University of Southern California Dissertations and Theses

Mechanisms of retinal degeneration caused by genetic and environmental factors

(USC Thesis Other)

Mechanisms of retinal degeneration caused by genetic and environmental factors

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content MECHANISMS OF RETINAL DEGENERATION
CAUSED BY GENETIC AND ENVIRONMENTAL
FACTORS

by
Tian Wang

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOURTHEN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GMCB)

May 2013

Copyright 2013 Tian Wang
ii

Dedication

To My Dearest Parents, Husband and Daughter
献给我最亲爱的父母，先生和女儿
iii

Acknowledgements

I would like to send my sincere gratitude to all those who helped me to make this thesis
possible. First and foremost I offer my sincerest gratitude to my mentor, Dr. Jeannie
Chen. I am so grateful for her unconditional support, patient guidance and invaluable
suggestions throughout my study and writing of this thesis. In addition to the
extraordinary laboratory environment that she provided me, she also granted me the
freedom to develop my own idea, and set the highest standards of science and ethics
for me to reach for as a graduate student.
My sincere thanks go to my Ph.D. dissertation committee members, Dr. Alapakkam
Sampath and Dr. Gage Crump, for their encouragement and insightful comments. I
also would like to thank my guidance committee members, Dr. Le Ma and Dr. Roger
Duncan, for their help on the initiation and progression of my study.
I want to thank Dr. Francis Concepcion for being congenial colleagues and collaborators
to work with and numerous enlightening discussions. My thanks are also due to my
current and former laboratory members for providing me with an amiable environment
and support for my work, including Dr. Jiayan Chen, Dr. Wen Mao, Dr. Hormoz
Moaven, Dr. Brain Soreghan, Chia-ling Hsieh, Helen He and Yun Yao.
iv

Table of Contents

Dedication .......................................................................................................................... ii
Acknowledgements .......................................................................................................... iii
List of Tables .................................................................................................................. viii
List of Figures ................................................................................................................... ix
Abstract ............................................................................................................................. xi
Chapter 1 ........................................................................................................................... 1
Overview of Photoreceptors, Phototransduction Cascade, Retinal Degeneration
and Light Damage
1.1 Retina and Photoreceptor Cells ............................................................................. 1
1.1.1 Retina .......................................................................................................... 1
1.1.2 Rod and Cone Photoreceptors ..................................................................... 3
1.2 Phototransduction Signaling in Rod Photoreceptors ............................................. 5
1.2.1 Rod Structure ............................................................................................... 5
1.2.2 Rod Phototransduction Cascade .................................................................. 6
1.3 Phototransduction Deactivation in Rod Photoreceptors ........................................ 9
1.3.1 Rhodopsin Inactivation ............................................................................... 9
1.3.2 Retinoid Cycle ........................................................................................... 10
1.3.3 Transducin Inactivation and Restoration of cGMP Concentration ........... 11
1.4 Retinal Degeneration ........................................................................................... 12
1.4.1 Retinitis Pigmentosa .................................................................................. 12
1.4.2 PDE6 Mutant Mouse Models .................................................................... 13
1.5.3 Retinal Remodeling ................................................................................... 16
1.5 Light-induced Retinal Degeneration ................................................................... 21
1.6 Endoplasmic Reticulum (ER) Stress in Retinal Degeneration ............................ 25
1.7 Thesis Outline ...................................................................................................... 28

v

Chapter 2 ......................................................................................................................... 30
Evaluation of Cellular Targets in the Induction of Photoreceptor Cell Death by
Elevated cGMP
2.1 Abstract ................................................................................................................ 30
2.2 Introduction ......................................................................................................... 30
2.3 Materials and Methods ........................................................................................ 34
2.3.1 Animals ..................................................................................................... 34
2.3.2 Retinal Morphology .................................................................................. 34
2.3.3 cGMP ELISA ............................................................................................ 35
2.3.4 Western Blot Analysis ............................................................................... 35
2.4 Results ................................................................................................................. 37
2.4.1 Ablation of CNG Channels Delayed PDE6 Mutations-induced Loss of
Phototransduction Proteins ................................................................................. 37
2.4.2 Retinal Degeneration cauded by PDE6 Mutations was delayed in the
CNG Null Background ....................................................................................... 39
2.4.3 Long Term Rescue of Photoreceptor Viability of CNG Null
Background on PDE6 Mutations in vivo ........................................................... 44
2.4.4 High cGMP Concentrations in Double Mutant Mice ................................ 44
2.5 Discussion ............................................................................................................ 47

Chapter 3 ......................................................................................................................... 47
Two Different Pathways of Light-induced Retinal Degeneration
3.1 Abstract ................................................................................................................ 50
3.2 Introduction ......................................................................................................... 51
3.3 Materials and Methods ........................................................................................ 54
3.3.1 Light Exposure .......................................................................................... 54
3.3.2 Retinal Morphology .................................................................................. 54
3.3.3 Western Blot Analysis ............................................................................... 55
3.3.4 cGMP and cAMP ELISA .......................................................................... 56
3.4 Results ................................................................................................................. 57
3.4.1 Detection of Photoreceptor Apoptosis in Light-exposed Arr-/- and
Balb/C Mice ....................................................................................................... 57
vi

3.4.2 Distinct Profiles of Phototransduction Protein Degradation in Two
Light-induced Retinal Degenerations ................................................................. 60
3.4.3 Increased Ubiquitination was an Early Event in Constitutive
Phototransduction-induced Retinal Degeneration .............................................. 61
3.4.4 ER Stress Activation in Constitutive Phototransduction-induced Retinal
Degeneration ...................................................................................................... 63
3.4.5 Early Activation of ER Stress in Low Light Exposed RK-/- Retinas ....... 66
3.4.6 Transducin Knockout Prevented Constitutive Phototransduction-
induced Retinal Degeneration ............................................................................ 68
3.5 Discussion ............................................................................................................ 70

Chapter 4 ......................................................................................................................... 77
Restoring CNGB1 Expression with Cre-mediated Recombination in a CNGB
-/-

Mouse Line
4.1 Abstract ................................................................................................................ 77
4.2 Introduction ......................................................................................................... 78
4.3 Materials and Methods ........................................................................................ 81
4.3.1 Ethics Statement ........................................................................................ 81
4.3.2 Generation of CNGB
-/-
and CNGB1
CaM
mice ......................................... 81
4.3.3 Generation of Transgenic ER
TM
-Cre-ER
TM
-IRES-mCherry Mice ............ 82
4.3.4 Light and Electron Microscopy and Retinal Morphology ........................ 82
4.3.5 Immunocytochemistry ............................................................................. 823
4.3.6 Western Blot Analysis ............................................................................... 84
4.4 Results ................................................................................................................. 85
4.4.1 Reversible Ablation of CNG-  Gene ........................................................ 85
4.4.2 CNGB1
-/-
Mice Show Slow Progressed Retinal Degeneration ................. 88
4.4.3 Neural Remodeling Markers Activated During Retinal Degeneration
Process in CNGB1
-/-
Mice .................................................................................. 90
4.5 Future work ......................................................................................................... 93
4.5.1 Generation of CNGB1
-/-
/ ER
TM
-Cre-ER
TM
-IRES-mCherry Mice ............. 93
4.5.2 Characterization of CNGB1
CaM
-dependent Recovery of Retinal
Degeneration ...................................................................................................... 94
4.5.3 Investigation of the Time Limit for the Retinal Recovery at Different
Degeneration Phases .......................................................................................... 95

vii

Conclusion and Future Perspective ............................................................................... 96
Bibliography .................................................................................................................... 99

viii

List of Tables
Table 1.1 Comparison of PDE6 mutant mouse models 14
Table 1.2 Constitutively activated signaling mutants 24
Table 2.1 cGMP concentration in double mutant and control mice 46

ix

List of Figures
Figure 1.1 Retinal structure and morphology 2
Figure 1.2 Rod and Cone photoreceptors in mammalian retina 4
Figure 1.3 Phototransduction cascade in the rod photoreceptor cell 8
Figure 1.4 Schematic representations of the three stages of retinal degeneration 17
Figure 1.5 Schematic representations of major components’ contribute to light-induced
photoreceptor apoptosis 23
Figure 1.6 The three branches of the UPR 26
Figure 2.1 Protein levels in double mutant and control mice 38
Figure 2.2 CNG null background rescued retinal degeneration in Pde6g-/-mice. 40

Figure 2.3 CNG null background rescued retinal degeneration in rd10 mice. 41

Figure 2.4 Long term rescue effects of CNG ablation on retinal degeneration caused
by PDE6 mutants. 42
Figure 2.5 The rescue of CNG ablation on PDE6 mutant-induced retinal degeneration
was incomplete. 43
Figure 3.1 Retinal morphology of light exposed Arr-/-, Balb/C and C57 mice 58
Figure 3.2 Distinct degradation profiles of phototransduction proteins in light
exposed Arr-/, Balb/C and C57 mice 59
Figure 3.3 Overload of UPS in the early phase of constitutive phototransduction
induced retinal degeneration 62
Figure 3.4 Activation of UPR in the early phase of constitutive phototransduction
induced retinal degeneration 64
x

Figure 3.5 Early up-regulations of ER stress sensors and increase of protein
ubiquitination in light exposed Arr-/- retinas 65
Figure 3.6 Early up-regulations of ER stress sensors and increase of protein
ubiquitination in light exposed RK-/- retinas 67
Figure 3.7 Transducin null background prevented constitutive phototransduction
induced retinal degeneration 69
Figure 3.8 Involvement of ROS and PKA signaling in bright light induced retinal
degeneration 71
Figure 4.1 Generation of mice strains 80
Figure 4.2 Characterization of CNGB1
-/-
and CNGB1
CaM
mice 86
Figure 4.3 Morphological and functional analysis of CNGB1
CaM
mice 87
Figure 4.4 Retinal morphology of CNGB1
-/-
mice 89
Figure 4.5 Neuronal remodeling markers activated in CNGB1
-/-
retinas 91
Figure 4.6 Bipolar cell and synaptic ribbon morphology in WT, CNGB1
CaM
and
CNGB1
-/-
retinas 92
Figure 4.7 Experiment design I 93
Figure 4.8 Experiment design II 95

xi

Abstract

Retinitis pigmentosa (RP) is a set of hereditary retinal diseases characterized by
progressive retinal degeneration and eventual photoreceptor cell death, affecting 1 in
4000 for a total of about 15 million people in the world. There is currently no
treatment for this debilitating retinal degenerative disorder. To develop clinical
treatments, two fundamental questions need to be answered: first, what are the
underlying mechanisms; and second, what is the optimum time window for rescue
approaches. In this thesis, I strived to answer these questions using multiple in vivo
models.
Approximately 8% of autosomal recessive RP cases have been linked to mutations in
the rod cGMP phosphodiesterase 6 (PDE6) complex. The elevated [cGMP], due to the
reduced or abolished PDE activity, is believed to cause photoreceptor cell death. In
this thesis, the roles of two cGMP targets: cyclic nucleotide gated (CNG) channels and
protein kinase G (PKG), were investigated in vivo. Our results show that genetic
ablation of CNG channels but not PKG significantly delayed the phototransduction protein
losses and improved photoreceptor viability in two PDE6 mutant mice, supporting the
hypothesis that elevated cGMP opens excessive numbers of CNG channels leading to
uncontrolled influx of Ca
2+
and Na
+
which further initiates photoreceptor cell death.
xii

Thus targeting rod CNG channels could be a promising approach to treat cGMP-
induced retinal degeneration.
Additionally, excessive light exposure is an environmental factor that modulates the
rate of retinal degeneration in inherited retinopathies and age-related macular
degeneration. Previous studies show that light damage to the retina occurs through at
least two distinct pathways: 1) bright light exposure kills photoreceptor cells in a
transducin-independent manner accompanied by AP-1 induction, and 2) retinal
degeneration induced by low light exposure depends on activation of the
phototransduction cascade but not AP-1 induction. However, the underlying
mechanisms are unclear. In this thesis, we demonstrated that ER stress proteins were
markedly up-regulated in the early phase of constitutive phototransduction-induced
retinal degeneration. These upregulations coincided with the increase of global
ubiquitination preceding the photoreceptor apoptosis, suggesting that the activation of
unfolded protein response might be the common initiator of retinal degeneration
induced by constitutive phototransduction activation.
Finally, retinal degenerative disorders initiated by death of photoreceptor cells are
followed by neural remodeling that includes neuronal death, cell migration and
rewiring of retinal circuits. Actual status of retinal remodeling at different phases
determine the practical intervention window and are therefore crucial to all retinal
rescue approaches. In this thesis, it was shown that the CNGB
-/-
mice is an ideal model
xiii

that recapitulates retinal degeneration patterns as seen in human retinitis pigmentosa,
and could thus be an appropriate platform for a systematic study of the potential of
functional recovery after neural remodeling events.
1

Chapter 1
Overview of Photoreceptors, Phototransduction Cascade, Retinal
Degeneration and Light Damage

1.1 Retina and Photoreceptor Cells
1.1.1 Retina
Sight begins in the retina. This innermost tissue of the eye specifically absorbs photons
from the environment, converts light signals via a chemical and electrical cascade into
nerve impulses which are then transmitted to the visual cortex to generate visual images.
Vertebrates’ retinas are composed of five major types of neurons: photoreceptors, bipolar
cells, horizontal cells, amacrine cells, and ganglion cells, and highly conserved in a basic
laminar organization (Fig. 1.1).
The only neurons that are photosensitive in the retina are photoreceptors. The other types
of neuronal cells are involved in processing and transmitting signals originated from the
photoreceptors to the brain. Outside photoreceptors lines retinal pigment epithelium
(RPE), a monolayer of polarized epithelial cells. RPE is essential for photoreceptor
function and survival and its functions include supplying small molecules, uptaking and
transporting retinoids and performing routine phagocytosis to remove decayed disks of
photoreceptors. Under light microscope, 10 layers can be recognized on a retinal vertical
2

section (Rodieck 1998). Four of them are belong to photoreceptors: outer segment (OS),
inner segment (IS), outer nuclear layer (ONL) and fiber layer (synapse layer). The
thickness of ONL is the classical index to reflect the health of the retina and used to
measure the severity of retinal degeneration (a detailed description in chapter 1.4).

Exterior
Interior
Figure 1.1. Retinal structure and morphology. The retina can be recognized as 10
layers. They are RPE: retinal pigment epithelium; OS: outer segment; IS: inner
segment; ONL: outer nuclear layer; fiber layer; OSL: outer synaptic layer; INL:
inner nuclear layer; ISL: inner synaptic layer; GCL: ganglion cell layer, and optic
fiber layer. Measurement of the thickness of outer nuclear layer (ONL) can be used
to quantify the degree of retinal degeneration. (From “The First Step in Seeing” and
“Gray’s Anatomy”).
3

1.1.2 Rod and Cone Photoreceptors
There are two types of photoreceptors in vertebrates, rods and cones, named after their
respective outer segment (OS) shapes (Fig. 1.2). Rod outer segment contains
approximately 1000 flattened stacked membranous discs formed by the internalization of
the plasma membrane. These disks are pinched off and exist separately in rods, whereas
the disks in cones are invaginated and attached to the outer membrane.
Besides the morphology differences, rods and cones also function distinctly. Rods
express only one type of visual pigment: rhodopsin with the maximal spectra sensitivity
at 498nm, and are 100 times more sensitive to a single photon than cones (Okawa and
Sampath 2007). In contrast there are three types of cones in human depending on the
opsins expressed: red opsin (560nm), green opsin (530nm) and blue opsin (414nm); and
two types of murine cones with short-wavelength opsin (360nm) and medium-
wavelength opsin (510nm), respectively. Though cones are less sensitive to light, they
provide color vision with more spatial and temporal resolution. Collectively, human
vision is subdivided into three categories depending on the photoreceptors involved:
scotopic vision up to 10
-2
lux illumination occurs when only rods are stimulated; mesopic
vision occurs between 10
-2
-10
-1
lux when both rods and cones are operating; while
photopic vision occurs between 10
-1
-10
5
lux when only cones are functioning due to the
saturation of rods activity (Rodieck 1998).

4

Figure 1.2. Rod and Cone photoreceptors in
mammalian retina. (a) A human retinal section
showing three neuronal cell layers: outer
nuclear layer (ONL) containing the nucleus of
rods and cones; inner nuclear layer (INL)
containing the nucleus of bipolar, horizontal
and amacrine and Muller glial cells; ganglion
cell layer (GCL). (b) Diagram of rod and cone
structure. (c) Scan EM showing the outer
segments. (Courtesy of Dr. Shiming Chen)
5

Accounting for 95% of total photoreceptor population (Curcio, Sloan et al. 1990; Oyster
1999), rods are not only responsible for dim light vision and also important to sustain the
existence of cones. This latter trait is supported by a common observation in patients
suffering from retinitis pigmentosa (RP, a detailed description in chapter 1.4): cone cell
death occurs secondarily to rod cell apoptosis that is induced by rod-specific mutations
(Sancho-Pelluz, Arango-Gonzalez et al. 2008). Possible mechanisms include loss of
structural or trophic support; releasing toxic substances that negatively affect the
neighboring cells by dying rods; and/or changes in oxygen consumption levels resulting
in a fatal increase in oxidative stress as a function of rod degeneration. In summary, this
latter role of rods is very important to the overall health of the retina.

1.2 Phototransduction Signaling in Rod Photoreceptors
1.2.1 Rod Structure
The rod photoreceptor cell can be described as having three sections (Fig 1.2): the outer
segment (OS), the inner segment (IS) and the cell body (ONL and fiber layer). The
phototransduction machinery is lining on the stacked disks of the outer segment, while
the ion channels/pumps responsible for the rod’s membrane potential localize along the
outer segment membrane. The inner segment contains other key proteins involved in the
phototransduction cascade as well as the cellular organelles such as the Golgi complex,
6

endoplasmic reticulum and the mitochondria. The cell body contains the nucleus and the
synapse where voltage-gated ion channels regulate the release of neurotransmitter at the
synaptic terminal.

