered
the flash 5mm from the surface of the cornea and a background light (200cd/m
2
), with
spectral peaks at 485, 530, 543nm and minimal transmission below 400nm, was used.
Dark-adapted maximum responses were measured using the non-attenuated light
stimulus. The b-wave amplitude was measured from the trough of the a-wave to the peak
of the b-wave.
2.1.3 Eyecup Embedding and Sectioning
All mice were euthanized by overdose of carbon dioxide inhalation either at mid-
day under fluorescent lights or in total darkness. Retinas were dissected either under
room light using a dissecting scope or in total darkness with infrared goggles and an
infrared equipped dissecting microscope. Eyes from each genotype and age examined
were enucleated, marked for orientation (if in the light) and fixed with 4%
paraformaldehyde in phosphate buffered saline (PBS) for at least 2 hours. Each eyecup
was washed twice in PBS for 5 minutes then dehydrated in a solution of 30% (wt/vol)
31
sucrose in PBS overnight at 4ºC. Each eyecup was then embedded in OCT media (Sakura
Fineteck, Torrance, CA) (Zhu and Craft, 2000a) and frozen in liquid nitrogen. Retinal
cryosections at 8μm thickness were cut along the vertical meridian through the optic
nerve and were placed on poly (L-lycine)-coated glass slides.
2.1.4 Retinal Histology
In order to examine the morphological changes associated with a decrease in b-
wave amplitude in Nrl
-/-
Grk1
-/-
mice in comparison to Nrl
-/-
controls, six mice of both
genotypes at 1, 5 and 9 months of age were used. Because the decrease in cone function
by ERG analysis is light independent, retinal sections used in Hematoxylin and Eosin
(H&E) studies were from mice born and maintained in ambient cyclic light.
Briefly, retinal sections were pre-rinsed in double distilled water (ddH
2
0) then
dipped in Harris Hematoxylin for 45 seconds followed by a wash in ddH
2
0. Slides were
subsequently dipped in acid alcohol (70% ethanol 1% HCl) for 1 minute, washed in
ddH2O, then dipped 5 times in ammonia water (0.3% NH4OH) and washed. Slides were
then dipped in Eosin-Phloxyine for 1 minute, then dehydrated in a series of 95% ethanol
and 100% ethanol followed by 6 minutes in Xylene. Once the slides were dried,
mounting medium was applied and slides were cover-slipped.
2.1.5 Immunohistochemistry (IHC)
Immunohistochemistry (IHC) is a well established technique for the identification
and localization of proteins expressed within the mouse retina. For all
immunohistochemistry experiments, our established protocol was followed with minor
modifications (Zhu and Craft, 2000a). Frozen retinal sections of Nrl
-/-
and Nrl
-/-
Grk1
-/-
32
mice were blocked in IHC blocking buffer (10% normal goat serum, 1% bovine serum
albumin (BSA) and 0.2% Triton X-100 in PBS) for 30 min, then incubated with the
mouse anti-Arrestin4 (Arr4) rabbit polyclonal antibody (pAB mCAR-LUMIJ) or the
mouse anti-S or M-opsin peptide rabbit polyclonal antibodies as described previously
(Zhu et al., 2003). All three antibodies were diluted to 1:1000 in PBS and incubated for 1
hour at room temperature. Animals were similarly stained using the mouse anti-VEGF
antibody (Abcam) at a dilution of 1:200 in PBS incubated overnight at 4°C. After three 5
minute washes in PBS, the slides were probed with pAB mCAR-LUMIJ and pAB S- or
M-opsin were incubated in fluorescein anti-rabbit IgG (1:200; Vector Laboratories) for 1
hour at room temperature. Slides were either mounted with Vectashield Mounting
Medium for fluorescence with DAPI (Vector Laboratories) or incubated with the
monomeric cyanine nucleic acid stain To-Pro®3 iodide (1:2,000 Invitrogen) and mounted
with Vectashield Mounting Medium and cover-slipped.
Slides prepared for IHC or H&E were photographed with a digital camera (SPOT
Model SP401-115, software version 3.5.0, Diagnostic Instruments, Inc., Sterling Heights,
MI) mounted to a Leica DMR Fluorescent Microscope (Leica Microsystems, Wetzlar
Gmbh, Wetzlar, Germany) (Zhu et al., 2005).
2.1.6 TUNEL Analysis of Apoptosis
Functional cone photoreceptor deficit in Nrl
-/-
and Nrl
-/-
Grk1
-/-
mice was evident
by 3 months; therefore, in order to verify cone apoptosis prior to outer nuclear layer
(ONL) thinning, five 1 month old mice from control C57Bl/6J, Nrl
-/-
and Nrl
-/-
Grk1
-/-
genotypes each and three post natal (PN) day 21 mice from C57Bl/6J, Nrl
-/-
and Nrl
-/-
33
Grk1
-/-
genotypes were born and maintained in ambient cyclic light and euthanized in the
light. Both eyes were fixed and embedded as described above for retinal histology. Three
adjacent sections cut through the optic nerve along the vertical meridian were used from
each eye. Apoptotic cells were visualized by means of the terminal deoxynucleotidyl
transferase-mediated dUTP nick-end labeling (TUNEL) assay using the DeadEnd
TM
Fluorometric TUNEL System (Promega), following the manufacturer’s instructions (Li et
al., 2003). After labeling, the slides were mounted with Vectashield Mounting Medium
for fluorescence with DAPI nuclear stain (Vector Laboratories). Apoptotic cells were
quantified by counting TUNEL positive cells from the entire section, and the average
number of apoptotic cells per section was calculated from 3 sections of all eyes from each
genotype. Statistical analysis was performed using the One-way ANOVA with
Bonferonni’s correction for repeated measures.
2.1.7 Endothelial Cell Staining
To further characterize changes in retinal morphology, fluorescein-labeled GSL I
– isolectin B
4
(Vector Laboratories) was utilized as a marker to selectively stain vascular
cells. Retinas from PN 21 and 1 month old and Nrl
-/-
and Nrl
-/-
Grk1
-/-
mice raised in
cycling light were dissected, fixed and sectioned as described above (Zhu and Craft,
2000b). Sections were dried then blocked with 1% bovine serum albumin (BSA) in PBS
for 30 min. In a humidified chamber, slides were incubated with isolectin B
4
(1:1000) for
1 hour at room temperature, washed in PBS, then mounted with Vectashield Mounting
Medium for fluorescence with DAPI (Vector Laboratories) and cover slipped. For
colocalization studies with VEGF, slides were incubated in a mixture of the mAB VEGF
34
antibody (1:200) and isolectin B
4
(1:100) followed by incubation with the Alexa fluor 568
goat anti mouse IgG secondary (1:250; Invitrogen).
.
Slides were photographed using a
Zeiss Confocal Laser Scan Microscope (LSM 510).
2.1.8 Fluorescein Angiography (FA)
Fluorescein angiography (FA) staining was employed to visualize changes in
retinal vasculature with increasing age in Nrl
-/-
, Nrl
-/-
Grk1
-/-
, C57Bl/6J and Grk1
-/-
and
mice. Nrl
-/-
and Nrl
-/-
Grk1
-/-
mice were examined from 1, 3, 5 and 7 months of age and
C57Bl/6J and Grk1
-/-
mice were examined from 1 and 7 months of age. Prior to
anesthesia, pupils were dilated with topical phenylephrine HCl (2.5%) and tropicamide
(0.5%). Following dilation, the mice were anesthetized with an intraperitoneal injection
of ketamine HCl (100mg/kg body weight) and xylazine HCl (10 mg/kg body weight) and
kept on a heating pad for the duration of the experiment. The mice were placed in a
lateral recumbence with the visual axis of the eye upward. The focal axis of the hand held
Kowa Genesis Small Animal Camera was aligned with the visual axis of the mouse. An
intraperitoneal injection of 0.05ml of 10% sodium fluorescein dye solution (Akorn,
Buffalo Grove, IL) was administered at the same time the dataphot (internal time) on the
Kowa Vk02 2.11 Digital Imaging for Genesis program was initiated. Time lapse digital
photos were taken of the center, top, bottom, left, and right quadrant for every time point
at approximately 1, 3, 5 and 7 minutes post injection.
2.1.9 Affymetrix
TM
GeneChip Microarray and Ingenuity Pathway Analysis
Transcriptional variability when apoptosis was documented and prior to severe
morphological changes was examined in retinas from 1 month old light-adapted Nrl
-/-
and
35
Nrl
-/-
Grk1
-/-
mice. Retinas were homogenized and total RNA isolated using a monophasic
phenol and guanidine isothiocyanate solution extraction per the manufacturer’s
instructions (TRIzol, Invitrogen). Retinas at this age also have a similar ONL thickness,
which allowed for comparable mRNA levels between the two strains. RNA purity and
concentration was determined using spectrophotometry A
260
/A
280
ratios. Affymetrix
TM
Mouse Genome 430 2.0 GeneChips (Affymetrix, Inc. Santa Clara CA) were used for
hybridization. RNA isolation and microarray hybridization were performed in triplicate
for statistical and biological relevance. The original Affymetrix raw intensity files were
imported into Partek Genomic Suite (Partek, Inc.) and pre-processed using the
software implemented GCRMA background correction (Wu and Irizarry, 2005) and
quantile normalization algorithms (Bolstad et al., 2003). The log2 transformed data was
then subjected to a 2-way mixed-model factorial ANOVA analysis and the list of
differentially expressed genes between the two genotypes was subsequently generated
(p<0.005 coupled with fold changes >1.2 or <-1.2). Transcripts with statistically
significant differences and annotated function were categorized using Ingenuity Pathway
Analysis (IPA) (Ingenuity Systems). Statistical significance of canonical pathways was
conducting using Fisher’s Exact Test with a p value threshold of 0.05. Ratios are the
number of molecules identified from the data set per pathway. Scores for top networks
were based on the hypergeometric distribution and calculated with the right tailed
Fisher’s Exact Test by IPA.
36
2.1.10 Real Time Quantitative Polymerase Chain Reaction (qPCR)
To verify the microarray results for Pituitary tumor transforming gene 1 (Pttg1)
and Kallikrein2 (Klk2), retinas were collected for RNA isolation from light-adapted 1
month old Nrl
-/-
and Nrl
-/-
Grk1
-/-
mice using First-Strand cDNA Synthesis (Invitrogen).
Briefly, 1μg of total RNA was reverse transcribed into first strand cDNA using
SuperScript™ III Reverse Transcriptase (200U/μl) plus RNase OUT™ (40U/μl) and
50μM Oligo(dT)
20
random hexamer primers and incubated at 50ºC for 50 minutes. All
cDNA synthesis reactions were treated with RNase H and stored at -20ºC. Primers used
for qPCR are as follows:
+mPttg1 CCCTTTCTATCATGGGAATCTG
-mPttg1 GGCAATTCAACATCCAGAGTG
+mKlk2 GGCTGGGGCAGCATTAACCAGT
-mKlk2 TCATTAGGATGGAGCTTGATGGACAC
+mβ-Actin ATGGAATCCTGTGGCATCCA
-m-β-Actin CGCTCAGGAGGAGCAATGAT.
Real Time qPCR was performed by monitoring RT
2
SYBR Green dye
fluorescence using the LightCycler
®
Systems for Real-Time PCR (Roche Applied
Science). The PCR reaction included 1μl of cDNA corresponding to 2-4ng of total RNA,
SYBR Green, ddH
2
O, and 10μm of PCR primer pairs. β-Actin was used as the reference
gene. Cycling conditions were 95ºC for 10 minutes, followed by 45 cycles of 95ºC for 15
seconds and 60ºC for 1 minute for quantification. The melting curve was calculated using
1 cycle of 95ºC for 1 minute and 65ºC for 2 minutes followed by continuous acquisition
37
at 97ºC and cooling at 40ºC. Melting curve analysis confirmed the absence of primer
dimers.
2.1.11 Isoelectric Focus (IEF) and Immunoblot Analysis of PGI
Protein expression profiles were originally conducted on Nrl
-/-
and Nrl
-/-
Grk1
-/-
retinas using two-dimensional gel electrophoresis (2DE) and Ettan
TM
DIGE overlay
technology. Retina samples from age matched Nrl
-/-
and Nrl
-/-
Grk1
-/-
mice revealed two
distinct isoelectric points (PI) for a 63 kDA protein, later identified by Mass
Spectrometry (MS) as Phosphoglucose Isomerase (PGI) [Craft & Brown, unpublished
observation]. To confirm these results, one dimensional gel electrophoresis was
performed using an Ampholine PAGplate gel (pH range 4.0-6.5, GE Healthcare) and a
Multiphor II Isolectric Focusing Apparatus (LKB/Pharmacia) per the manufacturer’s
instructions. The gel was pre-run for 30 minutes at 500V to establish the pH gradient, and
20ul per sample was added to loading strips on the basic side of the gel to allow for
adequate protein separation. The gel was run for 150 minutes and the pH levels across the
gel were measured with a surface electrode.
Immunoblot analysis was conducted by first transferring the proteins from the
PAGplate onto a PVDF membrane soaked in CAPS buffer (10mM CAPS, pH 11) using
blotting paper and a heavy weight overnight at 4ºC. The blot was blocked with TNT
blocking buffer (150mM NaCl, 0.5% Tween 20, 1% gelatin, and 0.05% Na Azide in
10mM TrisHCl pH 8.0) then probed with a rabbit polyclonal pAB PGI antibody (1:1000)
(sc33777; Santa Cruz Biotechnology) and a goat anti-rabbit pAB IgG (H+L)-HRP
38
conjugated (1:5,000; BioRad Laboratories) secondary antibody and visualized by an
Enhanced Chemiluminescence (ECL) Kit (Amersham, Arlington Heights, IL).
2.1.12 PGI Activity Assay
To determine PGI’s isomerization activity from mouse retinas, four retinas per
genotype of 1 month old Nrl
-/-
and Nrl
-/-
/Grk1
-/-
mice were homogenized in equal
volumes of homogenizing buffer (0.25M Tris HCl, pH 8.0, 0.5mM PMSF), and the
soluble protein fractions isolated via centrifugation. The isomerization activity of PGI
was measured using a reaction mixture consisted of 0.1 mol/L Tris (pH 8.5), 4.0 mmol/L
fructose 6-phosphate, 0.5 mmol/L NADP, and 1 unit/ml of glucose 6-phosphate
dehydrogenase. The mixture was placed in a light-path quartz cuvette and preincubated at
30ºC for 5 minutes to oxidize any glucose 6-phosphate that may have been present in
solution (Gracy and Tilley, 1975). The reaction was then initiated by the addition of
approximately 20ng of total protein containing the various isoform(s) of PGI and
incubated for 10 minutes. The formation of NADPH was measured
spectrophotometrically at an absorbance of 340nm. The results were normalized,
graphed, and slopes used to compare specific activity of PGI from the different retina
sources.
2.1.13 Molecular Identification and Sequence Analysis of PGI mRNAs
In order to determine strain specific nucleotide sequences of PGI from C57Bl/6J
and 129/SvJ founders, a Pubmed search was conducted using the available PGI clone
sequences from the National Center for Biotechnology Information (NCBI) database.
Figure 2.1 lists the different mRNA clone sequences from both C57Bl/6J (blue boxes)
39
and 129/SvJ (red box) mouse strains. The PGI sequences from each clone were aligned
using Vector NTI (Invitrogen) and variations in single nucleotide polymorphisms (SNPs)
were identified. PGI sequence amplification was performed using cDNA synthesize from
C57Bl/6J wildtype (WT), Nrl
-/-
, Nrl
-/-
Grk1
-/-
and Nrl
-/-
Arr
-/-
retinal mRNA using the First-
Strand cDNA Synthesis (Invitrogen) as described in section 2.1.10. The following
primers were used to amplify the full length PGI cDNA sequence:
+mPGI21: 5’- ATG GCT GCG CTC ACC CGG AAC-3’
-mPGI1654: 5’-TTA TTC TAG TTT GGT GTC CCG CTG-3’
The PGI PCR product was then cloned into a TOPO vector using the TOPO TA
Cloning Kit (Invitrogen) per the manufacturer’s instructions then the insert sequences
(USC DNA Sequencing and Genetics Analysis Core Laboratory). All sequences were
aligned in Vector NTI and the presence of published and unpublished SNPs recorded.
40
Figure 2.1: PGI mRNA Clone Sequences from Different Published Mouse Strains.
Information is acquired from the National Center for Biotechnology Information (NCBI)
database. Available < http://www.ncbi.nlm.nih.gov/gene/14751>.
41
2.2 Poly(ADP)Ribose Polymerase-1 (PARP) Activity in Grk1
-/-
Mice
2.2.1 Immunoblot Analysis of PARP and PAR
Retina homogenates from 3 light adapted (LA) 1 month old C57Bl/6J, Grk1
-/-
and
Arr1(A)
-/-
mice were isolated and homogenized and sonicated for 20 second pulses as
described in section 2.1.12. Light adaptation was 5 hours of exposure to room light (5
lux). A total of 25ug (Figure 4.2 and 4.3) of total protein from each sample were applied
onto wells of an 8% SDS-PAGE gel, electrophoresed, and transferred onto a PVDF
membrane. Immunoblot analysis was conducted using 5% nonfat milk in Tris Buffered
Saline with 1% Tween 20 (TBST) for blocking and for the antibody dilution buffer
(1:1,000) of the mouse anti-Poly(ADP)Ribose (PAR) polymer monoclonal antibody
(4335-MC-100; Trevigen). A goat anti-mouse IgG HRP conjugated secondary antibody
(1:5,000, BioRad) was diluted in 5% nonfat milk and incubated for 1 hour at room
temperature. All blots were washed three times following antibody incubation for 15
minutes each in TBST. Immunodetection was performed with exposure to ECL for 2
minutes prior to film exposure and development.
1 month old Grk1
-/-
and C57Bl/6J mice exposed to either 24 hours of total
darkness or 24 hours of fluorescent room light (5 lux) were euthanized and their retinas
harvested as described above. Three retinas per genotype were homogenized and
sonicated, and 13ug of total protein were applied onto an 8% SDS-PAGE gel,
electrophoresed, and immunoblotted for detection by ECL for Poly(ADP)Ribose
Polymerase (PARP) (1:1,000, clone C2-10; Trevigen) or the PAR polymer (4335-MC-
100; Trevigen). Gapdh was used as the loading control for all immunoblots. Briefly, all
42
blots were washed in TBST then blocked for 1 hour in 10% nonfat milk in TBST and
subsequently incubated with the Gapdh monoclonal antibody in 5% nonfat milk (1:8,000,
clone -71.1 Sigma) for 1 hour at room temperature. After three 15 minute washes in
TBST, the blots were incubated in 5% nonfat milk containing a goat anti-mouse IgG
HRP conjugated secondary antibody (1:5,000, Biorad) for one hour, washed, then
detected with ECL.
2.2.2 PAR Immunohistochemistry in Grk1
-/-
and C57Bl/6J Retinas
Retinal sections from 1, 2, an 4.5 month old Grk1
-/-
and C57Bl/6J controls were
immunostained for PAR moieties using the monoclonal anti-PAR polymer antibody
(1:1,000 clone 10HA; Trevigen). All sections were blocked using the IHC blocking
buffer described above. Briefly, sections from light adapted mice born and maintained in
total darkness were immunostained with the PAR antibody (1:1000) for 1 hour at room
temperature, washed in PBS, then incubated in a combined solution of the Alexa fluor
488 goat anti-mouse IgG (1:250) and the monomeric cyanine nucleic acid stain To-
Pro®3 iodide (1:2,000) in PBS for 1 hour at room temperature. The slides were
subsequently washed in PBS then mounted with Vectashield mounting medium and
cover-slipped.
2.2.3 TUNEL and PAR Colocalization
TUNEL staining was performed as described in section 2.1.6 on retinal sections
from mice exposed to either 24 hours of room light or 24 hours of total darkness. The
sections were then blocked as described in section 2.1.5 and immunostained with the
PAR antibody (1:1000) and incubated in 4ºC overnight. The slides were incubated in the
43
Alexa fluor 568 goat anti-mouse IgG (1:250) secondary antibody for 1 hour at room
temperature. Digital images were taken using a Zeiss Confocal Laser Scan Microscope
(LSM 510). The number of TUNEL and PAR positive cells from three sections of three
different mice were quantified and displayed graphically.
2.2.4 In Situ PARP Activity Assay
1 month old Grk1
-/-
and C57Bl/6J mice were exposed to either 24 hours of room
light or 24 hours of total darkness and their eyes enucleated. Eyecups were placed for 2
hours in a PARP activity reaction mixture containing 10mM MgCl
2
, 5μm 6-biotin-17-
nicotinamide-adenine-dinucleotide (bio-NAD
+
; Trevigen), and 1μM DTT in 1x PBS at
37ºC. Eyecups were then fixed for 30 minutes in 4% paraformaldehyde in phosphate
buffered saline (PBS), then washed, embedded and sectioned as described in section
2.1.3. Sections were blocked in normal goat serum blocking buffer for 30 minutes,
followed by 15 minutes incubations in biotin and avidin blocking solutions (Vector
Laboratories). Each section was then incubated in a 1:800 dilution of rhodamine
conjugated streptavidin for 2 hours (Vector Laboratories) to fluorescently label any
biotinylated NAD incorporated into the tissue samples. Slides were washed three times in
1xPBS for 15 minutes each then mounted with Vectashield mounting medium with DAPI
and cover-slipped. Pictures were taken using a Leica fluorescent light microscope with a
40x lens.
2.2.5 HT Colorimetric PARP Apoptosis ELISA Assay
An in vitro ELISA assay was performed on retina homogenates from three
different Grk1
-/-
and C57Bl/6J mice kept in either 24 hours of total darkness or 24 hours
44
of room light. The PARP Colorimetric ELISA Assay (Trevigen) was conducted per the
manufacturer’s instructions. Briefly, retinas were homogenized either under room light or
in the dark in 1x apoptosis buffer then centrifuged to pellet cellular debris. A PARP
standard curve was prepared using serial dilutions of the PARP enzyme from 10mU/well
to 0.1mU/well. Biological, as well as technical replicates were done in triplicate for
statistical significance. The PARP substrate cocktail containing NAD and activated DNA
was added with each sample to histone coated wells and incubated at room temperature
for 30 minutes. Each well was then washed in 1xPBS + 0.1% Triton X 100. To detect the
level of activated PAR groups attached to the histones, each well was incubated with the
mouse anti-PAR monoclonal antibody (1:1000) for 30 minutes, washed, and then
incubated in a goat anti-mouse secondary antibody (1:1000) for another 30 minutes.
TACS-Saphire™ was added to each well for colorimetric detection of the secondary
antibody and the reaction was terminated in 5% phosphoric acid and measured using a
plate reader at A
450nm
. A repeated measures ANOVA test using Bonferonni’s correction
was used in the statistical analyses.
45
2.3 Characterization of the Pttg1
-/-
Mouse Retina
2.3.1 Pttg1
-/-
Mice
Pituitary tumor transforming gene 1 knockout mice (Pttg1
-/-
) were kindly
provided by Dr. Shlomo Melmed, MD (Cedars-Sinai Research Institute, University of
California, Los Angeles) (Wang et al., 2001). Pttg1
-/-
mice were generated through the
microinjection of 129/SvJ embryonic stem cells carrying the targeting vector into the
blastocyst of a C57Bl/6J host. All Pttg1
-/-
mice were born and maintained in 12hr
light:12hr dark cycles.
2.3.2 Genotyping
Tails from Pttg1
-/-
mice were clipped and genomic DNA extracted using a
mixture of Direct PCR Lysis Reagent and Proteinase K (Viagen) incubated at 55ºC
overnight. Cell extracts were heated at 85ºC for 45 minutes to denature the Proteinase K.
Samples were then centrifuged to pellet cellular debris and 2μl of the supernatant were
added to 23μl of Taq PCR Core Kit per the manufacturer’s instructions (Qiagen). The
following primers for murine Pttg1 were used (Wang et al., 2001).
+mPttg2S: 5’-GGTTTCAACGCCACGAGTCG-3’
-mPttg1AS: 5’-CTGGCTTTTCAGTAACGCTGTTGAC-3’
Genomic DNA from each animal was also tested for the presence of the
Met450Leu RPE65 single nucleotide polymorphism (SNP) using the following primers
(Wenzel and Grimm 2001).
RPE65LEUforward: 5' – GAC/ACA/GGC/AGA/AAT/TTA/GTC/ACA/C -3'
RPE65LEUreverse: 5' – CCA/GAT/TTC/TTT/AGT/TTT/GAC/GTT/CAG -3'
46
RPE65METforward: 5' – CTT/GGT/CAT/AAG/CAG/CTC/TGT/AAG/A -3'
RPE65METreverse: 5' – GAC/CAC/AGA/AAT/TCA/ATT/CTG/GTG/C -3'
A common nonsense mutation screened for in animal models is the presence of a
DdeI restriction endonuclease enzyme site formed from a C to A transversion in codon
347 of exon 7 of the β subunit of the rod photoreceptor cGMP phosphodiesterase (PDE)
gene found in Retinal Degeneration1 (rd1) mice. The degeneration of rd1 mice is
attributable to a disorder of cyclic nucleotide metabolism involving a deficiency in PDE
(Pittler and Baehr, 1991). The insertion of the DdeI site is a known marker for retinal
degeneration susceptibility; therefore the presence of the DdeI site in Pttg1
-/-
mice was
screened using a restriction fragment length polymorphism (RFLP) pattern analysis.
Genomic DNA from Pttg1
-/-
mice was used in the amplification of the potential DdeI
insertion site using the following primers (Pittler and Baehr, 1991):
+mW149DdeIrd1: 5’CATCCCACCTGAGCTCACAGAAAG-3’
-mW150DdeIrd1: GCCTACAACAGAGGAGCTTCTAGC-3’
For DdeI enzyme digestion, genomic DNA was amplified with the primers listed
above and the digestion was carried out in a mixture containing 1x NEB buffer, 12μl of
PCR product, 5 units of DdeI enzyme (New England Biolabs), in ddH2O up to 40μl per
reaction. Following digestion, reaction products were separated by electrophoresis on a
2% agarose gel. Genomic DNA from rd1 mice was used as a positive control.
47
2.3.3 Electroretinography (ERG)
Pttg1
-/-
and C57Bl/6J mice born and maintained in ambient cyclic light were
examined at 9 months of age. At least 6 animals were recorded, consisting of 3 males and
3 female mice. See section 2.1.2 for more details on the ERG protocol.
2.3.4 Retinal Histology
For histological analysis, sections were stained with hematoxylin and eosin
(H&E) as described in section 2.1.4. Pttg1
-/-
and C57Bl/6J mice of 1, 5, and 9 months of
age were examined for changes in morphology associated with increasing age; including
outer nuclear layer (ONL) thinning.
2.3.5 Immunohistochemistry
The mouse anti-Arrestin4 (Arr4) polyclonal antibody (mCAR-LUMIJ) was used
to stain cones in Pttg1
-/-
and C57Bl/6J mice of 1.5 and 9 months of age as described in
section 2.1.5 with minor modifications. Whereas previous sections were processed and
nuclei stained with DAPI, the monomeric cyanine nucleic acid stain To-Pro®3 iodide
(1:2,000) in PBS was used instead and incubated with the secondary antibody. The slides
were subsequently washed then mounted with Vectashield mounting medium and cover-
slipped. Animals were examined at 1.5 and 9 months to detect changes in cone number
with increasing age. Images presented are from the superior region of the retina from
male mice.