1.2.2 Rod Phototransduction Cascade
Phototransduction cascade converts light signals carried by photons into downstream
chemical signals via a series of biochemical steps (Fig 1.3).
It begins with the absorption of a photon by visual pigment rhodopsin, the G-protein
coupled receptor (GPCR) with seven transmembrane -helices. Rhodopsin is composed
of two parts: a chromophore named 11-cis-retinal and an aproprotein opsin. These two
components are covalently linked together by a protonated Schiff base linkage at the 296
lysine residue (K296) of rhodopsin (Robinson, Cohen et al. 1992). Additionally, this
positively charged Schiff base linkage interacts with the negatively charged glutamate
(E113) residue at position 113 on rhodopsin to form a salt bridge (Sakmar, Franke et al.
1989). These interactions are very strong and the constrained “dark state” rhodopsin is
exceptionally stable.
Upon photon absorption, 11-cis-retinal is photoisomerized to all-trans-retinal, which
disrupts these intramolecular interactions (including salt bridge) and causes
conformational changes that leave rhodopsin in its active form known as Metarhdopsin
II (R*).
7

R* then binds to the heterotrimeric G-protein (transducin) and acts as a guanine-
nucleotide exchange factor (GEF) by catalyzing the substitution of GDP for GTP on the
transducing  subunit(Robinson, Cohen et al. 1992). This exchange releases the
transducin βγ dimer from the α-subunit, which in turn binds to the regulatory γ-subunit of
the effector enzyme phospodiesterase 6 (PDE6) complex and removes the inhibitory
constraint from the catalytic PDE6 or  subunits. The activated PDE  subsequently
hydrolyses 3’-5’ cyclic guanosine monophosphate (cGMP) to 5’GMP, resulting in the
decrease of intracellular cGMP concentration and the subsequent release of cGMP from
specific cyclic nucleotide-gated cation (CNG) channels. In the dark, ~3% of the cGMP-
gated channels are open (Yau and Baylor 1989) to form the “dark current” carried by the
influx of Na
+
, Ca
2+
and small amount of Mg
2+
(Yau and Nakatani 1984). This influx
current is balanced by the efflux by Na
+
/Ca
2+
K
+
exchangers in the OS and Na
+
/K
+
pumps
and K
+
pumps in the inner segment (IS) to maintain a depolarized membrane potential of
about -40mV in rod cells. In the light, the closure of the cGMP-gated channels due to the
decrease of cytosolic cGMP concentration precludes the Na
+
and Ca
2+
influx and thereby
results in the hyperpolarization of the photoreceptor cell. This hyperpolarization leads to
the closure of downstream voltage-gated Ca
2+
channels and a subsequent decrease in the
glutamate release at the synaptic terminal, which serves as chemical signals to the
secondary order neurons, i.e. bipolar cell , to trigger the signal transduction further along
the retina (Yau 1994; Burns and Baylor 2001; Lamb and Pugh 2006). Thus the
transformation of photon absorption to chemical signals is completed.
8

Figure 1.3. Phototransduction cascade in the rod photoreceptor cell. When rhodopsin is
activated (R*) by photon absorption, it stimulates the heterotrimeric G-protein, transducin
(G), by promoting the exchange of GDP for GTP within the α-subunit of Tr (G* α). The
binding of GTP to the T α subunit induces the release of βγ complex. The dissociated GTP-
bound T α then activates cGMP phosphodiesterase 6 (PDE6) by binding to PDE γ subunit.
This event, in turn, activates either PDE α- or β- catalytic subunit, which converts
cGMP (cGMP) to GMP, thereby lowering the intracellular [cGMP]. This condition
causes cGMP molecules to be released from cyclic nucleotide -gated cation channels
(CNGs), resulting in the closure of these channels and thereby preventing further entry
of Ca
2+
and Na
+
ions. This situation creates a hyperpolarization state across the entire
plasma membrane of the cell. Guanylyl cyclases (GCs) and guanylyl cyclase activating
proteins (GCAPs) then help the cell to restore the intracellular cGMP levels. The R* is
deactivated by the phosphorylation by RK and the binding of visual arrestin, and then
dephosphorylated and reconstituted with another 11-cis-retinal to form regenerated
rhodopsin. (Courtesy of Dr. F.A. Concepcion)
9

1.3 Phototransduction Deactivation in Rod Photoreceptors
The timely termination of phototransduction is equally important as its activation to
generate proper visual signals. This process requires quenches of the catalytic activities
of individual enzymes and final restoration of cGMP concentration to dark levels as
discussed below.

1.3.1 Rhodopsin Inactivation
The rhodopsin inactivation is completely achieved through two steps (Fig. 1.3). First, the
R* activity is partially deactivated due to multiply phosphorylation at Ser/Thr sites on its
carboxyl tail by rhodopsin kinase (RK) (Kuhn and Wilden 1987; Chen, Burns et al. 1999).
A minimum of three phosphorylation sites was required for response deactivation and all
six phosphorylation sites were needed for recovery with normal kinetics (Chen, Makino
et al. 1995; Mendez, Burns et al. 2000; Mendez, Krasnoperova et al. 2000). Once
phosphorylated, R*-P loses most of its activity, but still has some residual capacity for
transducin activation (Chen, Simon et al. 1999). The complete termination of the R*
activity requires the binding of visual arrestin (Arr) (Chen et al., 1995b), which
selectively interacts with light-activated phosphorylated rhodopsin (R*-P) with the
binding affinity 10-20 times higher than that with non-activated phosphorylated (Rh-P) or
with activated unphosphorylated species (R*) (Gurevich and Benovic 1993). Then, R*
hydrolyzes all-trans-retinal, arrestin dissociates, R*-P is dephosphorylated and finally
10

opsin binds a new 11-cis-retinal regenerated from a series of RPE-mediated reactions
knows as retinoid cycle (a detailed description in chapter 1.3.2) to regenerate rhodopsin.
Since both RK and Arr are required for complete deactivation of R*, it is not surprising
that the mutations of either genes lead to Oguchi disease, a congenital stationary night
blindness in humans caused by defective phototransduction deactivation(Yamamoto,
Sippel et al. 1997; Chen, Simon et al. 1999; Fain and Lisman 1999). Consistently, null
mutations of RK or Arr in mice result in retinal degeneration in light-dependent manner
that can be blocked by the transducin null background (Chen, Burns et al. 1999; Chen,
Simon et al. 1999), further confirming the prolonged phototransduction activation is toxic
to the photoreceptors (a detailed description in chapter 1.5).

1.3.2 Retinoid Cycle
The retinoid cycle is a complicated enzymatic process requiring both photoreceptors and
RPE cells, and contains four steps: photochemistry, removal of retinoid, reconversion of
retinoid and delivery of retinoid(Lamb and Pugh 2004). 1. Photochemistry: Upon photon
absorption, 11-cis-retinal is photoisomerized to all-trans-retinal, which converts
rhodopsin into R*. 2. Removal of retinoid: all-trans-retinal is then reduced to all-trans-
retinol by NADPH-dependent all-trans-retinol dehydrogenase (RDH). This reduction can
occur either when all-trans-retinal still non-covalently binds to opsin or after all-trans-
retinal is flipped into the cytoplasm by the ATP-binding cassette transporter rim protein
(ABCR) (Schadel, Heck et al. 2003). In either case, all-trans-retinol ends up in the
11

cytoplasm and is then carried by inter-photoreceptor retinol binding protein (IRBP) to
cross plasma membrane into RPE. 3. Reconversion of retinoid: Within RPE cytoplasm,
all-trans-retinol is chaperoned by cellular retinol binding protein (CRBP) and esterified
by lecithin retinol acyl transferase (LRAT). All-trans-retinyl ester is then guided by
RPE65 and isomerized to 11-cis-retinol by retinyl ester isomerohydrolase. The latter is
further oxidized to 11-cis-retinal by 11-cis RDH facilitated by cellular retinaldehyde
binding protein (CRALBP). 4. Delivery of retinoid: 11-cis-retinal then diffuses to
photoreceptor outer segment disc membrane possibly via IRBP and covalently binds to
opsin to form a new visual pigment.

1.3.3 Transducin Inactivation and Restoration of cGMP Concentration
Another key component of phototransduction inactivation is transducin deactivation,
which is actually the rate limiting step during photo-response recovery (Chen, Burns et al.
2000; Krispel, Chen et al. 2006). Activated Tr  has the intrinsic GTPase activity to
hydrolyze GTP back to GDP. This activity is accelerated by the G-protein signaling
(RGS) complex,  RGS9-1/G β
5
/R9AP, functioning as the GTPase activating protein
(GAP) (He, Cowan et al. 1998; Hu and Wensel 2002). Upon Tr  deactivation, PDE6 is
then released and rebinds to catalytic  subunits to reestablish the inhibitory interaction,
restoring PDE6 back to its low basal activity. Meanwhile, the decreased intracellular
[Ca
2+
] activates guanylyl cyclase activating proteins (GCAPs), which in turn stimulates
12

guanylyl cyclase (GCs) to upregulate its cGMP synthesis (Koutalos, Nakatani et al. 1995;
Koutalos and Yau 1996). The combination of accelerated GC activity and lowered PDE6
activity restores the intracellular [cGMP], leading to the reopening of the CNG channels
and eventually reestablishment of the depolarized dark-adapted state of the rod cell.

1.4 Retinal Degeneration
1.4.1 Retinitis Pigmentosa
Retinitis pigmentosa (RP) is the term given to a set of hereditary retinal diseases
characterized by progressive loss of rod and cone photoreceptors. As the most common
inherited form of retinal degeneration disorders, RP affects 1 in 4000 for a total of about
15 million people in the world(van Soest, Westerveld et al. 1999). In most cases, patients
with RP first experienced night blindness due to the defect of rod system, then a gradual
loss of peripheral vision correlated to the subsequent degeneration of cones, and
eventually complete vision loss. This disease is genetically heterogeneous and can be
inherited as an autosomal-dominant, autosomal-recessive or X-linked trait (Pierce 2001).
Until now, more than 45 genes have been identified for RP, and these genes account for
60% of all cases (Hartong, Berson et al. 2006). Most protein products of these genes are
expressed specially in photoreceptors, including components of phototransduction
cascade as well as proteins important for outer segment structure. Although different in
13

associated mutations, photoreceptors eventually die via apoptosis(Travis 1998). However,
the mechanisms by which these mutations lead to the apoptosis of photoreceptors are not
completely understood. Based on published studies, there are four major types of
degeneration mechanisms can be identified: disruption of photoreceptor outer segment
morphogenesis, constitutively activation of phototransduction cascade, overload of
photoreceptors’ metabolism and dysfunction of RPE cells. Yet much work still remains.

1.4.2 PDE6 Mutant Mouse Models
The heterotrtrameric PDE6 complex, made of ,  and two subunits regulating the
intracellular cGMP levels in response to light activation, is the key component of the
phototransduction cascade. Naturally occurring PDE6 mutations account for
approximately 8% autosomal RP cases (https://sph.uth.edu/retNet/) (McLaughlin, Ehrhart
et al. 1995; Hartong, Berson et al. 2006; Daiger, Bowne et al. 2007). Hence, the mouse
strains harboring PDE6 mutants are commonly used as models for RP.
The naturally occurring rd1 mouse model has been studied extensively for several decades.
This mouse strain harbors a loss-of-function mutation in the gene encoding for the -subunit
of rod PDE6 (Keeler 1924; Keeler 1966; Pittler and Baehr 1991). The photoreceptor
degeneration in rd1 mice begins at postnatal day 8 (P8) and progresses rapidly, leading to an
almost complete loss of the photoreceptor layer by 4 weeks even before the retina becomes
mature. Extremely high cGMP levels are found to precede the retinal degeneration (starting
14

at P5) and are correlated with deficient PDE6 activity (Farber and Lolley 1974; Farber and
Lolley 1976).
Another similar mouse strain is Pde6g-/-, first generated by Tsang et al (Tsang, Gouras et al.
1996) using a gene-targeting approach to disrupt PDE6 gene by Neo. This mutation results
in the absence of PDE6  expression and a rapid retinal degeneration progression similar as
that in rd1 mice. The photoreceptor outer segment fails to develop normally and is
disorganized at P10. A progressive loss of photoreceptor nuclei is observed from P14-P21,
and only a single row of photoreceptor nuclei remains by P21. The retina is completely
devoid of photoreceptor cells by 6 weeks. The PDE dimer is formed but lacks hydrolytic
activity, leading to an increase in cGMP levels preceding photoreceptor cell death.

Mouse Strain Mutation
Degeneration
Features
Onset of Rod
Degeneration
Recordable
ERG
Response
rd1
PDE -
subunit
nonsense
Rod-cone center-
to-periphery
gradient
P8 P14-16
rd10
PDE -
subunit
missense
Rod-cone center-
to-periphery
gradient
P18 P14-28
Pde6g-/-
PDE -
subunit
knockout
Rod-cone center-
to-periphery
gradient
P8 P14-21
Table 1.1 Comparison of PDE6 mutant mouse models
15

Another mutant PDE6 mouse model also widely used is rd10 (Chang, Hawes et al. 2007),
which harbors a missense point mutation in exon 13 of the -subunit of the rod PDE6
gene. Mice homozygous for the rd10 mutation show a much later onset and milder retinal
degeneration compared to rd1 or Pde6g-/-

mice. Loss of photoreceptors in the rd10 mouse
begins around P18, with peak photoreceptor death occurring at P25, and most
photoreceptor cells are lost by 8 weeks (Gargini, Terzibasi et al. 2007; Barhoum,
Martinez-Navarrete et al. 2008).
For all these PDE6 mouse models of RP, it has been hypothesized that the loss or
reduction of PDE6 enzyme activity leads to the high levels of cytosolic cGMP, which in
turn promote photoreceptor cell death (Fain and Lisman 1999; Doonan, Donovan et al.
2005). However the exact pathway underling the elevated cGMP levels and the onset of
photoreceptor death remains poorly understand. The dominant hypothesis is that the toxic
influx of Ca
2+
through the CNG channels that are gated open by cGMP is toxic to
photoreceptors. However, attempts to prevent photoreceptor death using Ca
2+
channel
blockers, i.e. D-cis-diltiazem, have shown controversial results (Frasson, Sahel et al.
1999; Pearce-Kelling, Aleman et al. 2001; Pawlyk, Li et al. 2002). Recent studies using
shRNA to knockdown GC or CNG channels have shown improvements of photoreceptor
survival and function in PDE6
H620Q
mutant mice (Tosi, Davis et al. 2011; Tosi, Sancho-
Pelluz et al. 2011), but the effect was incomplete and temporary.
16

Another hypothesis for this cGMP-mediated retinal degeneration is via a protein kinase G
(PKG) dependent pathway. There are three different isoforms of PKG: PKG1 , PKG1 ,
and PKG2(Hofmann, Feil et al. 2006). PKG1 is abundantly expressed in the eye, whereas
the PKG2 expression appears to be very low(Gamm, Barthel et al. 2000). PKGs are 100-
fold more sensitive than CNG channels to cGMP (Lincoln and Cornwell 1993), and their
excessive activation has been shown to cause neuronal cell death in many cases (Canals,
Casarejos et al. 2003; Canzoniero, Adornetto et al. 2006). cGMP analogues that
specifically inhibit PKG activity are shown to have rescue effects on the photoreceptor
degeneration of rd1 mice both in vitro and in vivo (Paquet-Durand, Hauck et al. 2009).
However, these cGMP analogues also affect CNG channels activity. Thus additional
studies are needed to confirm the involvement of PKG activation in cGMP-induced
retinal degeneration.

1.5.3 Retinal Remodeling
Current strategies to develop treatment for retinal degeneration assume that the inner
neuronal cells remain intact and the normal cell patterning and wiring are preserved even
after photoreceptor loss. However, when photoreceptors degenerate, secondary changes
also occur to other retinal cells, including neuronal cell death, migration and rewiring, in
a process termed as neural remodeling (Fig. 1.4) (Jones, Watt et al. 2003; Marc and Jones
2003; Marc, Jones et al. 2003; Jones and Marc 2005).
17

Figure 1.4. Schematic representations of the three stages of retinal degeneration (both
rod and cone photoreceptors in orange, rod and cone bipolar cells in light and dark blue,
ganglion cells in light and dark purple, a horizontal cell in olive, GABAergic amacrine
cells in red, glycinergic amacrine cells in green and Müller cell in yellow with the two
plexiform layers as horizontal bands). (a) Shows normal lamination and connectivity of
cell classes in the retina. (b) Reveals early photoreceptor and outer segment shortening
along with rod and cone neurite extensions projecting down into inner nuclear layer and
ganglion cell layer. Müller cells may also begin to hypertrophy in this stage. Stage 2
shown in (c) demonstrates a complete loss of photoreceptors and elaboration of a Müller
cell seal over the entire neural retina, sealing it off away from the remnant choroid. Early
stage 3 events ensue in (d) with the elaboration of neurite extensions from glycinergic
and GABAergic amacrine cells along with contributions from bipolar cells and ganglion
cells into complex tangles of processes called microneuromas that form outside the
normal lamination of the inner plexiform layer, sometimes merging with the inner
plexiform layer. (e) Demonstrates the final phase of retinal degeneration with
degeneration and cell death of many cell classes. Cellular translocation events also occur
in this stage with bi-directional movements of cells from the inner nuclear layer and
ganglion cell layer. (Jones and Marc 2005)

18

Several studies reported loss of inner nuclear layer cells, far more severe in peripheral
retina, in retinas from patients with RP including those were diagnosed with moderate
and severe forms of the disease(Stone, Barlow et al. 1992; Santos, Humayun et al. 1997;
Milam, Li et al. 1998; Humayun, Prince et al. 1999). Surviving rods, horizontal and
amacrine cells are shown to extend anomalous neurites and form aberrant circuitry
throughout the retina in patients (Fariss, Li et al. 2000; Jacobson, Sumaroka et al. 2007).
Consistently, analysis of rodent degeneration models also reveals similar changes in the
neural retina, some even preceding rod degeneration (Fletcher and Kalloniatis 1996;
Strettoi, Porciatti et al. 2002). Bipolar and horizontal cells axon terminals retract their
dendrites and lose synaptic connections as photoreceptors initiate cell death, followed by
glial and amacrine cell remodeling with ganglion cell sparing(Strettoi and Pignatelli 2000;
Strettoi, Pignatelli et al. 2003; Gargini, Terzibasi et al. 2007; Margolis, Newkirk et al.
2008; Mazzoni, Novelli et al. 2008; Puthussery and Taylor 2010). The cone circuits are
also defect as both cones and cone horizontal cells are found to sprouts new neuritis in
the young rd1 mouse (Fei 2002). Ultimately, bipolar and horizontal cells are lost. These
neuronal remodeling processes following photoreceptor death suggest a possible trophic
dependence of secondary order neurons on the presence of photoreceptors for
maintenance of their proper morphology and function.
There are three phases of retinal remodeling (Fig. 1.4) (Jones, Watt et al. 2003; Marc and
Jones 2003; Marc, Jones et al. 2003; Jones and Marc 2005): 1, photoreceptor stress; 2,
photoreceptor death and 3, complex neural remodeling. Phase 1 of retinal remodeling is
19

often manifested as photoreceptor phenotype deconstruction. The outer segments are
shortened, and the stressed photoreceptors begin to sprout neurites that bypass their
normal targets and extend as far as the ganglion cell layer, which are marked by
delocalized rhodopsin. These neurites will then be retracted before the photoreceptor cell
death. The failure of synaptic signaling also triggers the retraction of bipolar dendrites,
switching of synaptic targets by bipolar cells, and extension of horizontal cell into the
inner plexiform layer.
Phase 2 is characterized by photoreceptor death and formation of a distal fibrotic glial
seal composed of Müller cell distal processes. In this phase, a large amount of debris
resulting from photoreceptor cell death are removed by microglia, and the bystander
effects ultimately lead to the death of the remaining photoreceptors, eliminating light
mediated signaling to the neural retina. Another hallmark of this phase is a dramatic more
than 10-fold increase in glutamine expressed by Müller cell, suggesting that the metabolic
status and genetic profiles of these cells are apparently irreversibly altered (Marc, Murry
et al. 1998). Before the completion of phase 2, bipolar cells retract their dendrites and
horizontal cells send axonal processes into inner plexiform layer. Neuronal death may
also begin in phase 2.
Phase 3 is the final and more prolonged stage of remodeling, entailing a range of events
including, but not limit to, Müller cell hypertrophy, migration of RPE cells into the neural
retina, neuronal death, and formation of novel ectopic fascicles and microneuromas by
the survivor neurons in inner and outer plexiform layers. Microneuromas are tangles of
20

GABAergic amacrine cell, glycinergic amacrine cell, glutamatergic bipolar cell and
ganglion cell processes, ranging from 20 to over 100 m in diameter and containing
numerous synapses formed de novo. Both conventional and ribbon synapses are
abundant in microneuromas and display all common synaptic arrangements, though
infrequent instances of bipolar-bipolar contacts also occur.
The nature of remodeling by survivor neurons remains unclear. Existing hypotheses
include: 1, diminished glutamateric synaptic drive fails to maintain normal intracellular
Ca
2+
levels and triggers dendritic growth to seek new synaptic partners (Fiala, Spacek et
al. 2002); 2, retrograde trans-synaptic signaling by neurotrophin may play a key role in
neuronal survival (Quigley 1999; Yip and So 2000); 3, modification of cell adhesion
pathways promotes neuron migration and neurite extension, as in retinal detachment
(Lewis, Charteris et al. 2002).
The fact of neural remodeling influences all rescue strategies, especially since phase 1
remodeling begins before photoreceptor cell death. All tissue based approaches depend
explicitly upon preservation of the neural retinal architecture, and will be negatively
impacted by even minor changes in neural patterning. Additionally, in the late stage of
retinal degeneration, the formations of the glial seal and new anomalous circuitries are
more likely to corrupt the visual processing and preclude any retinal rescue approaches.
The practical intervention time is unclear, since RP arising from different primary defects
appears to progress at different rates. The actually effects of retinal remodeling on retinal
21

rescue approaches at different phases still need further investigation. This work is
attempted to contribute to fill this gap.