2.3.6 Fluorescein Angiography (FA)
Fluorescein Angiography (FA) staining was performed as described in section
2.1.8. Retinas from 7 month old male and female Pttg1
-/-
mice were examined for retinal
48
blood vessel leakage, along with an age matched C57Bl/6J control. Pictures were taken
of the central, inferior and superior regions of the retina for comparison.
49
2.4 Background Strain Variability in the Arrestin1 (Arr1
-/-
) Phenotype
2.4.1 Arr1(A)
-/-
and Arr1(B)
-/-
Mice
Arrestin null mice (Arr1
-/-
) generously provided by Jeannie Chen (USC) were
created from a mixture of C57Bl/6J and 129/SvJ founders and will be referred to as
Arr1(A)
-/-
for clarity. Arr1(B)
-/-
mice were the offspring of an F2 cross between Arr1(A)
-/-
mice with Arrestin 4 null animals (Arr4
-/-
), which were created in our laboratory and are
also a combined mixture of C57Bl/6J and 129/SvJ strains (Nikonov et al., 2008). The
animals were then backcrossed with heterozygous (Arr1
+/-
Arr4
+/-
) parents to preserve the
colony; therefore, Arr1(B)
-/-
mice are the offspring of an Arr1
-/-
Arr4
+/+
background strain
family. Arr1(A)
-/-
and Arr1(B)
-/-
mice were born and maintained in total darkness
throughout this study. The wildtype (WT) control animals used in this study were on a
mixed C57Bl/6J and 129/SvJ background.
2.4.2 TUNEL Analysis of Apoptosis
Changes in the number of TUNEL positive cells were measured in three mice
from C57Bl/6J controls and Arrestin knockout mice Arr1(A)
-/-
and Arr1(B)
-/-
from various
ages; including postnatal (P) day 22, 30, 45, and 60. Three animals were examined from
each age of each genotype for statistical significance. The TUNEL assay and
quantification were conducted as described in section 2.1.6.
2.4.3 Isoelectric Focusing and Immunoblot Analysis of Prdx6
C57Bl/6J control mice were born and maintained in cycling light and Arrestin
knockout mice Arr1(A)
-/-
and Arr1(B)
-/-
were born and maintained in the dark. One mouse
per genotype was euthanized and their retinas harvested at 1 month of age under room
50
light. Retinas were homogenized in 0.25M Tris HCl pH8.4 with protease inhibitors and
sonicated for 20 pulses then centrifuged to pellet cellular debris. Isoelectric focusing
(IEF) was performed using an Ampholine PAGplate gel (pH range 4.0-6.5, GE
Healthcare) and a Multiphor II Isolectric Focusing Apparatus (LKB/Pharmacia) per the
manufacturer’s instructions. The gel was pre-run for 30 minutes at 500V to establish the
pH gradient, and then 20μl per sample was added to loading strips on the basic side of the
gel to allow for adequate protein separation. The gel was run for 150 minutes and the pH
levels across the gel were measured using a surface electrode.
Immunoblot analysis was conducted by first transferring the proteins from the
PAGplate onto a PVDF membrane using blotting paper and a heavy weight overnight at
4
o
C. The blot was then air dried, re-wet with methanol and blocked with TNT blocking
buffer (150mM NaCl, 0.5% Tween 20, 1% gelatin, and 0.05% Na Azide in 10mM
TrisHCl pH 8.0) for 30 minutes. A mouse monoclonal anti-Prdx6 antibody (Abcam) was
prepared using 1% BSA in TBST and incubated at a dilution of 1:1000 in room
temperature for 1 hour. The blot was subsequently washed and blotted using the goat
anti-mouse HRP conjugated secondary antibody diluted in 1% BSA in TBST (1:10,000,
Bio-Rad Laboratories) then developed.
2.4.4 Allele Specific Genotype Analysis of Prdx6
Allele specific primers were designed using the protocol as described by You et
al, 2009. Briefly, the BatchPrimer3 design web application was employed to generate
allele specific primer sequences according to the Mouse Genome Informatics (MGI)
database including single nucleotide polymorphisms (SNP) of Prdx6 from both C57Bl/6J
51
and 129/SvJ mouse strains. The SNP located at amino acid 122 was previously linked to
resistance or susceptibility of atherosclerosis in mice according to their genetic
background (Phelan et al., 2002). Therefore, SNP sequences from C57Bl/6J and 129/SvJ
strains were uploaded into the BatchPrimer3 Allele Specific Polymerase Chain Reaction
(ASPCR) program and the following forward primers were obtained. A common forward
and common reverse primer was also designed to yield varying PCR product sizes.
+mASPCR_Prdx6_101A:GGATCCAGTCGAGAAGGATGA
+mASPCR_Prdx6_101C: GATCCAGTCGAGAAGGATGC
+m_Prdx6_Com: GGGTGGCTTCGGACAGTAAAACTA
–m_Prdx6_Com: TGTCCACCCTTCCTCTAACACA
The PCR product of the A or C allele along with the common reverse primer yield
a 300 basepair (bp) fragment, while the common forward and reverse primers yield a 516
bp fragment. Two independent PCR reactions were conducted per allele. Primers
+mASPCR_Prdxt_101A or +mASPCR_Prdx6_101C were added to equal concentrations
of +m_Prdx6_Com and –m_Prdx6_Com.
Mouse tails from respective animals were clipped and genomic DNA extracted
using a mixture of Direct PCR Lysis Reagent and Proteinase K (Viagen) incubated at
55
o
C overnight. Cell extracts were heated at 85
o
C for 45 minutes to denature the
Proteinase K. Samples were then centrifuged to pellet cellular debris and 2μl of the
supernatant was added to 23μl of Taq PCR Core Kit (Qiagen).
52
Figure 2.2: Allele Specific PCR (ASPCR) Primer Design for Prdx6. Two AS primers, one for
each allele of the SNPs, were designed. The AS primers contain one of two polymorphic
nucleotides at the 3’ end. A common forward and reverse primer was used in each PCR reaction.
The outcome of the ASPCR Genotyping is presented on the right. Adapted with permission You,
FM et al. BMC Bioinformatics, 2009.
2.4.5 Phospholipase A
2
Activity (PLA
2
) in Arr1(A)
-/-
and Arr1(B)
-/-
Retinas from P22 Arr1(A)
-/-
and Arr1(B)
-/-
mice were born and maintained in total
darkness and C57Bl/6J mice were born and maintained in cycling light and dark adapted
prior to euthanasia. The cPLA2 activity assay (Caymen Chemical, 765021) was
performed according to the manufacturer’s recommendations. Three retinas from each
genotype were homogenized in the recommended buffer (50nM Hepes, pH7.4, 1mM
EDTA) per gram of tissue. Following centrifugation, the supernatant from each sample
was used for each reaction mixture. In a 96 well plate, a combination of cPLA2 assay
buffer (160mM Hepes pH 7.4, 300mM NaCl, 20mM CaCl2, 8mM Triton X-100, 60%
glycerol, 2mg/ml BSA) and either a blank, bee venom control, or retina homogenate were
added in triplicate. The reaction was initiated by the addition of 200μl of substrate
solution (1.5mM Arachidonoyl Thio-PC in cPLA2 assay buffer). Following 60 minute
53
incubation at room temperature, the reaction was terminated by the addition of 10ul of
DTNB/EGTA to each well to stop enzyme catalysis and develop the reaction. The
colorimetric absorbance was then read at A
414nm
using a plate reader (BioRad
Laboratories). Absorbance values were calculated and statistical analysis performed using
a repeated measures ANOVA test with Bonferonni’s correction.
2.4.6 Association Mapping and Genome Wide Array Studies
A genome scan was performed as previously described (Haider et al., 2008) using
95 SSLP markers distributed across 19 autosomes (excluding X-chromosome) at
approximately 20-cM intervals. Fifty (50) Arr1(A)
-/-
samples, 47 Arr1(B)
-/-
samples and
control C5BL/6J and 129/SvJ samples were evaluated. Genomic DNA was isolated and
used for PCR amplification using the following protocol: 40ng of template DNA in a
10μl PCR reaction mixture containing 1.0μl PCR buffer (100mM Tris-HCl pH 8.8,
500mM KCl, 15mM MgCl
2
, and 0.01% w/v gelatin); 200μM each of a mixture of
dNTPs; 2.5pmol of each forward and reverse primer; and 0.25U Taq polymerase. The
reaction mixture was then incubated in 40 cycles of 94°C for 30 seconds, 53°C for 30
seconds, and 72°C for 30 seconds. Amplification products were electrophoresed at 200V
for 1 hour on a 4% agarose gel containing 0.5μg/ml ethidium bromide and visualized by
ultraviolet transillumination. C57Bl/6J and 129/SvJ control DNAs were included in each
set of reactions for proper sizing of strain-specific allele examination to ensure proper
genetic mapping. For genetic mapping of quantitative trait loci (QTL) in our two Arr1
-/-
populations, Windows QTL Cartographer V2.5 was used as a tool for summarizing the
mapping results obtained from the sequencing analysis. Each candidate gene with a LOD
54
score greater than 3 was identified as significant and annotated with its human ortholog,
human location, mammalian phenotype related to mutations in that gene, and human
disease and disease phenotypes according to the Expression Analysis Systemic Explorer
(EASE). EASE is a customizable, standalone Windows© desktop software application
that facilitates the biological interpretation of gene lists derived from our generated
marker data. The EASE program employed the Online Mendelian Inheritance in Man
(OMIM) and The Database for Annotation, Visualization and Integrated Discovery
(DAVID) V6.7 databases in the analyses.
The genomic DNA from Arr1(A)
-/-
and Arr1(B)
-/-
mice were divided into two
groups and association mapping was performed accordingly. One variable was created to
differentiate those susceptible from those that were not. This variable was used as the
outcome variable. Microsatellite markers were coded as 0, 1, and 2 for homozygous
wildtype, heterozygous and homozygous rare, respectively, for each observation.
Statistical analysis was performed using SAS 9.1 (SAS Institute Inc., Cary, NC). The
frequency procedure was used to observe the distribution of genotypes in the susceptible
mice compared to the resistant mice, and the chi square test was used to test for
significant differences using 2 degrees of freedom. The resulting p values were corrected
for multiple testing using the Bonferonni Correction. Additionally, in order to
corroborate findings obtained by chi-square, a linear regression model was employed to
predict susceptibility from genotype.
55
CHAPTER 3. LOSS OF G-PROTEIN COUPLED RECEPTOR KINASE (GRK1)
IN NRL
-/-
MICE LEADS TO NEOVASCULARIZATION, ENHANCED
INFLAMMATORY RESPONSE, AND LIGHT-INDEPENDENT CONE
DYSTROPHY
3.1 Introduction
Significant advances in bioinformatics have identified essential genetic links and
characterized basic molecular mechanisms driving the components of the visual G-
protein coupled receptor (GPCR) signal transduction cascade leading to rod
photoreceptor cell death. However, with a population of 3 to 5% cone photoreceptors in
the mouse retina, the manifestations of GPCR cascade disruption on cones have only
recently been studied with the help of the Neural retina leucine zipper knockout (Nrl
-/-
)
mouse model (Mears et al., 2001). In humans, a loss of function mutation in the NRL
gene leads to the autosomal recessive disorder, Enhanced S-Cone Syndrome (ESCS),
which causes an excess number of S cones and patients clinically present symptoms
including night blindness, variable loss of visual acuity, and visual field abnormalities
(Yoshida et al., 2004).
Because of a developmental cell switch from rod to short wave-length (SWL)
pigment photoreceptors, the Nrl transcription factor provides a unique opportunity to
study cone phototransduction mechanisms since rod phototransduction components
including rhodopsin are absent. Photopic electrophysiological analyses of Nrl
-/-
retinas
reveal that the amplitude of light-adapted electroretinography (ERG) responses elicited
by maximum stimulus are stable up to 31 weeks of age, suggesting cones survive without
56
rod function (Mears et al., 2001). Extensive ultrastructural, biochemical, and
electrophysiological characterization of the Nrl
-/-
mouse photoreceptors demonstrate that
the photoreceptors have many of the phenotypic hallmarks of cones (Daniele et al., 2005;
Mears et al., 2001; Nikonov et al., 2005). Results from gene expression profiling of the
Nrl
-/-
mouse retina with Affymetrix™ GeneChip technology further support the concept
that the rod precursors in the Nrl
-/-
retina differentiate as cones instead of rods and
therefore exhibit an enhanced S-cone population with the caveat that they are mutant
cones and different from those found in a wildtype (WT) retina (Yoshida et al., 2004).
Another essential regulatory component in the phototransduction cascade is the
serine/threonine G protein-coupled receptor kinase 1 (Grk1, rhodopsin kinase), which is
expressed in both rods and cones in rod-dominant human, monkey and mouse
(Lyubarsky et al., 2000; Weiss et al., 2001; Zhao et al., 1998; Zhu et al., 2003) and in
cone-dominant chicken (Zhao et al., 1999). Grk1 phosphorylation of light-activated
rhodopsin is required for normal phototransduction inactivation in vivo (Chen et al.,
1995; Hisatomi et al., 1998). Although the cone-specific Grk7 is expressed in various
species, (Chen et al., 2001; Hisatomi et al., 1998; Weiss et al., 1998; Weiss et al., 2001)
only Grk1 is expressed in the mouse photoreceptor. With the creation of a double
knockout mouse lacking both Grk1 and Nrl, the electrophysiological analyses of mouse
cone photoresponses clearly demonstrated that Grk1 plays a critical role in the
phosphorylation of S and M opsins, and the inactivation and normal recovery of cone
pigments (Lyubarsky et al., 2000; Nikonov et al., 2005; Zhu et al., 2003).
57
The Nrl
-/-
Grk1
-/-
mouse provides an invaluable model for not only the study of
cone GPCR signaling pathways, but also the molecular balance between cone
photoreceptor cell survival and death in inherited retinal diseases. Unlike the rod
photoreceptors of the Grk1
-/-
mouse, which degenerate rapidly when exposed to light due
to the inability to inactivate phototransduction, (Chen et al., 1999a) the cones lacking
Grk1 expression on the Nrl
-/-
background degenerate by apoptosis in the dark (Zhu et al.,
2006). The current study extends our initial observations and demonstrates that in
addition to a delayed photoresponse recovery, the absence of Grk1 expression also leads
to age-related and light independent cone dystrophy with abnormal vascularization in the
retinas of Nrl
-/-
Grk1
-/-
mice compared to Nrl
-/-
. To facilitate the identification of potential
genes involved in biological and disease pathways, we identified key molecular networks
to characterize the Nrl
-/-
Grk1
-/-
phenotype. Taken together, these data allow us to
hypothesize that aside from its known function in photoreceptor recovery, Grk1 plays a
functional role in the homeostasis of a healthy cone environment.
58
3.2 Results
3.2.1 Age-Related Light-Independent Cone Dystrophy
ERG analyses of retinal physiology and photopic function show that the b-wave
amplitudes of dark-adapted ERG responses elicited by maximum light stimulus are
largely stable until 7 months of age in Nrl
-/-
mice, regardless of the animals’
environmental lighting condition (Figure 3.1). In contrast, the photopic b-wave amplitude
decreases rapidly in Nrl
-/-
Grk1
-/-
mice in an age-dependent manner, with a 40% reduction
in b-wave amplitude by 3 months of age (Fig.ure 3.1B). Importantly, the average b-wave
amplitudes of 1 month old Nrl
-/-
Grk1
-/-
mice are similar to those of age matched Nrl
-/-
controls in the three environmental light conditions tested (Figure 3.1B). The ERG
results illustrate a defect in cone function and suggest that the degeneration of cone
photoreceptors in the Nrl
-/-
Grk1
-/-
retina are related to increasing age under different light
exposure, in contrast to the light-induced rod degeneration observed in the Grk1
-/-
mouse
retina (Chen et al., 1999a).
59
Figure 3.1: Maximum b-wave Amplitude of Dark-adapted ERG Responses in Nrl
-/-
(A) and
Nrl
-/-
Grk1
-/-
Mice (B). Mice were dark-adapted overnight before ERG recordings. Maximum
responses were elicited by high-intensity white fluorescent light. Note the rapid decrease of b-
wave amplitude (approximately 40% from 1 to 3 months) with increasing age in the Nrl
-/-
Grk1
-/-
mouse reared either in the dark, cyclic light or constant light.
Age-matched retinas of light adapted Nrl
-/-
and Nrl
-/-
Grk1
-/-
mice have a similar
photoreceptor outer nuclear layer (ONL) thickness at 1 month (Figure 3.2A,D); however,
the Nrl
-/-
Grk1
-/-
retinas had fewer rosettes and whorls at this age. At 5 months and older,
the ONL appears thinner in the Nrl
-/-
Grk1
-/-
mouse retina (Figure 3.2E,F) (Zhu et al.,
2006). Cone specific Arr4 immunoreactivity and H&E staining confirm that the thinning
of the retina in 5 and 9 month old Nrl
-/-
Grk1
-/-
mice is the result of cone photoreceptor
loss (Figure 3.2G-L).
Retinal cross sections from Nrl
-/-
and Nrl
-/-
Grk1
-/-
retinas were
immunohistologically stained with anti-S and anti-M opsin antibodies, which confirmed
our previous observations that both S and M cones degenerate in the Nrl
-/-
Grk1
-/-
mouse
retina in an age-dependent manner (Figure 3.3). By 9 months of age, a significant loss of
S- and M-opsin expressing cones is observed in the Nrl
-/-
Grk1
-/-
retinas when compared
60
to their age-matched Nrl
-/-
controls. Interestingly, more rosette structures are observed in
the ONL of the Nrl
-/-
retina, which are reduced with age faster in the Nrl
-/-
Grk1
-/-
retina
(Figure 3.3).
61
Figure 3.2: Age-Dependent Cone Photoreceptor Degeneration in the Nrl
-/-
and Nrl
-/-
Grk1
-/-
Mouse Retina. Retinal sections were stained with H&E (A-F) or immunostained with anti-
mCAR-LUMIJ followed by a fluorescein-conjugated anti-rabbit IgG secondary antibody (G-L).
Photographs were taken from the central inferior region of the retina.
62
Figure 3.3: Both S-opsin (A-F) and M-opsin (G-L) Expressing Cones Degenerate in the Nrl
-
/-
Grk1
-/-
Retina. Mice were born and maintained throughout their lifespan in total darkness. Mice
at the age of 1, 5 and 9 months were killed, and their eyes were fixed as described in Figure 3.2.
Photographs of the superior and inferior halves of the retinal section were taken separately at
lower magnification (5×10). The superior and inferior halves of the same section were then
aligned to assemble the entire cross section.
63
3.2.2 Apoptosis of Cone Photoreceptors
TUNEL analysis was performed on frozen retinal sections from postnatal PN21
and PN30 C57Bl/6J (C57), Nrl
-/-
and Nrl
-/-
Grk1
-/-
mice born and raised in cyclic ambient
light to further characterize photoreceptor degeneration. A representative picture of a
segment from PN21 Nrl
-/-
Grk1
-/-
, as well as PN30 Nrl
-/-
and Nrl
-/-
Grk1
-/-
retinal sections
demonstrate that apoptotic cells were observed only in the ONL of both Nrl
-/-
and Nrl
-/-
Grk1
-/-
mice (Figure 3.4A-C) and were statistically significantly different from Nrl
-/-
mice
at PN30 but not at PN21 (Figure 3.4D). Upon quantification of TUNEL positive cells
and a repeated measures ANOVA with Bonferonni correction, the Nrl
-/-
mouse retina was
found to have significantly more apoptotic cells than C57Bl/6J control retinas at both
PN21 and PN30, which is consistent with the slower retinal degeneration phenotype
(p<0.05) (Mears et al., 2001) (Figure 3.4). However, the Nrl
-/-
Grk1
-/-
retina shows a
statistically significantly greater number of apoptotic cells when compared to both Nrl
-/-
(p<0.01) and C57Bl/6J (p<0.001) controls at PN30 but was only significantly different
from C57Bl/6J mice at PN21. These results suggest that the cone photoreceptors of these
mice die through apoptosis and the degeneration is exacerbated following ablation of
Grk1 that is slightly higher at PN21, but is only significantly greater by PN30.
64
Figure 3.4: TUNEL Staining is Enhanced in Nrl
-/-
Grk1
-/-
. Retinal sections from P30 Nrl
-/-
(A)
and Nrl
-/-
Grk1
-/-
(C) and P21 Nrl
-/-
Grk1
-/-
(B) mice were processed for TUNEL staining. Shown
are images of the middle superior region of the retina. TUNEL positive apoptotic cells (green)
were counted under a Leica fluorescence light microscope. The total number of TUNEL positive
cells from 3 sections of each mouse was recorded, and the average count (mean ± SEM) was
calculated from 3 mice using a one way ANOVA of the same genotype (D). *P<0.05; **P<0.01;
***P<0.001. ONL, outer nuclear layer. OPL, outer plexiform layer. INL, inner plexiform layer.
Scale bar, 20μm.
65
3.2.3 Neovascularization
Endothelial cell staining using Isolectin B
4
and neovascularization testing using
FA were performed to analyze changes in retinal vasculature with increasing age.
Isolectin B
4
staining (green) was observed in the inner nuclear layer (INL) and inner
plexiform layer (IPL) in 1 month old Nrl
-/-
mice (Figure 3.5). The morphology and retinal
vasculature are similar to normal healthy retinas and are consistent throughout the entire
section of Nrl
-/-
controls. Age-matched Nrl
-/-
Grk1
-/-
mice reared under the same lighting
conditions, however, exhibit choroidal anastomoses that cross Bruch’s membrane and
infiltrate the retinal pigment epithelia (RPE). Blood vessels are present in the choroid,
INL, outer plexiform layer (OPL), IPL, and inner limiting membrane (ILM). In
comparison to the controls, Nrl
-/-
Grk1
-/-
mice have atrophied RPE cells and breaks in the
RPE monolayer coupled with anastomoses forming from both the choroid and the inner
retina (Figure 3.5). These studies demonstrate that by 1 month of age, Nrl
-/-
Grk1
-/-
retinas
have blood vessel infiltration that penetrates all retinal layers (Figure 3.5).
In order to correlate the observed photoreceptor TUNEL staining with choroidal
anastomoses, we performed Isolectin B
4
IHC on retinal sections of PN21 Nrl
-/-
Grk1
-/-
mice and compared them to PN30 Nrl
-/-
Grk1
-/-
sections. Figure 3.6 demonstrates that
overall, these mice have less blood vessel penetration from the choriocapillaris and have
an unbroken RPE monolayer, although signs of the initial penetration of blood vessels at
this age were present (Figure 3.6 top panel, top white arrow).
66
Figure 3.5: Retinal Anastomoses in Nrl
-/-
Grk1
-/-
Mice. Retinas were harvested from 1
month old Nrl
-/-
and Nr
-/-
Grk1
-/-
mice raised in cyclic light. The top panel is from an Nrl
-/-
Grk1
-/-
retina, the middle panel is from an Nrl
-/-
control, and the bottom panel is a
magnification of the RPE. Merged images presented below are of Nrl
-/-
(left) and Nrl
-/-
Grk1
-/-
(right) sections. Brightfield pictures demonstrate changes in the RPE structure
(arrows). DAPI is used as a nuclear stain (blue), and fluorescein labeled GSL I – isolectin
B
4
(green) show penetration of blood vessels into the retina from the choroid (arrows) in
Nrl
-/-
Grk1
-/-
retinas. C, choroid; RPE, retinal pigment epithelium; ONL, outer nuclear
layer; OPL, outer plexiform layer; IPL, inner plexiform layer; INL, inner nuclear layer.
Scale bar represents a length of 20μm. Images were taken with a Confocal Laser Scan
microscope using a 40x lens.
67
Figure 3.5, Continued: Retinal Anastomoses in Nrl
-/-
Grk1
-/-
Mice.
68
Figure 3.6: Retinal Anastomoses in Nrl
-/-
Grk1
-/-
Mice are Present Beginning at PN21.
Retinas were harvested from PN21 (top panel) and 1 month old (bottom panel) Nrl
-/-
Grk1
-/-
mice raised in cyclic light. Brightfield pictures demonstrate changes in the RPE
structure (arrows). DAPI is used as a nuclear stain (blue), and fluorescein labeled GSL I –
isolectin B4 (green) show penetration of blood vessels into the retina from the choroid
(arrows) in Nrl
-/-
Grk1
-/-
retinas. C, choroid; RPE, retinal pigment epithelium; ONL, outer
nuclear layer; OPL, outer plexiform layer; IPL, inner plexiform layer; INL, inner nuclear
layer. Scale bar represents a length of 20μm. Images were taken with a Confocal Laser
Scan microscope using a 40x lens.
69
Figure 3.6, Continued: Retinal Anastomoses in Nrl
-/-
Grk1
-/-
Mice are Present Beginning at PN21.
70
Vascular Endothelial Growth Factor (VEGF) is a hypoxia induced cytokine that is
strongly implicated in angiogenesis by virtue of its absolute requirement for retinal
vascularization and its expression in spatial and temporal conjunction with developing
retinal blood vessels (Gariano and Gardner, 2005). Therefore, IHC staining using an anti-
VEGF antibody on PN21 Nrl
-/-
and Nrl
-/-
Grk1
-/-
retinas was performed. The level of
VEGF expression in PN21 Nrl
-/-
Grk1
-/-
mice is more apparent than the staining pattern
observed in Nrl
-/-
mice of the same age (Figure 3.7). A control section without the
primary antibody was performed to confirm that staining is not due to secondary
background. These results indicate an enhanced level of VEGF expression in Nrl
-/-
Grk1
-/-
mice that begins as early as PN21. The section was focused on the nuclear plane;
however VEGF expression was localized throughout the retinal section but was highest in
the ILM located below the IPL.
71
Figure 3.7: VEGF Expression is Enhanced in Nrl
-/-
Grk1
-/-
Retinas Beginning at PN
21. Retinas from PN21 Nrl
-/-
Grk1
-/-
(top panel) and Nrl
-/-
(middle panel) mice were co-
stained with Isolectin-B4 (green) and VEGF (red) and DAPI (blue) was used for nuclei
staining. A C57Bl/6J mouse retinal section was used as a no primary antibody control
(bottom panel). The merged images demonstrate an enhanced level of VEGF expression
in Nrl
-/-
Grk1
-/-
retinas in the outer plexiform layer (OPL), inner nuclear layer (INL), and
inner plexiform layer (IPL) while minimal staining was observed in the ganglian cell
layer (GCL) below the IPL in the age matched Nrl
-/-
section. Scale bar, 10μm.
72
Figure 3.7, Continued: VEGF Expression is Enhanced in Nrl
-/-
Grk1
-/-
Retinas Beginning at PN 21.
73
Closer examination of VEGF expression in the endothelial cells of a PN21 Nrl
-/-
Grk1
-/-
retina is provided in Figure 3.8. The staining pattern indicates that VEGF levels
are enhanced and highly expressed in the blood vessels of these mice, particularly in the
branching segments of the vessel. Age matched Nrl
-/-
mice did not exhibit VEGF
expression within their blood vessels (Figure 3.7).
Figure 3.8: VEGF and IsolectinB4 Staining in a Retinal Blood Vessel of a PN21 Nrl
-/-
Grk1
-/-
Retina. The images are of the same area stained with DAPI nuclear stain in blue, Isolectin B4 in
green, VEGF staining in red and the merge of the three filters is presented at the bottom right.
VEGF expression is present near the sprouting blood vessel, particularly the branching segments.
74
Gross progressive changes that occur as a consequence of blood vessel
penetration were examined using FA on Nrl
-/-
and Nrl
-/-
Grk1
-/-
mice at 1, 3, 5 and 7
months of age. Nrl
-/-
mice exhibit no detectable leakage and have healthy retinal
vasculature at all ages examined (Figure 3.9). In contrast, age-matched Nrl
-/-
Grk1
-/-
mice
have a moderate level of leakage beginning at 1 month in the right upper quadrant
(arrows), and by 3 months, an excessive amount of leakage is observed that progresses
with age (Figure 3.9). FA staining on PN21 Nrl
-/-
Grk1
-/-
mice could not be performed due
to their small size. Leakage is first noticed in the right quadrant and spreads throughout
the retina by 5 months (data not shown). Conversely, the retinas of C57Bl/6J and Grk1
-/-
mice at both 1 and 7 months of age demonstrate healthy vasculature in all animals tested
(Figure 3.10).
Figure 3.9: Fluorescein Angiography (FA) Staining of Nrl
-/-
and Nrl
-/-
Grk1
-/-
Mice. Images
represent the right quadrant of the right eye of each animal at 1, 3, 5 and 7 months of age. The top
panel represents Nrl
-/-
Grk1
-/-
retinas and the bottom panel represents Nrl
-/-
controls. White arrows
indicate leakage of blood vessels in Nrl
-/-
Grk1
-/-
mice that begins at 1 month and progresses with
age. Retinas from age-matched Nrl
-/-
mice appear normal with healthy vasculature patterns.
Images were taken at approximately 7 minutes post injection of the fluorescein dye.
75
Figure 3.10: Fluorescein Angiography (FA) of C57Bl/6J and Grk1
-/-
Retinas. Images were
taken of the central portion of the right eye of each animal at 1 and 7 months of age. The top
panel represents Grk1
-/-
mice while the bottom panel represents C57Bl/6J controls. FA staining
patterns show no differences in retinal vasculature between C57Bl/6J and Grk1
-/-
retinas at both
ages examined.
3.2.4 Microarray and Ingenuity Pathway Analysis (IPA)
To identify potential genetic precursors and cellular pathways involved in the
cone dystrophy in the Nrl
-/-
Grk1
-/-
mouse, we performed microarray analysis in triplicate
and analyzed our results using Partek Genomic Suite and Ingenuity Pathway Analysis.
The data reveal statistically significant differences (p value ≤ 0.005) and average fold
changes (AFC) ≥ 1.2 of 422 mapped genes using a two-way ANOVA analysis. A list of
the genes with statistically significant up-regulation and an AFC of over 2.5 are listed in
Table 3.1, while Table 3.2 provides a list of statistically significantly down regulated
genes with an AFC less than -3.0. Expression of 10 known genes (Pituitary tumor
transforming gene 1, Pttg1; Tetratricopeptide repeat domain, 28Ttc28; Solute carrier
family 6 (proline IMINO transporter), member 20, Slc6a20; Ubxn4 UBX domain protein
4, Ubxn4; Fanconi anemia, complementation group C, Fancc; Adenylate cyclase-
76
associated protein 1, Cap1; Kallikrein, 2 Klk2; Sorbin and SH3 domain containing 1,
Sorbs; Coiled-coil domain containing 21, Ccdc21; Transmembrane protein 30A,
Tmem30A), were significantly increased in the Nrl
-/-
Grk1
-/-
in comparison to the Nrl
-/-
group. Upon examination of the transcripts with the highest fold increases, Pttg1 appears
twice with a 130.7 and 70.0 fold increase (Table 3.1). The Pttg1 gene encodes for a
transcription regulatory protein that can shuttle between the cytoplasm and nucleus with
alternative roles in sister chromatid segregation (van de Pavert et al., 2007a).
Interestingly, Crumbs homolog 1 (Crb1) transcript levels were decreased in Nrl
-/-
Grk1
-/-
mice approximately 6.3 fold when compared to Nrl
-/-
mice. This deregulation of Crb1 and
Pttg1 has been observed in two separate photoreceptor dystrophy models, the Crb1 and
Ras protein specific guanine nucleotide releasing factor 1 (RasGRF1) null mutant mice,
both of which demonstrate dystrophic retinas and are proposed models for retinal disease
studies (Fernandez-Medarde et al., 2009; van de Pavert et al., 2007a). Kallikrein 2
(Klk2), with a fold increase of 29.7, was also examined due to its serine protease and
auto-antigenic functions, and was recently identified as a major contributor in an
experimental model for autoimmune Keratoconjunctivitis Sicca (KCS) in Lewis Rats
(Jiang et al., 2009).
Other genes of interest that were significantly upregulated include the RNA
binding protein Embryonic lethal, abnormal vision, Drosophila-like-1 (Hu-antigen R)
(Elavl1) (AFC -6.99) known to play crucial roles in the stabilization and transport mRNA
including Vascular endothelial growth factor (VEGF) (Lu et al., 2009). Similarly, Usher
syndrome 1C (Autosomal recessive, severe) (Ush1C) (AFC-4.84) encodes for harmonin,
77
a PDZ domain containing protein with isoforms synthesized and expressed in
photoreceptors (Verpy et al., 2000; Williams et al., 2009). Patients with mutations in
USH1C have abnormal rod and cone ERGs and retinal degeneration at an early age.
78
Table 3.1: Comparison of Up-Regulated Trasncripts Between Nrl
-/-
and Nrl
-/-
Grk1
-/-
Retinas.
79
Table 3.2: Comparison of Down-Regulated Trasncripts Between Nrl
-/-
and Nrl
-/-
Grk1
-/-
Retinas.
80
Transcripts were categorized using Ingenuity Pathway Analysis according to
biological function with respect to diseases and disorders, molecular and cellular
functions, and physiological system development and function. Of these pathways,
neurological disease, cell signaling, and nervous system development and function were
ranked as the top categories according to p value and the number of molecules
categorized. The top canonical pathway identified was the Oncostatin M signaling
pathway (p value 3.51E-03), which activates the Signal transducer and activator of
transcription 1 (Jak-Stat) signaling machinery in some retinal degenerative disease
models (Samardzija et al., 2006; van de Pavert et al., 2007a). Other key canonical
pathways associated with the Nrl
-/-
Grk1
-/-
retinal dystrophy model include Synaptic Long
Term Potentiation Signaling, the Pentose Phosphate Pathway, Rac signaling, and Ciliary
Neurotrophic factor (Cntf) Signaling (Figure 3.11). The top networks identified with their
respective number of focus molecules are presented in Table 3.3. Of these identified
networks, the Inflammatory Disease, Inflammatory Response network was the most
relevant to our model system due to its known association with choroidal
neovascularization. The up-regulated transcripts from this network were organized into a
schematic diagram and are presented in Figure 3.12. Interestingly, up-regulation of Klk2
was identified in this pathway, as was the Oncostatin M signaling cascade with increased
transcription of Stat1 along with other inflammatory response genes.
81
Table 3.3: Biological Pathways Identified as Statistically Significant.
Table 3.4: Associated Functions of Top Networks.
82
Figure 3.11: Top Canonical Pathways Identified by IPA. Canonical pathways are presented on
the x axis, and the statistical significance of each pathway with a threshold of 0.05 is presented on
the y axis. Canonical pathways above and just below the threshold value are presented. The
orange line displays the ratio of up-regulated transcripts in the data set to the number of genes in
each pathway; revealing Oncostatin M Signaling to have the largest ratio, while Synaptic Long
Term Potentiation, CDK5 Signaling, and Rac Signaling among others, are above threshold for
statistical significance.
83
Figure 3.12: Schematic Representation of the Inflammatory Disease, Inflammatory
Response Network. Microarray data set analysis was used to generate a hypothetical network
based on up-regulated transcripts. Dotted lines represent indirect protein relationships, whereas
solid lines represent direct relationships; including protein protein interactions, phosphorylation,
activation, and ligand receptor binding. Molecules in pink represent significantly up-regulated
transcripts while green represent down regulated transcripts and uncolored molecules were not
differentially expressed but included for completeness.
84
3.2.5 Confirmation of Gene Expression Changes by Quantitative PCR
Quantitative PCR (qPCR) was performed for select genes of interest, Pttg1 and
Klk2, (Figure 3.13) to validate the microarray results, and similar significant increases in
both transcripts were observed in comparison to the β-Actin reference standard. Notably,
a similar trend in up-regulation of both transcripts was observed in Grk1
-/-
in comparison
to WT retinas.
Figure 3.13: Confirmation of Differential mRNA Expression of Klk22 and Pttg1 Using
Quantitative RT-PCR (qPCR). cDNAs from C57Bl/6J (C57), Grk1
-/-
, Nrl
-/-
and Nrl
-/-
Grk1
-/-
from 1 month old retinas were used for quantitative expression analysis, and β-Actin was a
reference standard using the Roche Light-Cycler® Real-Time PCR system. Statistically
significant up-regulation of Pituitary tumor transforming gene 1 (Pttg1) (A) and Kallikrein 22
(Klk22) (B) was observed in both Grk1
-/-
compared to C57 and Nrl
-/-
Grk1
-/-
compared to Nrl
-/-
.*P<0.05; **P<0.01.
85
3.2.6 Nrl
-/-
and Nrl
-/-
Grk1
-/-
Mice Have a Single Nucleotide Polymorphisms
(SNP) in Phosphoglucose Isomerase (PGI)
Preliminary characterization of the light independent age-related photoreceptor
degeneration in Nrl
-/-
Grk1
-/-
retinas was performed using Etan DIGE two dimensional gel
electrophoresis and mass spectrophotometry analyses, and identified Phosphoglucose
Isomerase (PGI) as a potential target for examination. Differing isoelectric mobility of
PGI and the absence of the kinase Grk1 in the double knockout animals lead us to
hypothesize that PGI was a target for phosphorylation by Grk1. Previous studies on PGI
identified critical phosphorylation residues that alter important physiologic and kinetic
functions for PGI in vitro (Yanagawa et al., 2005). To confirm the results from the 2D
gel, total retinal proteins were separated using one dimensional isoelectric focusing (IEF)
and mobility differences were identified using immunoblot analysis for PGI. Figure 3.14
represents the immunoblot and the isoelectric mobility of PGI of various retinal
homogenates. Whereas Nrl
-/-
retinas express a more acidic form of PGI (pH 8.4), Nrl
-/-
Grk1
-/-
retinas express a slightly more basic form (pH 8.7) as seen in Figure 3.14. Colony
control wildtype (WT), Grk1
-/-
and Nrl
-/-
Arr1
-/-
retinal proteins were provided for
comparison. Interestingly, WT retina homogenates have the basic form of PGI (PGIb) as
well as another basic isoform. The Nrl
-/-
Arr
-/-
retinas express a combination of the acidic
form of PGI (PGIa), PGIb, and a potentially unique isoform with a pH in between PGIa
and PGIb (Figure 3.14).
In order to determine if the variability in pH of PGI led to changes in protein
function in vitro, a PGI activity assay was conducted using total retinal proteins as a
86
substrate from Nrl
-/-
and Nrl
-/-
Grk1
-/-
mice. The formation of NADH as the byproduct of
the PGI catalyzed isomerization of fructose-6-phosphate to glucose-6-phosphate was
measured spectrophotometrically at an A
340nm
. The kinetic rates of the reaction from both
samples were graphed and demonstrate that the activity of PGI was similar between Nrl
-/-
and Nrl
-/-
Grk1
-/-
mice.
Changes in isoelectric mobility are typically due to post translational
modifications or single nucleotide polymorphisms (SNPs) translating into amino acid
residues of different pH levels. In an attempt to further explain the changes in isoelectric
mobility of PGI in our mouse models, the PGI cDNA sequences from mRNA of
C57Bl/6J (C57) or WT), Nrl
-/-
, Nrl
-/-
Grk1
-/-
and Nrl
-/-
Arr1
-/-
mice were PCR amplified,
cloned into individual TOPO TA cloning vectors and sequenced. All sequences were
aligned using Vector NTI and 34 unique SNPs were identified. The SNP corresponding
to amino acid reside 95 was consistently a guanine in Nrl
-/-
mice that translates to an
aspartate residue, which is more acidic, and an adenine in Nrl
-/-
Grk1
-/-
mice that translates
to an asparagine residue, which is more basic. The source of the unique forms of PGI
were traced back to the founding strains of mice used to generate both the Nrl
-/-
and Nrl
-/-
Grk1
-/-
knockout mice, C57Bl/6J and 129/SvJ (Pearce et al., 1995). Using the sequence
alignments from the NCBI database for these strains, along with the sequence results
from Nrl
-/-
and Nrl
-/-
Grk1
-/-
mice, it was concluded that the isoelectric mobility of PGI is
not due to a posttranslational modification, but a SNP which does not affect the activity
of PGI as an Embed Meyerhoff glycolytic pathway enzyme catalyzing the reversible
87
isomerization of fructose-6 phosphate to glucose-6 phosphate in glycoslysis (Yanagawa
et al., 2005).
Figure 3.14: Isoelectric Focusing of PGI in Various Retina Tissue Homogenates. Immunoblot
analysis of the IEF gel identified an acidic and basic form of PGI, later determined to be due to
the presence of a SNP at amino acid residue 95.
Figure 3.15: PGI Enzymatic Activity is Not Affected by Grk1. The kinetic studies of PGI
activity from tissue homogenates were performed and measured spectrophotometrically. The
graph demonstrates a similar kinetic activity rate for PGI in both Nrl
-/-
and Nrl
-/-
Grk1
-/-
tissue
homogenates.
88
3.3 Discussion
The absence of Grk1 in retinas leads to a rapid light dependent retinal
degeneration of rod photoreceptors due to a defective shutoff of rhodopsin in
phototransduction (Chen et al., 1999a). Moreover, Grk1
null mice have a delayed
recovery of photopic response in both the rod-dominant (Lyubarsky et al., 2000) and the
“all cone” Nrl
-/-
mouse retina (Nikonov et al., 2005; Zhu et al., 2006). In this study, we
observed for the first time that the cone photoreceptors from Nrl
-/-
mice lacking Grk1
expression degenerate with increasing age in a manner that is independent of their
environmental light exposure (Figure 3.1). Furthermore, we established that even when
the Nrl
-/-
Grk1
-/-
mice are maintained in darkness, their retinas exhibit cone functional
deficits as well as slower morphological changes leading to the inevitable loss of the
nuclei in the ONL (Figure 3.2A-L). The first indication of a defective photopic ERG
function was apparent at 3 months in all lighting conditions tested and led us to examine
the molecular and morphologic changes at 1 month when photopic ERG responses are
similar to Nrl
-/-
controls (Figure 3.1). Despite Grk1’s well characterized essential function
in phototransduction recovery, our data suggest alternative functions for Grk1, either
directly or indirectly, in the homeostasis and survival of cone photoreceptors. Our results
strongly suggest that even with a defective phototransduction shutoff pathway, the Nrl
-/-
mice are resistant to light mediated damage and the cone dystrophy is a manifestation of
an alternate mechanism.
In most retinal degeneration (rd) models, cone cell death is often secondary to rod
cell death, either due to the release of endotoxins from degenerating rods, environmental
89
alterations, or deprivation of a rod-derived trophic factor (Mohand-Said et al., 1998).
The loss of cone function, however, results in the more severe disability and reduction in
visual quality. We believe that the differences found between the Grk1
-/-
and Nrl
-/-
Grk1
-/-
phenotypes is a demonstration of the dynamic role and protective mechanisms rods
employ in the prevention of cone cell death.
Grk1 expression is essential for light dependent phosphorylation of short
wavelength (SWL) and medium wavelength (MWL) pigments in the mouse retina (Zhu
et al., 2003) and controls the rapid rate of cone recovery analogous to its rod function
(Lyubarsky et al., 2000; Nikonov et al., 2005; Zhu et al., 2006). Immunohistochemical
studies using antibodies for S- or M-opsin plus Arr4 prove that both SWL and MWL
expressing cones degenerate with increasing age (Figure 3.3). Furthermore, quantification
of TUNEL positive nuclei confirms that the cone photoreceptors have hallmarks of active
cell death that are significant by 1 month of age (Figure 3.4).
To further characterize the cone dystrophy of Nrl
-/-
Grk1
-/-
retinas, vascular cell
staining and fluorescein angiography (FA) experiments were performed. Nrl
-/-
Grk1
-/-
mice harbor severe breaks in the RPE with retinal anastomoses detectable by 1 month
(Figure 3.5), and exhibit retinal angiogenesis that progresses with age (Figure 3.6). FA
staining patterns show first signs of leakage in the right quadrant of the eye, which may
explain the wave of cell death in SWL and MWL expressing photoreceptors of these
mice (Figure 3.3). In contrast, Grk1
-/-
mice are similar to C57Bl/6J controls (Figure 3.10)
and do not exhibit a fluorescein dye leakage like Nrl
-/-
Grk1
-/-
retinas, even by 7 months.
This observation recapitulates our findings that the angiogenesis is specific to the loss of
90
Grk1 in Nrl
-/-
retinas and our observed dystrophy is most likely due to an increase in
retinal angiogenesis and not light exposure.
There are two known types of neovascularization in the retina: retinal
neovascularization (RNV) and choroidal neovascularization (CNV). Retinal
neovascularization is characterized by sprouting blood vessels that penetrate the inner
limiting membrane (ILM) and grow into the vitreous (Campochiaro, 2000); although in
some cases, the blood vessels can grow in the other direction, from the outer retina to the
subretinal space. The pathogenesis of RNV is better understood than that of CNV, with
ischemia, vascular endothelial cell growth factor (VEGF) expression, hyperoxia and
hypoxia-mediated gene regulation being key players in the characterization of the disease
(Campochiaro, 2000). In our Nrl
-/-
Grk1
-/-
mice, we observed an enhanced level of VEGF
expression beginning at PN21 that localized to both the blood vessels and inner limiting
membrane (Figure 3.7 and 3.8).
Alternatively to RNV, the molecular participants in the pathogenesis and
stimulation of CNV are less well understood. One possible contributor to CNV is
inflammation. Previous reports have indicated that deposits in and around Bruch’s
membrane may induce the inflammatory response, (Campochiaro, 2000) while another
attributes CNV to an abnormal extracellular matrix (ECM) resulting in diffuse thickening
of Bruch’s membrane (Green and Enger, 1993). During their initial characterization, the
Nrl
-/-
OS discs were found to often misalign and abnormally associate with the retinal
pigment epithelium (RPE) (Mears, et al. 2001), which may contribute to the weakened
Bruch’s membrane and increase susceptibility for choroidal blood vessel penetration
91
following an insult, in this case, loss of Grk1. We also observe an increase in the level of
inflammatory response genes in our cone dystrophy model at 1 month (Table 3.4 and
Figure 3.5), and a subsequent increase in blood vessel penetration into the retina that is
exacerbated with age (Figure 3.8). Following Isolectin B
4
staining, we identified blood
vessel penetration both from the choroid into the retina and the ILM, although it is
difficult to determine in which direction the anastomosis is growing in the ILM. Given
the results presented in this study, we hypothesize that the loss Grk1 on the Nrl
-/-
background stimulates angiogenesis that most closely resembles retinal angiomatous
proliferation (RAP), which is a variant form of CNV typified by intraretinal
neovascularization in the form of a retinal-retinal anastomosis and intraretinal
hemorrhage (Yannuzzi LA, 2001).
Microarray studies are an informative approach in understanding the potential
molecular pathways and mechanisms associated with the cone dystrophy and retinal
angiogenesis in our double knockout mice. Our analysis of the top molecular candidates
identified by IPA isolated two candidate genes, Pttg1 and Klk2, as interesting targets for
their role in cell cycle progression and inflammation, respectively, as well as their
previous identification in other retinal dystrophy mouse models (Fernandez-Medarde et
al., 2009; van de Pavert et al., 2007a). Pttg1 has the greatest fold increase in the Nrl
-/-
Grk1
-/-
retina (Table 3.1, Figure 3.13A), and was of interest due to its deregulation in both
Crb1 and RasGrf1 null mice (Fernandez-Medarde et al., 2009; van de Pavert et al.,
2007a). Crb1 mutant and null mice exhibit defects in retina morphology, focal retinal
disorganization, and humans with mutations in Crb1 develop Leber congenital amaurosis
92
(LCA) (van de Pavert et al., 2007a). RasGRF1 null mice, which have severe light
perception defects, also differentially express both Pttg1 and Crb1 genes among other
transcripts (Fernandez-Medarde et al., 2009). Nrl
-/-
Grk1
-/-
retinas similarly exhibit a
decrease in expression of Crb1 by over 6 fold (Table 3.2). Klk2, with an up-regulation of
over 29 fold that was validated by qPCR (Table 3.1 and Figure 3.13B), is an autoantigen
that was recently described as an inducer of Sjögren’s syndrome
(SS)-like
Keratoconjunctivitis Sicca (KCS) in Lewis rats (Jiang et al., 2009). Tissue Kallikreins
inhibit apoptosis and promote cell survival though the activation of the mitogen-activated
protein (MAP) kinases p44
ERK1
and p42
ERK2
(Erk1/2) signaling pathways (Liu et al.,
2009). We believe that the up-regulation of Klk2 and Pttg1 is noteworthy because of the
high AFC values for each as well as their prevalence in microarray studies from other
mouse models of retinal degenerative disease; nevertheless, the precise role and causal
mechanisms behind differential expression of these transcripts require further study.
Oncostatin M signaling was the top pathway identified (Figure 3.11) and is a well
characterized mechanism for both light mediated and inherited forms of retinal
degeneration (Samardzija et al., 2006). Previously, the retinal degeneration 1 (rd1)
mouse, with a mutation in the β-subunit of phosphodiesterase leading to a rapid
degeneration of photoreceptors, and the VPP mouse, a transgenic strain carrying three
rhodopsin mutations (V20G, P23H, P27L) leading to a slow degeneration, (Wu et al.,
1998) were examined for Jak-STAT signaling as independent forms of inherited retinal
degeneration (Samardzija et al., 2006). Phosphorylation of Stat3 and viral proto-
oncogene 1 (Akt), along with an induction of leukemia inhibitory factor (Lif), Cntf,
93
fibroblast growth factor-2 (Fgf-2), Osmr, Gp130, and Ch3l1 are some of the family
members of the Oncostatin pathway that are upregulated in both mouse models and can
be identified in the immune response pathway found in the Nrl
-/-
Grk1
-/-
retina (Table 3.1,
Table 3.2 and Figure 3.11) (Samardzija et al., 2006). It is unclear from the microarray and
qPCR data which cell types exhibit up or down regulation of the transcripts
characterized; however, we postulate that these changes in gene expression are somehow
associated in the cone photoreceptor degeneration observed in the Nrl
-/-
Grk1
-/-
mouse. We
understand the challenging caveats when identifying potential signal transduction
pathways using either microarray studies or proteomic approaches due to the volume of
information acquired using either technique. However, data presented here lays the
groundwork for further studies.
Grk1 is only one of at least seven members of the Grk superfamily expressed in
the mouse retina and pineal gland, including Grk2 (Penela et al., 2008). The ubiquitous
Grk2 may be a relevant modulator of inflammatory responses due to its ability to
attenuate chemokine induced migration in T cells and monocytes (Vroon et al., 2006).
Chronic down-regulation in Grk2 protein expression in immune cells leads to an aberrant
inflammatory response (Penela et al., 2008). Despite the limitations of studying a model
that does not mimic the natural structure of a rod dominant retina, the Nrl
-/-
Grk1
-/-
mouse
is an invaluable tool that demonstrates the relevance of Grk1 in cones and supports the
theory that Grk1 has alternative roles in the retina, analogous to Grk2, in regulation of
inflammatory response genes in maintaining healthy cone structure and function.
94
In conclusion, we have identified a retinal neovascularization phenotype that most
closely resembles RAP in AMD. Although future studies are required, our working
hypothesis is that Grk1 ablation in cones leads to a hypoxic or metabolically
compromised environment that subsequently stimulates increased blood vessel
penetration into the retina, leading to increased cone apoptosis. It is important to note that
the OS discs of Nrl
-/-
mice have been characterized to misalign and abnormally associate
with the RPE monolayer (Mears et al., 2001), which may facilitate blood vessel
penetration via a weakened Bruch’s membrane. Closer inspection of the membrane itself
must be conducted to further examine this possibility. Forthcoming studies will
extrapolate the causal relationship and relevance of Grk1 in the cone photoreceptor and
will provide a model for potential pharmacological interventions to either slow or rescue
photoreceptors from cell death.
95
CHAPTER 4. ACTIVATION OF POLY(ADP)RIBOSE POLYMERASE-1 (PARP)
CONTRIBUTES TO LIGHT-MEDIATED PHOTORECEPTOR
DEGENERATION IN GRK1
-/-
MICE
4.1 Introduction
Retinitis Pigmentosa (RP) is an inherited form of neurodegeneration that
specifically targets rod and cone photoreceptors. In 2010, RP was found to have a
worldwide prevalence of 1:3,500 and is regarded as the leading cause of blindness in
young individuals in the developed world (Herse, 2005). Several genetic mutations have
been implicated in the etiology of RP; however the pathological mechanisms behind the
photoreceptor dystrophy have yet to be resolved and effective treatments strategies are
currently lacking.
Oguchi disease is a form of RP characterized by congenital stationary night
blindness, fundus discoloration, abnormally slow dark adaptation after light exposure,
and characteristic electroretinography (ERG) abnormalities (Azam et al., 2009b). Human
populations with Oguchi disease exhibit loss of function mutations in phototransduction
recovery proteins G-protein coupled receptor kinase 1 (Grk1) and Arrestin 1 (Arr1),
leading to defective rhodopsin recovery and prolonged signal flow (Chen et al., 1999b).
Phosphorylation and deactivation of rhodopsin and cone opsins by Grk1 and Arr1,
respectfully, are crucial in maintaining the vitality of photoreceptors (Chen et al., 1999b;
Zhu et al., 2003). Loss of both recovery proteins in the mouse along with constant light
exposure lead to inhibition of the shutoff mechanism followed by DNA damage and
96
photoreceptor degeneration characterized by abnormal photoresponses and outer nuclear
layer (ONL) thinning.
In order to better understand the mechanisms leading to photoreceptor
degeneration in Oguchi disease, we examined another model for RP, the retinal
degeneration 1 (rd1) mouse. The rd1 mouse carries a loss of function mutation in the β
subunit of the rod photoreceptor cGMP phosphodiesterase 6 gene, (Bowes et al., 1990;
Pittler and Baehr, 1991) resulting in an cGMP accumulation and high levels of Ca
2+,
subsequently leading to apoptosis of rods (Lolley et al., 1994). Previous studies on the
rd1 mouse have identified the excessive activation of Poly(ADP)Ribose Polymerase-1
(PARP), an enzyme responsible for promoting DNA repair in their photoreceptor nuclei.