1.5 Light-induced Retinal Degeneration
RP affects 1 out of 4000 people worldwide; while age-related macular degeneration
(AMD) shows an even higher prevalence of about 10% of people over the age of 65 (Bok
2002) with a distinct trend of increase with aged population. Retinal degeneration in these
diseases is finally through photoreceptor (and RPE) apoptosis (Wenzel, Grimm et al.
2005), the mechanism of which mimics that of retinal damage caused by constant light
exposure. Excessive light exposure has also been reported to exacerbate the rate of
photoreceptor apoptosis in patients with these retinal diseases (Cruickshanks, Klein et al.
1993; Simons 1993; Mata, Weng et al. 2000). Consistently, several animal models for
human retinal degeneration show a higher light damage susceptibility than do controls,
thus light induced retinal degeneration has been used as a model for the study of RP
(Noell 1980; Sanyal, De Ruiter et al. 1980; Chen, Burns et al. 1999; Chen, Simon et al.
1999; Wenzel, Grimm et al. 2005).
Previous studies show that light damage to the retina occurs through at least two distinct
pathways: 1) bright light induced acute damage depends on rhodopsin regeneration and
transcriptional activator AP-1 activation (Hafezi, Steinbach et al. 1997; Grimm, Wenzel
22

et al. 2000; Wenzel, Grimm et al. 2000; Wenzel, Reme et al. 2001) in the absence of
phototransduction; 2) retinal degeneration induced by low light exposure in instances is
caused by constitutive activation of the phototransduction cascade but not AP-1
activation (Hao, Wenzel et al. 2002).
At higher light intensities, light induced photoreceptor cell death is initiated by photon-
activated rhodopsin (Fig. 1.5). Mice deficient in rhodopsin regeneration have been
reported to be more resistant to bright light-induced retinal damage (Danciger, Matthes et
al. 2000; Grimm, Wenzel et al. 2000). During light exposure period, the intracellular Ca
2+

levels increase (Donovan, Carmody et al. 2001) and reactive oxygen species (ROS) are
generated by photon-excited rhodopsin (Yang, Basinger et al. 2003) as an early event of
retinal damage. Application of the calcium channel blocker (Donovan and Cotter 2002)
and antioxidants (Specht, Organisciak et al. 2000; Tanito, Masutani et al. 2002; Tanito,
Nishiyama et al. 2002) prevented the photoreceptor apoptosis, confirming the roles of
Ca
2+
and ROS as initiators of bright light-induced photoreceptor degeneration. Neuronal
nitric-oxide synthase (nNOS) and GC are the possible downstream effectors of ROS
signaling (Donovan, Carmody et al. 2001). Finally, AP-1 is activated leading to full
activation of the apoptotic process. Mice lacking c-fos, an AP-1 component, are highly
resistant to bright light damage (Hafezi, Steinbach et al. 1997); whereas the absence of
transducin shows no protective effects, indicating that the phototransduction cascade is
not required (Hao, Wenzel et al. 2002).
23

Continuous
light
Arr or RK
null mutants
Rho or Tr
constitutively
active mutants
Figure 1.5. Schematic representations of major components’ contribute to light-induced
photoreceptor apoptosis. Induction Phase: Essential for light-induced apoptosis is the
visual pigment rhodopsin, its regeneration rate as it occurs in the visual cycle, and,
potentially, metabolic products (retinoids) arising after photon absorption by rhodopsin.
Death signal transduction I comprises an increase in intracellular calcium levels, lesions of
mitochondria, an increase in NO and ROS. Death signal transduction II comprises
activation of the transcription factor AP-1, which is essential for light-induced apoptosis in
our model. Execution: The actual execution is less clear at this stage, for example, the role
of caspases in light-induced apoptosis is controversial, possibly, other proteolytic systems
such as proteasomes and others are exclusively or additionally involved. Clearance of
apoptotic bodies is rather conspicuous morphologically, however, the ‘‘eat me’’ signaling
has not yet been explored. In acute light-induced apoptosis, cells of the pigment epithelium
as well as abundant macrophages (M) are observed phagocytosing photoreceptor remnants.
The end stage after the active phagocytic period appears as partial or total removal of
photoreceptors and remaining RPE cells depending on the damaging illuminant levels.
(Wenzel, Grimm et al. 2005)
24

Table 1.2 Constitutively activated signaling mutants. (Lem and Fain 2004)
25

On contrast, constitutive phototransduction activation leads to photoreceptor cell
apoptosis through different mechanisms, and can result from low light exposure when
arrestin or rhodopsin kinase, protein necessary for deactivating rhodopsin, is defective
(Xu, Dodd et al. 1997; Chen, Simon et al. 1999). Retinal light damage in mouse models
lacking arrestin or rhodopsin kinase can be prevented by removing rod transducin and
does not require the activation of AP-1(Hao, Wenzel et al. 2002). However, the
molecular pathways that trigger the photoreceptor apoptosis remain unclear. Constitutive
phototransduction activation is thought to be the cause of a subset of retinal disorders
(Table 1.2) (Rao, Cohen et al. 1994; Lisman and Fain 1995; Fain and Lisman 1999).
Elucidating the underlying molecular pathway also helps to identify similar mechanism
in these inherited human retinal dystrophies.

1.6 Endoplasmic Reticulum (ER) Stress in Retinal Degeneration
Recently, ER stress has been implicated in a wide variety of neurodegenerative diseases
such as Alzheimer disease, Huntington disease, Parkinson disease and amyotrophic
lateral sclerosis (Katayama, Imaizumi et al. 2004; Paschen and Mengesdorf 2005; Silva,
Ries et al. 2005; Turner and Atkin 2006), as well as retinal neurodegeneration conditions
such as diabetic retinopathy, retinitis pigmentosa (RP) and age-related macular
degeneration (AMD) (Rebello, Ramesar et al. 2004; Roybal, Yang et al. 2004; Lin,
Walter et al. 2008; Salminen, Kauppinen et al. 2010).
26

Figure 1.6 The three branches of the UPR. Three families of signal transducers (ATF6,
PERK, and IRE1) sense the protein-folding conditions in the ER lumen and transmit
that information, resulting in production of bZIP transcription regulators that enter the
nucleus to drive transcription of UPR target genes. Each pathway uses a different
mechanism of signal transduction: ATF6 by regulated proteolysis, PERK by
translational control, and IRE1 by nonconventional mRNA splicing. In addition to the
transcriptional responses that largely serve to increase the protein-folding capacity in
the ER, both PERK and IRE1 reduce the ER folding load by down-tuning translation
and degrading ER-bound mRNAs, respectively. ((Walter and Ron 2011))
27

ER is a cellular organelle responsible for folding and processing of membrane and
secretory proteins. It is also involved in intracellular calcium homeostasis and activation
of cell death signaling (Baumann and Walz 2001). The production of large amounts of
unfolded proteins exceeding the functional capacity of ER causes ER stress and activates
a collection of phylogenetically conserved signaling pathways collectively known as the
unfolded protein response (UPR) (Harding, Novoa et al. 2000; Travers, Patil et al. 2000;
Schroder and Kaufman 2005; Walter and Ron 2011). UPR activation increases ER
abundance by mediating expansion of the ER membrane, upregulating protein-folding
machinery and inhibition of protein translation to reestablish the hemostasis. Prolonged
activity of UPR, an indication that ER stress cannot be mitigated, is correlated with cell
apoptosis (Tabas and Ron 2011). There are three signaling arms distinguished by the
transmembrane proteins that are involved: IRE1 α, ATF6 α, and PERK (Fig.1.6) (Schroder
and Kaufman 2005). All three pathways are controlled by a key protein, GRP78/BiP, a
highly conserved member of the Hsp70 family chaperones. In normal conditions, the
bindings of GRP78 to these three ER stress sensors (IRE1 α, ATF6 α, and PERK) retain
these proteins in ER and quench them in inactive states. Upon accumulation of unfolded
proteins in the ER, GRP78 is competitively titrated from these three sensors which in turn
results in the activation of these proteins and initiates UPR.
Because neurons are highly susceptible to the toxic effects of misfolded proteins, ER
stress mediated cell apoptosis may have an important role in neuronal degeneration
diseases. Previous studies have shown that activation of ER stress has been observed in
28

some RP mouse models (Yang, Wu et al. 2007; Chiang, Messah et al. 2012; Kroeger,
Messah et al. 2012). Overexpression or induction of GRP78 levels was reported to
prevent these types of retinal degeneration (Inokuchi, Nakajima et al. 2009; Gorbatyuk,
Knox et al. 2010). However, the exact role of UPR in retinal apoptosis needs further
investigation.

1.7 Thesis Outline
In this these, my colleagues and I focused on the investigation of the pathological
molecular mechanisms involved in retinal degenerations caused by genetic and
environmental factors, and also the effects of retinal remodeling on retinal rescue
approaches.
In chapter 2, the study was focused on the mechanisms underlying cGMP-induced retinal
degeneration. We tested the roles of two cGMP targets in vivo: cyclic nucleotide gated
(CNG) channels and protein kinase G (PKG), by breeding two strains of PDE6 mutant
mice (Pde6g-/-and rd10) into the null background of either -CNG or PKG genes to
generate double mutant mice. Our results show that the genetic ablation of CNG channels
but not PKG significantly improved photoreceptor viability, thus implicating that CNG
channels play a key role in the cGMP-induced RP.
29

In chapter 3, we studied the mechanisms of light-induced retinal degeneration, by using
albino Balb/C and pigmented arrestin or rhodopsin kinase knockout (Arr-/- or RK-/-)
mice as models for bright light- and constitutive phototransduction-induced retinal
degeneration, respectively. We demonstrated that bright light-induced retinal
degeneration was triggered by ROS activation and PKA inhibition, whereas the activation
of unfolded protein response might be the common initiator of retinal degeneration
caused by constitutive phototransduction activation.
In chapter 4, we investigated the in vivo effects of retinal remodeling at different phases
on retinal rescue approaches. We generated a CNGB
-/-
mouse strain that is capable of
gene reactivation upon Cre-mediated recombination, and demonstrated that this strain is a
good model that recapitulates retinal degeneration patterns as seen in human retinitis
pigmentosa and allows for a systematic study of the potential of functional recovery after
neural remodeling events.

30

Chapter 2
Evaluation of Cellular Targets in the Induction of Photoreceptor Cell
Death by Elevated cGMP

2.1 Abstract
Several naturally occurring mutations of the phototransduction cascade proteins, such as
guanylate cyclase activating proteins (GCAPs) and phosphodiesterase 6 (PDE6), lead to
elevated cGMP levels prior to cell death. PDE6 mutations occur in humans and in several
mouse strains which in turn are commonly used as animal models for retinitis pigmentosa
(RP). The elevated [cGMP], due to reduced or abolished PDE activity, has different
potential cellular targets that could trigger cell death. We evaluated two of these targets:
protein kinase G (PKG) and the cGMP-gated (CNG) channels.
Two different strains of PDE mutant mice with different rates of retinal degeneration
were crossed with mice that either harbor the CNGb1 knockout or the Prkg1
tm1
knockout
genes. Retinal morphology was significantly improved when the PDE6 mutant Pde6g-/-
or the rd10 strain

was crossed into the CNGb1 null background despite elevated cGMP
levels. In contrast, no improvement in cell viability was detected in the Prkg1 null
background.
31

Our data suggest that elevated cGMP opens excessive numbers of CNG channels leading to
uncontrolled influx of Ca
2+
and Na
+
which subsequently initiates the cell death process.
Thus targeting rod CNG channels could be a promising approach to treat cGMP-induced
retinal degeneration.

2.2 Introduction
Retinitis pigmentosa (RP) is a set of hereditary retinal diseases characterized by early
symptoms of loss of night-time vision to loss of peripheral vision and eventually total
blindness. The clinical symptoms mirror initial rod cell death followed by cone cell death.
Approximately 8% of autosomal recessive RP cases have been linked to mutations in the
rod cGMP phosphodiesterase 6 (PDE6) enzyme (https://sph.uth.edu/retNet/), the key
effector enzyme in the phototransduction cascade. The tetrameric PDE6 holoenzyme
consists of the catalytic subunits and ß and two inhibitory subunits. The absence of
any of these subunits leads to the instability and degradation of the other subunits,
leading to a functional null phenotype. 
Multiple mouse strains harboring PDE6ß have been extensively used as animal models
for RP. These include rd1(Keeler 1924; Keeler 1966; Bowes, Li et al. 1990; Pittler and
Baehr 1991; Punzo and Cepko 2007) and Pde6g
-/-
(Tsang, Gouras et al. 1996), both of
which are functional null mutants with rapid rates of retinal degeneration that is evident
by postnatal day 14 (p14). The rd10 strain contains a R560C missense mutation and is a
32

hypomorph with a slower degeneration rate (Chang, Hawes et al. 2007). In the
photoreceptor cell, the cGMP concentrations are controlled by the dynamic equilibrium
of their synthesis by the basal activity of guanylyl cyclase (GC) and their hydrolysis by
PDE6. In the dark, these two processes are well-balanced and cGMP maintains ~3% of
the cyclic nucleotide-gated ion (CNG) channels in the open state to form the “dark
current” carried by influx of Na
+
and Ca
2+
. Upon light absorption, visual pigment
rhodopsin activates transducin (Tr), which in turns stimulates PDE6 activity to hydrolyze
cGMP. The decline of cGMP concentrations leads to the closure of CNG channels and
reduction of the dark current. Thus, loss or reduced function of PDE causes an abnormal
accumulation of cGMP within photoreceptors prior to onset of photoreceptor cell death
(Fain and Lisman 1999; Doonan, Donovan et al. 2005). However, the mechanism of how
elevated cGMP levels lead to photoreceptor death is still not fully understood.
One hypothesis of the cGMP-induced retinal degeneration is that the uncontrolled influx
of Ca
2+
through the CNG channels that are gated open by cGMP is toxic to
photoreceptors. However, attempts to prevent photoreceptor death using Ca
2+
channel
blockers such as D-cis-diltiazem, have shown controversial results (Frasson, Sahel et al.
1999; Pearce-Kelling, Aleman et al. 2001; Pawlyk, Li et al. 2002). Recent studies show
that the knockdown of GC and CNG channel by shRNA approaches improve the survival
and function of photoreceptors in PDE6
H620Q
mutant (Tosi, Davis et al. 2011; Tosi,
Sancho-Pelluz et al. 2011), but the effect is incomplete and temporary.
33

In addition to the CNG channel, another target of cGMP is protein kinase G (PKG).
There are three different isoforms of PKG: PKG1 , PKG1 , and PKG2(Hofmann, Feil et
al. 2006). PKG1 is abundantly expressed in the eye, whereas the PKG2 expression
appears to be very low(Gamm, Barthel et al. 2000). PKGs are 100-fold more sensitive
than CNG channels to cGMP (Lincoln and Cornwell 1993) and their excessive activation
has been shown to cause neuronal cell death (Canals, Casarejos et al. 2003; Canzoniero,
Adornetto et al. 2006). cGMP analogues that specifically inhibit PKG activity is shown
to have a rescue effect on the photoreceptor degeneration of rd1 mice both in vitro and in
vivo (Paquet-Durand, Hauck et al. 2009). However, these cGMP analogues also affect
CNG channels activity. Thus additional studies are necessary to demonstrate the
involvement of PKG activation in cGMP-induced retinal degeneration.
In this study, we bred two strains of mice bearing PDE6 mutations but with different rates
of retinal degeneration (Pde6g
-/-
and rd10) into the knockout background of either -CNG
or PKG genes to generate double mutant mice. These double mutant mice were examined
to test the involvement of CNG channels and/or PKG in cGMP-induced retinal
degeneration.

34

2.3 Materials and Methods
2.3.1 Animals
All experimental procedures were performed in accordance with regulations established
by the National Institutes of Health, as well as with Society for Neuroscience Policy on
Animal Use in Neuroscience Research. Two strains of mice bearing PDE6 mutations
(Pde6g-/-and rd10) were bred into the knockout background of either -CNG or PKG
genes to generate double mutant mice, termed as Cngb1-/-Pde6g-/-, Cngb1-/-rd10,
Prkg1-/-Pde6g-/-

and Prkg1-/-rd10.

2.3.2 Retinal Morphology
The eyes were enucleated, dissected and embedded into epoxy resin as described
previously (Concepcion, Mendez et al. 2002). The superior pole of the cornea was
cauterized for orientation before enucleation. The epon-embedded eyes were sectioned
along vertical meridian into 1 μm sections and stained with Richardson stain. Images
were acquired on an Axioplan2 microscope (Zeiss). The thickness of the outer nuclear
layer (ONL) was measured based on a previously described method (Chen, Shi et al.
2006). Briefly, retinal section was viewed by a microscope (40× objective) attached to a
camera lucida; and measurements were taken with the aid of a graphics tablet (WACOM,
Vancouver, WA) and the Axiovision LE Rel. 4.1. imaging software (Carl Zeiss Inc.). A
35

stage micrometer (Klarmann Rulings, Litchfield, NH) was used for calibration. Each
hemisphere - determined by the optic nerve - was divided into ten equal segments from
the optic nerve to either the superior or inferior tip, and three measurements were taken
and averaged for each segment. Due to the thinness of the outer nuclear layer at the optic
nerve location, determination of the ten equal segments for each hemisphere excluded the
first 100 µm from the optic nerve site.

2.3.3 cGMP ELISA
The retinas were dissected and immediately froze in liquid N
2
.
The frozen retinas were then homogenized in 150ul 6% trichloroacetic acid on ice
followed by 6 times extraction with 1000ml water-saturated ether. The aqueous extract
was dried in a vacuum centrifuge (UVS 400 Universal Vacuum system). The total cGMP
amount was determined by cGMP ELISA Biotrack system (GE healthcare #RPN226)
following manufacturer’s instructions.

2.3.4 Western Blot Analysis
The retina was dissected and homogenized in 150ul buffer (150mM NaCl, 50mM Tris
pH8.0, 0.1% NP-40, 0.5% deoxycholic acid) containing 0.1mM PMSF and Complete
36

mini protease inhibitor (Roche #11836153001). DNase I (30U, Roche) was added and
incubated at room temperature for 30min. The total protein amount of each sample was
determined by the BCA
TM
Protein Assay Kit (Thermo Scientific #23227). An equal
amount of retinal homogenate from each sample was electrophoresed on 4-12% Bis-Tris
SDS-PAGE Gel (Invitrogen) followed by transfer to nitrocellulose membrane (Whatman
#10402480) and incubated overnight at 4°C with the following primary antibodies: rabbit
anti-PDE polyclonal antibody (1:1000 Cytosignal PAB-06800), rabbit anti-ROS-GC1
polyclonal antibody (1:500 Santa Cruz, sc50512), mouse Anti-G
t
α-1 antibody (1:5000
EMD4Biosciences 371740 ), rabbit polyclonal anti-GCAP1 antibody (1:2000 generated
by our lab), rabbit polyclonal anti-GCAP2 antibody (1:1000 generated by our lab), mouse
anti-CNG  antibody (1:500 a generous gift from Dr. R. Molday), rabbit anti-phospho-
VASP (Ser239) antibody (1:500 Cell signaling #3114) and mouse anti-Actin antibody
(1:5000 Millipore MAB1501). The membranes were then incubated with fluorescently
labeled secondary antibodies (1:10,000 Li-Cor P/N926-31081) at room temperature for 1
hour and detected by Odyssey infrared imaging system. Densitometric scanning of each
band was followed by quantitative analysis using Odyssey 2.1 software.

37

2.4 Results
2.4.1 Ablation of CNG Channels Delayed PDE6 Mutations-induced Loss of
Phototransduction Proteins
To study the involvement of CNG channels and PKG in cGMP-induced retinal
degeneration, Cngb1
-/-
Pde6g
-/-
, Cngb1
-/-
rd10, Prkg1
-/-
Pde6g
-/-
and Prkg1
-/-
rd10 double
mutant mice were generated. We first confirmed the absence of PKG activity using VASP,
a specific substrate of PKG. As can be seen in Fig. 1, phospho-VASP was present in
control C57 and Pde6g
-/-
at postnatal day 14 (P14), but was absent in the age-matched
Prkg1
-/-
Pde6g
-/-
double mutant retinas, indicating the absence of PKG activity. Similarly,
no -CNG subunit was detected in the retinal homogenates from mice with CNG null
background.
Increased cGMP levels cause photoreceptor cell death in PDE6 mutant mice and manifest
in the decrease of phototransduction protein levels and reduce of outer nuclear layer
(ONL) thickness. To test the effects of the deletion of CNG channels or PKG on the
PDE6 mutants, we first assessed the levels of GC and PDE, enzymes directly responsible
for [cGMP] as well as transducin (Tr). As shown in Fig.1, protein levels of GC, PDE and
Tr were indistinguishable between Pde6g
-/-
and Prkg1
-/-
Pde6g
-/-
or between rd10 and
Prkg1
-/-
rd10. In contrast, these protein levels were noticeably higher in Cngb1-/-Pde6g-/-

and Cngb1-/-rd10 retinas when compared to the age-matched Pde6g-/-

and rd10 retinas,
38

indicating that the loss of these phototransduction proteins caused by PDE6 mutants was
alleviated in the absence of CNG channels but not PGK.
Figure 2.1 Protein levels in double mutant and control mice.
Right. Western blots of phototransduction proteins from age-matched (2 weeks old)
Cngb1-/-Pde6g-/-, Prkg1-/-Pde6g-/-, Cngb1-/-, Pde6g-/-and C57 retinas. Left.
Western blots of phototransduction proteins from age-matched (3 weeks old)
Cngb1-/-rd10, Prkg1-/-rd10, Cngb1-/-, rd10 and C57 retinas. Protein levels of p-
VASP and -CNG were detected to confirm the null background of PKG and CNG
channels, respectively. Protein levels of GC, PDE and Tr were examined to assess
the status of retinal degeneration.

39

2.4.2 Retinal degeneration caused by PDE6 Mutations was delayed in the CNG Null
Background
To further determine whether the ablation of CNG channels or PKG could prolong
photoreceptor cell survival in these two PDE6 mutant mice, retinal sections from age-
matched double mutant mice and control Pde6g-/- or rd10 mice were compared. As shown
in Fig 2 A, at two weeks the outer segment failed to develop and reduction of photoreceptor
layers was observed Pde6g-/-

mice, a result consistent with the loss of phototransduction
proteins seen in Fig 1; at three weeks only a single row of photoreceptor nuclei remained.
This time course and degree of retinal degeneration were indistinguishable to those of age-
matched Prkg1-/-Pde6g-/-

mice, indicating that PKG null background provided no
protection of photoreceptor cell death to the Pde6g-/-

mutant in vivo. In contrast, retinal
morphology was substantially improved in Cngb1-/-Pde6g-/-

mice: the rod outer
segments were much more elaborate and the number of surviving photoreceptor cell was
noticeably more when compared to those of age-matched Pde6g-/-mice at P14 and P21
(Fig. 2 A). The quantification of the ONL thickness across the entire span of the retina
further confirmed the remarkable rescue effects on photoreceptor numbers by CNG null
background (Fig. 2B). At P21, Cngb1-/-Pde6g-/-

mice still had about 40% photoreceptors
remaining, and the ONL was six times thicker compared to that of age-matched Pde6g-/-

mice.

40

A
B
Pde6g-/-
Cngb1-/-
Pde6g-/-
Prkg1-/-
Pde6g-/-
Cngb1-/-Pde6g-/-

Prkg1-/-Pde6g-/-

Pde6g-/-
2W
3W
ONL Thickness ( m) ONL Thickness ( m)
S
S
I
0
I
0
Retinal Region
Figure 2.2 CNG null background rescued retinal degeneration in Pde6g-/-mice.
A. Retinal morphology of age-matched Cngb1-/-Pde6g-/-, Prkg1-/-Pde6g-/-and
Pde6g-/-mice. Photoreceptor morphological structures were better preserved in
Cngb1-/-Pde6g-/-mice at the same age. B. Quantification of ONL thickness from
mice in A further confirmed the rescue effects of CNG null background on
photoreceptor numbers in Pde6g-/-retinas. S, superior ( −10); I, inferior (10); 0,
optic nerve position.

41

Cngb1-/-rd10 Prkg1-/-rd10
rd10

A
B
ONL Thickness ( m)
S 0 I
Retinal Region
Figure 2.3 CNG null background rescued retinal degeneration in rd10 mice.
A. Retinal morphology of 3 weeks-old Cngb1-/-rd10, Prkg1-/-rd10 and rd10 mice.
Photoreceptor morphological structures were better preserved in Cngb1-/-rd10 mice
at 3 weeks old. B. Quantification of ONL thickness from mice in A further
confirmed the rescue effects of CNG null background on photoreceptor numbers in
rd10 retinas. S, superior ( −10); I, inferior (10); 0, optic nerve position.

Cngb1-/-rd10

Prkg1-/-rd10

rd10

42

Cngb1-/-Pde6g-/-
Cngb1-/-rd10

rd10
Cngb1-/-rd10

B
wks
ONL Thickness ( m)
Figure 2.4 Long term rescue effects of CNG ablation on retinal degeneration
caused by PDE6 mutants. A. Retinal morphology of Cngb1-/-Pde6g-/-and Cngb1-/-
rd10 mice at 4 weeks, 6 weeks and 8 weeks old. B. ONL thickness in the central
portions of Cngb1-/-Pde6g-/-, Pde6g-/-, Cngb1-/-rd10 and rd10 retinas show the
rescues on photoreceptor viability of CNG null background at different ages.

A
Pde6g-/-

Cngb1-/-Pde6g-/-

ONL Thickness ( m)
43

Cngb1-/-Pde6g-/-
Cngb1-/-
Pde6g-/-

Cngb1-/-rd10
Cngb1-/-
ONL Thickness ( m)
Retinal Region
Retinal Region
S
S
0
I
I
4W
0
Figure 2.5 The rescue of CNG ablation on PDE6 mutant-induced retinal
degeneration was incomplete. Quantification of ONL thickness of Cngb1-/-Pde6g-/-
, Cngb1-/-rd10 and age-matched Cngb1-/- and Pde6g-/-mice. When comparing to
Cngb1-/- mice at 4 weeks, there were more photoreceptor loss in Cngb1-/-Pde6g-/-
and Cngb1-/-rd10 retinas. S, superior ( −10); I, inferior (10); 0, optic nerve position.

44

The rd10 mice have a slower rate of degeneration than Pde6g-/-. At three weeks of age
about half of the photoreceptor cells remain, and the outer segment is short but present (Fig.
3). Morphometric measurements across the entire span of the retina show that the
degeneration appears to be more severe in the central portion than the peripheral portion of
the retina (Fig. 3 B). Consistent with the results observed in Pde6g-/- mice, the
progression of retinal degeneration in Prkg1-/-rd10 mice was similar to that of rd10 mice
at P21 (Fig. 3). The retinal structure of Cngb1-/-rd10 mice, however, was better preserved:
the ONL was 20% thicker compared to that of rd10and Prkg1-/-rd10 retinas and the degree
of degeneration was similar in the central and peripheral portions of the retina (Fig. 3 B).
Thus CNG, but not PKG, appears to be the cellular target of elevated cGMP-induced
retinal degeneration.

2.4.3 Long Term Rescue of Photoreceptor Viability of CNG Null Background on PDE6
Mutations in vivo
To further study the rescue effects of CNG null background on these two PDE6 mutant
mice, we examined the retinal morphology of the Cngb1-/-Pde6g-/-

and Cngb1-/-rd10
double mutant mice at older ages (Fig 4). As seen in Fig. 2, most photoreceptor cells are
lost by 3 weeks in Pde6g-/-

retinas. This stage is reached by 4 weeks in the rd10 retinas
(Barhoum et al., 2008). Remarkably, at 4 weeks, there were still 30% of photoreceptors
remaining in Cngb1-/-Pde6g-/-

mice and 70% of photoreceptors remaining in Cngb1-/-
45

rd10 mice assessed by the thickness of ONL. At 8 weeks, the percentages of the survival
photoreceptors were reduced to about 10% in Cngb1-/-Pde6g-/-mice and to 40% in
Cngb1-/-rd10 mice, respectively. Quantification of the ONL thickness in the central
portions of retinas further confirm the rescue on photoreceptor viability of CNG null
background on PDE6 mutant mice at different ages (Fig. 4 B).
Although retinal structures were remarkably improved in Cngb1-/-Pde6g-/-

mice compared
to age-matched Pde6g-/-

mice, the rescue of retinal degeneration of CNG ablation was
not complete (Fig. 5). When comparing to Cngb1-/- mice at 4 weeks, there were more
photoreceptor loss in Cngb1-/-Pde6g-/ -mice. Better rescue effects were observed in
Cngb1-/-rd10 mice, but there was still more severe retinal degeneration in the central
portion of the retina observed compared to that of Cngb1-/- mice at 4 weeks.
Collectively, the rescue of ablation of CNG channels on PDE6 mutant-induced retinal
degeneration was significant and long term, but still incomplete.

2.4.4 High cGMP Concentrations in Double Mutant Mice
Cyclic GMP concentrations in whole retinas in all these mouse strains were quantified by
ELISA (Table 1). As expected, cGMP concentrations of Pde6g-/-

and rd1 retinas were
higher than those of age-matched C57 control retinas, confirming the reduced PDE6
activity in these PDE6 mutant mice. cGMP concentrations of Prkg1-/-Pde6g-/-retinas
were at similar levels as those of age-matched Pde6g-/- retinas. In contrast, cGMP
46

concentrations in Cngb1-/-Pde6g-/- retinas were much higher, about 3-fold of that of age-
matched Pde6g-/-and 4-fold of that of C57 controls. This may be due to the relative
higher GC activities stimulated by the extremely low [Ca
2+
]

in the absence of CNG
channels in the Cngb1-/-Pde6g-/- retinas.
Taken together, although the cGMP concentrations were still at pathological high levels
in CNG null background mice, the loss of phototransduction proteins and retinal
degeneration caused by PDE6 mutants were significantly delayed, implying that the
elevated cGMP is tolerated in the absence of CNG. These data strongly support CNG as
the downstream target of elevated cGMP-induced retinal degeneration. In contrast, no
protective effect was observed in PKG null background, indicating that PKG is not
involved in the cell death signaling initiated by cGMP.
Table 2.1 cGMP concentrations in double mutant and control mice.

Mouse Strains
cGMP
pm
Protein
mg
cGMP/Protein
pm/mg
C57 2w 7.03 0.2 35.15
Rd1 12d 8.362348 0.13 64.32575
Pde6g-/- 2w 4.68 0.1 46.8
Prkg1-/-Pde6g-/- 2w 3.13 0.1 31.3
Cngb1-/-Pde6g-/- 2w 23.9079 0.2 119.5395
Cngb1-/-Pde6g-/- 3w 26.69076 0.2 133.4538
47

2.5 Discussion
Mouse strains harboring PDE6 mutants have been extensively used as models for retinitis
pigmentosa. The abolished or reduced activity of PDE6 result in the abnormal elevation
in cytosolic cGMP levels, which in turn promotes photoreceptor cell death (Doonan et al.,
2005; Fain & Lisman, 1999).
Our results show the importance of CNG channels in cGMP-induced retinal degeneration.
These observations were consistent with previous reports using shRNA knockdown of
Cnga1 to improve photoreceptor survival in PDE6
H620Q
mutant mice (Tosi, Davis, et al.,
2011; Tosi, Sancho-Pelluz, et al., 2011), and were also in agreement with results on
improved photoreceptor survival by treatment of different types of calcium channel
blockers (Takano et al., 2004). The rescue effects of the ablation of CNG channels were
apparent in both Pde6g-/-

and rd10 mice, suggesting that the CNG channels were the
common effectors in cGMP-induced retinal degeneration. In contrast, the ablation of
PKG did not show any protective effects in these two PDE6 mutants in vivo, which was
contradicted with the previous reports that the inhibition of PKG activity by cGMP
analogues has a rescue effect on the photoreceptor degeneration of rd1 and rd2 mice in
vivo (Paquet-Durand et al., 2009). The reason could be that the PKG inhibitor Rp-8-Br-
PET-cGMPS (a cGMP analogue) used in that study also inhibits CNG channels activity
(Wei, Cohen, Yan, Genieser, & Barnstable, 1996), and the inhibition of CNG channels
could be the real effector of rescue effects on retinal degeneration in rd mice of that study.
48

This possibility was confirmed in the present study by the respective deletion of the CNG
and PKG genes. Additionally, the PKG activator 8-pCPT-PET-cGMP is a potent blocker
of CNG channels (Wei, Cohen, Genieser, & Barnstable, 1998). The deletion of CNG
gene causes retinal degeneration although with a later onset and slower rate compared to
PDE6 single mutants (Huttl et al., 2005). Thus the block of CNG channels in wildtype
animals could lead to photoreceptor death by mechanisms that are not related to PDE6
activity. Our data clearly exclude the involvement of PKG pathway in cGMP-induced retinal
degeneration in vivo and supported that cGMP toxicity is not directly responsible for early
initiation of the cell-death pathway. Rather, the involvement of CNG channels and possibly the
uncontrolled Ca
2+
/Na
+
cation entry are needed to produce the degeneration phenotype. Since
cGMP and Ca
2+
may be common initiators triggering cell death in other neurodegenerative
disease, this thorough investigation may significantly advance our understanding of
pathological processes of these diseases.
Are there other effectors in cGMP-induced retinal degeneration? Although the removal of
CNG channels in PDE6 mutants remarkably improved the retinal degeneration phenotype,
the rescue effect was incomplete. There were still more photoreceptor losses comparing
to that of age-matched Cngb1-/- mice, indicating the involvement of other pathways.
Additionally, in the course of crossing mouse lines for these experiments, we identified a
subset of mice with much milder PDE6-associated retinal degeneration progression
independent of CNG or PKG genotypes. These mice are being examined to identify
49

additional genetic factors. The existence of other factors clearly indicates the complexity
of the mechanisms underlying cGMP-induced retinal degeneration.
Our data show that the genetic ablation of CNG channels in Pde6g-/-and rd10 mice
significantly rescued retinal degeneration phenotype despite elevated cGMP levels.
Combined with our results showing that the ablation of PKG did not have any protective
effects in these two PDE6 mutant mice in vivo, our data strongly supported the
hypothesis that uncontrolled Ca
2+
/Na
+
cation entry instead of cGMP toxicity is directly
responsible for the early initiation of photoreceptor cell death. Because both RP and AMD are
characterized by an initial loss of rod photoreceptors and reduced PDE6 function is a
common cause in many cases of photoreceptor degenerations, targeting rod CNG
channels may be a promising approach to treat cGMP-induced retinal degeneration and
humans with photoreceptor diseases.
50

Chapter 3
Two Different Pathways of Light-induced Retinal Degeneration

3.1 Abstract
Excessive light exposure is an environmental factor that modulates the rate of retinal
degeneration in inherited retinopathies and age-related macular degeneration. Previous
studies show that light damage to the retina occurs through at least two distinct pathways:
1) bright light exposure that kills photoreceptor cells in a transducin-independent manner
accompanied by AP-1 induction; and 2) retinal degeneration induced by low light
exposure in instances where the absence of arrestin or rhodopsin kinase leads to
constitutive activation of the phototransduction cascade does not require AP-1. In order
to better understand the underlying mechanisms, we investigated the molecular events
that occur in these two pathways of light damage using albino Balb/C and pigmented
arrestin or rhodopsin kinase knockout (Arr-/- or RK-/-) mice as models for bright light-
and constitutive phototransduction-induced retinal degeneration, respectively. Our study
demonstrated that, the ER stress sensors including glucose-regulated protein-78 (GRP78),
phosphor-eukaryotic initiation factor 2 (p-eIF2 ), activating transcription factor 3
(ATF3) and activating transcription factor 4 (ATF4) were significantly upregulated in
constitutive phototransduction- but not in bright light-induced retinal damage. The
upregulation of these proteins coinciding with the increased general ubiquitination
51

preceded photoreceptor apoptosis. Thus the unfolded protein response is possibly the
initiator of retinal degeneration induced by constitutive phototransduction activation and
could be a strong candidate in the treatment of retinal degenerative diseases.