Through an NAD
+
dependent mechanism, PARP is responsible for the addition of
Poly(ADP)Ribose (PAR) moieties to itself and other proteins following adherence to
damaged DNA in an attempt to recruit proteins for base excision repair (BER) (Rouleau
et al., 2010a). The rd1 mouse was found to have excessively activated PARP beginning
at post natal (PN) day 11 that correlates with TUNEL positive staining in the ONL.
Therefore, the relevance of PARP expression and activation in mouse models for Oguchi
disease was assessed.
PARP is a nuclear protein comprised of an amino terminal DNA binding domain
harboring two zinc fingers important for binding to single and double strand breaks
(Gradwohl et al., 1990; Hassa et al., 2008), a central auto-modification domain with
glutamate and lysine residues for accepting (ADP)Ribose moieties (Altmeyer et al., 2009;
Tao et al., 2009) and a C-terminal catalytic domain sequentially transferring
97
(ADP)Ribose subunits from NAD
+
to protein acceptors. Following DNA nicks or breaks,
PARP is quickly recruited to the damaged region and its activity is increased up to 500
fold, synthesizing long protein conjugated branches of PAR chains (Hassa et al., 2008).
Following the addition of PAR groups onto itself, PARP recruits hundreds of other
proteins, such as X-ray repair complementing defective repair in Chinese hamster cells 1
(XRCC1), activator of DNA BER.
PARP family proteins are often regarded as the “guardians of the genome” since
they promote DNA repair whenever the DNA is compromised (Scott et al., 2004) and
thus continuously protect the cell. The PARP superfamily is classified based on their
functional domains or functions, but all of them share the common PARP catalytic
domain. PARP-1 and PARP-2 are DNA dependent, while tankyrases PARP-5, CCCH-
type PARP-12 and PARP-13, and tiPARP and macroPARPs, PARP-9, PARP-14, and
PARP-15 are located elsewhere and function as mono-ADP-ribosyltransferases (Rouleau
et al., 2010b; Yanagawa et al., 2007). PARP-1 is the most abundant PARP in the nucleus
and it, along with PARP-2, is the only PARP known to bind DNA, become activated by
DNA damage, and is implicated in repair and maintenance mechanisms for genomic
integrity (Yanagawa et al., 2007). PARP-1 has also been shown to localize to the
mitochondria and contribute to apoptosis inducing factor release and cell death (Du et al.,
2003). Due to the location and functional specificity of the various PARPs, the
experiments performed here are focused on PARP-1 (and will be referred to as PARP) as
the main mediator of ADP-ribosylation and activity in the mouse retina.
98
Figure 4.1: Structural and Functional Characteristics of PARP-1. (A) PARP-1 is shown with
its DNA-binding (DBD), auto-modification (AD) and catalytic domains. The PARP signature
sequence (yellow box) comprises the sequence most conserved among PARPs. Crucial residues
for NAD
+
binding (histidine; H, tyrosine; Y) and for polymerase activity (glutamic acid; E) are
indicated. (B) Consequences of PARP-1 activation by DNA damage. PARP-1 detects DNA
damage through its DBD. This activates PARP-1 to synthesize PAR (yellow beads) on acceptor
proteins, including histones and PARP-1. Owing to the dense negative charge of PAR, PARP-1
loses DNA affinity, allowing the recruitment of repair proteins to the damaged DNA (blue and
purple circles). PAR glycohydrolase (PARG) and possibly ADPribose hydrolase (ARH3)
hydrolyze PAR. ADP ribose is further metabolized by the pyrophosphohydrolase NUDIX
enzymes into AMP, raising AMP:ATP ratios, which activate the metabolic sensor AMP-activated
protein kinase (AMPK). NAD
+
is replenished at the expense of phoshoribosylpyrophosphate
(PRPP) and ATP. ATM, ataxia telangiectasia-mutated; BER base excision repair, BRCT, BRCA1
carboxy-terminal repeat motive; DNA-PKcs, DNA-protein kinase catalytic subunit; DSB, double-
strand break; HR, homologous recombination, NHEJ, non-homologous end joining, ; NLS,
nuclear localization signal; PP, inorganic pyrophosphate, SSB, single-strand break; Zn, zinc
finger. Reprinted with permission: Rouleau, M., et al., 2010, Nature Reviews.
A
B
99
The correlation between PARP activation with photoreceptor degeneration in the
rd1 mouse led us to investigate the possible association of PARP with the light-mediated
photoreceptor degeneration in mouse models for Oguchi disease, Arr1 and Grk1
knockout mice. Here we combined in situ activity analysis along with PARP and PAR
expression studies to show a definitive correlation between PARP activity and the light-
mediated photoreceptor degeneration that was exclusive to Grk1
-/-
and absent in Arr1
-/-
animals.
100
4.2 Results
4.2.1 PARP and PAR Expression in Grk1
-/-
, Arr1
-/-
and C57Bl/6J Mice
Total retinal protein from Arrestin 1 (Arr1) and G-protein coupled receptor kinase
1 (Grk1) knockout mice with known light-dependent photoreceptor degeneration were
examined for changes in Poly(ADP) ribosylation (PAR) following light adaptation (LA).
Mice were tested at 1 month because their ONL thickness at this age is comparable to
C57Bl/6J control retinas following light exposure. Initial studies testing the levels of
PAR moieties in light adapted retinas demonstrate a significant increase in PAR levels in
Grk1
-/-
mice but only a slight change in Arr1
-/-
retinas (Figure 4.2). Immunoblot analysis
revealed an enhanced level of PAR moiety addition to proteins at approximately 78kD-
170kD, which is in the molecular weight range of Poly(ADP) Ribose Polymerase-1
(PARP) in its full length and cleaved forms. Due to the presence of an enhanced level of
PARP activity as measured by PAR expression in Grk1
-/-
following light exposure, we
further tested our theory that PARP activation is a contributing factor to the light-
mediated photoreceptor degeneration in these animal models of RP.
PARP-1 is the best known variant of the PARP isoforms and is relevant for
consideration in pathophysiology and experimental treatment strategies (Jagtap and
Szabo, 2005). All immunostaining experiments were conducted using an anti-PARP-1
specific antibody (clone C2-10, Trevigen); for clarification, PARP-1 will be referred to as
PARP.
101
Figure 4.2: PAR Expression in Light Adapted Grk1
-/-
, Arr1
-/-
and C57Bl/6J Retinas. Each
well represents 25ug of total protein from 1 month old animals. Although a slight increase in
PAR expression was observed in Arr1
-/-
, Grk1
-/-
retinas had the more significant increase in
expression between 78 and 170kD. GAPDH was used as a loading control.
Immunohistochemistry (IHC) experiments were performed on Grk1
-/-
mice at 1, 2,
and 4.5 months to detect changes in PAR expression with increasing age. Figure 4.3A
represents the level of PAR expression in 1 month old Grk1
-/-
retinas, while Figure 4.3B
demonstrates the level of PAR expression in light adapted mice at various ages. Our
studies denote that PAR expression was most prevalent in 1 month old light adapted
Grk1
-/-
mice; however, we hypothesize that if all mice were exposed to 24 hours of
continuous light, an enhanced PAR expression pattern would be observed at all ages.
White arrows indicate the location of PAR expression within photoreceptor nuclei in 1
month old samples.
102
Figure 4.3: PAR Expression is Enhanced at 1 Month in Light Adapted Grk1
-/-
Retinas. (A) PAR immunoblot on 1 month old Grk1
-/-
and C57Bl/6J retina homogenates.
The black arrow indicates the approximate location of Parp-1. GAPDH is shown as the
loading control. (B) PAR immunostaining on retinal sections from C57Bl/6J mice at 1
month and Grk1
-/-
at 1, 2, and 4.5 months of age. The white arrows indicate the presence
of PAR staining in the ONL that is most prevalent in light adapted 1 month old Grk1
-/-
mice. C57Bl/6J retinas processed without primary antibody is provided as a negative
control.
103
Figure 4.3, Continued: PAR Expression is Enhanced at 1 Month in Light Adapted Grk1
-/-
Retinas.
104
Our initial studies of PAR expression lead us to further examine the changes in
protein expression and activation of PARP in Grk1
-/-
and C57Bl/6J controls after
exposure to 24 hours of room light. Because Grk1
-/-
mice degenerate significantly under
these conditions, we hypothesized that the increase in apoptosis will correlate to PARP
activity and expression (Chen et al., 1999a). Immunoblot analysis of PARP and PAR in
control and experimental animals demonstrate an increased level of PARP expression in
Grk1
-/-
mice left in 24 hours of room light, which is significantly greater than their
counterparts left in 24 hours of complete darkness. C57Bl/6J mice were exposed to the
same treatment conditions and show no changes in PARP expression. Although PARP
expression is slightly greater in Grk1
-/-
dark mice when compared to C57Bl/6J controls, it
is significantly enhanced in Grk1
-/-
mice exposed to light for 24 hours (Figure 4.4). PARP
activation is coupled with its cleavage by Caspase-3 (Rouleau et al., 2010a). Figure 4.4
thus suggests that light treated Grk1
-/-
mice have an increase in the level of the PARP
cleavage product at 85kDa (lower black arrow). Similarly, by loading only 13ug of total
protein per sample, the level of PAR moieties is less prevalent in Figure 4.4 than is
observed in Figure 4.2, however the level of Poly(ADP)ribosylation of PARP itself is
enhanced when compared to Grk1
-/-
dark samples . The black arrow of the PAR
immunoblot represents the location of PARP-1.
105
Figure 4.4: PARP and PAR Expression in Dark and Light Treated Grk1
-/-
and C57Bl/6J
Mice. (A) Mice were exposed to either 24 hours of total darkness or room light and probed for
PARP. The top arrow corresponds to the uncleaved form (116kDa) while the bottom arrow
indicates the cleaved fraction (85kDa). Grk1
-/-
mice exposed to 24 hours of room light have
higher levels of PARP expression. (B) The same retinal samples were probed for PAR and an
enhanced level of PAR expression was observed at the molecular weight corresponding to the full
length PARP. GAPDH was used as a loading control.
4.2.2 Correlation of PARP Activity with Cell Death
Immunoblot experiments demonstrate an increase in PARP expression and
activation through PAR staining in Grk1
-/-
mice kept in 24 hours of light. The association
between PARP and PAR expression with light exposure suggests a relationship between
PARP activity and cell death. We therefore performed TUNEL assays to determine the
amount of dying cells in the retinas of all mice following exposure to either 24 hours of
darkness or 24 hours of light. To test for PARP activity, we performed a dual localization
study with TUNEL staining followed by incubation with the PAR antibody. As shown in
Figure 4.5, C57Bl/6J (WT) mice did not have an increase in TUNEL or PAR staining in
106
both conditions, which was similar to Grk1
-/-
mice kept in 24 hours of darkness. In
contrast, Grk1
-/-
mice exposed to 24 hours of light had a significant increase in the
number of TUNEL positive cells within the ONL, along with an increased number of
PAR stained nuclei. Colocalization of TUNEL with PAR is prevalent in light exposed
Grk1
-/-
mice (Figure 4.5 insert). The number of TUNEL positive cells with PAR stained
nuclei was quantified and shown in Figure 4.5. Accordingly, PAR staining is
approximately half that of the number of TUNEL positive cells, demonstrating a strong
correlation between PAR expression and cell death.
107
Figure 4.5: PAR IHC and TUNEL Staining. TUNEL positive cells are indicated in
green and PAR immunofluorescence in red. C57Bl/6J mice exposed to either 24 hours of
room light or total darkness. Grk1
-/-
mice exposed to the same conditions are presented on
the right. DAPI nuclear staining in blue is shown for orientation and morphology. ONL,
outer nuclear layer; IPL, inner plexiform layer; INL, inner nuclear layer. Images were
taken with a Confocal Laser Scan microscope using a 40x lens. Scale bar, 10μm.
108
Figure 4.5, Continued: PAR IHC and TUNEL Staining.
109
Figure 4.6: Quantification of TUNEL and PAR Positive Cells. Three retinal sections from
three different mice per genotype were used for quantification. Green bars represent the average
number of TUNEL positive cells per section while the red bar represents the number of PAR
stained nuclei per section. Approximately 50% of TUNEL positive cells exhibited expression of
PAR in their nuclei.
4.2.3 Enhanced PARP Cleavage and Activity Analysis
The importance of PARP activity for light mediated Grk1
-/-
photoreceptor
degeneration was addressed by an ELISA colorimetric assay (Trevigen), by which the
level of cleaved PARP is measured as an indicator for its activation. The results obtained
from the colorimetric kit are indicated as absorbance values read at 450nm on a plate
reader. Cleavage of PARP promotes apoptosis be preventing DNA repair-induced
survival and by blocking energy depletion-induced necrosis (D'Amours et al., 2001). The
incorporation of biotinylated PAR groups onto histone coated wells was used as a
measure for PARP activity from homogenized tissue (Figure 4.7). Retinas from 3 mice
exposed to both light and dark conditions per genotype were used and reveal a decrease
110
in the addition of PAR groups onto the histones in 24 hour light exposed Grk1
-/-
mice,
representing cleavage of PARP and enhanced activation in vivo.
Figure 4.7: PARP Activity ELISA Assay. Retinas from 3 mice per genotype and condition were
utilized. Black bars represent C57Bl/6J mice and blue bars represent Grk1
-/-
mice. Dark and light
conditions indicate 24 hours of exposure. A nonparametric ANOVA with Bonferonni’s correction
was performed and indicates a statistically significant decrease in PAR addition in light exposed
Grk1
-/-
mice compared to those kept in the dark and C57Bl/6J mice kept in the light. ** P< 0.01.
111
Finally, PARP activity was measured directly using an in situ assay. The
incorporation of biotinylated NAD was measured from mice exposed to 24 hours of room
light and total darkness from both Grk1
-/-
and C57Bl/6J controls. The incorporation of
biotin was observed following incubation with a rhodamine labeled avidin secondary
antibody. The red nuclei indicate PARP activity, which was only observed in Grk1
-/-
mice
exposed to 24 hours of room light. These observations confirm our PAR IHC and
TUNEL colocalization staining experiments in that they demonstrate the addition of PAR
is correlated to increased PARP activity. The ELISA assay examines changes in PARP,
and with a decrease in the full length form, we can conclude that the activation of PARP
in the Grk1
-/-
light induced photoreceptor degeneration is correlated to enhanced PARP
expression and activity in the photoreceptor nuclei.
112
Figure 4.8: In Situ PARP Activity Assay. Mice exposed to both 24 hours of light and
dark conditions were examined for incorporation of biotinylated NAD+, a direct measure
of PARP activity. Where dark and light treated C57Bl/6J mice and dark treated Grk1
-/-
mice had little to no PARP activity, Grk1
-/-
mice kept in 24 hours of light have marked
PARP activity as indicated by red stained nuclei and marked by white arrows in the ONL.
DAPI nuclear stain was used for morphology and orientation and the scale bar represents
a distance of 10μm.
113
Figure 4.8, Continued: In Situ PARP Activity Assay.
114
4.3 Discussion
This study demonstrates for the first time that the light mediated photoreceptor
degeneration of Grk1
-/-
mice involves PARP enzymatic activity. PARP expression as well
as its product PAR, were dramatically increased in the ONL of Grk1
-/-
mice exposed to
24 hours of room light but not when the mice were kept in the dark. We also correlated
PARP expression with dying TUNEL positive photoreceptors, indicating an involvement
of excessive PARP activity in light mediated Grk1
-/-
photoreceptor apoptosis. TUNEL
and PAR positive nuclei were located near the outer limiting membrane and throughout
the ONL. With over 50% of the cones localizing to the top portion of the ONL, we
predict that both rod and cone photoreceptors are degenerating in these mice (Carter-
Dawson et al., 1978).
Protein expression of PARP and PAR were increased in the light exposed Grk1
-/-
retinas when compared to those kept in total darkness and both C56Bl/6J control groups.
This demonstrates that the light mediated DNA damage previously reported in Grk1
-/-
mice is affecting both the level of PARP expression and its function within the mouse
retina. The accumulation of PAR within a given cell is due to the balance between PARP
and Poly(ADP)Ribose Glycohydrolase (PARG) activity. Therefore, the accumulation of
PARP in Grk1
-/-
light exposed retinas correlates with the theory that the light induced
DNA damage is affecting PARP transcription or stability level. Future studies using
quantitative RT-PCR will be performed to determine whether the increase in protein
expression is due to enhanced transcription, mRNA translation, decreased PARG activity
or a combination of the three. Because PARP expression and PAR addition to proteins is
115
enhanced, we cannot yet conclude if the PARP enzymes are more active per se, or if the
elevated level of PARP protein is contributing to its enhanced activation. Several recent
reports have shown changes in PARP-1 expression under pathophysiological conditions,
including up-regulation in chronic heart failure (Pillai et al., 2005). Nevertheless, the
data presented here demonstrate that not only is PARP expression increased in Grk1
-/-
mice following light exposure, but the overall activity is also enhanced most notably at 1
month.
These findings necessitated the extended analyses from protein expression to
biochemical enzymatic function within the mouse retina. We therefore performed PARP
activity assays to determine if in fact the activity level of PARP is also enhanced. In situ
experiments using biotinylated NAD
+
as the substrate identified marked photoreceptor
nuclei within the ONL of light exposed Grk1
-/-
mice. This study demonstrates that the
light mediated photoreceptor dystrophy is due not only to increased PARP expression,
but its enhanced level of activation.
The colorimetric ELISA was used to demonstrate that not only is PARP
expression and activation increased in the Grk1
-/-
retina, but it is cleaved by caspases
following light exposure. PARP expression levels in C57Bl/6J and dark treated Grk1
-/-
mice were also found to be comparable to one another using the PARP ELISA assay. The
level of PARP cleavage, a direct correlation to its activation, was identified only in the
Grk1
-/-
light treated samples, indicated by a decrease in the level of PAR groups added to
the histones located on each strip well. This demonstrates that there is a comparable level
116
of expression and activity of PARP in the C57Bl/6J mice that, when given the
appropriate substrate and conditions, function normally.
PARP is primarily a nuclear protein and has been previously shown to be
excessively activated in the rd1 mouse (Paquet-Durand et al., 2007). The fact that a
similar level of PARP activation in light treated Grk1
-/-
mice was observed is interesting
because the rd1 mouse is a well studied RP model that also carries a loss of function
mutation in a critical phototransduction protein, the β-subunit of PDE6 (Bowes and
Farber, 1987; Farber and Lolley, 1974). The mutation in PDE6 leads to cGMP
accumulation and subsequently, abnormally high levels of Ca
2+
in the photoreceptors
leading to apoptotic like rod cell death. Knockout mice for phototransduction recovery
proteins, such as Arr1 and Grk1, are believed to have low Ca
2+
levels due to their
inability to regulate the deactivation mechanism and have prolonged release of Ca
2+
ions.
It is important to note, however, that the PARP association with photoreceptor cell death
was exclusive to light treated Grk1
-/-
mice and not in those lacking a functional Arr1. The
direct causative relationship between Grk1 loss and PARP activation has yet to be
elucidated; however the data presented here provide the foundation for future mechanism
development studies.
The quantification of TUNEL positive and PAR positive stained nuclei further
supports this theory that excessive PARP activation results from the increase in DNA
damage of Grk1
-/-
photoreceptors. The approximate 50% co-localization of TUNEL with
PAR staining implies that PARP is activated subsequent to DNA damage caused by over
activation of phototransduction. However, excess PARP activation has been shown to
117
have several deleterious effects, mainly due to the significant consumption of NAD
+
through the formation of PAR moieties leading to energy depletion and eventual cell
death. We hypothesize that the DNA damage induced by the defective phototransduction
recovery mechanism may be the primary culprit for the enhanced expression and
activation of PARP-1, and energy depletion due to excessive PARP-1 activation may be
secondary and just as detrimental to photoreceptor survival.
The mechanism by which loss of function mutations in phototransduction proteins
leading to cell death associated with PARP activation has several therapeutic
implications. Experiments employing the selective PARP inhibitor, PJ34 (N-(6-Oxo-5,6-
dihydrophenanthridin-2-yl)-N,N-dimethylacetamide.HCl) will further assess the
therapeutic possibilities in human retinal diseases like Oguchi (Azam et al., 2009a).
Retinal explant studies using PJ34 will delineate the role of PARP inhibition in the
protection of light mediated photoreceptor dystrophy of Grk1
-/-
mice. In summary, the
results presented here provide new insights into the mechanisms of retinal photoreceptor
degeneration, particularly degeneration associated with defective phototransduction
recovery mechanisms and Grk1, fostering our attempts to find therapeutics for human
disease.
118
CHAPTER 5. CHARACTERIZATION OF THE PITUITARY TUMOR
TRANSFORMING GENE 1 (PTTG1) NULL MOUSE RETINA
5.1 Introduction
Upon examination of the gene expression differences between Nrl
-/-
and Nrl
-/-
Grk1
-/-
retinas, we found that the transcript receiving the two highest average fold change
values of 130 and 70 in experimental Nrl
-/-
Grk1
-/-
mice was Pituitary tumor transforming
gene 1 (Pttg1). Pttg1 was first identified in rat pituitary tumors and according to
sequence homology was determined to be the vertebrate securin involved in sister
chromatid segregation during mitosis (Wang et al., 2001). Over expression of Pttg1 has
been reported in a variety of endocrine-related tumors, including pituitary, thyroid,
breast, ovarian, and uterine tumors, as well as nonendocrine-related cancers involving the
central nervous system, pulmonary and gastrointestinal systems (Wang et al., 2001).
Pttg1 is considered a proto-oncogene for its role in NIH3T3 cell transformation, tumor
formation and correlation with tumor invasiveness and metastasis. Together with its
functions in cell replication, Pttg1 plays important roles in DNA damage/repair and organ
development and metabolism. More recently, Pttg1 was shown to mediate endothelial
cell survival and angiogenesis through its regulation by phospho-Akt (Caporali et al.,
2008b).
Despite its well characterized role in sister chromatid segregation and cell cycle
progression, the murine Pttg1 knockout is surprisingly viable and fertile (Wang et al.,
2001). The phenotypic characteristics of these mice currently include testicular and
splenic hypoplasia, thymic hyperplasia, thrombocytopenia, aberrant cell cycle
119
progression, chromosome instability, and premature centromere division (Wang et al.,
2001). Pttg1
-/-
mice also have severely impaired glucose homeostasis and pancreatic beta
cell proliferation leading to diabetes, particularly in male mice over 5 months of age
(Wang et al., 2003).
The mouse Pttg1 gene shares 70% homology to human PTTG1; therefore, the
Pttg1
-/-
mouse is a good model for the characterization of the protein in physiological
functions for implications of human disease. Due to the significant up-regulation of the
Pttg1 transcript in Nrl
-/-
Grk1
-/-
retinas with a known light independent photoreceptor
degeneration, deregulation in other retinal dystrophy models (Fernandez-Medarde et al.,
2009; van de Pavert et al., 2007b), along with the characterized diabetes found in older
male mice, we hypothesized that Pttg1 plays a critical role in the structure and function of
the mouse retina. With this in mind, characterization studies were performed using
Hematoxylin and Eosin (H&E), immunohistochemistry (IHC), and electroretinography
(ERG). Also, 7 month old male and female Pttg1
-/-
mice were examined for diabetic
retinopathy and neovascularization. This study examines the functional and morphologic
characteristics associated with the loss of Pttg1 in the mouse retina in an effort to further
understand its transcriptional variability in retinal dystrophy models.
120
5.2 Results
5.2.1 Pttg1
-/-
Mice Lack the DdeI Mutation for Rdle and are Met450Met for
Rpe65
Upon receipt of the 6 Pttg1
-/-
mice from Cedars Sinai Hospital, tails were clipped
and genotyped for the absence of Pttg1. A C57Bl6/J mouse was used as the positive
control (PC). Figure 5.1A represents the results of the genotyping, which confirm that the
animals were true knockouts for Pttg1.
To verify that the Pttg1
-/-
mice all have the same variant of the Rpe65 gene
product at amino acid residue 450, Met450 specific and Leu450 specific primers were
used. Figure 5.1B shows the PCR products of both primer sets, alongside appropriate
positive controls. According to Figure 5.1, all of the Pttg1
-/-
mice were Met450Met for
Rpe65 and are therefore not susceptible to light damage (Kim et al., 2004).
All six animals were screened for the presence of the nonsense mutation in the β
subunit of the phosphodiesterase gene (β-PDE) containing two DdeI sites. Following
PCR amplification of the sequence containing the potential DdeI site, the PCR products
were subject to DdeI digestion and electrophoresed on a 2% agarose gel. Figure 5.1C
demonstrates that the DdeI sites are absent from the 6 Pttg1
-/-
mice. Genomic DNA from
rd1 mice were used as a positive control. The absence of a PCR product in these animals
represents the insertion of a DdeI site (Pittler and Baehr, 1991).
121
Figure 5.1: Genotyping of the 6 Pttg1
-/-
Parents. (A) The six mice were confirmed as true
knockouts for Pttg1. The positive control (PC) used was the genomic DNA of a C56Bl/6J mouse.
(B) Genotyping for the Rpe65 SNP indicates that all 6 mice are Met450Met. (C) Digestion with
the DdeI restriction enzyme shows a lack of the DdeI transversion mutation found in rd1 mice.
Genomic DNA from C57Bl/6J and RDLE mice were used as negative and positive controls for
the DdeI mutation, respectively.
5.2.2 Healthy Cone Function and Cone Number in Pttg1
-/-
Mice
In order to determine the possible relevance of Pttg1 in the murine retina, the
morphology of Pttg1
-/-
retinas were examined with increasing age for changes in outer
nuclear layer (ONL) thickness using Hematoxylin and Eosin (H&E) staining. As shown
in Figure 5.2, the ONL thickness in Pttg1
-/-
mice at 1, 5, and 9 months of age are similar
and comparable to C57Bl/6J controls of the same age. This further confirms that the
absence of Pttg1 is not necessary for photoreceptor number and viability.
A
B
C
122
Figure 5.2: Hematoxylin and Eosin (H&E) Staining Indicate Healthy Retinal Morphology
and Normal ONL Thickness in Pttg1
-/-
. All mice were reared in cycling light and light adapted
prior to euthanasia. The ONL thickness is indicated by white arrows. Images were taken of the
central inferior region of the retina. The scale bar represents a length of 10μm.
Alteration to cone cell function was analyzed in Pttg1
-/-
mice in comparison to age
matched C57Bl/6J (WT) controls in both male and female mice at 7 months of age.
According to the maximum b-wave amplitudes plotted in Figure 5.3, there was no
statistically significant difference in cone function in Pttg1
-/-
mice when compared to
controls. This demonstrates that the expression of Pttg1 is not essential for normal cone
function.
123
Figure 5.3: Maximum b-wave Amplitude of Dark-Adapted ERG Responses in WT and
Pttg1
-/-
Mice. Mice were dark-adapted overnight before ERG recordings. Maximum responses
were elicited by high-intensity white fluorescent light. Both sets of animals were 9 months old
and show similar ERG recordings demonstrating normal cone b-wave light-adaptation function.
To further extrapolate the potential importance of Pttg1 for murine cone
photoreceptors, cone Arrestin 4 immunohistochemistry (IHC) staining was conducted in
1.5 and 9 month old animals to examine changes associated with increasing age.
According to Figures 5.4 and 5.5 below, it is apparent that the cone numbers are
comparable in both Pttg1
-/-
and C57Bl/6J mice at both ages examined. The staining
pattern for cone cells is similar across the entire retinal sections shown (Figure 5.4) and
the staining pattern of the superior region of the retina show similar cone cell numbers
and ONL thickness as indicated by the white arrows.
124
Figure 5.4: Arrestin 4 IHC Staining in Pttg1
-/-
Mice is Similar to C57Bl/6J at 1.5 and 9
Months. Mice were born and maintained in cycling light and euthanized under room light. Each
section represents a 7um section through the optic nerve. Arr4 staining is indicated in green
fluorescence. Images were taken under a Leica Fluorescence Light Microscope with a 20x lens.
Figure 5.5: Mouse Arrestin 4 Staining in the Superior Region of Pttg1
-/-
and C57Bl/6J Mice.
Staining patterns indicate a similar number of cone cells between Pttg1
-/-
mice and C57Bl/6J
controls at both 1.5 and 9 months of age. White arrows demonstrate no change in ONL thickness.
125
5.2.3 Pttg1
-/-
Male and Female Mice Have Normal FA Staining Patterns
The disruption of Pttg1 in mice has been shown to severely impair glucose
homeostasis leading to diabetes in late adulthood, especially in males associated with
nonautoimmune insulinopenia and reversed alpha/beta cell ratio (Wang et al., 2001).
Pttg1
-/-
animals therefore exhibit sexually dimorphic diabetes mellitus (Wang et al.,
2001). Based on these observations, we hypothesized that male Pttg1
-/-
mice over 5
months of age had defects in vasculature similar to diabetic retinopathy. Diabetic
retinopathy is caused by damage to retinal blood vessels through the formation of
microaneurysms, retinal hemorrhage, or retinal neovascularization (Wang et al., 2003).
In order to examine changes in retinal vasculature associated with sexually
dimorphic Pttg1
-/-
mediated diabetes, fluorescein angiography (FA) staining was
performed on 7 month old Pttg1
-/-
male and female mice. C57Bl/6J mice at both ages
were used for comparison. The central, superior and inferior regions of the retina are
provided in Figure 5.6. The images show no apparent changes in vasculature associated
with leakage or breaks. This led us to conclude that the diabetic phenotype observed in
older male Pttg1
-/-
mice does not affect the retina or exhibit a phenotype similar to
diabetic retinopathy up to 7 months of age.
126
Figure 5.6: Pttg1
-/-
Male and Female Mice Have Normal Retinal Vasculature. The
Fluorescein Angiography (FA) images represent the central, superior and inferior regions of the
retinas from 7 month old animals. Both male and female Pttg1
-/-
mice were compared to a male
C57Bl/6J healthy control.
127
5.3 Discussion
A known cell cycle regulatory protein and oncogene, Pttg1 was an interesting
target for investigation due to its deregulation in various retinal dystrophy models
including the Nrl
-/-
Grk1
-/-
mouse. Microarray and quantitative PCR studies on Ras protein
specific guanine nucleotide releasing factor 1 (RasGRF1) and Crumbs homolog 1 (Crb1)
null mice, which also exhibit retinal deficits, have significant deregulation of Pttg1 in
their gene transcription analyses (Fernandez-Medarde et al., 2009; van de Pavert et al.,
2007b). Pttg1 null mice were thus acquired with the intent to further elucidate the
functional relevance of Pttg1 in the structure and function of the murine retina.
According to our retinal morphology studies, Pttg1
-/-
mice are comparable to
C57Bl/6J controls, with slightly smaller ONL thickness at 1 month that is maintained up
to 9 months of age. By rearing the mice in 12hr light:12hr dark cycles, we were able to
test for the presence of light mediated photoreceptor cell death. Because the ONL
thickness of these mice were stable up to 9 months, it is clear that the photoreceptor cells
are viable and not susceptible to light mediated cell death.
Mice lacking the Neural retina leucine zipper transcription factor (Nrl
-/-
) have
retinas lacking rods but express photoreceptors that share morphological, molecular and
electrophysiological characteristics with cones (Daniele et al., 2005). In the initial
microarray studies performed on Nrl
-/-
mice, Pttg1 was identified as significantly down
regulated by 10.3 fold when compared to wildtype controls (Oh et al., 2008a). We
therefore postulated that the loss of Pttg1 influenced cone cell function and number with
age. Our examination of cone functional ERGs along with cone arrestin (Arr4) IHC
128
revealed that Pttg1
-/-
mice have photopic b wave amplitudes similar to controls (Figure
5.3) and healthy cone cell numbers (Figure 5.4) that persist up to 9 months.
We performed FA staining on older male and female mice to detect signs of
diabetic retinopathy at an age where animals were previously described to exhibit
characteristics of diabetes mellitus. However, in our FA experiments in both male and
female mice at 7 months, we observed normal staining patterns and the absence of any
leakage or damage to blood vessels (Figure 5.6). In conclusion, Pttg1
-/-
male mice over 5
months exhibit diabetes due to their inefficient pancreatic beta cell turnover, however
their retinas function normally, have healthy morphology, and do not exhibit a diabetic
retinopathy phenotype.
The retina is a complex layered structure composed of several neurons in an
interconnected arrangement maintained by the expression and regulation of many genes.
To further elucidate the factors that maintain a healthy retina, we identified Pttg1 as a
novel target in the homeostasis of retinal structure and function. The results presented
here demonstrate that the loss of Pttg1 does not manifest into a retinal degenerative
phenotype and its expression is not essential in the maintenance of normal retinal
function. Despite the lack of Pttg1 leading to diabetes mellitus, the animals examined
here lacked neovascularization or a phenotype similar to diabetic retinopathy.
Deregulation of Pttg1 must not be minimized however, because the over expression of
the gene transcript may potentially lead to retinal neovascularization and photoreceptor
dystrophy. Future studies on retinal disease models with enhanced Pttg1expression
crossed with the Pttg1 null mouse may help to identify Pttg1 as an important modifier or
129
regulator of retinal degeneration. According to our studies, Pttg1 does not seem to be the
primary insult leading to photoreceptor cell death; however, the more likely hypothesis is
that it is secondary to the loss of function for other proteins critical to retinal function.
For example, Pttg1 has been shown to stimulate the production of angiogenic growth
factors such as basic fibroblast growth factor (bFGF) and enhance the stability of
endothelial cells in solid tumors. The enhanced expression of Pttg1 may be a secondary
effecter of the retinal neovascularization phenotype observed in the Nrl
-/-
Grk1
-/-
retina,
although further examination is required (Caporali et al., 2008a). Whether or not the
variability in Pttg1 expression is a consequence of or contributor to retinal degeneration
has yet to be determined. Although numerous mechanisms of action have been identified
for Pttg1, these studies provide the basis for further extrapolation of the relevance of this
multifunctional protein in the murine retina.
130
CHAPTER 6. MOUSE BACKGROUND STRAIN VARIABILITY IN THE
ARRESTIN 1 NULL (ARR1
-/-
) PHENOTYPE
6.1 Introduction
Mutations in genes encoded by phototransduction components have been linked
to photoreceptor cell death in numerous animal models. Loss of function phenotypes
caused by mutations in genes involved in recovery, including G-protein coupled receptor
kinase 1 (GRK1) and Arrestin 1 (ARR1), lead to a constitutive signal flow and underlie
retina disorders such as Oguchi disease, a form of Retinitis Pigmentosa (RP)
characterized by congenital stationary night blindness (Chen et al., 1999b).
ARR1 plays a key role in the recovery of the visual phototransduction cascade by
binding to light-activated, phosphorylated rhodopsin and preventing its interaction with
the G-protein transducin. Mice lacking a functional Arr1 show greatly prolonged light
responses and eventual light-dependent photoreceptor degeneration. Excessive light
exposure accelerates this cell death, with a 30% reduction in rod photoreceptor outer
nuclear layer (ONL) following 1 week of constant light exposure (Chen et al., 1999b).
However, recent examination of retinas from the colony of Arr1
-/-
mice in our laboratory
has lead to the identification of a light-independent cone photoreceptor dystrophy, which
revealed significant cone loss by postnatal day (P) 60 (Brown et al., 2010).
To address the discrepancy between data comparing these phenotypic variations,
we examined the genetic backgrounds of Arr1
-/-
mice. Two separate mouse colonies: the
original knockout (KO) mice kindly provided by Dr. Jeannie Chen (denoted as Arr1(A)
-/-
), and our KO mice (Arr1
-/-
Arr4
+/+
, denoted as Arr1(B)
-/-
) that are the offspring of an F2
131
cross between the original Arr1
-/-
(A) mice with Arrestin 4 null animals (Arr4
-/-
) (Nikonov
et al., 2008). Both KO mice are on a combined genetic mixture of C57Bl/6J and 129/SvJ
strains.
To further characterize the phenotypic variability leading to cone dystrophy
between colonies, protein expression profiles were conducted on Arr1(A)
-/-
and Arr1(B)
-/-
retinas using two-dimensional gel electrophoresis (2DE) and Ettan
TM
DIGE overlay
technology. In doing so, we identified two distinct isoelectric points (PI) for a 24 kDa
protein, later identified by Mass Spectrometry (MS) as Peroxiredoxin6 (Prdx6) (Craft &
Brown, unpublished observation). A more acidic form of Prdx6 was expressed in the
Arr1(A)
-/-
retina, in contrast to the more basic form in the Arr1(B)
-/-
retina.
Peroxiredoxins are a family of thiol-dependent peroxidases that reduce peroxides
using conserved cysteine residues in their catalytic centers (Ho et al., 2010). Of the six
members identified in mammalian tissue, Prdx6 is the sole member to include 1 cysteine
redox-active residue and a unique phospholipase A
2
activity (PLA
2
) (Ho et al., 2010).
Prdx6 is therefore a bifunctional enzyme, acting as both a peroxidase and phospholipase
catalyzing the hydrolysis of the sn-2 fatty acyl ester bond of glycerophospholipids to
produce free fatty acids and lysophopholipids (Hooks and Cummings, 2008). Although
the physiologic role of Prdx6 is largely unknown, studies performed on various mouse
strains revealed a unique single nucleotide polymorphism (SNP) at amino acid residue
122 to be associated with increased susceptibility for oxidative damage and
atherosclerosis (Iakoubova et al., 1997). We therefore studied this protein as a potential
132
modifier in the susceptibility to oxidative stress, as well as examine the coding sequence
to verify the source of the isoelectric mobility differences between the Arr1
-/-
colonies.
In addition to the unique isoelectric mobility of Prdx6 between Arr1
-/-
(A) and
Arr1(B)
-/-
mice, additional modifier genes may be involved in the phenotype; therefore,
linkage analysis (Neena Haider, University of Nebraska) and genome wide association
studies (Margaret DeAngelis, Harvard Medical School) were performed to examine the
genetic variability in mice that lead to the dissimilarity in the cone dystrophy phenotypes.
These techniques provide a high throughput but comprehensive approach to measure
linkage disequilibrium in identifying candidate genes associated with a particular disease
or condition. Genomic DNA from ~50 mice from both Arr1(A)
-/-
and Arr1(B)
-/-
colonies
were analyzed and compared with C57Bl/6J and 129SVj. The pilot association mapping
study with microsatellite markers identified 10 potential modifier resistant candidate loci.
The arrays, along with our analyses of Prdx6, provide us with a better understanding of
the link between genetic variability and the light-independent age related photoreceptor
degeneration in our animal models. Our analysis will further provide us with a better
understanding of potential modifier genes associated with retinal degenerative diseases
such as atrophic Age-related Macular Degeneration (AMD).
133
6.2 Results
6.2.1 Light-Independent Photoreceptor Apoptosis
TUNEL staining was performed and quantified on dark adapted Arr1(A)
-/-
and
Arr1(B)
-/-
mice at postnatal days (P) 22, 45, and 60. A statistically significant increase in
the number of TUNEL positive cells was observed in Arr1(A)
-/-
and Arr1(B)
-/-
mice
compared to age matched WT controls at P45 but not at P22. It is interesting to note that
at P45, Arr1(B)
-/-
mice had significantly greater TUNEL positive cells than Arr1(A)
-/-
mice, indicating that they are more susceptible to light independent photoreceptor
degeneration at this age. By P60, the level of TUNEL positive cells in Arr1(B)
-/-
mice
was still significantly greater than WT; however, the Arr1(A)
-/-
retinas had minimal cell
death and were similar to colony control WT. From the age groups examined, it is
apparent that Arr1(A)
-/-
mice have a peak in photoreceptor apoptosis at P45, which
decreases to WT levels by P60. The Arr1(B)
-/-
mice have an increase in TUNEL positive
cells by P45 and maintain active cell death even at P60. This further supports the theory
that retinas from Arr1(B)
-/-
mice are more susceptible to light-independent cell death than
the Arr1(A)
-/-
.
134
WT P22
P22
-/-
Arr1(A)
P22
-/-
Arr1(B)
WT P45
P45
-/-
Arr1(A)
P45
-/-
Arr1(B)
WT P60
P60
-/-
Arr1(A)
P60
-/-
Arr1(B)
0
20
40
60
80
**
***
**
ns
***
***
ns
ns
ns
TUNEL-Positive Cells
Figure 6.1: Quantification of TUNEL Positive Cells from WT, Arr1(A)
-/-
and Arr1(B)
-/-
Mice
with Increasing Age. Three mice from each genotype per age were used. Values were obtained
by counting the number of TUNEL positive cells on three sections from three different animals.
**P<0.01, *** P<0.001.
6.2.2 Arr1(A)
-/-
and Arr1(B)
-/-
Mice Harbor Unique Prdx6 Isoforms
The Prdx6 mRNA was RT-PCR amplified from cDNA and the fragments were
subcloned from both Arr1(A)
-/-
and Arr1(B)
-/-
retinas and their nucleotide sequences
compared revealing a single nucleotide polymorphism (SNP) at the genetic location
corresponding to the translated amino acid residue number 122. Whereas the Arr1(A)
-/-
mice have an adenine as the central base pair of the codon corresponding to position 122,
Arr1(B)
-/-
mice have a cytosine, translating to an aspartic acid and alanine residue,
respectfully. Figure 6.2 demonstrates the isoelectric focusing (IEF) analysis from retinal
135
samples of each genotype, including mice that were heterozygous for the Prdx6 SNP.
Allele Specific Polymerase Chain Reaction (ASPCR) genotyping for the Prdx6 SNP was
used as the primary genetic indicator distinguishing the two groups.
Figure 6.2: Isoelectric Focusing and ASPCR SNP Genotype Analysis for Prdx6. Isoelectric
focusing and immunoblot analysis of Prdx6 identifies two main isoforms with amino acid residue
122 as either Alanine or Aspartic Acid, which correlates with the genomic ASPCR/SNP analysis
identifying either the Adenine (A) or Cytosine (C) (C/C=homozygous susceptible/C57Bl/6J,
A/A=homozygous resistant/129/SvJ, A/C=heterozygous susceptible).
6.2.3 Prdx6 from Arr1(A)
-/-
and Arr1(B)
-/-
Have Comparable PLA
2
Activity
Prdx6 has two known functions: 1) Peroxidase activity involved in reducing
hydrogen peroxide, peroxynitrite and various organic hydroperoxides, and 2)
Phospholipase A
2
(PLA
2
) activity catalyzing the hydrolysis of the sn-2 fatty acyl ester
136
bond of glycerophospholipids to produce free fatty acids and lysophospholipids (Hooks
and Cummings, 2008). While current studies are undergoing to investigate possible
changes in peroxidase activity of Prdx6 from Arr1(A)
-/-
and Arr1(B)
-/-
mice, PLA
2
activity
was examined from total retinal proteins and the reaction product measured
colorimetrically (Caymen Chemical). Figure 6.3 is a graphical representation of the levels
of PLA
2
activity from the two groups, along with a C57Bl/6J control and Bee Venom
positive control per the manufacturer’s instructions. According to this test, there was no
statistically significant difference between the Prdx6 122A and 122D isoforms from both
retinal samples and when compared to C57Bl/6J retinas. Although the phospholipase
activity of Prdx6 is not affected by the SNP, this does not rule out the possibility that the
peroxidase activity is not affected and may be responsible for the susceptibility of the
retina to photoreceptor cell death.
Figure 6.3: Phospholipase A
2
Activity. Total retinal protein homogenates from WT, Arr1(A)
-/-
and Arr1(B)
-/-
mice were used in biological and technical triplicates per genotype. Statistical
analysis revealed no significant difference between all genotypes examined.
137
6.2.4 Quantitative Trait Locus (QTL) Mapping and Genome Wide
Association Studies (GWAS)
Although the role of Prdx6 as a potential modifier gene in the light-independent
photoreceptor dystrophy in Arr1(A)
-/-
and Arr1(B)
-/-
mice is exciting, alternative
explanations could be responsible for the phenotypic variation. To test alterative
hypothesis, we explored alternative modifier genes using QTL mapping and genome
wide association studies. Genomic DNA was isolated from tail clips of Arr1(A)
-/-
and
Arr1(B)
-/-
each, as well as C57Bl/6J and 129/SvJ mice. The QTL mapping and LOD
score analyses revealed 22 markers across 16 chromosomes with statistically significant
LOD scores above a threshold value of 3.0 that were further scrutinized for chromosomal
location, gene identification, mammalian phenotype and human disease association using
the EASE/DAVID and OMIM databases. Of the 22 microsatellite markers that identified
statistically significantly different regions between the Arr1(A)
-/-
and Arr1(B)
-/-
, the QTL
loci allowed us to identify multiple candidate genes (QTG) within those respective
regions. A list of all the candidate genes identified is presented in Appendix B, and the
genetic markers associated with significant LOD scores are presented in Figure 6.4, along
with the marker names and locations in Table 6.2. Some of the genes identified by the
linkage analysis include signal transducer and activator of transcription 1 and 4 (Stat1,
Stat4), located on chromosome 1, which are key transcription factors in the Jak/STAT
signaling pathway linked to various forms of retinal degeneration in mice and humans
(Samardzija et al., 2006). Genes identified with annotated mammalian phenotypes
associated with retinal degeneration or abnormality include; Beta Arrestin 1 (Arrb1),
138
Ornithine amino transferase (Oat), Retinoblastoma like 2 (Rbl2), Cryptochrome 1
(photolyase-like) (Cry1), Apolipoprotein B (Apob), A Disintegrin and Metallopeptidase
Domain 17 (Adam17), Lysosomal Trafficking Regulator (Lyst), Serine peptidase
inhibitor (Serpin), Pairded Box Gene 2 (Pax2), Hermansky-Pudlak Syndrome (Hps6) and
(Slc). These QTGs are of interest because linkage analyses differences were identified as
statistically significant and may play a role in the dark-mediated photoreceptor cone
degeneration observed in Arr1(B)
-/-
mice.
A genome wide association study (GWAS) was performed in conjunction with
the linkage analysis as a strategy for fine-mapping candidate genes under previously
detected QTL peaks containing hundreds of genes. Our GWAS analysis used the same
genomic DNA from the linkage study and identified 10 microsatellite markers on 7
chromosomes that were statistically significant following a chi squared test with
Bonferonni correction and logistic regression in SAS. Employing both statistical methods
M7 (Chr. 1); M9 (Chr. 2); M22 (Chr. 4); M42, M43 (Chr. 8); M59, M60 (Chr. 12); M69,
M70 (Chr. 14); M83 (Chr. 17) were significant between the groups (p <0.05). Table 6.3
provides a list of the microsatellite markers, along with their respective chromosomal
locations and gene names for those that were identified as statistically significant. These
results were compared to the results of a genome-scan meta-analysis (GSMA) performed
on 6 independent Age-related Macular Degeneration (AMD) genome screens (Fisher et
al., 2005). The comparison study identified chromosome regions 1q23, 16q12.2, 2p24-
p23, and 2p23.3 to have a previously reported linkage to AMD and correspond to genes
Fc receptor IgG low affinity IV and IIb (Fcgr4, Fcgr2b), Retinoblastoma-like 2 (Rbl2),
139
Apolipoprotein B (Apob), Dystrobrevin beta (Dtnb), Matrilin 3 (Matn) and the Member
RAS oncogene family (Rab10). Most of these genes were also identified in our linkage
analysis study (Appendix B). Taken together, these candidate genes provide an
interesting springboard for future work on modifier genes and their functional
significance in photoreceptor disease susceptibility.
140
Figure 6.4: LOD Score Graph of the Linkage Analysis Performed on Arr1(A)
-/-
and
Arr1(B)
-/-
Genomic DNA. A threshold value of 3 was designated for this dataset, and
peaks above this value were considered statistically significant. LOD score values
represent the mapping of Mendelian traits. The smaller triangles located on the X axis
correspond to the respective locations of genetic markers used for screening. Regions of
statistical significance were examined more closely to link genes of interest to the marker
locations. The genes identified with their murine and human chromosomal locations, as
well as mammalian phenotypes associated with defects in these genes, can be found in
Appendix B.
141
Figure 6.4, Continued: LOD Score Graph of the Linkage Analysis Perfomed on Arr1(A)
-/-
and Arr1(B)
-/-
Genomic DNA.
142
Table 6.1: Chromosomal Locations and LOD Scores for Quantitative Trait Loci (QTL).
Trait Chrom. Position LR Additive Dominant R2 LOD1_L LOD1_R LOD2_L LOD2_R
1 1 0.1471 17.49 -0.3132 -0.3128 0.042898 0.142 0.149 0.142 0.15
1 1 0.2831 17.61 -0.3135 -0.3134 0.042926 0.278 0.285 0.278 0.288
1 2 0.0401 14 0.3222 -0.3102 0.293095 0.03 0.044 0.018 0.047
1 2 0.2471 70.62 0.4619 0.0155 0.513726 0.235 0.257 0.234 0.258
1 2 0.4221 13.47 0.3147 -0.3006 0.26905 0.417 0.431 0.417 0.442
1 2 0.6351 14.77 0.2981 -0.299 0.216691 0.63 0.652 0.627 0.668
1 3 0.0001 19.32 0.3025 0.3024 0.096129 0 0.026 0 0.044
1 3 0.5741 18.12 -0.3397 0.34 0.319246 0.569 0.58 0.569 0.587
1 3 0.6161 18.7 0.3191 -0.3203 0.28894 0.611 0.616 0.611 0.616
1 4 0.2881 28.93 -0.3329 0.2806 0.328071 0.283 0.299 0.283 0.318
1 5 0.0001 82.36 0.3986 -0.3981 0.788926 0 0.015 0 0.029
1 5 0.3251 13.51 0.2921 -0.2932 0.195563 0.32 0.338 0.32 0.335
1 6 0.4431 15.85 -0.039 0.5506 0.158018 0.384 0.508 0.337 0.565
1 7 0.4801 67.27 0.3949 -0.3951 0.989064 0.47 0.48 0.465 0.481
1 7 0.5951 66.15 0.3908 -0.391 0.65716 0.59 0.6 0.59 0.6
1 8 0.0201 13.03 -0.3061 0.3059 0.200523 0.014 0.028 0.007 0.035
1 8 0.3651 58.17 -0.3881 -0.388 0.098705 0.357 0.365 0.35 0.366
1 8 0.5601 66.12 -0.3877 -0.3876 0.123968 0.555 0.57 0.555 0.57
1 9 0.0151 15.05 0.2994 -0.3004 0.221546 0.01 0.015 0.007 0.015
1 9 0.3201 38.21 0.4736 0.0236 0.438082 0.315 0.425 0.315 0.33
1 10 0.3401 17.04 0.3139 -0.3134 0.26962 0.335 0.344 0.331 0.349
1 11 0.0451 15.72 0.312 -0.3085 0.262006 0.035 0.05 0.023 0.055
1 11 0.6771 14.48 0.2961 -0.2976 0.210024 0.672 0.684 0.672 0.682
1 12 0.0001 96.4 0.4726 -0.0274 0.653035 0 0.015 0 0.024
1 13 0.0001 27.14 0.2966 -0.3179 0.248739 0 0.04 0 0.075
1 16 0.1901 18.65 0.3197 -0.3186 0.294389 0.185 0.197 0.185 0.206
1 17 0.0621 48.87 -0.38 -0.2453 0.133176 0.06 0.073 0.06 0.083
1 19 0.3601 65.01 0.3705 -0.3708 0.57395 0.355 0.373 0.355 0.384
143
Table 6.2: Primer Names and Genetic and Physical Locations for Linkage Analysis Studies.
Primer Name Genetic
Location (cM)
Physical
Location (MB)
Primer Name Genetic
Location (cM)
Physical
Location
(MB)
D1Mit231 12 20.86 D9Mit182 55 101.43
D1Mit213 25.7 43.39 D9Mit215 63 115.98
D1Mit303 34.8 62.95 D10Mit282 12 20.65
D1Mit24 41 74.46 D10Mit198 32.47 51.71
D1Mit49 54.5 89.1 D10Mit42 44 82.12
D1Mit98 67 128.48 D10Mit233 62 113.82
D1Mit268 83.40 159.29 D11Mit71 1.1 6.83
D1Mit36 92.3 171.14 D11Mit260 34.5 61.61
D2Mit149 7 13.49 D11Mit219 43 72.13
D2Mit433 31.7 57.15 D11Mit124 57.8 99.05
D2Mit274 52.5 114.28 D11Mit61 70 112.37
D2Mit277 69 123.26 D12Mit182 2 10.88
D2Mit343 84.2 169.1 D12Mit109 19 43.15
D2Mit263 92 162.18 D12Mit156 34 80.56
D3Mit203 11.2 26.84 D12Nds2 59 115.13
D3Mit151 18.5 31.14 D13Mit16 10 20.39
D3Mit22 33.7 69.52 D13Mit139 32 51.86
D3Mit103 51.1 107.27 D13Mit191 45 85.05
D3Mit84 71.8 143.42 D13Mit213 59 109.04
D3Mit147 79.4 148.41 D14Mit11 0.7 12.92
D4Mit1 6.3 17.82 D14Mit141 15 47.38
D4Mit108 12.1 38.32 D14Mit203 28.3 N/A
D4Mit178 35.5 66.84 D14Mit193 40 71.92
D4Mit166 44.5 93.54 D14Mit165 52 106.98
D4Mit232 71 144.65 D14Mit75 54 N/A
D5Mit267 24 N/A D15Mit175 9.9 9.2
D5Mit113 42 77.68 D15Mit6 13.7 38.47
D5Mit239 58 107.84 D15Mit85 16.4 40.15
D5Mit95 68 125.31 D15Mit107 49 84.22
D6Mit138 0.68 4.45 D15Mit193 57.9 97.77
D6Mit123 29 56.88 D16Mit131 4.3 7.32
D6Mit105 45 107.75 D16Mit4 27.3 36.24
D6Mit339 65.5 136.37 D16Mit139 43.1 65.67
D6Mit15 74 146.38 D16Mit71 70.65 97.13
D7Mit76 3.4 19.58 D17Mit164 16.3 3.92
D7Mit246 15 31.19 D17Mit176 22.5 42.88
D7Mit176 27 70.36 D17Mit93 44.5 74.15
D7Mit250 37 86.52 D17Mit123 56.7 93.6
D7Mit262 49.9 114.51 D18Mit19 2 5.04
D7Mit103 63.5 136.36 D18Mit149 24 45.16
D8Mit141 6 12.57 D18Mit142 47 75.55
D8Mit205 30 51.92 D18Mit4 57 84.3
D8Mit50 41 91.88 D19Mit78 5 7.67
D8Mit120 63 120.87 D19Mit86 20 26.4
D8Mit56 73 131.54 D19Mit88 34 37.33
D9Mit250 5 8.39 D19Mit90 41 42.3
D9Mit206 20 40.32 D19Mit71 54 59.67
D9Mit107 40 73.32
144
Marker cM Mouse
Gene
Human
Gene
Human
Location
Linkage GWAS Linkage
M7 1-36 92.3 Fcgr4 FCGR3
A
1q23 Yes Yes Fisher et
al. 2005
Fcgr2b FCGR2
B
1q23 Yes Yes Fisher et
al. 2005
M9 D2-433 31.7 n/a n/a n/a n/a n/a n/a
M22 4-178 35.5 n/a n/a n/a n/a n/a n/a
M42 8-50 41 Rb12 RBL2 16q12.2 No Yes Fisher et
al. 2005
M43 8-120 63 n/a n/a n/a n/a n/a n/a
M59 12-182 2 Apob APOB 2p24-p23 Yes Yes Fisher et
al. 2005
Dtnb DTNB 2p24 Yes Yes Fisher et
al. 2005
Matn3 MATN3 2p24-p23 Yes Yes Fisher et
al. 2005
Rab10 RAB10 2p23.3 Yes Yes Fisher et
al. 2005
M60 12-109 19 n/a n/a n/a n/a n/a n/a
M69 14-203 28.3 n/a n/a n/a n/a n/a n/a
M70 14-193 40 Hr HR 8p21.2 No Yes No
M83 17-176 22.5 Bysl BYSL 6p21.1 No No No
Mut MUT 6p21.3 No No No
Table 6.3: Microsatellite Markers Identified from the Genome Wide Array and
Association Mapping Analysis.