3.2 Introduction
Exposure to excessive light exacerbates the rate of photoreceptor apoptosis in several
retinal diseases and has been used as a model for the study of retinal degeneration (Noell
1980; Sanyal, De Ruiter et al. 1980; Chen, Burns et al. 1999; Chen, Simon et al. 1999;
Wenzel, Grimm et al. 2005). Two different mechanisms have been implicated in light
damage depending on the light intensities applied: bright light induced acute damage
depends on rhodopsin regeneration and transcriptional activator AP-1 activation but is
independent of phototransduction (Hafezi, Steinbach et al. 1997; Grimm, Wenzel et al.
2000; Wenzel, Grimm et al. 2000; Wenzel, Reme et al. 2001); in contrast, damage can
also occurs from constitutive phototransduction but not AP-1 activation (Hao, Wenzel et
al. 2002).
At higher light intensities, intracellular Ca
2+
levels increase (Donovan, Carmody et al.
2001) and reactive oxygen species (ROS) are generated by photon-excited rhodopsin
(Yang, Basinger et al. 2003) as early events of retinal damage. Application of the calcium
channel blockers (Donovan and Cotter 2002) and antioxidants (Specht, Organisciak et al.
2000; Tanito, Masutani et al. 2002; Tanito, Nishiyama et al. 2002) prevented the
52

photoreceptor apoptosis, confirming the role of Ca
2+
and ROS as initiators of bright light-
induced photoreceptor degeneration. Neuronal nitric-oxide synthase (nNOS) and GC are
the possible downstream effectors of ROS signaling (Donovan, Carmody et al. 2001).
Finally, AP-1 is activated leading to full activation of the apoptotic process (Hafezi,
Steinbach et al. 1997).
On the other hand, constitutive phototransduction activation is thought to be the cause of
certain forms of retinal disorders (Rao, Cohen et al. 1994; Lisman and Fain 1995; Fain
and Lisman 1999), and can result from low light exposure when arrestin or rhodopsin
kinase, protein necessary for deactivating rhodopsin, is defective (Xu, Dodd et al. 1997;
Chen, Simon et al. 1999). Retinal light damage in mouse models lacking arrestin or
rhodopsin kinase can be prevented by removing rod transducin (Hao, Wenzel et al. 2002).
The underlying molecular pathways, however, remain unclear.
Recently, endoplasmic reticulum (ER) stress has been implicated in a wide variety of
neurodegenerative diseases such as Alzheimer disease, Huntington disease, Parkinson
disease and amyotrophic lateral sclerosis (Katayama, Imaizumi et al. 2004; Paschen and
Mengesdorf 2005; Silva, Ries et al. 2005; Turner and Atkin 2006), as well as retinal
neurodegeneration conditions such as diabetic retinopathy, retinitis pigmentosa (RP) and
age-related macular degeneration (AMD) (Rebello, Ramesar et al. 2004; Roybal, Yang et
al. 2004; Lin, Walter et al. 2008; Salminen, Kauppinen et al. 2010). ER is a cellular
organelle involved in folding and processing of membrane and secretory proteins. It is
also involved in intracellular calcium homeostasis and activation of cell death signaling
53

(Baumann and Walz 2001). The production of large amounts of unfolded proteins in the
lumen of ER causes ER stress and activates a set of phylogenetically conserved responses
collectively known as the unfolded protein response (UPR) (Harding, Novoa et al. 2000;
Travers, Patil et al. 2000; Schroder and Kaufman 2005). There are three signaling arms
distinguished by the transmembrane proteins that sense the prolonged protein folding
stress in the ER: activating transcription factor 6 (ATF6), the inositol requiring kinase 1
(IRE1), and double-stranded RNA-activated protein kinase (PERK) (Schroder and
Kaufman 2005). All three pathways are controlled by a key protein, GRP78/BiP, a highly
conserved member of the Hsp70 family chaperones. GRP78 binds to each of the ER
stress sensors (IRE1 α, ATF6 α, and PERK) and initiates UPR when the amounts of
misfolded proteins exceed the functional capacity of ER. Previous studies have shown
that activation of ER stress has been observed in some RP mouse models (Yang, Wu et al.
2007; Chiang, Messah et al. 2012; Kroeger, Messah et al. 2012). Overexpression or
induction of GRP78 protein levels was also reported to prevent these types of retinal
degeneration (Inokuchi, Nakajima et al. 2009; Gorbatyuk, Knox et al. 2010).
In this study, we investigated whether UPR is involved in two different light induced
retinal degenerations in vivo by exposing Balb/C and pigmented arrestin or rhodopsin
kinase knockout (Arr-/- or RK-/-) mice to 5000 lux white light. In addition, we examined
which pathway of UPR is activated during this process.

54

3.3 Materials and Methods
3.3.1 Light Exposure
All experimental procedures were performed in accordance with regulations established
by the National Institutes of Health, as well as with Society for Neuroscience Policy on
Animal Use in Neuroscience Research. Arr-/-, RK-/-, Balb/C and Arr-/-Tr-/- mice were
born and reared in darkness to avoid light-dependent retinal degeneration. C57Bl/6 mice
were reared in 12hr/12hr light/dark cycle and moved to darkness 1 week before the light
exposure.
Experiments were performed with 4 week-old mice, at which age no retinal
degenerations were observed in dark reared Arr-/-, RK-/-, Balb/C and Arr-/-Tr-/- mice.
Mice were exposed to diffuse cool white fluorescent light at luminescence level of 5000
lux without pupillary dilation, and then sacrificed at the following time points: 3 hr, 4 hr,
5 hr, 12 hr and 36 hr after light onset and after 36 hr of darkness that followed 12 hr light
exposure.

3.3.2 Retinal Morphology
The eye was enucleated, dissected and embedded into epoxy resin as described
previously (Concepcion, Mendez et al. 2002). The superior pole of the cornea was
cauterized for orientation before enucleation. The epon-embedded eyes were sectioned
55

along vertical meridian into 1 μm sections and stained with Richardson stain. Images
were acquired on an Axioplan2 microscope (Zeiss, 60× objective).

3.3.3 Western Blot Analysis
The retina was dissected and homogenized in 150ul buffer (150mM NaCl, 50mM Tris
pH8.0, 0.1% NP-40, 0.5% deoxycholic acid) containing 0.1mM PMSF, Complete mini
protease inhibitor (Roche #11836153001) and 50 M sodium fluoride (NaF). DNase I
(30U, Roche) was then added and incubated at room temperature for 30min. The total
protein amount of each sample was determined by the BCA
TM
Protein Assay Kit
(Thermo Scientific #23227). An equal amount of retinal homogenate from each sample
was electrophoresed on 4-12% Bis-Tris SDS-PAGE Gel (Invitrogen) followed by transfer
to nitrocellulose membrane (Whatman #10402480) and incubated overnight with the
following primary antibodies: rabbit anti-PDE polyclonal antibody (1:1000 Cytosignal
PAB-06800), rabbit anti-ROS-GC1 polyclonal antibody (1:500 Santa Cruz, sc50512),
mouse Anti-G
t
α-1 antibody (1:5000 EMD4Biosciences 371740 ), rabbit polyclonal anti-
GCAP1 antibody (1:2000 generated by our lab), rabbit polyclonal anti-GCAP2 antibody
(1:1000 generated by our lab), mouse anti-nNOS monoclonal antibody (1:1000 BD
#610308 ), mouse anti-ubiquitin monoclonal antibody (1:1000 Sigma U0508), mouse
anti-GRP78/Bip monoclonal antibody (1:1000 BD #610978), rabbit anti-phospho-
eIF2 (Ser51) polyclonal antibody (1:1000 Cell signaling #9721), rabbit anti-ATF3
56

antibody (1:1000 Sigma Aldrich HPA001562), rabbit anti-CREB-2 antibody (1:500 Santa
Cruz, sc200), rabbit anti-ATF6  antibody (1:500 Santa Cruz, sc22799), rabbit anti-
IRE1  antibody (1:1000 Cell signaling #3294) and mouse anti-Actin antibody (1:5000
Millipore MAB1501). The membranes were then incubated with fluorescently labeled
secondary antibodies (1:10,000 Li-Cor P/N926-31081) at room temperature for 1 hour
and detected by Odyssey infrared imaging system. Densitometric scanning of each band
was followed by quantitative analysis using Odyssey 2.1 software.
The proteasome binding proteins were isolated by Proteasome Isolation Kit (Calbiochem
#539176) following the manufactory protocols and detected by the western blot analysis
as described above.

3.3.4 cGMP and cAMP ELISA
The retinas were dissected and immediately froze in liquid N
2
. The frozen retinas were
then homogenized in 150ul 6% trichloroacetic acid on ice followed by 6 times extraction
with 1000ml water-saturated ether. The aqueous extract was dried in a vacuum centrifuge
(UVS 400 Universal Vacuum system). The total cGMP and cAMP amount were
determined by cGMP ELISA Biotrack system (GE healthcare #RPN226) and cAMP
ELISA Biotrack system (GE healthcare #RPN225), respectively.

57

3.4 Results
3.4.1 Detection of Photoreceptor Apoptosis in Light-exposed Arr-/- and Balb/C Mice
To access the process of retinal degeneration at different light intensities, we exposed
Arr-/-, C57 and Balb/C mice to cool white fluorescent light at luminescence level of 5000
lux without papillary dilation. The eye pigmentation in Arr-/- and C57 mice reduced the
amount of light reaching the retina by two orders of magnitude relative to that in albino
Balb/C mice, giving an effective intensity of about 50 lux (Hao, Wenzel et al. 2002).
In Arr-/- mice, pyknotic nuclei were observed as the first sign of apoptosis after 12 hr of
light exposure (Fig.3.1). The photoreceptor cell apoptosis became more evident after 36
hr of light exposure: thinning and disorganization of outer nuclear layer (ONL) and
membrane disruption of outer and inner segments were apparent. After 36 hr of dark
recovery, significant photoreceptor cell loss was observed as the reduction of the ONL
thickness, further confirming 12 hr light exposure was sufficient to induce the
photoreceptor cell apoptosis in Arr-/- mice. In Balb/C mice, after 12 hr of light exposure,
decrease of photoreceptor cell numbers and vesiculated outer and inner segment were
detected. Although retinal damage seemed to occur faster, the degrees of retinal
degeneration after 36 hr of light exposure or dark recovery were similar to those of Arr-/-
mice. Such damages were absent in C57 mice.

58

Control 12h   36h 12h+36h Dark
Fig.3.1. Retinal morphology of light exposed Arr-/-, Balb/C and C57 mice. 4
weeks-old Arr-/-, Balb/C and C57 mice were exposed to 5000 lux white light for
12 hours or 36 hours. Some mice were dark adapted for 36 hours after 12 hours
of light exposure (Dark Recovery). Pyknotic nuclei were observed in 12 hours
of low light exposed Arr-/- retinas. After 36 hours of light exposure, ONL was
disorganized and numerous apoptotic cells can be seen. After dark recovery,
ONL became aligned again but was thinner than that of unexposed retina
(control). Retinas from Balb/C mice show similar degenerative changes. No
changes in retinal morphology were detected in C57 Mice.
59

Fig.3.2. Distinct degradation profiles of phototransduction proteins in light
exposed Arr-/, Balb/C and C57 mice.
Relative Density to Control
60

Collectively, though exposed to different effective light intensities, photoreceptor
apoptosis started by 12 hr light exposure in Arr-/- and Blab/C mice and progressed with
comparable degrees of retinal damages for longer periods.

3.4.2 Distinct Profiles of Phototransduction Protein Degradation in Two Light-induced
Retinal Degenerations
Low light-induced retinal degeneration is phototransduction cascade dependent, but not
bright light-induced retinal damage (Hao, Wenzel et al. 2002). To determine the roles of
phototransduction proteins in the initiation of the photoreceptor apoptosis, western blots
were used to examine the protein levels after different light exposure periods. In Arr-/-
mice, PDE and GCAP2 levels were more sensitive to light exposure comparing to the
other phototransduction proteins. Their amounts decreased to about 50% after 12 hr of
light exposure (Fig. 3.2), preceding the massive photoreceptor cell apoptosis, and
continued to reduce to about 30% after 36 hr of light exposure. GC, GCAP1 and Tr
protein levels did not change for the first 12 hr, but decreased to about 40% after 36 hr of
light exposure. All these five protein did not recover to original levels after 36 hr of dark
recovery, probably due to the decrease of photoreceptor cell numbers.
In contrast, all these five phototransduction protein levels reduced to similar degrees at 12
hr and 36 hr of light exposure or dark recovery in Balb/C mice, consistent with the loss of
61

photoreceptor cells. Collectively, distinct phototransduction protein degradation profiles
were observed in the initiation phase of constitutive phototransduction- but not bright
light-induced retinal degeneration.
In light exposed C57 retinas, slightly decrease of PDE and GCAP2 levels were observed
after 12 and 36 hr of light exposure. PDE and GC levels were found to reduce after 36 hr
of dark recovery. All these changes were less severe comparing to those in the Arr-/- or
Balb/C mice and might due to the self-protective adaptation to continued light exposed
condition.

3.4.3 Increased Ubiquitination Was an Early Event in Constitutive Phototransduction-
induced Retinal Degeneration
Impairment of ubiquitin-proteasome system (UPS) has been implicated in many types of
retinal degeneration (Illing, Rajan et al. 2002; Saliba, Munro et al. 2002). To analyze the
involvement of UPS, the global ubiquitination levels and proteasome-associated proteins
were examined by western blots (Fig.3.3). There was about two-fold increase of global
ubiquitination in Arr-/- mice after 12 hr of light exposure, coincident with the degradation
of phototransduction proteins in the early phase. This elevation reduced back to the
control level after 36 hr of light exposure. In contrast, no changes in ubiquitination were
observed in light exposed Balb/C mice.
62

0
0.5
1
1.5
2
2.5
Arr C12h 36h DR
Ubi
  Arr Balb/C
C   12h   36h   DR C   12h   36h DR
A
PDE
  Arr Balb/C
C 12h 36h   DR   C   12h 36h   DR
Fig.3.3. Overload of UPS in the early phase of constitutive phototransduction
induced retinal degeneration. (A) Light exposure leads to global increase in
levels of ubiquitinated proteins in Arr-/- retinas but not in Balb/C retinas at
12hr of light exposure. (B) Proteasomes were isolated in light-exposed Arr-/-
and Blab/C retinas, PDE and GCAP2 was found to bind to proteasome after
12hr of light exposure in Arr-/- mice.
B
GCAP2
C   12h   36h   DR
Arr
Balb/C
Relative Density to Control
63

We also checked whether phototransduction proteins from Fig. 3.2 were targeted to the
proteasome and found that PDE and GCAP2 bound to the proteasome at 12 hr light
exposed retinas from Arr-/- mice, confirming their degradations were through UPS. The
other phototransduction proteins were not detected to bind to the proteasome (Data not
shown). Taken together, the degradation of large amount of proteins was selectively via
UPS and possibly overloaded the system in the initiation phase of constitutive
phototransduction-induced retinal degeneration.

3.4.4 ER Stress Activation in Constitutive Phototransduction-induced Retinal
Degeneration
Overloaded UPS and increased misfolded protein in ER lumen can activate ER stress and
lead to cell apoptosis (Yang, Wu et al. 2008; Gorbatyuk, Knox et al. 2010). To examine
this possibility, three major arms of ER stress markers were analyzed by western blot.
These include PERK, IRE1  and ATF6 and GRP78 that binds and regulates their activity.
In Arr-/- mice (Fig. 3.4), the expressions of GRP78 and proteins downstream from PERK
(p-eIF2, ATF3 and ATF4) were markedly up-regulated at 12 hr of light exposure and
returned back to control level after 36 hr of light exposure or dark recovery, coincident
with the time-dependent manner of ubiquitination, suggesting that their activations
occurred exclusively at the early phase of constitutive phototransduction-induced retinal
degeneration.
64

0
1
2
3
4
5
6
7
C12h 36h DR
GRP78
ATF3
ATF4
p‐eIF2a
ATF6
IRE1a
Arr
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
C12h 36h DR
Balb/C

Fig.3.4. Activation of UPR in the early phase of constitutive phototransduction
induced retinal degeneration. GRP78, p-eIF2 , ATF3 and ATF4 were up-
regulated after 12hr of light exposure in Arr-/- retinas. Only ATF4 is mildly
increased in Balb/C retinas.
Arr   Balb/C
C 12h   36h   DR C 12h   36h   DR
Relative Density to Control
65

0
2
4
6
8
10
12
Control 3h4h5h
Arr
GRP78
ATF3
ATF4
p‐eIF2a
ATF6
IRE1a
0
0.5
1
1.5
1234
Balb/C

Balb/C
C   3h 4h 5h
  Arr
C   3h   4h   5h
Fig. 3.5. Early up-regulation of ER stress sensors and increase of protein
ubiquitination in light exposed Arr-/- retinas. Up-regulations of ATF3, ATF4 and
p- eIF2  were early events in the unfolded protein response (UPR).

Relative Density to Control
66

The level of IRE1  increased slightly at 36 hr of light exposure, indicating a later
activation. ATF6  expressions did not change during the entire period. In contrast, in
Balb/C mice, only ATF4 protein levels show a mild increase at 12 hr of light exposure.
To further refine the time course of ER stress activation, we exposed Arr-/- and Balb/C
mice for shorter time periods (Fig. 3.5). p-eIF2 , ATF3 and ATF4 protein levels were
upregulated for more than 2 folds in Arr-/- mice after 3 hr of light exposure , and the
upregulations continued after 4 and 5 hr of light exposure. The global ubiquitination also
increased similarly, while the IRE1  and ATF6 expressions did not change at the
same periods. No changes in these ER stress markers or ubiquitination were detected in
Blab/C retinas.
Collectively, the activation of UPR via GRP78/PERK pathway by overloaded UPS
played an important role in the initiation of photoreceptor cell death of constitutive
phototransduction- but not bright light-induced retinal degeneration.

3.4.5 Early Activation of ER Stress in Low Light Exposed RK-/- Retinas
RK-/- mice were reported to show similar low light induced retinal degeneration as Arr-/-
mice previously. To demonstrate the ER stress activation is the common mechanism for
constitutive phototransduction-induced retinal degeneration, we exposed RK-/- mice
under the same conditions as described for Arr-/- mice.
67

ATF3
ATF4
Ubi
Actin
C   12h   36h DR
GRP78
IRE1 
ATF6
p‐eIF2 
C   3h 4h   5h
Fig. 3.6. Early up-regulation of ER stress sensors and increase of protein
ubiquitination in light exposed RK-/- retinas.
68

Similar to the results obtained from Arr-/- mice, p-eIF2 , ATF3 and ATF4 levels were
markedly upregulated after 3 hr of light exposure (Fig.3.6), and continued to increase up
to 12 hr and declined by 36 hr of light exposure. These time-dependent changes were also
observed for global ubiquitination. GRP78 expression was elevated at 12 hr same as in
Arr-/- retinas. No changes were observed for IRE1  or ATF6 levels.
These results, in combination with our results showing the early activation of UPR in
Arr-/- mice, suggested that the UPR was the common initiator for constitutive
phototransduction-induced retinal degeneration.

3.4.6 Transducin Knockout Prevented Constitutive Phototransduction-induced Retinal
Degeneration
To further clarify the role of UPR in constitutive phototransduction-induced retinal
degeneration, we studied the effects of rod transducin null background on constitutive
phototransduction-induced UPR activation. The absence of rod transducin has been
shown previously to block the constitutive phototransduction-induced retinal degradation
in Arr-/- mice, thus we hypothesize UPR would not be activated in light exposed Arr-/-
Tr-/- retinas. Analysis of retinal sections (Fid.3.7) confirmed that no photoreceptor
apoptosis was observed in Arr-/-Tr-/- mice after same periods of light exposure.

69

Ubi
0
1
Conrol 12h 36h DR
C   12h 36h   DR
C   12h 36h DR
Proteasome
pull down
PDE
GRP78
p‐eIF2 
Fig.3.7. Transducin null background prevented constitutive phototransduction
induced retinal degeneration. (A) Retinal morphology of Arr-/-Tr-/- mice was
unchanged after light exposure. (B) Phototransduction proteins levels and (C)
ubiquitinated protein levels in Arr-/- and Arr-/-Tr-/- retinas after light exposure. (D)
PDE was not associated with proteasome and GRP78 and p-eIF2  were not up-
regulated in Arr-/-Tr-/- retinas after light exposure.
C
C 12h 36h DR
A
C   12h 36h DR
0
0.2
0.4
0.6
0.8
1
1.2
1.4
control 0.5d 1.5d DR
B
D
Relative Density to control
C   12h   36h   DR
70

There were no increase in global ubiquitination and proteasome associated proteins, nor
any decrease of phototransduction protein levels. No changes of GRP78 or p-eIF2 
levels were detected. Collectively, the absence of transducin blocked the massive
degradations of downstream proteins caused by continuous phototransduction activation
and prevented UPR activation and retinal degeneration.

3.5 Discussion
Our results provide evidence that at least two separate molecular pathways can be
initiated by light in photoreceptor cells. In bright light, retinal degeneration was triggered
by ROS activation and PKA inhibition (Supplementary Fig 3.8). The nNOS levels
increased progressively during the light exposure period in Balb/C mice, consistent with
previous reports (Donovan, Carmody et al. 2001; Wenzel, Grimm et al. 2005), but
showed little difference in Arr-/- mice at the same time points. Significant decrease of
cAMP concentrations was also observed in Balb/C mice at 12 hr of light exposure. PKA
activation is anti-apoptotic (Racz, Gallyas et al. 2007) and inhibits pro-apoptotic
signaling molecules such as JNK1/2 and p38MARK. The inhibition of PKA activates c-
Jun/c-Fos signal that is required for bright light-induced retinal degeneration (Krishnan,
Lee et al. 2008). This early decrease in cAMP concentrations observed in our study
confirmed the inhibition of PKA pathway in the initial phase of bright light-induced
photoreceptor apoptosis.

71

nNOS
  Arr Balb/C
C   12h 36h DR   C   12h   36h DR
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0
0.2
0.4
0.6
0.8
1
1.2
control 0.5d 1.5d DR
C 12h   36h   DR

cGMP concentration pmol/ug cAMP concentration pmol/ug
Arr‐/‐
C57
Balb/C
Arr‐/‐
C57
Balb/C
A
B
Supplementary Fig 3.8. Involvement of ROS and PKA signaling in bright
light induced retinal degeneration. (A) nNOS expressions were upregulated
in light exposed Balb/C but not Arr-/- mice. (B). cAMP concentrations
decreased in Balb/C mice but increased in Arr-/- mice after light exposure.
cGMP concentrations reduced more significant in light exposed Arr-/-
mice.
72

Whereas cAMP concentrations were increased in Arr-/- mice for all the three time points
studied, suggesting that PKA might function as a protective pathway in constitutive
phototransduction-induced retinal degeneration.
In contrast, the ER stress markers, including GRP78, p-eIF2 , ATF3 and ATF4 were
markedly up-regulated in the first 12 hours in both Arr-/- and RK-/- mice. These
upregulations coincided with the increase of global ubiquitination that preceded
photoreceptor apoptosis, suggesting the activation of UPR might be the common initiator
of constitutive phototransduction-induced retinal degeneration.
Distinct decrease of PDE and GCAP2 protein levels were observed in the 12 hr light-
exposed Arr retinas and proteasome-bond PDE and GCAP2 were also detected
exclusively at this time point. Constitutive phototransduction activation leads to high
percentage of total transducin binding to PDE  subunits, leaving PDE  subunits in
continued unstable states, and also results in low cytosolic Ca
2+
concentrations that
renders GCAP2 proteins in unstable Ca
2+
-free configurations. These unstable structures
of PDE  and GCAP2 proteins caused by constant phototransduction activation may
lead to their selective degradations by proteasome pathway. The consequent large amount
of protein degradations possibly overloaded UPS and eventually activated UPR by the
interactions of these two systems. The protein degradations in light-exposed Balb/C
retinas were not through UPS, possibly directly by activated apoptotic caspases, further
confirming the degradations by UPS were selective and pre-apoptosis.
73

The UPR is initiated by the release of GRP78 from the sensor proteins (IRE1 α, ATF6 α,
and PERK) to bind to the misfolded protein in ER lumen, which results in the activation
of the sensors and downstream signals (Schroder and Kaufman 2005). From the previous
studies, UPR is activated in many models of dominant rhodopsin mutations which cause
large amount of protein sequestration in the ER (Saliba, Munro et al. 2002; Shinde,
Sizova et al. 2012) . Overexpression or induction of GRP78 levels was reported to
prevent these types of retinal degeneration (Inokuchi, Nakajima et al. 2009; Gorbatyuk,
Knox et al. 2010). Thus UPR activation could be common mechanisms for retinal
degeneration caused by abnormal protein accumulations in ER.
In our studies, we found the protein levels of p-eIF2, ATF3 and ATF4 were elevated in
the early phase of photoreceptor death in light exposed Arr-/- and RK-/- mice, which
were consistent with a previous report by our lab showing that ATF4 and ATF3 mRNA
levels were increased by 2.63 fold and 4.1 fold respectively in Arr-/- mice after 3 hr of
light exposure (Roca, Shin et al. 2004). Upon PERK activation, eIF2is phosphorylated
and results in the increased production of ATF4 protein, which in turn upregulates ATF3
expression (Jiang, Wek et al. 2004). ATF4 is a key mediator that controls cell metabolism,
apoptosis, and targets multiple genes, including CHOP and GADD34, both of which are
related to ER-induced cell apoptosis (Kim, Emi et al. 2006). This transcription factor has
been implicated in retinal degeneration caused by rhodopsin mutants (Kunte, Choudhury
et al. 2012), oxidative stress response of RPE cells (Miyamoto, Izumi et al. 2011) and
diabetic retinopathy(Zhong, Li et al. 2012). Interestingly, we also observed a mild
74

increase of ATF4 levels in Balb/C mice after 12 hr of light exposure. This increase was
late onset, not detected in the early phase of photoreceptor death, i.e. from 3 hr to 5 hr,
and may be in response to the oxidative stress which was activated in bright light exposed
Balb/C retinas. Additional changes in the other ER sensors or ubiquitination were not
observed, suggesting that ER stress did not play a major role in bright light-induced
retinal degeneration. Combined with our results showing the involvement of ATF4 in
constitutive phototransduction-induced retinal degeneration, the thorough investigation of
this protein may significantly advance our understanding of retinal response to different
stresses.
IRE1 α is another highly conserved UPR pathway mediating cell apoptosis and has been
implicated in a Drosophila model for retinal degeneration (Ryoo, Domingos et al. 2007).
Our results show that the protein levels IRE1 α did not change for the first 12 hr but
increase at 36 hr light exposed Arr-/- retinas, implying a later activation of IRE1 α
signaling and possible interactions of different UPR pathways. The different phases of
UPR activation clearly indicate the complexity of the mechanisms of constitutive
phototransduction-induced retinal degeneration.
Recently, it has been reported that the UPR is activated in bright light exposed albino
Balb/C mice (Yang, Wu et al. 2008). The protein levels of GRP78 and p-PERK were
shown to mildly increase after 24 hr of light exposure, and continued to be up-regulated
following dark recovery and peaked after 24 hr of dark recovery. The p-eIF2  levels
were found to be decreased after light exposure and elevated after dark recovery. The
75

discrepancy may due to the different time point examined. In the previous study, it is
shown that the photoreceptor apoptosis in light exposed Balb/C mice were already
apparent at 24 hr of light exposure, which is similar to our results. Thus the initiation of
the apoptosis should occur much earlier before the visible photoreceptor apoptosis. The
late upregulation of GRP78 coincident with massive photoreceptor death may be the
secondary signaling or the self-recovery pathway after dark recovery. Additionally, the
initial decrease of p-eIF2 also suggested that the UPR is not activated at the initiation
phase of bright light-induced photoreceptor death, which is consistent with our
observation.
Although it is well accepted that the constitutive phototransduction-induced retinal
degeneration depends on transducin, but there are always questions about the transducin-
independent pathways (Hao, Wenzel et al. 2002). In the previous study, light exposed
Arr-/-Tr-/- mice still lose roughly 10% of their photoreceptors. However, we did not
observe any retinal degeneration or phototransduction protein degradations in our light
exposed Arr-/-Tr-/- mice. One explanation is that the different phenotypes may be due to
the different genetic backgrounds when generating double mutant mice by crossbreeding,
thus the comparison of our Arr-/-Tr-/- mice with the ones that are still mildly affected by
light exposure may help to identify additional genetic factors that affect susceptibility to
light damage.
In conclusion, the mechanisms of light induced retinal degeneration are complex,
depending on environmental differences as well as genetically diversities. Our results
76

support the hypothesis that the UPR activation is the common mechanism for retinal
degeneration caused by constitutive phototransduction activation. Therefore, modulators
of ER stress could be strong candidates as therapeutic agents in the treatment of these
types of retinal degeneration diseases.
77

Chapter 4
Restoring CNGB1 Expression with Cre-mediated Recombination in a
CNGB
-/-
Mouse Line

4.1 Abstract
Most retinal degenerative disorders in humans are initiated by death of photoreceptor
cells. This is then followed by neural remodeling that includes neuronal death, cell
migration and rewiring of retinal circuits. How these changes negatively impact
therapeutic efforts to restore vision function is not known. Here, we generated a
CNGB
-/-
mouse line that is capable of gene reactivation upon Cre-mediated
recombination. The retraction of bipolar dendrites and loss of synaptic connections were
observed together with photoreceptor cell death. The Müller cells were also stressed and
became hypertrophic during the retinal degeneration process. Thus this CNGB
-/-
mice is
an ideal model that recapitulates retinal degeneration patterns as seen in human retinitis
pigmentosa, and could thus be an appropriate platform for a systematic study of the
potential of functional recovery after neural remodeling events.

78

4.2 Introduction
Retinitis pigmentosa (RP) is a set of hereditary retinal diseases characterized by
progressive loss of rod and cone photoreceptors, affecting 1 in 4000 for a total of about
15 million people in the world (van Soest, Westerveld et al. 1999). There is currently no
treatment for this debilitating retinal degenerative disorder. Current strategies to
developing clinical treatment for retinal degeneration, i.e. gene replacement therapy and
retinal transplantation, depend explicitly upon preservation of the neural retina. However,
when photoreceptors degenerate, secondary changes also occur in other retinal cells,
including neuronal cell death, migration and rewiring, in a process termed as neural
remodeling (Jones, Watt et al. 2003; Marc and Jones 2003; Marc, Jones et al. 2003; Jones
and Marc 2005).
There are three phases of retinal remodeling: 1, photoreceptor stress; 2, photoreceptor
death and 3, complex neural remodeling. Although subtle, neuronal cells start to remodel
immediately after photoreceptor cell are stressed. Bipolar dendrites begin to retract and
switch synaptic targets before visible photoreceptor cell death. Horizontal cells migrate
and send axonal processes into inner plexiform layer. Müller cells become hypertrophy
and form distal fibrotic glial seal after the loss of most photoreceptor cells. In the late
stage of retinal degeneration, thousands of new anomalous circuitries are formed and
more likely to corrupt the visual processing and preclude any retinal rescue approaches.
79

Thus the investigation of the exact effects of retinal remodeling at different phases is
crucial to all retinal rescue approaches.
Interestingly, when photoreceptor cells die, neural dendrites do not develop properly.
These neuronal remodeling processes suggest a possible trophic dependence of secondary
order neurons on the presence of photoreceptors for maintenance of their proper
morphology. Thus the preservation of photoreceptors, even relatively late in the course of
retinal degeneration may still have a positive effect on inner retinal remodeling.
Several mutations of CNGB1 gene have been identified with RP patients (Bareil, Hamel
et al. 2001; Kondo, Qin et al. 2004). Moreover, CNGB1
-/-
mice has been reported to
exhibit similar retinal degeneration patterns analogous to human RP caused by defective
CNG channels with a substantially delayed process of photoreceptor loss (Huttl,
Michalakis et al. 2005). In this study, we generated a CNGB
-/-
mouse line that is capable
of gene reactivation upon Cre-mediated recombination, and demonstrated this CNGB
-/-

mice is a good model that recapitulates retinal degeneration patterns as seen in human
retinitis pigmentosa. Thus we will use this mouse model to study the potential of
functional recovery after neural remodeling events.

80

A
C
Figure 4.1 . Generation of mice strains. A. Diagram of the CNG 1
CaM
protein.
The 14 amino acid residues in the Ca
2+
/CaM binding site were removed by the
targeting vector by homologous recombination. B. Generation of CNGB1
-/-
mice.
The neolox cassette was inserted into the intron adjacent to exon 20 that contains
the Ca
2+
/CaM binding site (*). The inserted neolox sequence disrupted splicing of
the CNGB1 gene, and the silenced CNGB1 gene can be reactivated by removal of
the neolox cassette by Cre mediated recombination. C. Construct of ER
TM
-Cre-
ER
TM
-IRES-mCherry.
Rho promoter
1.7kb
IERS ER
TM
-Cre-ER
TM

mCherry
MP1
81

4.3 Materials and Methods
4.3.1 Ethics Statement
All experimental procedures were performed in accordance with regulations established
by the National Institutes of Health, as well as with Society for Neuroscience Policy on
Animal Use in Neuroscience Research.

4.3.2 Generation of CNGB
-/-
and CNGB1
CaM
mice
A CNGB1 genomic fragment was obtained by long-range PCR with 129sv mouse
embryonic stem (ES) cell DNA as template (Fig.4.1). A targeting vector was constructed
whereby 14 amino acid residues (LQELVKMFKERTE) were deleted within the CaM
binding site contained within exon 20.This mutation was flanked by 5’ (1.6 kb) and 3’
(6.2 kb) arms. The Loxp-flanked neomycin selection (LNL) cassette was inserted into
intron 19, and a thymidine kinase cassette was cloned 5’ to the targeting vector, thus
offering both positive and negative selection to the integrated DNA. The construct was
electroporated into 129sv mouse ES cells. G418 and FIAU resistant colonies were picked,
expanded and analyzed by Southern hybridization. Clones that had undergone
homologous recombination were injected into C57BL/6 blastocysts. High degree
chimeras (>95%) were bred to C57BL/6 to obtain mice heterozygous for the insertion.
The presence of LNL cassette for the drug selection eliminates the expression of CNGB1
82

gene, possibly due to the disruption of intron splicing, resulting in complete knockout of
the CNGB1 gene. This Strain is termed as CNGB
-/-
bellow. These mice were then bred
with the Msx2-cre line to remove the neomycin cassette to generate CNGB1
CaM
mice
(Chen, Woodruff et al. 2010).

4.3.3 Generation of Transgenic ER
TM
-Cre-ER
TM
-IRES-mCherry Mice
The internal ribosome entry site (IRES) bicistronic construct was made for expressing
inducible ER
TM
-Cre-ER
TM
gene (Addgene 13777) and mCherry reporter driven by 1.7kb
Rhodopsin promoter (Fig.4.1). The construct was purified by the CsCl
2
gradient method.
The DNA fragment was released from its vector by double digestion of AseI/AflII
enzymes, and separated in a 0.8% agarose gel and extracted by the QIAEXII Gel
Extraction Kit (Qiagen, Valencia, CA). This DNA fragment was then microinjected into
fertilized eggs of donor B6D2F1 females to generate transgenic ER
TM
-Cre-ER
TM
-IRES-
mCherry mice (Norris Transgenic Core facility, Keck School of Medicine of USC, Los
Angeles, CA).

4.3.4 Light and Electron Microscopy and Retinal Morphology
The eye was enucleated, dissected and embedded into epoxy resin as described
previously (Concepcion, Mendez et al. 2002). The epon-embedded eyes were sectioned
83

into 1 μm or 60 nm sections using an ultramicrotome (Leica Ultracut UCT, Leica
Microsystems, Bannockburn, USA) for LM and EM, respectively. Electron micrographs
were obtained on a JEOL JEM 2100 microscope. For retinal morphometry the eyecups
were sectioned at or near the vertical meridian as determined by the optic nerve, and the
outer nuclear layer thickness was measured based on a previously described method
(Chen, Shi et al. 2006). Briefly, retinal section was viewed by a microscope (40×
objective) attached with a camera lucida; and measurements were taken with the aid of a
graphics tablet (WACOM, Vancouver, WA) and the Axiovision LE Rel. 4.1. imaging
software (Carl Zeiss Inc.). A stage micrometer (Klarmann Rulings, Litchfield, NH) was
used for calibration. Each hemisphere - determined by the optic nerve - was divided into
ten equal segments from the optic nerve to either the superior or inferior tip, and three
measurements were taken and averaged for each segment. Due to the thinness of the
outer nuclear layer at the optic nerve location, determination of the ten equal segments
for each hemisphere excluded the first 100 µm from the optic nerve site.

4.3.5 Immunocytochemistry
The superior pole of the mouse eye was marked by cauterization before enucleation.
Cornea and lens were removed, and the remaining eyecups were fixed in 4%
paraformaldehyde and 2.5% glutaraldehyde in 0.1M cacodylate buffer, infiltrated with 30%
sucrose overnight, and embedded in O.C.T. (Tissue-Tek, Sakura Finetech, Torrance, CA)
84

as described previously (Concepcion, Mendez et al. 2002). 10 μm thick frozen retinal
sections were obtained using a cryostat (Leica, Nussloch, Germany) at -20°C. Sections
were air dried and treated with 0.1mg/ml proteinase K (Roche, Germany). After blocking
with PBS containing 1% BSA, 5% normal goat serum, and 0.3% Triton X-100, the
sections were incubated first with the following primary antibodies: mouse monoclonal
R2-12N against rhodopsin ( 1:500 gifts from Dr. P. A. Hargrave, University of Florida,
Gainesville, FL), mouse anti-CNG  antibody (1:80 a generous gift from Dr. R. Molday),
rabbit anti-GFAP antibody (1:500 Millipore AB5804), mouse anti-CtBP2 antibody
(1:2000 BP transduction #612044) and mouse anti-PKC  antibody (1:500 Santa cruz sc-
8393); and then with FITC or Texas Red conjugated secondary antibodies (1:400 Vector
Laboratories, Burlingame, CA). Images were acquired on an Axioplan2 microscope
(Zeiss, Oberkochen, Germany). All images for each section were taken at the same
detection gain unless indicated.