145
6.3 Discussion
Our laboratory has identified a unique phenotypic difference between retinas from
Arr1(A)
-/-
and Arr1(B)
-/-
mice, which have the identical Arr1 knockout genotype but
during subsequent breeding, inherited different alleles from either C57Bl/6J or 129/SvJ
strains. The phenotypic variations include increased retinal TUNEL staining compared to
WT colony controls by P45 in both dark adapted Arr1(A)
-/-
and Arr1(B)
-/-
mice; however,
Arr1(B)
-/-
mice have an even greater level of TUNEL staining than Arr1(A)
-/-
mice at this
age. By P60, the level of TUNEL staining is significantly greater in Arr1(B)
-/-
mice, but
similar to WT controls in Arr1(A)
-/-
mice. Previous work performed on Arr1(A)
-/-
mice
have demonstrated that when these mice were reared in the dark, the retinal morphologic
structure was indistinguishable from that in normal wildtype controls (Chen et al.,
1999b). The results presented here demonstrate that when Arr1(A)
-/-
mice are reared in
the dark, they exhibit an increase in TUNEL staining at P45, but not at P60. In the
Arr1(B)
-/-
strain, however, an increase in TUNEL staining was present at P45, and
perhaps earlier; exhibiting a light-independent cone dystrophy confirming previously
published results (Brown et al., 2010).
Peptide analysis of differentially expressed proteins from two-dimensional gel
electrophoresis (2DE) initially identified Prdx6 as a potential modifier gene in the
characterization of the phenotypic variability between the two Arr1
-/-
groups.
Theoretically, these mice harbor the identical genetic knockout; however they also have
unique isoforms for Prdx6, a known peroxidase important in the protection of cellular
components from oxidative damage (Iakoubova et al., 1997). We identified the variation
146
for the isoelectric mobility difference in the amino acid residue at amino acid position
122 of the protein sequence by one-dimensional isoelectric focusing and confirmed this
result with allele specific PCR genotyping experiments. This method allowed for the
isolation and tracking of the SNP between generations and will be used to further study
the relevance of the Prdx6 SNP in determining disease susceptibility or resistance in
these mice. A colorimetric PLA2 assay on retinal homogenates from both Arr1(A)
-/-
and
Arr1(B)
-/-
groups revealed that the Prdx6 SNP does not affect its PLA2 activity in these
mice. This does not however exclude the possibility that the oxidative stress protection
mechanism of Prdx6 is not affected. Current studies are ongoing to examine the levels of
oxidation in these strains, as well as microarray mRNA expression changes to further
characterize the light-independent retinal degeneration phenotype of Arr1(B)
-/-
mice.
Association mapping for quantitative traits was performed to identify genetic
variations that associate with quantitative levels of complex traits in our two Arr1
-/-
populations. The genes identified from the linkage analysis revealed interesting QTL
regions that isolated various genes with mammalian phenotypes of retinal abnormalities
(Appendix B). Genome wide association studies (GWAS) are commonly used to identify
genetic links for disease susceptibility. A GWAS analysis was performed for Arr1(A)
-/-
and Arr1(B)
-/-
mice to identify potential targets to characterize our observed disease
variability. These results identified 10 microsatellite markers on 7 chromosomes, 7 of
which were previously reported in linkage analysis studies of AMD genome screens
(Fisher et al., 2005). Fc receptor IgG low affinity IV and IIb (Fcgr4, Fcgr2b) are both
located in chromosomal region 1q23 and function as IgG receptor reported to contribute
147
to autoimmune diseases, although its specific role remains uncertain (Syed et al., 2009).
The Retinoblastoma-like 2 (Rbl2) gene located at chromosome region 16q12.2 was also
identified in the AMD GSMA and acts as s tumor suppressor and is a potent inhibitor of
E2F-mediated trans-activation, cell cycle regulation, proliferation and differentiation
(Simpson et al., 2009). Apolipoprotein B (Apob), Dystrobrevin beta (Dtnb), Matrilin 3
(Matn3) and the Member RAS oncogene family (Rab10) were significant and are located
in chromosome region 2p24-p23. Interestingly, the Apolipoprotein E4 allele (ApoE4)
gene has been associated with a reduced risk of AMD in a large cohort study (Zareparsi
et al., 2004). While ApoE and ApoB are both found in chylomicrons and very low
density lipoproteins, both were associated in a linkage analysis study on coronary artery
disease (Corbo et al., 1999). Although ApoB is an interesting target, it is located in a
different chromosomal region than ApoE, which has been associated with AMD. Dtnb,
Matn3 and Rab10 function as a motor protein receptor, in collagen fibrillogenesis, and
protein trafficking, respectively. The chromosomal region identified for these genes were
all previously reported in the GSMA study of AMD patients. The human chromosomal
location of PRDX6 is 1q25.1, and although markers near the respective murine
chromosomal region did not isolate Prdx6 as a potential QTL, we believe it may still be
an interesting target for investigation due to its previously identified role in disease
resistance and susceptibility. Linkage analysis is a useful tool for large scale data
analysis; however, we have identified a SNP using ASPCR genotyping that provides a
more specific method of isolating and characterizing our observed strain dependent
variability.
148
Further scrutiny into the QTL chromosomal regions and oxidative stress studies in
our Arr1(A)
-/-
and Arr1(B)
-/-
mice will generate evidence for their role in a disease
phenotype similar to atrophic AMD. Discovering novel resistant modifier genes in a well
characterized mouse model allows for the translation to potential markers for atrophic
AMD and other photoreceptor degenerative diseases in humans. Studies such as this one
on two well characterized strains with related genotypes but varying phenotypes can be
utilized in human syntenic loci studies to target novel candidate genes for screening and
potential therapeutic treatment of photoreceptor dystrophy.
149
CHAPTER 7. CONCLUSION
To better understand the molecular and biochemical mechanisms of retinal
disease progression and induction, and to develop better therapeutic strategies to treat
photoreceptor degeneration, appropriate animal models are continuously sought after and
tested to best mimic human disease. Genetic engineering of and inherited mutations in
laboratory mice are valuable tools in the study of various biological functions and
relevance for genes and their respective permutations. This work is an extensive
examination of four unique genetic knockout mice that exhibit individual phenotypic
characteristics in their retinal function, morphology, and disease homology. Investigation
of each of the genes discussed were conducted to provide a better understanding for their
physiologic functions in the mouse retina, and to potentially identify their significance to
human vision and retinal disease.
The roles of phototransduction recovery proteins in the homeostasis of retinal
function, both in light and dark environments, were examined using G protein coupled
receptor kinase-1 knockout (Grk1
-/-
) animals. The affects of Grk1 loss on a pure cone
retina was studied in the double knockout Nrl
-/-
Grk1
-/-
, lacking both Grk1 and the Nrl
transcription factor that drives rod development. When compared to Nrl
-/-
controls that
express Grk1, Nrl
-/-
Grk1
-/-
mice exhibit a light independent but age-related cone
dystrophy and harbor a weakened Bruch’s membrane that leads to blood vessel
penetration similar to retinal angiomatous proliferation (RAP). As these mice age, they
develop neovascularization similar to both choroidal and retinal forms, possibly due to a
hypoxic or poorly metabolic environment as a consequence to the loss of Grk1. Gene
150
expression studies at 1 month of age revealed an enhanced inflammatory response in the
Nrl
-/-
Grk1
-/-
mice, most likely due to the blood vessel penetration observed at this age.
This animal model provides a useful tool in both the examination of alternative functions
for Grk1 in cone photoreceptors, and is useful for the study of inherited retinal
angiogenesis. Further studies on these animals to determine the causative relationship
between Grk1 loss in cones and a potentially hypoxic retina leading to blood vessel
penetration will provide interesting information of the alternative functions for this
critical phototransduction recovery protein.
Examination of the Grk1
-/-
mouse, which exhibit a well characterized light
mediated photoreceptor degeneration, led to the identification of the enhanced expression
and activity of Poly(ADP)ribose Polymerase-1 (PARP) in these retinas. A mediator of
DNA damage recovery proteins as well as a marker for apoptosis, PARP activity was
similarly enhanced in the rd1 mouse. When Grk1
-/-
mice are exposed to 24 hours of direct
room light, they exhibit massive photoreceptor apoptosis. The in situ and in vitro studies
presented here reveal that the light induced cell death is mediated by the PARP pathway.
Future work employing the selective PARP inhibitor, PJ34 (N-(6-Oxo-5,6-
dihydrophenanthridin-2-yl)-N,N-dimethylacetamide.HCl) with retinal explant studies will
further assess the therapeutic possibilities in human retinal diseases like Oguchi’s disease
(Azam et al., 2009a).
A critical tumor progression marker and component of cell cycle progression,
Pituitary Tumor Transforming Gene-1 (Pttg1) was recently identified in several retinal
disease models. We also identified an up-regulation of Pttg1 in Nrl
-/-
Grk1
-/-
retinas by
151
both quantitative PCR (qPCR) and microarray analyses. To determine the relevance of
Pttg1 within the retina, Pttg1
-/-
mice were acquired and their retinas analyzed for
morphologic and functional changes. Despite their deregulation in Nrl
-/-
Grk1
-/-
mice and
others, Pttg1
-/-
mice have relatively normal electroretinography, physiology and
morphology when examined up to 9 months of age. Although male Pttg1
-/-
mice over 5
months have diabetes, they lack a diabetic retinopathy phenotype characterized by blood
vessel weakening and leakage within the retina. The examination of Pttg1
-/-
mice reveals
that the loss of this protein is not detrimental to the retina; however this does not
eliminate the possibility that its over-expression may be a contributing factor to
photoreceptor degeneration.
Another phototransduction recovery protein studied was Arrestin 1 (Arr1), or
visual Arrestin. A well studied knockout mouse, the Arr1
-/-
was first characterized to have
light mediated photoreceptor degeneration, but those that are kept in the dark were
similar to their wildtype counterparts. We have found that mice with a unique blend of
genetic information from two popular mouse strains, C57Bl/6J and 129/SvJ carry
modifier genes that affect their respective photoreceptor susceptibility to cell death.
Whereas the two Arr1
-/-
mice populations studied (Arr1(A)
-/-
and Arr1(B)
-/-
) have the
identical Arr1 knockout, the Arr1(B)
-/-
have a light independent cone dystrophy that
worsens with age. Linkage analysis and genome wide association studies (GWAS) on
populations of these mice identified potential modifier regions which were similarly
published in meta-analyses of patients with AMD. Although Peroxiredoxin 6 (Prdx6) was
marked as a potential modifier for its unique SNP isoforms and their previously
152
published links to atherosclerosis susceptibility in different mouse strains, further
research is ongoing to identify exact modifiers that would make one strain more
susceptible to photoreceptor dystrophy than another strain with the same genotype.
Mouse models for retinal degeneration exist as either genetically developed or
inherited. Where some mice degenerate due to light exposure, others exhibit
photoreceptor degeneration as a ramification of other means, such as neovascularization
or hypoxic environments. Nevertheless, genetic knockout mice are crucial in the
examination of genes and their functions within the retina. It is only when these mice are
extensively scrutinized with repeated measures in various conditions that novel functional
roles and potential mechanisms for once characterized genes are identified. Here, we
demonstrate that mice lacking both Nrl and Grk1 have a unique morphology
characterized by blood vessel proliferation, mice lacking Grk1 degenerate following a
particular pathway, while those lacking Pttg1 are relatively healthy and normal, and mice
without Arr1 expression exhibit a unique degeneration phenotype. Taken together, these
results provide us with a better understanding for the contributions to photoreceptor
homeostasis each of these genes and their gene products provide. The goal of this work is
to characterize animal models that most closely mimic human disease to facilitate the
design of potential therapeutic targets with our enhanced understanding of the genetic and
proteomic components that constitute a healthy retina. Without genetic knockout
technologies, high throughput screening, and proteomics, our understanding of human
diseases would be extremely limited. Therefore, continued efforts to advance retinal
153
disease studies are not only imperative but essential to our understanding of the eye and
the preservation of the gift of sight.
154
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168
APPENDIX A. A COMPREHENSIVE LIST OF DIFFERENTIALLY
EXPRESSED GENES BETWEEN NRL
-/-
GRK1
-/-
AND NRL
-/-
RETINAS
A Comprehensive List of Differentially Expressed Genes Between Nrl
-/-
Grk1
-/-
and Nrl
-/-
Retinas
Symbol Entrez Gene Name p-value Fold
Change
Probe
Set ID
PTTG1 pituitary tumor-transforming 1 2.85E-05 130.741 1438390
_s_at
PTTG1 pituitary tumor-transforming 1 2.13E-06 70.022 1424105
_a_at
CAP1 CAP, adenylate cyclase-associated protein 1 (yeast) 2.39E-05 32.933 1417461
_at
KLK2 kallikrein-related peptidase 2 4.01E-05 29.665 1425182
_x_at
CAP1 CAP, adenylate cyclase-associated protein 1 (yeast) 4.13E-05 23.502 1417462
_at
LANCL3 LanC lantibiotic synthetase component C-like 3
(bacterial)
1.43E-04 19.525 1437268
_at
A330076H08RIK RIKEN cDNA A330076H08 gene 1.69E-03 9.575 1457557
_at
NUDT19 nudix (nucleoside diphosphate linked moiety X)-type
motif 19
7.10E-04 7.046 1434216
_a_at
DHDH dihydrodiol dehydrogenase (dimeric) 1.32E-03 6.802 1453487
_at
4833408G04RIK RIKEN cDNA 4833408G04 gene 4.20E-03 6.083 1454409
_at
ACTG2 (includes
EG:72)
actin, gamma 2, smooth muscle, enteric 1.29E-03 4.155 1416454
_s_at
OSBPL3 oxysterol binding protein-like 3 2.57E-04 4.146 1438724
_at
ACTR1A ARP1 actin-related protein 1 homolog A, centractin
alpha (yeast)
1.98E-03 4.016 1457970
_at
LGALS4 lectin, galactoside-binding, soluble, 4 5.43E-04 3.48 1451336
_at
AY512938 cDNA sequence AY512938 4.15E-03 3.468 1458551
_at
CYP26A1 cytochrome P450, family 26, subfamily A, polypeptide
1
1.80E-03 3.374 1419430
_at
OCIAD1 OCIA domain containing 1 1.29E-03 3.339 1424952
_at
NUDT19 nudix (nucleoside diphosphate linked moiety X)-type
motif 19
2.96E-04 3.337 1432332
_a_at
UBLCP1 ubiquitin-like domain containing CTD phosphatase 1 4.69E-03 3.165 1429606
_at
SLC6A20 solute carrier family 6 (proline IMINO transporter),
member 20
1.32E-05 3.117 1427221
_at
2810488G03RIK RIKEN cDNA 2810488G03 gene 1.74E-03 2.927 1429068
_at
GSTO1 glutathione S-transferase omega 1 2.09E-03 2.728 1456036
_x_at
MYOM2 myomesin (M-protein) 2, 165kDa 2.13E-03 2.714 1457435
_x_at
ANKRD11 ankyrin repeat domain 11 1.28E-03 2.694 1436967
_at
ITGA6 integrin, alpha 6 1.45E-03 2.531 1422444
_at
MIOS missing oocyte, meiosis regulator, homolog
(Drosophila)
2.89E-04 2.488 1424803
_at
ADARB2 adenosine deaminase, RNA-specific, B2 (RED2 1.22E-03 2.484 1458112
169
homolog rat) _at
RBPMS RNA binding protein with multiple splicing 4.42E-03 2.459 1455936
_a_at
ZNF99 zinc finger protein 99 2.34E-03 2.447 1442654
_at
C8ORF79 chromosome 8 open reading frame 79 1.94E-03 2.426 1443902
_at
C030014A21RIK RIKEN cDNA C030014A21 gene 1.81E-03 2.408 1453897
_at
CCND2 cyclin D2 1.33E-03 2.4 1434745
_at
B830008H07RIK RIKEN cDNA B830008H07 gene 2.32E-03 2.341 1457791
_at
SSR1 signal sequence receptor, alpha 4.59E-03 2.335 1417764
_at
ASF1B ASF1 anti-silencing function 1 homolog B (S.
cerevisiae)
2.63E-03 2.267 1423714
_at
SPOCK1 sparc/osteonectin, cwcv and kazal-like domains
proteoglycan (testican) 1
3.61E-03 2.226 1419672
_at
A430108E01RIK RIKEN cDNA A430108E01 gene 1.84E-03 2.203 1438049
_at
A2M alpha-2-macroglobulin 1.91E-03 2.199 1434719
_at
PCOLCE procollagen C-endopeptidase enhancer 8.70E-05 2.191 1437165
_a_at
PPP1R3C protein phosphatase 1, regulatory (inhibitor) subunit
3C
1.09E-03 2.185 1433691
_at
MYOF myoferlin 1.72E-04 2.179 1427318
_s_at
VPS37A vacuolar protein sorting 37 homolog A (S. cerevisiae) 8.60E-04 2.165 1429363
_at
MTRR 5-methyltetrahydrofolate-homocysteine
methyltransferase reductase
8.64E-05 2.083 1452110
_at
NAV2 (includes
EG:78286)
neuron navigator 2 5.84E-04 2.063 1435981
_at
OSBPL6 oxysterol binding protein-like 6 2.22E-03 2.043 1457881
_at
6820431F20RIK cadherin 11 pseudogene 4.23E-04 2.038 1452997
_at
NUP62 nucleoporin 62kDa 4.60E-03 1.972 1415926
_at
SLC7A2 solute carrier family 7 (cationic amino acid
transporter, y+ system), member 2
2.98E-03 1.969 1436555
_at
FAM189A1 family with sequence similarity 189, member A1 3.83E-03 1.926 1433945
_at
GSTO1 glutathione S-transferase omega 1 2.14E-03 1.904 1416531
_at
SORBS1 sorbin and SH3 domain containing 1 5.27E-05 1.896 1425826
_a_at
CD44 CD44 molecule (Indian blood group) 2.96E-04 1.845 1434376
_at
9630005C17RIK RIKEN cDNA 9630005C17 gene 3.69E-03 1.837 1430268
_at
FTL ferritin, light polypeptide 1.63E-03 1.833 1418364
SLC26A7 solute carrier family 26, member 7 2.18E-03 1.829 1436279
_at
A930009L07RIK RIKEN cDNA A930009L07 gene 2.33E-03 1.819 1445124
_at
LYPLAL1 lysophospholipase-like 1 1.01E-03 1.816 1451490
_at
CADPS2 Ca++-dependent secretion activator 2 6.48E-04 1.815 1451499
_at
170
WDR60 WD repeat domain 60 3.47E-03 1.77 1436396
_at
ZFP36 zinc finger protein 36, C3H type, homolog (mouse) 3.27E-04 1.763 1452519
_a_at
CD44 CD44 molecule (Indian blood group) 5.54E-04 1.758 1423760
_at
RWDD4A RWD domain containing 4A 2.57E-03 1.727 1457983
_s_at
TTC28 tetratricopeptide repeat domain 28 5.16E-06 1.727 1435456
_at
OSMR oncostatin M receptor 3.56E-03 1.724 1418674
_at
FOXP4 forkhead box P4 1.78E-03 1.722 1431088
_at
TTR transthyretin 2.22E-03 1.711 1454608
_x_at
SAMD4A sterile alpha motif domain containing 4A 4.94E-03 1.709 1436356
_at
DAPK1 death-associated protein kinase 1 4.40E-03 1.703 1426915
_at
SEPN1 (includes
EG:57190)
selenoprotein N, 1 1.82E-03 1.696 1426680
_at
RET ret proto-oncogene 2.04E-03 1.672 1436359
_at
SPON1 spondin 1, extracellular matrix protein 2.13E-03 1.668 1442613
_at
SLCO3A1 solute carrier organic anion transporter family,
member 3A1
4.82E-03 1.655 1448918
_at
RCOR3 (includes
EG:55758)
REST corepressor 3 1.02E-03 1.648 1428343
_at
CHI3L1 chitinase 3-like 1 (cartilage glycoprotein-39) 2.52E-04 1.646 1451537
_at
HIST1H1C histone cluster 1, H1c 5.34E-04 1.644 1436994
_a_at
MTUS1 microtubule associated tumor suppressor 1 6.16E-04 1.639 1454824
_s_at
APLP1 amyloid beta (A4) precursor-like protein 1 4.97E-03 1.63 1416134
_at
TTR transthyretin 1.40E-03 1.602 1459737
_s_at
CSF1R colony stimulating factor 1 receptor 4.94E-03 1.589 1419872
_at
PCNP PEST proteolytic signal containing nuclear protein 7.30E-04 1.583 1452735
_at
PNPLA6 patatin-like phospholipase domain containing 6 1.76E-03 1.577 1416517
_at
MGLL monoglyceride lipase 4.86E-03 1.575 1426785
_s_at
LOC729505 hypothetical LOC729505 9.24E-04 1.571 1433507
ANXA1 annexin A1 8.48E-04 1.568 1448213
_at
PCDHB15 protocadherin beta 15 3.96E-03 1.567 1418941
PCNXL2 pecanex-like 2 (Drosophila) 1.04E-03 1.552 1455559
_at
C11ORF87 chromosome 11 open reading frame 87 3.17E-04 1.551 1444026
_at
NLGN3 neuroligin 3 1.08E-03 1.549 1436135
_at
GRIA1 glutamate receptor, ionotropic, AMPA 1 1.95E-03 1.546 1435239
_at
GCN1L1 GCN1 general control of amino-acid synthesis 1-like
1 (yeast)
2.25E-03 1.544 1433713
_at
171
SH3RF1 SH3 domain containing ring finger 1 7.31E-04 1.544 1445178
_at
SMAD3 SMAD family member 3 3.65E-03 1.535 1454960
_at
RWDD4A RWD domain containing 4A 2.32E-03 1.523 1424243
_at
6820431F20RIK cadherin 11 pseudogene 4.78E-04 1.521 1457673
_at
VAV3 vav 3 guanine nucleotide exchange factor 2.34E-03 1.514 1448600
_s_at
DAPK1 death-associated protein kinase 1 4.73E-03 1.509 1427358
_a_at
KIAA1958 KIAA1958 4.35E-03 1.503 1436852
_at
TMTC1 transmembrane and tetratricopeptide repeat
containing 1
1.19E-03 1.502 1435261
_at
APLP1 amyloid beta (A4) precursor-like protein 1 2.69E-04 1.5 1435857
_s_at
FAIM2 Fas apoptotic inhibitory molecule 2 4.42E-03 1.498 1455410
_at
STAT1 signal transducer and activator of transcription 1,
91kDa
1.13E-03 1.486 1440481
_at
TGM2 transglutaminase 2 (C polypeptide, protein-
glutamine-gamma-glutamyltransferase)
3.01E-03 1.483 1455900
_x_at
PALM paralemmin 3.36E-03 1.48 1423967
_at
BTRC beta-transducin repeat containing 7.75E-04 1.478 1457305
_at
ECE2 endothelin converting enzyme 2 5.38E-04 1.477 1424561
_at
CHL1 cell adhesion molecule with homology to L1CAM
(close homolog of L1)
4.36E-03 1.472 1435190
_at
ZFP36L2 zinc finger protein 36, C3H type-like 2 3.94E-03 1.47 1437626
_at
CPNE8 copine VIII 2.87E-03 1.467 1431146
_a_at
LTBP3 latent transforming growth factor beta binding protein
3
4.06E-03 1.461 1418049
_at
COL4A2 collagen, type IV, alpha 2 3.01E-03 1.454 1424051
_at
IGFBP4 insulin-like growth factor binding protein 4 4.82E-04 1.451 1437405
_a_at
PTPN14 protein tyrosine phosphatase, non-receptor type 14 2.42E-03 1.451 1455359
_at
P4HA2 prolyl 4-hydroxylase, alpha polypeptide II 5.59E-04 1.447 1417149
_at
6720469N11RIK RIKEN cDNA 6720469N11 gene 1.45E-03 1.442 1434797
_at
TEAD1 TEA domain family member 1 (SV40 transcriptional
enhancer factor)
1.61E-03 1.442 1429556
_at
NFIX nuclear factor I/X (CCAAT-binding transcription
factor)
1.63E-03 1.441 1436364
_x_at
C8ORF41 chromosome 8 open reading frame 41 2.63E-03 1.44 1424361
_at
PHACTR4 phosphatase and actin regulator 4 3.23E-03 1.438 1434258
_s_at
FXYD7 FXYD domain containing ion transport regulator 7 2.22E-03 1.433 1419200
_at
MAB21L2 mab-21-like 2 (C. elegans) 2.27E-03 1.428 1418934
_at
PPT1 palmitoyl-protein thioesterase 1 1.41E-03 1.428 1422467
172
_at
TSPAN6 tetraspanin 6 4.02E-03 1.428 1416872
_at
SENP7 SUMO1/sentrin specific peptidase 7 3.49E-03 1.425 1436860
_at
CNTNAP4 contactin associated protein-like 4 9.64E-04 1.422 1419044
_at
LRTM2 leucine-rich repeats and transmembrane domains 2 4.36E-03 1.404 1433744
_at
ADCY8 adenylate cyclase 8 (brain) 2.04E-03 1.401 1418754
_at
GM10397 predicted gene 10397 1.02E-03 1.398 1435740
_at
HOMER2 homer homolog 2 (Drosophila) 2.38E-04 1.397 1424367
_a_at
NFIX nuclear factor I/X (CCAAT-binding transcription
factor)
2.76E-03 1.393 1436363
_a_at
PCDHB12
(includes
EG:56124)
protocadherin beta 12 1.69E-03 1.393 1420443
_at
CELSR2 cadherin, EGF LAG seven-pass G-type receptor 2
(flamingo homolog, Drosophila)
2.31E-03 1.38 1435336
_at
SHANK2 SH3 and multiple ankyrin repeat domains 2 1.91E-03 1.38 1442823
_at
ZSWIM6 zinc finger, SWIM-type containing 6 4.43E-03 1.379 1451819
_at
PLXNA4 plexin A4 1.49E-03 1.377 1441371
_at
CD47 CD47 molecule 1.30E-03 1.373 1419554
_at
MAF v-maf musculoaponeurotic fibrosarcoma oncogene
homolog (avian)
2.16E-03 1.368 1447849
_s_at
RAI1 retinoic acid induced 1 4.03E-03 1.367 1453200
_at
MICAL3 (includes
EG:194401)
microtubule associated monoxygenase, calponin and
LIM domain containing 3
1.79E-04 1.363 1434221
_at
DAG1 dystroglycan 1 (dystrophin-associated glycoprotein 1) 2.87E-03 1.36 1456131
_x_at
CTSH cathepsin H 1.54E-03 1.359 1443814
_x_at
SLC2A3 solute carrier family 2 (facilitated glucose
transporter), member 3
2.61E-03 1.359 1455898
_x_at
RNASE4 ribonuclease, RNase A family, 4 1.80E-03 1.358 1422603
CSF1R colony stimulating factor 1 receptor 1.73E-03 1.354 1419873
_s_at
SCGN secretagogin, EF-hand calcium binding protein 1.79E-03 1.353 1451534
_at
NDRG1 N-myc downstream regulated 1 3.85E-04 1.352 1420760
_s_at
IQSEC3 IQ motif and Sec7 domain 3 8.97E-04 1.345 1437912
_at
PPM1L protein phosphatase 1 (formerly 2C)-like 1.28E-03 1.345 1435787
_at
SDK2 sidekick homolog 2 (chicken) 3.78E-03 1.342 1430425
_at
PGBD5 piggyBac transposable element derived 5 1.54E-04 1.339 1460570
_at
SPON1 spondin 1, extracellular matrix protein 1.23E-03 1.336 1441226
_at
TNIK TRAF2 and NCK interacting kinase 9.50E-04 1.335 1430579
_at
ARHGEF17 Rho guanine nucleotide exchange factor (GEF) 17 2.14E-03 1.334 1419938
173
_s_at
RPH3A rabphilin 3A homolog (mouse) 7.11E-04 1.331 1434635
_at
B230114P17RIK RIKEN cDNA B230114P17 gene 2.52E-03 1.328 1456295
_at
ANKRD6 ankyrin repeat domain 6 1.03E-03 1.326 1437217
_at
IGSF3 immunoglobulin superfamily, member 3 3.13E-03 1.326 1455049
_at
IQGAP1 IQ motif containing GTPase activating protein 1 7.27E-04 1.324 1417380
_at
MALT1 mucosa associated lymphoid tissue lymphoma
translocation gene 1
3.45E-03 1.319 1456429
_at
AIFM3 apoptosis-inducing factor, mitochondrion-associated,
3
4.95E-03 1.308 1434742
_s_at
A930004D18RIK RIKEN cDNA A930004D18 gene 2.49E-03 1.303 1439514
_at
ANK1 ankyrin 1, erythrocytic 2.01E-03 1.296 1419421
_at
EPHA3 EPH receptor A3 3.74E-04 1.294 1455426
_at
NACC2 NACC family member 2, BEN and BTB (POZ)
domain containing
3.03E-03 1.294 1429582
_at
2900006B11RIK RIKEN cDNA 2900006B11 gene 1.07E-03 1.293 1430253
_at
SLC6A17
(includes
EG:388662)
solute carrier family 6, member 17 1.33E-03 1.293 1436137
_at
ZNF99 zinc finger protein 99 4.60E-03 1.291 1438491
_x_at
IGFBP4 insulin-like growth factor binding protein 4 2.32E-03 1.29 1437406
_x_at
GNA11 guanine nucleotide binding protein (G protein), alpha
11 (Gq class)
3.87E-03 1.289 1434254
_at
STK35 serine/threonine kinase 35 3.75E-03 1.288 1439802
1700049G17RIK RIKEN cDNA 1700049G17 gene 4.11E-03 1.284 1440171
GLCCI1 glucocorticoid induced transcript 1 3.71E-03 1.284 1426218
IL6ST interleukin 6 signal transducer (gp130, oncostatin M
receptor)
3.98E-03 1.282 1452843
_at
SORD sorbitol dehydrogenase 2.26E-03 1.281 1426584
_a_at
APC2 adenomatosis polyposis coli 2 4.15E-03 1.28 1455231
_s_at
PLAGL1 pleiomorphic adenoma gene-like 1 1.55E-04 1.28 1426208
_x_at
APCDD1 adenomatosis polyposis coli down-regulated 1 2.08E-03 1.279 1454822
_x_at
SAMD14 sterile alpha motif domain containing 14 2.53E-03 1.273 1454630
_at
OPLAH 5-oxoprolinase (ATP-hydrolysing) 7.53E-04 1.272 1424359
_at
STIM1 stromal interaction molecule 1 8.72E-04 1.267 1448320
_at
ARFGAP1 ADP-ribosylation factor GTPase activating protein 1 2.66E-03 1.265 1427245
_at
KCND2 potassium voltage-gated channel, Shal-related
subfamily, member 2
8.62E-04 1.265 1422834
_at
CAMK2G calcium/calmodulin-dependent protein kinase II
gamma
1.22E-04 1.264 1423942
_a_at
174
PLXNB1 plexin B1 3.86E-03 1.26 1435254
_at
GNG10 guanine nucleotide binding protein (G protein),
gamma 10
7.82E-04 1.259 1450649
_at
FASN fatty acid synthase 1.31E-03 1.258 1423828
_at
FOXP4 forkhead box P4 1.45E-04 1.258 1429719
_at
TPM3 tropomyosin 3 2.00E-03 1.256 1427260
_a_at
TMEM68 transmembrane protein 68 6.23E-04 1.254 1423649
_at
FNDC3B fibronectin type III domain containing 3B 2.16E-03 1.252 1433833
_at
CADM1 cell adhesion molecule 1 1.56E-03 1.25 1417378
_at
OMG oligodendrocyte myelin glycoprotein 1.93E-03 1.25 1418212
_at
PLXNA3 plexin A3 2.99E-03 1.249 1420996
_at
TMEM201 transmembrane protein 201 1.69E-03 1.247 1454868
_at
3100003L13RIK RIKEN cDNA 3100003L13 gene 1.12E-03 1.246 1432065
_at
GPI glucose phosphate isomerase 3.03E-03 1.245 1434814
_x_at
CAPN2 calpain 2, (m/II) large subunit 8.03E-04 1.244 1416257
_at
KLHDC10 kelch domain containing 10 3.57E-03 1.24 1433997
TRIP6 thyroid hormone receptor interactor 6 2.54E-03 1.24 1449041
_a_at
ARL6IP1 ADP-ribosylation factor-like 6 interacting protein 1 3.70E-03 1.239 1423818
_a_at
RPS6KA4 ribosomal protein S6 kinase, 90kDa, polypeptide 4 4.47E-03 1.239 1448498
_at
SIDT2 SID1 transmembrane family, member 2 2.86E-03 1.235 1426940
_at
ARHGEF11 Rho guanine nucleotide exchange factor (GEF) 11 1.89E-04 1.232 1434926
_at
NDST1 N-deacetylase/N-sulfotransferase (heparan
glucosaminyl) 1
1.91E-03 1.229 1460436
_at
LAMP2 lysosomal-associated membrane protein 2 2.15E-03 1.228 1434503
_s_at
HUNK hormonally up-regulated Neu-associated kinase 4.01E-03 1.224 1418260
_at
ATP1A1 ATPase, Na+/K+ transporting, alpha 1 polypeptide 1.52E-03 1.219 1451071
_a_at
IQSEC1 IQ motif and Sec7 domain 1 6.29E-04 1.218 1452327
_at
SYNJ2 synaptojanin 2 7.29E-05 1.216 1452344
_at
AGTPBP1 ATP/GTP binding protein 1 5.33E-04 1.215 1418332
_a_at
OCRL oculocerebrorenal syndrome of Lowe 1.83E-04 1.209 1438396
_at
TRNP1 TMF1-regulated nuclear protein 1 2.89E-03 1.204 1453008
_at
FLYWCH1 FLYWCH-type zinc finger 1 3.87E-04 1.203 1426982
_at
MAPK8IP3 mitogen-activated protein kinase 8 interacting protein
3
2.72E-03 1.203 1425975
_a_at
TMEM132D transmembrane protein 132D 3.60E-04 1.203 1446492
_at
175
KCNAB1 potassium voltage-gated channel, shaker-related
subfamily, beta member 1
1.86E-03 -1.20 1448468
_a_at
ANKZF1 ankyrin repeat and zinc finger domain containing 1 1.56E-03 -1.202 1419818
_x_at
RNF144B ring finger protein 144B 3.46E-03 -1.203 1443252
_at
C16ORF80 chromosome 16 open reading frame 80 2.15E-04 -1.204 1419462
_s_at
RBP3 retinol binding protein 3, interstitial 4.93E-03 -1.206 1457855
_at
UBLCP1 ubiquitin-like domain containing CTD phosphatase 1 4.97E-03 -1.208 1415790
_at
UBE2F ubiquitin-conjugating enzyme E2F (putative) 6.34E-04 -1.209 1429568
_x_at
C10ORF104 chromosome 10 open reading frame 104 2.51E-03 -1.212 1419994
_s_at
TMEM70 transmembrane protein 70 1.71E-03 -1.215 1424541
_at
PPIG peptidylprolyl isomerase G (cyclophilin G) 4.13E-03 -1.218 1435341
_at
CDK2AP1 cyclin-dependent kinase 2 associated protein 1 4.10E-03 -1.22 1435509
CLN5 ceroid-lipofuscinosis, neuronal 5 4.42E-03 -1.22 1426886
9230114K14RIK RIKEN cDNA 9230114K14 gene 4.38E-03 -1.223 1456643
_at
TALDO1 transaldolase 1 2.02E-03 -1.223 1425129
_a_at
ZNHIT1 zinc finger, HIT type 1 2.69E-03 -1.224 1425531
_at
BCAT2 branched chain aminotransferase 2, mitochondrial 3.84E-03 -1.227 1425764
_a_at
HMGN5 high-mobility group nucleosome binding domain 5 4.54E-03 -1.227 1418152
_at
PEX11A peroxisomal biogenesis factor 11 alpha 1.85E-03 -1.228 1419365
_at
TIMM44 translocase of inner mitochondrial membrane 44
homolog (yeast)
3.02E-03 -1.23 1448801
_a_at
MANEAL mannosidase, endo-alpha-like 3.41E-03 -1.237 1455202
_at
MPP5 membrane protein, palmitoylated 5 (MAGUK p55
subfamily member 5)
1.52E-03 -1.239 1450113
_at
NEGR1 neuronal growth regulator 1 2.95E-03 -1.239 1456392
_at
TMEM60 transmembrane protein 60 9.68E-04 -1.24 1428000
_at
RPAP3 RNA polymerase II associated protein 3 2.29E-03 -1.244 1448555
_at
UBXN4 UBX domain protein 4 1.38E-05 -1.244 1426484
_at
1110033J19RIK ribosomal protein S4, Y-linked 2 8.63E-04 -1.246 1452730
_at
MPHOSPH10 M-phase phosphoprotein 10 (U3 small nucleolar
ribonucleoprotein)
5.43E-04 -1.246 1429080
_at
C12ORF4 chromosome 12 open reading frame 4 2.96E-03 -1.247 1449437
_at
NPHP3 nephronophthisis 3 (adolescent) 1.72E-04 -1.247 1433545
_s_at
SLC41A3
(includes
EG:54946)
solute carrier family 41, member 3 1.12E-04 -1.249 1425439
_a_at
C030044B11RIK RIKEN cDNA C030044B11 gene 2.25E-03 -1.25 1429264
_at
TIMM10 translocase of inner mitochondrial membrane 10 3.08E-03 -1.256 1417499
176
homolog (yeast) _at
TMEM30A transmembrane protein 30A 6.11E-05 -1.257 1448339
_at
5330438D12RIK RIKEN cDNA 5330438D12 gene 1.48E-03 -1.26 1437265
_at
RNF160 ring finger protein 160 4.39E-03 -1.263 1452612
_at
SLC7A6OS solute carrier family 7, member 6 opposite strand 1.69E-03 -1.263 1429596
_at
CPSF2 cleavage and polyadenylation specific factor 2,
100kDa
2.32E-03 -1.264 1431089
_at
ZNF259 zinc finger protein 259 4.04E-03 -1.265 1419281
_a_at
ARRDC3 arrestin domain containing 3 3.36E-03 -1.267 1454617
_at
G6PD glucose-6-phosphate dehydrogenase 1.13E-03 -1.267 1422327
_s_at
STK3 serine/threonine kinase 3 (STE20 homolog, yeast) 2.65E-03 -1.268 1418512
_at
EIF3E eukaryotic translation initiation factor 3, subunit E 1.79E-03 -1.276 1460432
_a_at
ESF1 ESF1, nucleolar pre-rRNA processing protein,
homolog (S. cerevisiae)
3.12E-04 -1.278 1434942
_at
PHYHD1 phytanoyl-CoA dioxygenase domain containing 1 2.65E-03 -1.28 1428394
_at
CNOT6L CCR4-NOT transcription complex, subunit 6-like 3.23E-03 -1.282 1451723
_at
CCNG1 cyclin G1 4.97E-03 -1.284 1450017
_at
MRPL45 mitochondrial ribosomal protein L45 4.30E-03 -1.286 1423492
_at
TBCE tubulin folding cofactor E 8.31E-04 -1.286 1428282
_at
PNRC2 (includes
EG:55629)
proline-rich nuclear receptor coactivator 2 4.42E-03 -1.287 1416186
_at
TUBE1 tubulin, epsilon 1 7.26E-04 -1.288 1443481
_at
KLHL23 kelch-like 23 (Drosophila) 4.72E-03 -1.294 1435743
_at
NHP2 NHP2 ribonucleoprotein homolog (yeast) 2.73E-03 -1.295 1416605
_at
4933439C10RIK RIKEN cDNA 4933439C10 gene 2.66E-03 -1.299 1452696
_a_at
SDF4 stromal cell derived factor 4 2.37E-03 -1.3 1448367
_at
COPG2 coatomer protein complex, subunit gamma 2 2.59E-03 -1.304 1448761
_a_at
NT5E 5'-nucleotidase, ecto (CD73) 6.86E-04 -1.304 1428547
_at
AMN1 antagonist of mitotic exit network 1 homolog (S.
cerevisiae)
3.48E-03 -1.311 1453358
_s_at
AGBL3 ATP/GTP binding protein-like 3 3.44E-03 -1.312 1452619
_a_at
SURF6 surfeit 6 3.93E-03 -1.312 1416864
_at
BBS10 Bardet-Biedl syndrome 10 2.46E-03 -1.316 1430170
_at
SERINC4 serine incorporator 4 1.51E-04 -1.316 1426035
_at
2010107G12RIK RIKEN cDNA 2010107G12 gene 5.28E-05 -1.319 1455449
_at
4932415G12RIK RIKEN cDNA 4932415G12 gene 8.22E-04 -1.321 1428376
177
_at
MGC42105 serine/threonine-protein kinase NIM1 2.07E-04 -1.323 1434297
_at
HIST3H2A histone cluster 3, H2a 3.32E-03 -1.324 1455712
_at
MFAP1 microfibrillar-associated protein 1 1.80E-03 -1.331 1449444
_a_at
TMEM136
(includes
EG:235300)
transmembrane protein 136 2.19E-03 -1.332 1457701
_at
GNPNAT1 glucosamine-phosphate N-acetyltransferase 1 3.26E-03 -1.334 1423158
_at
HPS3 Hermansky-Pudlak syndrome 3 3.03E-03 -1.335 1450647
_at
GSTM4 glutathione S-transferase mu 4 4.74E-03 -1.339 1424835
OPTN optineurin 3.50E-04 -1.341 1435679
_at
DCTN6 dynactin 6 6.00E-04 -1.342 1416499
_a_at
TMEM158 transmembrane protein 158 2.69E-03 -1.348 1428074
_at
AKR1B1 aldo-keto reductase family 1, member B1 (aldose
reductase)
4.50E-04 -1.356 1448319
_at
PAQR4 progestin and adipoQ receptor family member IV 4.74E-03 -1.36 1423101
_at
DMD dystrophin 1.75E-03 -1.362 1417307
_at
1700080G18RIK RIKEN cDNA 1700080G18 gene 2.73E-03 -1.363 1430055
_at
MTHFS 5,10-methenyltetrahydrofolate synthetase (5-
formyltetrahydrofolate cyclo-ligase)
1.72E-03 -1.363 1460257
_a_at
5830469G19RIK RIKEN cDNA 5830469G19 gene 3.45E-03 -1.371 1430089
_at
TRPS1 trichorhinophalangeal syndrome I 3.36E-03 -1.381 1434286
_at
DHRS4 dehydrogenase/reductase (SDR family) member 4 4.16E-04 -1.384 1451559
_a_at
1700007L15RIK RIKEN cDNA 1700007L15 gene 9.44E-04 -1.385 1435403
_at
ZDHHC17 zinc finger, DHHC-type containing 17 3.34E-03 -1.388 1458363
_at
AGPAT5 1-acylglycerol-3-phosphate O-acyltransferase 5
(lysophosphatidic acid acyltransferase, epsilon)
3.70E-03 -1.392 1434287
_at
GM10374 predicted gene 10374 4.36E-03 -1.394 1436357
_at
DUSP19 dual specificity phosphatase 19 1.96E-03 -1.396 1448922
_at
MAP7D2 MAP7 domain containing 2 1.77E-03 -1.396 1431403
_a_at
C9ORF46 chromosome 9 open reading frame 46 1.12E-03 -1.418 1460361
_at
DTD1 D-tyrosyl-tRNA deacylase 1 homolog (S. cerevisiae) 1.39E-03 -1.419 1451040
_at
PIH1D1 PIH1 domain containing 1 8.79E-04 -1.423 1452726
_a_at
ERO1L ERO1-like (S. cerevisiae) 4.72E-03 -1.43 1419029
_at
TTLL11 tubulin tyrosine ligase-like family, member 11 6.37E-04 -1.435 1430064
_at
GPR135 G protein-coupled receptor 135 4.23E-03 -1.446 1459274
_at
C1ORF192 chromosome 1 open reading frame 192 3.43E-03 -1.449 1429181
178
_at
5033425G24RIK RIKEN cDNA 5033425G24 gene 2.95E-04 -1.452 1430204
_at
C22ORF23 chromosome 22 open reading frame 23 3.63E-03 -1.454 1422774
_at
PGAP2 post-GPI attachment to proteins 2 1.27E-03 -1.458 1424614
_at
HRASLS HRAS-like suppressor 2.68E-04 -1.459 1428991
_at
WWC2 WW and C2 domain containing 2 1.37E-03 -1.459 1448611
_at
HGSNAT heparan-alpha-glucosaminide N-acetyltransferase 3.76E-03 -1.461 1450868
_at
KRT222 keratin 222 9.66E-04 -1.473 1457354
_at
DUSP4 dual specificity phosphatase 4 1.40E-03 -1.475 1428834
_at
PRPF31 PRP31 pre-mRNA processing factor 31 homolog (S.
cerevisiae)
2.64E-04 -1.483 1457083
_at
GPR135 G protein-coupled receptor 135 9.08E-04 -1.485 1441944
_s_at
AKAP9 A kinase (PRKA) anchor protein (yotiao) 9 3.47E-03 -1.487 1455151
_at
ALDH16A1 aldehyde dehydrogenase 16 family, member A1 6.70E-04 -1.487 1423731
_at
TRIP10 thyroid hormone receptor interactor 10 4.80E-03 -1.491 1418092
_s_at
EFCAB7 EF-hand calcium binding domain 7 4.05E-03 -1.493 1451349
_at
TRAPPC5 trafficking protein particle complex 5 8.54E-04 -1.497 1448999
_at
CCDC51 coiled-coil domain containing 51 1.56E-03 -1.507 1452891
_at
ERAP1 endoplasmic reticulum aminopeptidase 1 4.33E-03 -1.517 1416942
_at
2310047D07RIK RIKEN cDNA 2310047D07 gene 4.04E-04 -1.525 1458587
_at
DSTN destrin (actin depolymerizing factor) 3.05E-03 -1.532 1417124
_at
C2ORF77 chromosome 2 open reading frame 77 1.71E-03 -1.542 1454000
_s_at
CCDC21 coiled-coil domain containing 21 5.39E-05 -1.542 1432391
_at
CCDC127 coiled-coil domain containing 127 3.39E-03 -1.547 1455248
_at
SIRT2 sirtuin (silent mating type information regulation 2
homolog) 2 (S. cerevisiae)
5.24E-04 -1.554 1423507
_a_at
BPNT1 3'(2'), 5'-bisphosphate nucleotidase 1 2.92E-03 -1.573 1449211
_at
SNAPC1 small nuclear RNA activating complex, polypeptide 1,
43kDa
2.72E-03 -1.574 1429335
_at
6430590I03RIK melanoma antigen family F, 1 4.51E-03 -1.578 1429479
_at
2810025M15RIK RIKEN cDNA 2810025M15 gene 2.03E-03 -1.581 1428452
_at
CD99L2 CD99 molecule-like 2 3.57E-04 -1.581 1456746
_a_at
ZFP14 zinc finger protein 14 homolog (mouse) 4.37E-03 -1.586 1441619
_at
SDCCAG8 serologically defined colon cancer antigen 8 1.51E-03 -1.589 1431203
_at
179
TATDN3 TatD DNase domain containing 3 1.68E-03 -1.608 1428769
_at
GM11696 predicted gene 11696 6.10E-04 -1.613 1458888
_at
HIST1H2BL histone cluster 1, H2bl 2.20E-04 -1.631 1455095
_at
RAPGEF5 Rap guanine nucleotide exchange factor (GEF) 5 2.58E-03 -1.646 1455840
_at
KLHL36 kelch-like 36 (Drosophila) 3.74E-03 -1.658 1425123
_at
DFNB31 deafness, autosomal recessive 31 2.96E-03 -1.675 1436485
_s_at
PTOV1 prostate tumor overexpressed 1 1.81E-03 -1.685 1416945
_at
FANCC Fanconi anemia, complementation group C 1.60E-05 -1.69 1450861
_at
MCCC2 methylcrotonoyl-Coenzyme A carboxylase 2 (beta) 2.32E-03 -1.72 1428021
_at
SMAD5 SMAD family member 5 2.29E-03 -1.723 1451873
_a_at
C19ORF2 chromosome 19 open reading frame 2 2.96E-03 -1.727 1433913
_at
VPS36 vacuolar protein sorting 36 homolog (S. cerevisiae) 1.61E-03 -1.743 1424417
_at
GAS7 growth arrest-specific 7 2.48E-03 -1.752 1417859
_at
RIOK1 RIO kinase 1 (yeast) 3.22E-03 -1.771 1440893
_at
AGA aspartylglucosaminidase 4.10E-03 -1.778 1434665
_at
SLC25A32 solute carrier family 25, member 32 1.50E-03 -1.782 1453149
_at
ESF1 ESF1, nucleolar pre-rRNA processing protein,
homolog (S. cerevisiae)
4.45E-03 -1.799 1453727
_at
ANKRD32 ankyrin repeat domain 32 5.63E-04 -1.806 1423577
_at
PTPRF protein tyrosine phosphatase, receptor type, F 4.08E-03 -1.823 1420843
_at
WDR78 WD repeat domain 78 3.52E-03 -1.823 1434793
_at
EPM2AIP1 EPM2A (laforin) interacting protein 1 2.29E-04 -1.838 1425624
_at
ABHD5 abhydrolase domain containing 5 3.15E-03 -1.843 1417566
_at
QARS glutaminyl-tRNA synthetase 1.54E-03 -1.851 1453237
_at
SLC7A3 solute carrier family 7 (cationic amino acid
transporter, y+ system), member 3
3.15E-03 -1.854 1417022
_at
C9ORF21 chromosome 9 open reading frame 21 2.42E-04 -1.858 1434441
_at
ERI1 exoribonuclease 1 4.36E-03 -1.867 1418827
_at
CWC22 CWC22 spliceosome-associated protein homolog (S.
cerevisiae)
3.58E-03 -1.875 1424609
_a_at
LYNX1 Ly6/neurotoxin 1 2.09E-03 -1.894 1441952
_x_at
CWC22 CWC22 spliceosome-associated protein homolog (S.
cerevisiae)
2.17E-03 -1.917 1424607
_a_at
TMEM20 transmembrane protein 20 1.84E-03 -1.923 1435452
_at
PGGT1B protein geranylgeranyltransferase type I, beta subunit 1.43E-03 -1.934 1438230
_at
180
ZNF593 zinc finger protein 593 1.21E-03 -1.948 1447703
_x_at
FAM194A family with sequence similarity 194, member A 5.65E-04 -1.951 1458320
_at
FRMPD2 FERM and PDZ domain containing 2 1.97E-04 -1.98 1458506
_at
ASNS asparagine synthetase 1.82E-04 -2.168 1433966
_x_at
ZFP386 zinc finger protein 386 (Kruppel-like) 4.95E-03 -2.177 1451146
_at
RRM2B ribonucleotide reductase M2 B (TP53 inducible) 1.55E-03 -2.199 1437222
_x_at
5830433G22RIK RIKEN cDNA 5830433G22 gene 2.83E-03 -2.202 1431266
_at
C14ORF149 chromosome 14 open reading frame 149 4.71E-03 -2.204 1424692
_at
POP4 processing of precursor 4, ribonuclease P/MRP
subunit (S. cerevisiae)
1.90E-03 -2.221 1448419
_at
NME1 non-metastatic cells 1, protein (NM23A) expressed in 1.11E-03 -2.224 1424110
_a_at
BTRC beta-transducin repeat containing 3.65E-03 -2.236 1446925
_at
PFKFB2 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase
2
2.03E-04 -2.259 1422091
_at
ASNS asparagine synthetase 1.47E-04 -2.287 1451095
_at
C8ORF41 chromosome 8 open reading frame 41 4.23E-03 -2.414 1457906
_at
PPP1R14C protein phosphatase 1, regulatory (inhibitor) subunit
14C
8.21E-04 -2.419 1417701
_at
PISD phosphatidylserine decarboxylase 3.66E-03 -2.42 1426387
_x_at
UEVLD UEV and lactate/malate dehyrogenase domains 4.05E-03 -2.574 1455551
_at
PCP2 Purkinje cell protein 2 6.07E-04 -2.58 1424944
_at
THG1L tRNA-histidine guanylyltransferase 1-like (S.