4.3.6 Western Blot Analysis
The retina was dissected and homogenized in 150ul buffer (150mM NaCl, 50mM Tris
pH8.0, 0.1% NP-40, 0.5% deoxycholic acid) containing 0.1mM PMSF and Complete
mini protease inhibitor (Roche #11836153001). DNase I (30U, Roche) was added and
incubated at room temperature for 30min. The total protein amount of each sample was
determined by the BCA
TM
Protein Assay Kit (Thermo Scientific #23227). An equal
85

amount of retinal homogenate from each sample was electrophoresed on 4-12% Bis-Tris
SDS-PAGE Gel (Invitrogen) followed by transfer to nitrocellulose membrane (Whatman
#10402480) and incubated overnight with the following primary antibodies: rabbit anti-
PDE polyclonal antibody (1:1000 Cytosignal PAB-06800), rabbit anti-ROS-GC1
polyclonal antibody (1:500 Santa Cruz, sc50512), mouse Anti-G
t
α-1 antibody (1:5000
EMD4Biosciences 371740 ), mouse anti-CNG  antibody (1:500 a generous gift from Dr.
R. Molday), and mouse anti-Actin antibody (1:5000 Millipore MAB1501). The
membranes were then incubated with fluorescently labeled secondary antibodies
(1:10,000 Li-Cor P/N926-31081) at room temperature for 1 hour and detected by
Odyssey infrared imaging system.

4.4 Results
4.4.1 Reversible Ablation of CNG-  Gene
To confirmed the reversible ablation of the CNG-  gene in CNGB1
-/-
and CNGB1
CaM

mice, we first assessed the CNG-  protein levels by western blots in these mice. As
shown in Fig. 4.2, the CNG-  protein was present in wildtype and CNGB1
CaM
mice with
similar levels, but completely absent in the CNGB1
-/-
mice. ICC results also confirmed
the normal localization of CNG- 
CaM
protein in the outer segments as that in wildtype
mice. Additionally, the levels of phototransduction proteins, i.e. PDE, GC, were similar
86

in wildtype and CNGB1
CaM
mice, but decreased moderately in CNGB1
-/-
mice at 1
month old age consistent with the retinal degeneration process.

GC
PDE
Actin
CNG  
B
Figure 4.2 Characterization of CNGB1
-/-
and CNGB1
CaM
mice. A. Western blot of
retinal extracts from 4-week-old wildtype, CNGB1
CaM
and CNGB1
-/-
mice, at
which age the outer nuclear layer thickness is similar between CNGB1
-/-
and
wildtype mice. B. CNG-  immunofluorescence in retinas from 1-month-old
wildtype, CNGB1
CaM
and CNGB1
-/-
mice. The CNG 1
CaM
showed correct
localization at the outer segment.
A
WT CNGB1
CaM
CNGB1
‐/‐

WT CNGB1
CaM
CNGB1
‐/‐

87

WT
Figure 4.3. Morphological and functional analysis of CNGB1
CaM
mice. A.
Retinal morphology of age-matched wildtype and CNGB1
CaM
mice. B.
CNGB1
CaM
mice did not exhibit any detectable defect in light adaptation in single
cell recording as we previously reported (Chen, Woodruff et al. 2010).
.
CNGB1
CaM

1M 3M 6M 9M 12M
A
B
88

Furthermore, we examined the retinal morphology and function of CNGB1
CaM
mice. As
shown in Fig 4.3, CNGB1
CaM
mice maintained normal retinal structure up to 12 months.
No shortened or disorganized outer segments or reduced photoreceptor numbers were
observed. Additionally, CNGB1
CaM
mice did not exhibit any detectable defect in light
adaptation in single cell recording as we previously reported (Chen, Woodruff et al.
2010).
Collectively, the presence of Loxp-flanked neomycin selection cassette eliminated the
expression of CNGB1 gene and could be removed by Cre-mediated recombination to
generate CNGB1
CaM
mice, which show no differences both morphologically and
functionally from wildtype mice.

4.4.2 CNGB1
-/-
Mice Show Slow Progressed Retinal Degeneration
To analyze the retinal degeneration process in CNGB1
-/-
mice, we examined the retinal
morphology of CNGB1
-/-
mice at different ages. At 1 month old (Fig.4.4), the outer
segments of CNGB1
-/-
mice began to shorten, but the ONL was mostly intact. EM
sections also show that the outer segment discs were also severely disorganized in
CNGB1
-/-
mice but stacked normally in CNGB1
CaM
mice.
89

A
Figure 4.4. Retinal morphology of
CNGB1
-/-
mice. A. Retinal
morphology of CNGB1
CaM
mice was
similar to that of wildtype mice,
while CNGB1
-/-
mice exhibited
progressive retinal degeneration.
ONL: outer nuclear layer; IS: inner
segment; OS: outer segment. B. EM
section of 3 months old
CNGB1
CaM
mice (left) and 1 month
old CNGB1
-/-
mice (right). The
CNGB1
CaM
mice retinal morphology
was similar to age-matched wildtype
mice. In contrast, the outer segment
CNGB1
-/-
retina was disorganized.
Longitudinally arranged discs are
often seen in outer segments of
CNGB1
-/-
mice (arrows).
B
CNGB1
CaM
(3M)

CNGB1
‐/‐
(1M)
90

At 3 months old, the photoreceptor nuclei in the ONL of CNGB1
-/-
mice reduced to 1-3
rows compared to the normal 10-12 rows in wildtype mice. At 6 months old,
photoreceptor degeneration was essentially complete; no remaining photoreceptors were
visible at this age in CNGB1
-/-
mice. Collectively, the CNGB1
-/-
mice show retinal
degeneration with a later onset and slow degeneration process.

4.4.3 Neural Remodeling Markers Activated During Retinal Degeneration Process in
CNGB1

Mice
In order to use CNGB1
-/-
mice as model to study retinal remodeling, we tested several
neural remodeling markers during the retinal degeneration process. An increase in GFAP
reactivity was observed in CNGB1
-/-
mice at 1 month old (Fig.4.5), indicating the early
activation of Müller cells even before the photoreceptor cell death. This activity persisted
as a function of retinal degeneration. Rhodopsin mis-localization was also observed in
CNGB1
-/-
mice during the whole degeneration period, but not in CNGB1
CaM
and
wildtype mice. The dendrites of bipolar cells appeared to retract as the number of
photoreceptor cell started to decrease in CNGB1
-/-
mice at 1 month old (Fig.4.6). At 3
months old, some bipolar cells even appeared to migrate outside of their usual layer. The
synaptic ribbon also decreased progressively in CNGB1
-/-
mice. At 3 months old, less
than 10% of synaptic ribbons still remained. No degenerative changes were detected in
the CNGB1
CaM
mice.
91

Combined with our results showing the slow progress of retinal degeneration in
CNGB1
-/-
mice, our data proved that the CNGB1
-/-
mice was a good model for a
systematic study of the potential of functional recovery after neural remodeling events.
Figure 4.5. Neuronal remodeling markers activated in CNGB1
-/-
retinas. An
increase in GFAP reactivity (top) was observed in the CNGB1
-/-
retina at an early
age (1 month), indicating Müller cell activation. This activity persisted as a
function of retinal degeneration. Rhodopsin mis-localization (bottom) was also
observed in CNGB1
-/-
retinas but not WT or CNGB1
CaM
retinas.
WT(3M) CNGB1
CaM
(3M)

CNGB1
‐/‐
(1M) CNGB1
‐/‐
(2M) CNGB1
‐/‐
(3M)
92

Figure 4.6. Bipolar cell and synaptic ribbon morphology in WT, CNGB1
CaM
and
CNGB1
-/-
retinas. Bipolar cell was visualized by PKC staining (top). As shown
above, the dendrites of bipolar cell appeared to retract as the number of
photoreceptor cells decreased in CNGB1
-/-
retina. At 3 months some bipolar cells
even appeared to migrate outside of their usual stratification. The synaptic ribbon
(bottom) also decreased progressively in CNGB1
-/-
retina. At 3 months less than
10% of synaptic ribbons still remained. Degenerative changes were not detected in
the CNGB1
CaM

retina.
PKC
Ribeye
WT(3M) CNGB1
CaM
(3M)

CNGB1
‐/‐
(1M) CNGB1
‐/‐
(3M)
93

4.5 Future work
4.5.1 Generation of CNGB1
-/-
/ ER
TM
-Cre-ER
TM
-IRES-mCherry Mice
We will breed CNGB1
-/-
mice with ER
TM
-Cre-ER
TM
-IRES-mCherry
+
mice to generate
ER
TM
-Cre-ER
TM
-IRES-mCherry
+
/CNGB1
-/-
mice. The fusion ER
TM
-Cre-ER
TM
protein
remains in the cytoplasm unless exposed to estrogen antagonist 4-OH-tamoxifen (4-OH-
TM), which causes the translocation of this fusion protein to the nucleus to catalyze site-
specific recombination of DNA between two LoxP sites. Accordingly, we will
administrate 4-OH-tamoxifen (TM) on these mice to confirm the TM-induced
CNGB1
CaM
expression by: 1) LNL-specific PCR to confirm that the removal of LNL
cassette; and 2) western blot analysis to test the presence of CNG-  protein.

Figure 4.7 Experiment design I
94

4.5.2 Characterization of CNGB1
CaM
-dependent Recovery of Retinal Degeneration
To study the possibility of functional recovery retinal degeneration in CNGB1
-/-
mice, we
will dose 1-month-old ER
TM
-Cre-ER
TM
-IRES-mCherry
+
mice with TM to induce
CNGB1
CaM
protein expression as illustrated in Figure 4.7.
The rationale of using mice at 1 month-old is: 1), 1 month-old CNGB1
-/-
mice show
detectable but mild retinal degeneration, and could thus serve as an ideal time point to
start; 2), CNGB1
-/-
mice undergo continuous retinal degeneration during 1 to 3 months ,
allowing the detection of even milder changes of the degeneration speeds and degrees
during this period; and 3), the retinal degeneration is not complete at 3 months, making
the whole period meaningful to study the effects of neural remolding in the early stages.
We will compare the morphological structure of retinas, the biomarkers of retinal
remodeling and retinal function of drug-treated mice with control mice, to test whether
the degeneration process or neural remodeling can be stopped or attenuated, and whether
the retinal function can be recovered after mild degeneration.
This study will provide meaningful information for the development for the recovery
interventions of retinal degeneration, and the effects of neural remodeling on retinal
rescue approaches.

95

4.5.3 Investigation of the Time Limit for the Retinal Recovery at Different Degeneration
Phases

Is there a practical intervention window for retinal rescue before the damages become
irreversible? Will the process of neuronal remodeling eventually preclude any rescue
approaches after certain stages? To answer these questions, we will extend the same set
of experiments with 2-month-old and 3-month-old CNGB1
-/-
/ ER
TM
-Cre-ER
TM
-IRES-
mCherry
+
mice as described above in Figure 4.8 to investigate whether the recovery of
retinal degeneration would be affected by more severe retinal damage and neural
remolding.
Our studies will advance our knowledge in the progression of the retinal degeneration
and neural remolding and also reveal the practical intervention window for functional
recovery for retinal degenerative diseases.
Figure 4.8 Experiment design II
96

Conclusion and Future Perspective
Retinal degenerative disorders, such as retinitis pigmentosa and age-related macular
degeneration, severely reduce patients’ qualities of life and show a distinct trend of
increase in prevalence with the aged population in this decade. The investigation of the
underlying mechanisms of these disorders provides solid fundamental and valuable clues
for the development of clinical interventions.
In chapter 2, it is demonstrated that genetic ablation of CNG channels but not PKG
significantly improved photoreceptor viability in two PDE6 mutant mice, thus supporting the
notion that elevated cGMP opens excessive numbers of CNG channels
+
which cause the cell
death process possibly via uncontrolled influx of Ca
2+
and Na. In order to further confirm the
role of Ca
2+
in cGMP-induced retinal degeneration, cytosolic Ca
2+
concentrations could be
measured by Ca
2+
indicators in double mutant and control photoreceptor cells. The
downstream death pathways activated by increased Ca
2+
concentrations also need more
investigation. In this study, we proved that targeting rod CNG channels could be a
promising approach to treat cGMP-induced retinal degeneration. Thus, specific rod CNG
channels inhibitors, such as DAG, could be tested. In addition to retinal degenerative
disorders, cGMP and Ca
2+
may be common initiators triggering cell death in other
neurodegenerative diseases. Future investigation of the regulation of these second
messengers may also significantly advance the understanding of pathological processes
of these diseases. Additionally, in the course of crossbreeding mice for these experiments,
97

we identified a subset of mice with much milder PDE6-associated retinal degeneration
progression independent of CNG or PKG genotypes. These mice are being examined to
identify additional genetic factors involved in the pathway.
In chapter 3, it is demonstrated that ER stress proteins were markedly up-regulated in the
early phase of constitutive phototransduction-induced retinal degeneration, indicating that
the activation of unfolded protein response might be the common initiator of retinal
degeneration induced by constitutive phototransduction activation. Hence, the light
damage sensitivity needs to be measured on photoreceptor specific GRP78 knockout
mice to further confirm the requirement of unfolded protein response as the initiator of
constitutive phototransduction activation-induced retinal degeneration. Drugs specifically
targeting ER stress sensors, such as valproic acid and guananbenz, are being tested to see
the effects on this type of retinal degeneration. In addition, UPR is also activated in many
models of dominant rhodopsin mutations which cause large amount of protein
sequestration in the ER, thus further investigation of the ER-stress pathway and the
downstream activation of death signals may reveal underlying common mechanisms of
retinal degeneration caused by various genetic mutations.
In chapter 4, it is shown that the CNGB
-/-
mice is an ideal model that recapitulates retinal
degeneration patterns as seen in human retinitis pigmentosa, and could be further used as
a platform for a systematic study of the potential of functional recovery after neural
remodeling events. This part of study would provide more comprehensive understanding
98

of the practical intervention time window and the role of neural remolding on therapeutic
efforts to restore vision function.
Taken together, from multiple aspects, these studies expand and deepen our
understanding of the mechanisms of retinal degeneration caused by both genetic and
environmental factors. They will not only provide the directional guidance for future
basic studies by unraveling distinct common pathways, but also gain tremendous
translational and clinical significance by identifying multiple therapeutic targets and
optimum intervention windows.

99

Bibliography
Bareil, C., C. P. Hamel, et al. (2001). "Segregation of a mutation in CNGB1 encoding the beta‐
subunit of the rod cGMP‐gated channel in a family with autosomal recessive retinitis
pigmentosa." Hum Genet 108(4): 328‐334.

Barhoum, R., G. Martinez‐Navarrete, et al. (2008). "Functional and structural modifications
during retinal degeneration in the rd10 mouse." Neuroscience 155(3): 698‐713.

Baumann, O. and B. Walz (2001). "Endoplasmic reticulum of animal cells and its organization
into structural and functional domains." Int Rev Cytol 205: 149‐214.

Bok, D. (2002). "New insights and new approaches toward the study of age‐related macular
degeneration." Proc Natl Acad Sci U S A 99(23): 14619‐14621.

Bowes, C., T. Li, et al. (1990). "Retinal degeneration in the rd mouse is caused by a defect in the
beta subunit of rod cGMP‐phosphodiesterase." Nature 347(6294): 677‐680.

Burns, M. E. and D. A. Baylor (2001). "Activation, deactivation, and adaptation in vertebrate,
photoreceptor cells." Annu Rev Neurosci 24: 779‐805.

Canals, S., M. J. Casarejos, et al. (2003). "Nitric oxide triggers the toxicity due to glutathione
depletion in midbrain cultures through 12‐lipoxygenase." J Biol Chem 278(24): 21542‐
21549.

Canzoniero, L. M., A. Adornetto, et al. (2006). "Involvement of the nitric oxide/protein kinase G
pathway in polychlorinated biphenyl‐induced cell death in SH‐SY 5Y neuroblastoma
cells." J Neurosci Res 84(3): 692‐697.

Chang, B., N. L. Hawes, et al. (2007). "Two mouse retinal degenerations caused by missense
mutations in the beta‐subunit of rod cGMP phosphodiesterase gene." Vision Res 47(5):
624‐633.

Chen, C. K., M. E. Burns, et al. (2000). "Slowed recovery of rod photoresponse in mice lacking the
GTPase accelerating protein RGS9‐1." Nature 403(6769): 557‐560.

Chen, C. K., M. E. Burns, et al. (1999). "Abnormal photoresponses and light‐induced apoptosis in
rods lacking rhodopsin kinase." Proc Natl Acad Sci U S A 96(7): 3718‐3722.

Chen, J., C. L. Makino, et al. (1995). "Mechanisms of rhodopsin inactivation in vivo as revealed by
a COOH‐terminal truncation mutant." Science 267(5196): 374‐377.
100

Chen, J., G. Shi, et al. (2006). "Stable rhodopsin/arrestin complex leads to retinal degeneration in
a transgenic mouse model of autosomal dominant retinitis pigmentosa." J Neurosci
26(46): 11929‐11937.

Chen, J., M. I. Simon, et al. (1999). "Increased susceptibility to light damage in an arrestin
knockout mouse model of Oguchi disease (stationary night blindness)." Invest
Ophthalmol Vis Sci 40(12): 2978‐2982.

Chen, J., M. L. Woodruff, et al. (2010). "Channel modulation and the mechanism of light
adaptation in mouse rods." J Neurosci 30(48): 16232‐16240.

Chiang, W. C., C. Messah, et al. (2012). "IRE1 directs proteasomal and lysosomal degradation of
misfolded rhodopsin." Mol Biol Cell 23(5): 758‐770.

Concepcion, F., A. Mendez, et al. (2002). "The carboxyl‐terminal domain is essential for
rhodopsin transport in rod photoreceptors." Vision Res 42(4): 417‐426.

Cruickshanks, K. J., R. Klein, et al. (1993). "Sunlight and age‐related macular degeneration. The
Beaver Dam Eye Study." Arch Ophthalmol 111(4): 514‐518.

Curcio, C. A., K. R. Sloan, et al. (1990). "Human photoreceptor topography." J Comp Neurol
292(4): 497‐523.

Daiger, S. P., S. J. Bowne, et al. (2007). "Perspective on genes and mutations causing retinitis
pigmentosa." Arch Ophthalmol 125(2): 151‐158.

Danciger, M., M. T. Matthes, et al. (2000). "A QTL on distal chromosome 3 that influences the
severity of light‐induced damage to mouse photoreceptors." Mamm Genome 11(6):
422‐427.

Donovan, M., R. J. Carmody, et al. (2001). "Light‐induced photoreceptor apoptosis in vivo
requires neuronal nitric‐oxide synthase and guanylate cyclase activity and is caspase‐3‐
independent." The Journal of biological chemistry 276(25): 23000‐23008.

Donovan, M., R. J. Carmody, et al. (2001). "Light‐induced photoreceptor apoptosis in vivo
requires neuronal nitric‐oxide synthase and guanylate cyclase activity and is caspase‐3‐
independent." J Biol Chem 276(25): 23000‐23008.