cerevisiae)
4.62E-03 -2.627 1432393
_a_at
FZD6 frizzled homolog 6 (Drosophila) 3.35E-03 -2.631 1417301
_at
ZNF658 zinc finger protein 658 6.61E-04 -2.674 1439181
_at
2310058N22RIK RIKEN cDNA 2310058N22 gene 1.35E-03 -2.758 1429215
_at
UTP18 UTP18, small subunit (SSU) processome component,
homolog (yeast)
3.72E-03 -3.114 1447266
_at
FKBP9 FK506 binding protein 9, 63 kDa 4.93E-03 -3.196 1437687
_x_at
TM2D2 TM2 domain containing 2 3.46E-04 -3.249 1456663
_x_at
NEK3 NIMA (never in mitosis gene a)-related kinase 3 1.56E-03 -3.326 1418947
_at
MOGAT1 monoacylglycerol O-acyltransferase 1 2.54E-03 -3.35 1419504
_at
RNLS renalase, FAD-dependent amine oxidase 3.40E-03 -3.51 1453180
_at
ALS2 amyotrophic lateral sclerosis 2 (juvenile) 9.83E-04 -3.813 1417784
_at
4933439C20RIK phosphatidylserine decarboxylase, pseudogene 1 6.42E-05 -3.843 1435353
_a_at
PIK3AP1 phosphoinositide-3-kinase adaptor protein 1 2.20E-03 -4.017 1429831
FUT10 (includes fucosyltransferase 10 (alpha (1,3) fucosyltransferase) 3.29E-03 -4.601 1437388
181
EG:84750) _at
C8ORF41 chromosome 8 open reading frame 41 3.77E-03 -4.65 1435309
_at
4632415L05RIK RRS1 ribosome biogenesis regulator homolog
pseudogene
3.91E-03 -4.689 1419611
_at
FKBP9 FK506 binding protein 9, 63 kDa 1.02E-03 -4.767 1423677
_at
USH1C Usher syndrome 1C (autosomal recessive, severe) 1.19E-03 -4.839 1450001
_a_at
AI605517 expressed sequence AI605517 2.29E-03 -6.042 1457797
_at
ESCO1 establishment of cohesion 1 homolog 1 (S.
cerevisiae)
2.18E-04 -6.056 1424324
_at
CRB1 crumbs homolog 1 (Drosophila) 1.03E-03 -6.324 1441330
_at
ELAVL1 ELAV (embryonic lethal, abnormal vision,
Drosophila)-like 1 (Hu antigen R)
5.84E-04 -6.986 1440464
_at
YIPF4 Yip1 domain family, member 4 3.40E-03 -8.031 1426417
_at
C4ORF47 chromosome 4 open reading frame 47 2.31E-05 -8.472 1453168
_at
MAP2K7 mitogen-activated protein kinase kinase 7 1.36E-03 -9.381 1457182
_at
DCDC2B (includes
EG:149069)
doublecortin domain containing 2B 2.18E-03 -9.736 1444714
_at
ABCB10 ATP-binding cassette, sub-family B (MDR/TAP),
member 10
2.15E-03 -11.188 1416403
_at
RBM45 RNA binding motif protein 45 1.97E-03 -12.899 1437904
_at
MTMR7 myotubularin related protein 7 1.67E-03 -19.189 1447831
_s_at
9430027B09RIK RIKEN cDNA 9430027B09 gene 2.64E-03 -21.358 1454232
_at
SKIV2L2 superkiller viralicidic activity 2-like 2 (S. cerevisiae) 1.48E-04 -28.664 1447517
_at
ZFP874 zinc finger protein 874 2.13E-04 -29.67 1434171
_at
ZNF26 zinc finger protein 26 3.73E-04 -30.101 1444092
_at
GRK1 G protein-coupled receptor kinase 1 9.36E-04 -72.593 1421361
_at
182
APPENDIX B. GENE IDENTIFICATION AND CATEGORIZATION OF
REGIONS WITH SIGNIFICANT LOD SCORES IN ARR1(A)
-/-
AND ARR1(B)
-/-
GENOMIC DNA
Chrom. Marker QTL
Approx
Cm
Loc.
Gene Gene
Loc
Gene
Phys. Loc
Human
Gene
Human
Loc
Mammalian
Phenotype
1 D1Mit213 25.8-
27.3
Gas10 25.9
Gls 25.9 52220292-
52290076 (-)
GLS 2q32-
q34
Stat1 25.9 52176282-
52218704 (+)
STAT1 2q32.2
Stat4 25.9 52065088-
52164028 (+)
STAT4 2q32.2-
q32.3
Ercc5 26.6 44204692-
44238105 (+)
ERCC5 13q33
Nab1 27 52514737-
52557204 (-)
NAB1 2q32.3-
q33
Inpp1 27.1 52846257-
52874532 (-)
INPP1 2q32
1 D1Mit24 39.8-41 Smarc
al1
40 72629825-
72679708 (+)
SMAR
CAL1
2q34-
q35
Arpc2 40.3 74283071-
74314783 (+)
ARPC2 2q36.1
Gpbar
1
40.4 74325174-
74326163 (+)
GPBAR
1
2q35
Vil1 40.8 74455950-
74482133 (+)
VIL1 2q35-
q36
Ihh 40.8 74991892-
74998225 (-)
IHH 2q33-
q35
absent enteric
neurons (details)
Cryba
2
40.8 74936508-
74939717 (-)
CRYB2 2q34-
q36
Speg 41 75371872-
75428895 (+)
SPEG 2q35
Slc4a
3
41 75542841-
75558747 (+)
SLC4A
3
2q36 absent enteric
neurons
(details)abnormal
optic nerve
morphology (details);
blindness (details);
abnormal retinal
vasculature
morphology (details);
abnormal eye
electrophysiology
(details);
abnormal retinal
apoptosis (details);
abnormal retinal rod
bipolar cell
morphology (details)
Ptprn 41 75243616-
75260783 (-)
PTPRN 2q35-
q36
1 D1Mit36 91-93 Fcgr4 92.29 172949051-
172959892 (+)
FCGR3
A
1q23
183
Adamt
s4
92.3 173180552-
173190768 (+)
ADAMT
S4
1q21-
q23
Fcgr2
b
92.3 172890689-
172906678 (-)
FCGR2
B
1q23
Fcgr3 92.3 172981301-
172989493 (-)
FCGR2
A
1q23
Mpz 92.4 173080842-
173091261 (+)
MPZ 1q23.3 neuron degeneration
(details)
Apoa2 92.6 173155185-
173156510 (+)
APOA2 1q21-
q23
2 D2Mit433 30-33.3 Neb 30 51992182-
52194318 (-)
NEB 2q22
Rif1 31 51928362-
51977903 (+)
RIF1 2q23.3
Cacnb
4
31.5 52283845-
52532350 (-)
CACNB
4
2q22-
q23
abnormal eye
electrophysiology
(details)
Tbr1 32 61640987-
61652171 (+)
TBR1 2q24
Kif5c 32.5 49474818-
49630298 (+)
KIF5C 2q23.1 increased sensory
neuron number
(details)
Gpd2 33 57090046-
57223130 (+)
GPD2 2q24.1
3 D3Mit203 11.0-14 Car3 11.7 14863538-
14872381 (+)
CA3 8q13-
q22
Hltf 12 19957811-
20018376 (+)
HLTF 3q25.1q
26.1
Pik3ca 12.4 32296593-
32365029 (+)
PIK3CA 3q26.3
Gyg 12.5 20021970-
20055216 (-)
GYG1 3q24-
q25.1
Hps3 12.5 19895945-
19935315 (-)
HPS3 3q24 abnormal eye
pigmentation
(details);
abnormal retinal
pigmentation
(details)
Cldn1
1
12.6 31048868-
31063246 (+)
CLDN1
1
3q26.2-
q26.3
Cpb1 13 20148163-
20174655 (-)
CPB1 3q24
Mfn1 13 32428387-
32478161 (+)
MFN1 3q26.33
Skil 13 30993980-
31021499 (+)
SKIL 3q26
3 D3Mit84 68.5-
69.2
Unc5c 68.5 141128528-
141497888 (+)
UNC5C 4q21-
q23
Nfkb1 68.9 135247619-
135354511 (-)
NFKB1 4q24
Odf2l 69 144781564-
144816851 (+)
ODF2L 1p22.3
3 D3Mit84 72.5-
73.1
Clca1 72.5 144392641-
144423941 (-)
CLCA1 1p31-
p22
Cyr61 72.9 145309942-
145312944 (-)
CYR61 1p31-
p22
Lmo4 73.1 143851494-
143868184 (-)
LMO4 1p22.3
4 D4Mit178 34.9-
39.6
Rgs3 35 62220881-
62364052 (+)
RGS3 9q32
Ptprd 38 75587143-
77857865 (-)
PTPRD 9p23-
24.3
184
Tyrp1 38 80480027-
80497623 (+)
TYRP1 9p23 retinal ganglion cell
degeneration
(details)
Mpdz 38.6 80924409-
81088719 (-)
MPDZ 9p24-
p22
Nfib 38.6 81936077-
82351654 (-)
NFIB 9p24.1
Plin2 38.9 86294290-
86315964 (-)
PLIN2 9p22.1 delayed dark
adaptation (details);
abnormal retinol
metabolism (details);
abnormal eye
electrophysiology
(details)
6 D6Mit105 42.2-
48.1
Cntn3 43.5 102113300-
102414661 (-)
CNTN3 3p26
Il5ra 46 106660378-
106699031 (-)
IL5RA 3p26-
p24
Timp4 46 115195634-
115201867 (-)
TIMP4 3p25
Gmcl1 47.3 86641762-
86683372 (-)
GMCL1 2p13.3
Itpr1 48 108163112-
108501103 (+)
ITPR1 3p26-
p25
Ogg1 48 113276966-
113285062 (+)
OGG1 3p26.2
7 D7Mit262 49.8-
51.8
Trim6 49.8 111367307-
111383666 (+)
Trim6 11p15.4
Hbb-
bh1
49.96 110990150-
110991678 (-)
HBG1 11p15.5
Arrb1 50 106683976-
106755281 (+)
Arrb1 11q13 abnormal rod
electrophysiology
(details);
abnormal cone
electrophysiology
(details);
abnormal retinal rod
cell outer segment
morphology (details)
Art1 50 109250257-
109259662 (+)
Art1 11p15
Art5 50 109245393-
109251359 (-)
Art5 11p15.4
Cckbr 50 112574334-
112584852 (+)
Cckbr 11p15.4
Clns1
a
50 104845144-
104867389 (+)
Clns1a 11q13.5
-q14
Phox2
a
50 108967004-
108971236 (+)
Phox2a 11q13.2
Scube
2
50 116942204-
117009193 (-)
Scube2 11p15.3
Stim1 50 109416338-
109585374 (+)
Stim1 11p15.5
Swap
70
50 117365275-
117427017 (+)
Swap7
0
11p15
Tpp1 50 112893532-
112900743 (-)
Tpp1 11p15
Trpc2 50 109214025-
109245378 (+)
Trpc2 11p15.4
-p15.3
Ucp2 50 107641846-
107650529 (+)
Ucp2 11q13
Ucp3 50 107621499-
107634941 (+)
Ucp3 11q13
185
Prcp 50.3 100023807-
100082495 (+)
Prcp 11q14
Adm 50.5 117771183-
117773333 (+)
Adm 11p15.4
Tmem
41b
50.5 117115701-
117130443 (-)
Tmem4
1b
11p15.4
Zfp14
3
50.5 117205216-
117238906 (+)
Zfp143 11p15.4
Smpd
1
51 112702939-
112706901 (+)
Smpd1 11p15.4
-p15.1
Tub 51.45 116154339-
116177974 (+)
Tub 11p15.5 decreased retinal
photoreceptor cell
number (details);
abnormal retinal
photoreceptor layer
(details);
abnormal
photoreceptor outer
segment morphology
(details);
abnormal
photoreceptor inner
segment morphology
(details);
abnormal retinal
pigment epithelium
morphology (details)
abnormal eye
electrophysiology
(details)
abnormal retinal
neuronal layer
morphology (details)
abnormal retinal
apoptosis (details)
retinal photoreceptor
degeneration
(details)
thin retinal outer
nuclear layer
(details)
retinal outer nuclear
layer degeneration
(details)
short photoreceptor
inner segment
(details)
photoreceptor outer
segment
degeneration
(details)
absent photoreceptor
outer segment
(details)
Lmo1 51.5 116282085-
116314027 (-)
Lmo1 11p15
Nup98 51.5 109282717-
109358634 (-)
Nup98 11p15.5
Eif4g2 51.52 118214082-
118226544 (-)
Eif4g2 11p15
Mrvi1 51.52 118011780-
118125975 (-)
Mrvi1 11p15
Stk33 51.52 116422737-
116582595 (-)
Stk33 11p15.3
186
Trim6
6
51.54 116592520-
116651648 (-)
Trim66 11p15.4
Rpl27
a
51.55 116662661-
116665881 (+)
Rpl27a 11p15
St5 51.55 116667425-
116760661 (-)
St5 11p15
7 D7Mit103 62.6-65 Oat 63 139749162-
139768029 (-)
Oat 10q26 abnormal retina
morphology (details);
retinal degeneration
(details);
decreased retinal
photoreceptor cell
number (details);
abnormal retinal
pigment epithelium
morphology (details);
abnormal eye
electrophysiology
(details);
thin retinal outer
nuclear layer
(details);
disorganized
photoreceptor inner
segment (details);
short photoreceptor
inner segment
(details);
absent photoreceptor
outer segment
(details);
short photoreceptor
outer segment
(details)
Uros 63 140877926-
140901755 (-)
Uros 10q25.2
-q26.3
Hmx2 65 138697576-
138700096 (+)
Hmx2 10q25.2
-q26.3
Hmx3 65 138686477-
138688439 (+)
Hmx3 10q26.1
3
8 D8Mit50 40.8-
42.9
Rbl2 40.99 93593956-
93647743 (+)
Rbl2 16q12.2 abnormal retina
morphology (details);
retina hypoplasia
(details);
abnormal horizontal
cell morphology
(details);
abnormal retinal
neuronal layer
morphology (details);
abnormal retinal
apoptosis (details);
absent retinal bipolar
cells (details);
retinoblastoma
(details);
absent retinal
ganglion cell (details)
Rpgrip
1l
41.8 93740929-
93837161 (-)
Rpgrip1
l
16q12.2
Fto 41.9 93837431-
94192338 (+)
Fto 16q12.2
Abcc1
2
42 89006159-
89104585 (-)
Abcc12 16q12.1
187
Irx3 42.1 94322424-
94325273 (-)
Irx3 16q12.2
8 D8Mit120 61.6-
63.5
Plcg2 62 120022191-
120159042 (+)
Plcg2 16q24.1
9 D9Mit107 36.7-40 Anxa2 37 69301427-
69339602 (+)
Anxa2 15q21-
q22
Zfp42
6
37 20272993-
20297190 (-)
Znf426 19p13.2
Mapk6 38 75234712-
75257805 (-)
Mapk6 15q21
Tmod
2
38 75413428-
75459132 (-)
Tmod2 15q21.1
-q21.2
Tmod
3
38 75345926-
75407464 (-)
Tmod3 15q21.1
-q21.2
Lipc 39 70646059-
70782402 (-)
Lipc 15q21-
q23
Rnf11
1
39 70273236-
70351532 (-)
Rnf111 15q21
Igdcc3 40 64988996-
65033679 (+)
Igdcc3 15q22.3
-q23
Nedd4 40 72510364-
72597649 (+)
Nedd4 15q
Spg21 40 65308778-
65335262 (+)
Spg21 15q21-
q22
Tpm1 40 66870397-
66897213 (-)
Tpm1 15q22.1
10 D10Mit42 45.7-
46.2
Cry1 46 84594448-
84647798 (-)
Cry1 12q23-
q24.1
retinal degeneration
(details);
decreased retinal
cone cell number
(details);
absent retinal rod
cells (details);
absent photoreceptor
outer segment
(details)
11 D11Mit61 68.7-69 Abca8
b
69 109793504-
109857159 (-)
Abca8 17q24
Cpsf4l 69 113559486-
113571331 (-)
Cpsf4l 17q25.1
Sstr2 69 113480656-
113487319 (+)
Sstr2 17q24 abnormal retinal rod
bipolar cell
morphology (details)
12 D12Mit18
2
0-6 Cenpo 1 4196004-
4234294 (-)
Cenpo 2p23.3
Osr1 1 9581303-
9587705 (+)
Osr1 2p24.1
Sdc1 1 8778129-
8800521 (+)
Sdc1 2p24.1
Apob 2 8017208-
8023641 (+)
Apob 2p24-
p23
abnormal retinal
pigment epithelium
morphology (details);
abnormal eye
electrophysiology
(details)
Dtnb 2 3572523-
3781505 (+)
Dtnb 2p24
Matn3 2 8954735-
8978834 (+)
Matn3 2p24-
p23
Rab10 2 3249429-
3309959 (-)
Rab10 2p23.3
188
Fam4
9a
2.5 12283149-
12383169 (+)
Fam49
a
2p24.2
Kcns3 2.5 11097008-
11157179 (-)
Kcns3 2p24
Adam
17
3 21329370-
21379493 (-)
Adam1
7
2p25 abnormal eye
morphology (details)
Kif3c 4 3365132-
3406494 (+)
Kif3c 2p23
Mycn 4 12942902-
12948720 (-)
Mycn 2p24.1
Pomc 4 3954951-
3960618 (+)
Pomc 2p23.3
Cmpk
2
6 27154080-
27164702 (+)
Cmpk2 2p25.2
E2f6 6 16817834-
16833558 (+)
E2f6 2p25.1
Klf11 6 25336236-
25347639 (+)
Klf11 2p25
Ntsr2 6 16660276-
16667042 (+)
Ntsr2 2p25.1
13 D13Mit16 0-13 Larp4
b
5 9093151-
9172332 (+)
Larp4b 10p15.3
Mtr 5 12278803-
12350362 (-)
Mtr 1q43
Gpr13
7b
6 13450883-
13485854 (-)
Gpr137
b
1q42-
q43
Prl2c2 6 13088391-
13097597 (-)
Actn2 7 12361693-
12433027 (-)
Actn2 1q42-
q43
Chrm3 7 9875859-
10360049 (-)
Chrm3 1q43
Gng4 7 13876326-
13920164 (+)
Gng4 1q42.3
Lyst 7 13682676-
13869707 (+)
Lyst 1q42.1-
q42.2
abnormal eye
pigmentation
(details);
abnormal retinal
layer morphology
(details);
abnormal retinal
pigmentation
(details);
reduced eye
pigmentation
(details);
abnormal retinal
pigment epithelium
morphology (details)
Nid1 7 13529958-
13604521 (+)
Nid1 1q43
Ryr2 7 11645370-
12199212 (-)
Ryr2 1q42.1q
43
Sfrp4 7 19715052-
19724690 (+)
Sfrp4 7p14.1
189
Tbce 7 14090216-
14131905 (-)
Tbce 1q42.3
Akr1c
6
8 4433552-
4456776 (+)
Akr1c1 10p15-
p14
Arid4b 8 14155499-
14291634 (+)
Arid4b 1q42.1-
q43
Ggps1 8 14145948-
14155661 (-)
Ggps1 1q43
Gpx5 8 21378298-
21384600 (-)
Gpx5 6p22.1
Heatr1 8 12487642-
12531160 (+)
Heatr1 1q43
Hecw
1
8 14318705-
14615495 (-)
Hecw1 7p14.1-
p13
Lgals8 8 12531682-
12557211 (-)
Lgals8 1q42-
q43
Marck
sl1-
ps4
9 4247981-
4248271 (+)
Slc17
a1
9 23959619-
23987599 (+)
Slc17a
1
6p23-
p21
Vps41 9 18809168-
18958672 (+)
Vps41 7p14-
p13
Amph 10 19040240-
19242782 (+)
Amph 7p14-
p13
Inhba 10 16106308-
16119044 (+)
Inhba 7p15-
p13
Phf2 10 48897122-
48966252 (-)
Phf2 9q22.31
Prss1
6
10 22094045-
22101611 (-)
Prss16 6p21
Rala 10 17972409-
18036073 (-)
Rala 7p15-
p13
Tcrg-
C
10 19436427-
19444212 (+)
Tarp 7p15-
p14
Trim2
7
10 21271314-
21286593 (+)
Trim27 6p22
Txndc
3
11.5 19736947-
19789663 (-)
Txndc3 7p14.1
Btn1a
1
12 23548861-
23557770 (-)
Btn1a1 6p22.1
Hist1h
3f
12 23636334-
23637181 (+)
Hist1h3
f
6p22.2
Serpin
b1a
12 32933961-
32943054 (-)
SerpinB
1
6p25
Tdp2 12 24923548-
24934022 (+)
Tdp2 6p22.3-
p22.1
Serpin
b1-
ps1
12.1 32951828-
32955039 (-)
Serpin
b1c
12.2 32973702-
32990030 (-)
Serpin
b6b
12.3 33057182-
33070936 (+)
Serpin
b9
12.4 33095119-
33109826 (+)
Serpinb
9
6p25
Serpin
b9b
12.5 33119285-
33133753 (+)
Serpin
b1b
12.6 33175994-
33186249 (+)
190
Serpin
b9c
12.7 33241144-
33251611 (-)
Serpin
b9d
12.8 33284828-
33294998 (+)
Serpin
b9e
12.9 33341481-
33352715 (+)
Gpld1 13 25035021-
25082622 (+)
Gpld1 6p22.3-
p22.2
Serpin
b9f
13 33415946-
33427239 (+)
Tpi-
rs8
13 25547591-
25548298 (+)
16 D16Mit4 23.2-
24.2
Drd3 23.3 43762355-
43822952 (+)
Drd3 3q13.3
17 D17Mit17
6
22.4-23 Bysl 22.5 47738186-
47748167 (-)
Bysl 6p21.1
Mut 22.5 41071656-
41097848 (+)
Mut 6p12.3
Crisp2 22.8 40901683-
40943952 (-)
Crisp2 6p21-
qter
17 D17Mit12
3
53.7-
59.7
Pigf 54.3 87396596-
87424746 (-)
Pigf 2p21-
p16
Abcg5 54.5 85057574-
85082263 (-)
Abcg5 2p21
Abcg8 54.5 85082471-
85099672 (+)
Abcg8 2p21
Ttc7 55.7 87682226-
87781109 (+)
Ttc7a 2p21
Rhoq 56 87362422-
87399409 (+)
Rhoq 2p21
19 D19Mit90 40.6-44 Hps1 42 42829595-
42854468 (-)
Hps1 10q23.1
-q23.3
abnormal eye
pigmentation
(details);
abnormal retina
morphology (details)
Nkx2-
3
42 43686815-
43690382 (+)
Nkx2-3 10q24.2
Slc25
a28
42 43738312-
43749371 (-)
Slc25a
28
10q23-
q24
Abcc2 43 43856682-
43912441 (+)
Abcc2 10q24
Fbxw4 43 45652744-
45734802 (-)
Fbxw4 10q24
Pax2 43 44831884-
44910517 (+)
Pax2 10q24 abnormal eye
development
(details);
abnormal retina
morphology (details);
abnormal optic nerve
morphology (details);
abnormal optic nerve
innervation (details);
abnormal retinal
pigmentation
(details);
abnormal retinal
pigment epithelium
morphology (details);
abnormal retinal
neuronal layer
morphology (details);
abnormal retinal
nerve fiber layer
morphology (details);
191
decreased retinal
ganglion cell number
(details);
abnormal retinal
blood vessel
morphology (details);
abnormal retinal
blood vessel pattern
(details)
Scd1 43 44468945-
44482043 (-)
Scd2 43 44368578-
44381350 (+)
Scd 10q24.3
1
Tlx1 43 45225205-
45231432 (+)
Tlx1 10q24
Wnt8b 43 44567963-
44587007 (+)
Wnt8b 10q24
Hps6 44 46077998-
46080643 (+)
Hps6 10q24.3
2
abnormal eye
pigmentation
(details);
reduced eye
pigmentation
(details);
abnormal pigment
epithelium of the eye
(details)
Pkd2l
1
44 44222127-
44266932 (-)
Pkd2l1 10q24
Abstract (if available)
Abstract
G-protein coupled receptor kinase 1 (Grk1) is essential for light-activated opsin phosphorylation in phototransduction shutoff, and genetic defects cause Oguchi's disease, a form of Retinitis Pigmentosa (RP). To elucidate the recovery function of cone pigments, we combined Grk1-/- murine knockouts with the Neural retina leucine zipper (Nrl-/-), which have an enhanced S-cone phenotype. We observed that with increasing age and independent of light, the retinas of Nrl-/-Grk1-/- when compared to Nrl-/- developed progressive cone degeneration and decreased cone protein expression. The degeneration initially occurs in the central inferior quadrant and spreads with retinal pigment epithelia (RPE) atrophy. Endothelial cell specific immunohistochemistry and fluorescein angiography (FA) revealed progressive changes in retinal neovascularization in the Nrl-/-Grk1-/- at 1 month of age, prior to the onset of significant cone functional deficits and ONL thinning. Vascular Endothelial Growth Factor (VEGF) expression was also observed in the inner retina and within blood vessels at post natal (PN) 21 of these mice. To further delineate the cone degeneration phenotype, we performed microarray analyses, observed statistically significant changes in retinal transcript levels of >400 genes, and examined these candidates with Ingenuity Pathway Analysis (IPA). The Oncostatin M Signaling pathway was the top canonical pathway, and inflammatory disease/response genes were one of the top networks identified. These data demonstrate that the loss of a functional Grk1 on the Nrl-/- background exacerbates age-related cone dystrophy in a light-independent manner, mediated partly through the inflammatory response pathway leading to retinal neovascularization.
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Asset Metadata
Creator
Yetemian, Rosanne Marie
(author)
Core Title
Elements of photoreceptor homeostasis: investigating phenotypic manifestations and susceptibility to photoreceptor degeneration in genetic knockout models for retinal disease
School
Keck School of Medicine
Degree
Doctor of Philosophy
Degree Program
Genetic, Molecular and Cellular Biology
Publication Date
07/31/2010
Defense Date
05/26/2010
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
Degeneration,knockout,OAI-PMH Harvest,photoreceptor,retina
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Craft, Cheryl M. (
committee chair
), Chen, Jeannie (
committee member
), Hinton, David R. (
committee member
), Ma, Le (
committee member
), Sampath, Alapakkam P. (
committee member
)
Creator Email
yetemian@gmail.com,yetemian@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-m3238
Unique identifier
UC152138
Identifier
etd-Yetemian-3922 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-358642 (legacy record id),usctheses-m3238 (legacy record id)
Legacy Identifier
etd-Yetemian-3922.pdf
Dmrecord
358642
Document Type
Dissertation
Rights
Yetemian, Rosanne Marie
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Repository Name
Libraries, University of Southern California
Repository Location
Los Angeles, California
Repository Email
cisadmin@lib.usc.edu
Tags
knockout
photoreceptor
retina