Donovan, M. and T. G. Cotter (2002). "Caspase‐independent photoreceptor apoptosis in vivo
and differential expression of apoptotic protease activating factor‐1 and caspase‐3
during retinal development." Cell Death Differ 9(11): 1220‐1231.
101

Doonan, F., M. Donovan, et al. (2005). "Activation of multiple pathways during photoreceptor
apoptosis in the rd mouse." Invest Ophthalmol Vis Sci 46(10): 3530‐3538.

Fain, G. L. and J. E. Lisman (1999). "Light, Ca2+, and photoreceptor death: new evidence for the
equivalent‐light hypothesis from arrestin knockout mice." Invest Ophthalmol Vis Sci
40(12): 2770‐2772.

Farber, D. B. and R. N. Lolley (1974). "Cyclic guanosine monophosphate: elevation in
degenerating photoreceptor cells of the C3H mouse retina." Science 186(4162): 449‐451.

Farber, D. B. and R. N. Lolley (1976). "Enzymic basis for cyclic GMP accumulation in degenerative
photoreceptor cells of mouse retina." J Cyclic Nucleotide Res 2(3): 139‐148.

Fariss, R. N., Z. Y. Li, et al. (2000). "Abnormalities in rod photoreceptors, amacrine cells, and
horizontal cells in human retinas with retinitis pigmentosa." Am J Ophthalmol 129(2):
215‐223.

Fei, Y. (2002). "Cone neurite sprouting: an early onset abnormality of the cone photoreceptors
in the retinal degeneration mouse." Mol Vis 8: 306‐314.

Fiala, J. C., J. Spacek, et al. (2002). "Dendritic spine pathology: cause or consequence of
neurological disorders?" Brain Res Brain Res Rev 39(1): 29‐54.

Fletcher, E. L. and M. Kalloniatis (1996). "Neurochemical architecture of the normal and
degenerating rat retina." J Comp Neurol 376(3): 343‐360.

Frasson, M., J. A. Sahel, et al. (1999). "Retinitis pigmentosa: rod photoreceptor rescue by a
calcium‐channel blocker in the rd mouse." Nat Med 5(10): 1183‐1187.

Gamm, D. M., L. K. Barthel, et al. (2000). "Localization of cGMP‐dependent protein kinase
isoforms in mouse eye." Invest Ophthalmol Vis Sci 41(9): 2766‐2773.

Gargini, C., E. Terzibasi, et al. (2007). "Retinal organization in the retinal degeneration 10 (rd10)
mutant mouse: a morphological and ERG study." J Comp Neurol 500(2): 222‐238.

Gorbatyuk, M. S., T. Knox, et al. (2010). "Restoration of visual function in P23H rhodopsin
transgenic rats by gene delivery of BiP/Grp78." Proc Natl Acad Sci U S A 107(13): 5961‐
5966.

Gorbatyuk, M. S., T. Knox, et al. (2010). "Restoration of visual function in P23H rhodopsin
transgenic rats by gene delivery of BiP/Grp78." Proceedings of the National Academy of
Sciences of the United States of America 107(13): 5961‐5966.
102

Grimm, C., A. Wenzel, et al. (2000). "Protection of Rpe65‐deficient mice identifies rhodopsin as a
mediator of light‐induced retinal degeneration." Nat Genet 25(1): 63‐66.

Gurevich, V. V. and J. L. Benovic (1993). "Visual arrestin interaction with rhodopsin. Sequential
multisite binding ensures strict selectivity toward light‐activated phosphorylated
rhodopsin." J Biol Chem 268(16): 11628‐11638.

Hafezi, F., J. P. Steinbach, et al. (1997). "The absence of c‐fos prevents light‐induced apoptotic
cell death of photoreceptors in retinal degeneration in vivo." Nat Med 3(3): 346‐349.

Hao, W., A. Wenzel, et al. (2002). "Evidence for two apoptotic pathways in light‐induced retinal
degeneration." Nat Genet 32(2): 254‐260.

Hao, W., A. Wenzel, et al. (2002). "Evidence for two apoptotic pathways in light‐induced retinal
degeneration." Nature genetics 32(2): 254‐260.

Harding, H. P., I. Novoa, et al. (2000). "Regulated translation initiation controls stress‐induced
gene expression in mammalian cells." Mol Cell 6(5): 1099‐1108.

Hartong, D. T., E. L. Berson, et al. (2006). "Retinitis pigmentosa." Lancet 368(9549): 1795‐1809.

He, W., C. W. Cowan, et al. (1998). "RGS9, a GTPase accelerator for phototransduction." Neuron
20(1): 95‐102.

Hofmann, F., R. Feil, et al. (2006). "Function of cGMP‐dependent protein kinases as revealed by
gene deletion." Physiol Rev 86(1): 1‐23.

Hu, G. and T. G. Wensel (2002). "R9AP, a membrane anchor for the photoreceptor GTPase
accelerating protein, RGS9‐1." Proc Natl Acad Sci U S A 99(15): 9755‐9760.

Humayun, M. S., M. Prince, et al. (1999). "Morphometric analysis of the extramacular retina
from postmortem eyes with retinitis pigmentosa." Invest Ophthalmol Vis Sci 40(1): 143‐
148.

Huttl, S., S. Michalakis, et al. (2005). "Impaired channel targeting and retinal degeneration in
mice lacking the cyclic nucleotide‐gated channel subunit CNGB1." J Neurosci 25(1): 130‐
138.

Illing, M. E., R. S. Rajan, et al. (2002). "A rhodopsin mutant linked to autosomal dominant
retinitis pigmentosa is prone to aggregate and interacts with the ubiquitin proteasome
system." The Journal of biological chemistry 277(37): 34150‐34160.
103

Inokuchi, Y., Y. Nakajima, et al. (2009). "Effect of an inducer of BiP, a molecular chaperone, on
endoplasmic reticulum (ER) stress‐induced retinal cell death." Invest Ophthalmol Vis Sci
50(1): 334‐344.

Jacobson, S. G., A. Sumaroka, et al. (2007). "Evidence for retinal remodelling in retinitis
pigmentosa caused by PDE6B mutation." Br J Ophthalmol 91(5): 699‐701.

Jiang, H. Y., S. A. Wek, et al. (2004). "Activating transcription factor 3 is integral to the eukaryotic
initiation factor 2 kinase stress response." Mol Cell Biol 24(3): 1365‐1377.

Jones, B. W. and R. E. Marc (2005). "Retinal remodeling during retinal degeneration." Exp Eye
Res 81(2): 123‐137.

Jones, B. W., C. B. Watt, et al. (2003). "Retinal remodeling triggered by photoreceptor
degenerations." J Comp Neurol 464(1): 1‐16.

Katayama, T., K. Imaizumi, et al. (2004). "Induction of neuronal death by ER stress in Alzheimer's
disease." J Chem Neuroanat 28(1‐2): 67‐78.

Keeler, C. (1966). "Retinal degeneration in the mouse is rodless retina." J Hered 57(2): 47‐50.

Keeler, C. E. (1924). "The Inheritance of a Retinal Abnormality in White Mice." Proc Natl Acad Sci
U S A 10(7): 329‐333.

Kim, R., M. Emi, et al. (2006). "Role of the unfolded protein response in cell death." Apoptosis
11(1): 5‐13.

Kondo, H., M. Qin, et al. (2004). "A homozygosity‐based search for mutations in patients with
autosomal recessive retinitis pigmentosa, using microsatellite markers." Invest
Ophthalmol Vis Sci 45(12): 4433‐4439.

Koutalos, Y., K. Nakatani, et al. (1995). "Characterization of guanylate cyclase activity in single
retinal rod outer segments." J Gen Physiol 106(5): 863‐890.

Koutalos, Y. and K. W. Yau (1996). "Regulation of sensitivity in vertebrate rod photoreceptors by
calcium." Trends Neurosci 19(2): 73‐81.

Krishnan, J., G. Lee, et al. (2008). "Characterization of phototransduction gene knockouts
revealed important signaling networks in the light‐induced retinal degeneration."
Journal of biomedicine & biotechnology 2008: 327468.

104

Krispel, C. M., D. Chen, et al. (2006). "RGS expression rate‐limits recovery of rod
photoresponses." Neuron 51(4): 409‐416.

Kroeger, H., C. Messah, et al. (2012). "Induction of endoplasmic reticulum stress genes, BiP and
chop, in genetic and environmental models of retinal degeneration." Invest Ophthalmol
Vis Sci 53(12): 7590‐7599.

Kuhn, H. and U. Wilden (1987). "Deactivation of photoactivated rhodopsin by rhodopsin‐kinase
and arrestin." J Recept Res 7(1‐4): 283‐298.

Kunte, M. M., S. Choudhury, et al. (2012). "ER stress is involved in T17M rhodopsin‐induced
retinal degeneration." Invest Ophthalmol Vis Sci 53(7): 3792‐3800.

Lamb, T. D. and E. N. Pugh, Jr. (2004). "Dark adaptation and the retinoid cycle of vision." Prog
Retin Eye Res 23(3): 307‐380.

Lamb, T. D. and E. N. Pugh, Jr. (2006). "Phototransduction, dark adaptation, and rhodopsin
regeneration the proctor lecture." Invest Ophthalmol Vis Sci 47(12): 5137‐5152.

Lem, J. and G. L. Fain (2004). "Constitutive opsin signaling: night blindness or retinal
degeneration?" Trends Mol Med 10(4): 150‐157.

Lewis, G. P., D. G. Charteris, et al. (2002). "Animal models of retinal detachment and
reattachment: identifying cellular events that may affect visual recovery." Eye (Lond)
16(4): 375‐387.

Lin, J. H., P. Walter, et al. (2008). "Endoplasmic reticulum stress in disease pathogenesis." Annu
Rev Pathol 3: 399‐425.

Lincoln, T. M. and T. L. Cornwell (1993). "Intracellular cyclic GMP receptor proteins." FASEB J
7(2): 328‐338.

Lisman, J. and G. Fain (1995). "Support for the equivalent light hypothesis for RP." Nat Med
1(12): 1254‐1255.

Lyubarsky, A. L., B. Falsini, et al. (1999). "UV‐ and midwave‐sensitive cone‐driven retinal
responses of the mouse: a possible phenotype for coexpression of cone
photopigments." J Neurosci 19(1): 442‐455.

Marc, R. E. and B. W. Jones (2003). "Retinal remodeling in inherited photoreceptor
degenerations." Mol Neurobiol 28(2): 139‐147.

105

Marc, R. E., B. W. Jones, et al. (2003). "Neural remodeling in retinal degeneration." Prog Retin
Eye Res 22(5): 607‐655.

Marc, R. E., R. F. Murry, et al. (1998). "Amino acid signatures in the detached cat retina." Invest
Ophthalmol Vis Sci 39(9): 1694‐1702.

Margolis, D. J., G. Newkirk, et al. (2008). "Functional stability of retinal ganglion cells after
degeneration‐induced changes in synaptic input." J Neurosci 28(25): 6526‐6536.

Mata, N. L., J. Weng, et al. (2000). "Biosynthesis of a major lipofuscin fluorophore in mice and
humans with ABCR‐mediated retinal and macular degeneration." Proc Natl Acad Sci U S
A 97(13): 7154‐7159.

Mazzoni, F., E. Novelli, et al. (2008). "Retinal ganglion cells survive and maintain normal
dendritic morphology in a mouse model of inherited photoreceptor degeneration." J
Neurosci 28(52): 14282‐14292.

McLaughlin, M. E., T. L. Ehrhart, et al. (1995). "Mutation spectrum of the gene encoding the beta
subunit of rod phosphodiesterase among patients with autosomal recessive retinitis
pigmentosa." Proc Natl Acad Sci U S A 92(8): 3249‐3253.

Mendez, A., M. E. Burns, et al. (2000). "Rapid and reproducible deactivation of rhodopsin
requires multiple phosphorylation sites." Neuron 28(1): 153‐164.

Mendez, A., N. V. Krasnoperova, et al. (2000). "Functional study of rhodopsin phosphorylation in
vivo." Methods Enzymol 316: 167‐185.

Milam, A. H., Z. Y. Li, et al. (1998). "Histopathology of the human retina in retinitis pigmentosa."
Prog Retin Eye Res 17(2): 175‐205.

Miyamoto, N., H. Izumi, et al. (2011). "Transcriptional regulation of activating transcription
factor 4 under oxidative stress in retinal pigment epithelial ARPE‐19/HPV‐16 cells."
Invest Ophthalmol Vis Sci 52(3): 1226‐1234.

Noell, W. K. (1980). "Possible mechanisms of photoreceptor damage by light in mammalian
eyes." Vision Res 20(12): 1163‐1171.

Okawa, H. and A. P. Sampath (2007). "Optimization of single‐photon response transmission at
the rod‐to‐rod bipolar synapse." Physiology (Bethesda) 22: 279‐286.

Oyster, C. W. (1999). The human eye : structure and function. Sunderland, Mass., Sinauer
Associates.
106

Paquet‐Durand, F., S. M. Hauck, et al. (2009). "PKG activity causes photoreceptor cell death in
two retinitis pigmentosa models." J Neurochem 108(3): 796‐810.

Paschen, W. and T. Mengesdorf (2005). "Endoplasmic reticulum stress response and
neurodegeneration." Cell Calcium 38(3‐4): 409‐415.

Pawlyk, B. S., T. Li, et al. (2002). "Absence of photoreceptor rescue with D‐cis‐diltiazem in the rd
mouse." Invest Ophthalmol Vis Sci 43(6): 1912‐1915.

Pearce‐Kelling, S. E., T. S. Aleman, et al. (2001). "Calcium channel blocker D‐cis‐diltiazem does
not slow retinal degeneration in the PDE6B mutant rcd1 canine model of retinitis
pigmentosa." Mol Vis 7: 42‐47.

Pierce, E. A. (2001). "Pathways to photoreceptor cell death in inherited retinal degenerations."
Bioessays 23(7): 605‐618.

Pittler, S. J. and W. Baehr (1991). "Identification of a nonsense mutation in the rod
photoreceptor cGMP phosphodiesterase beta‐subunit gene of the rd mouse." Proc Natl
Acad Sci U S A 88(19): 8322‐8326.

Punzo, C. and C. Cepko (2007). "Cellular responses to photoreceptor death in the rd1 mouse
model of retinal degeneration." Invest Ophthalmol Vis Sci 48(2): 849‐857.

Puthussery, T. and W. R. Taylor (2010). "Functional changes in inner retinal neurons in animal
models of photoreceptor degeneration." Adv Exp Med Biol 664: 525‐532.

Quigley, H. A. (1999). "Neuronal death in glaucoma." Prog Retin Eye Res 18(1): 39‐57.

Racz, B., F. Gallyas, Jr., et al. (2007). "Effects of pituitary adenylate cyclase activating polypeptide
(PACAP) on the PKA‐Bad‐14‐3‐3 signaling pathway in glutamate‐induced retinal injury in
neonatal rats." Neurotoxicity research 12(2): 95‐104.

Rao, V. R., G. B. Cohen, et al. (1994). "Rhodopsin mutation G90D and a molecular mechanism for
congenital night blindness." Nature 367(6464): 639‐642.

Rebello, G., R. Ramesar, et al. (2004). "Apoptosis‐inducing signal sequence mutation in carbonic
anhydrase IV identified in patients with the RP17 form of retinitis pigmentosa." Proc
Natl Acad Sci U S A 101(17): 6617‐6622.

Robinson, P. R., G. B. Cohen, et al. (1992). "Constitutively active mutants of rhodopsin." Neuron
9(4): 719‐725.
107

Roca, A., K. J. Shin, et al. (2004). "Comparative analysis of transcriptional profiles between two
apoptotic pathways of light‐induced retinal degeneration." Neuroscience 129(3): 779‐
790.

Rodieck, R. W. (1998). The first steps in seeing. Sunderland, Mass., Sinauer Associates.

Roybal, C. N., S. Yang, et al. (2004). "Homocysteine increases the expression of vascular
endothelial growth factor by a mechanism involving endoplasmic reticulum stress and
transcription factor ATF4." J Biol Chem 279(15): 14844‐14852.

Ryoo, H. D., P. M. Domingos, et al. (2007). "Unfolded protein response in a Drosophila model for
retinal degeneration." EMBO J 26(1): 242‐252.

Sakmar, T. P., R. R. Franke, et al. (1989). "Glutamic acid‐113 serves as the retinylidene Schiff
base counterion in bovine rhodopsin." Proc Natl Acad Sci U S A 86(21): 8309‐8313.

Saliba, R. S., P. M. Munro, et al. (2002). "The cellular fate of mutant rhodopsin: quality control,
degradation and aggresome formation." Journal of cell science 115(Pt 14): 2907‐2918.

Saliba, R. S., P. M. Munro, et al. (2002). "The cellular fate of mutant rhodopsin: quality control,
degradation and aggresome formation." J Cell Sci 115(Pt 14): 2907‐2918.

Salminen, A., A. Kauppinen, et al. (2010). "Endoplasmic reticulum stress in age‐related macular
degeneration: trigger for neovascularization." Mol Med 16(11‐12): 535‐542.

Sancho‐Pelluz, J., B. Arango‐Gonzalez, et al. (2008). "Photoreceptor cell death mechanisms in
inherited retinal degeneration." Mol Neurobiol 38(3): 253‐269.

Santos, A., M. S. Humayun, et al. (1997). "Preservation of the inner retina in retinitis pigmentosa.
A morphometric analysis." Arch Ophthalmol 115(4): 511‐515.

Sanyal, S., A. De Ruiter, et al. (1980). "Development and degeneration of retina in rds mutant
mice: light microscopy." J Comp Neurol 194(1): 193‐207.

Schadel, S. A., M. Heck, et al. (2003). "Ligand channeling within a G‐protein‐coupled receptor.
The entry and exit of retinals in native opsin." J Biol Chem 278(27): 24896‐24903.

Schroder, M. and R. J. Kaufman (2005). "ER stress and the unfolded protein response." Mutat
Res 569(1‐2): 29‐63.

Schroder, M. and R. J. Kaufman (2005). "The mammalian unfolded protein response." Annu Rev
Biochem 74: 739‐789.
108

Shinde, V. M., O. S. Sizova, et al. (2012). "ER stress in retinal degeneration in S334ter Rho rats."
PLoS One 7(3): e33266.

Silva, R. M., V. Ries, et al. (2005). "CHOP/GADD153 is a mediator of apoptotic death in
substantia nigra dopamine neurons in an in vivo neurotoxin model of parkinsonism." J
Neurochem 95(4): 974‐986.

Simons, K. (1993). "Artificial light and early‐life exposure in age‐related macular degeneration
and in cataractogenic phototoxicity." Arch Ophthalmol 111(3): 297‐298.

Specht, S., D. T. Organisciak, et al. (2000). "Continuing damage to rat retinal DNA during
darkness following light exposure." Photochem Photobiol 71(5): 559‐566.

Stone, J. L., W. E. Barlow, et al. (1992). "Morphometric analysis of macular photoreceptors and
ganglion cells in retinas with retinitis pigmentosa." Arch Ophthalmol 110(11): 1634‐1639.

Strettoi, E. and V. Pignatelli (2000). "Modifications of retinal neurons in a mouse model of
retinitis pigmentosa." Proc Natl Acad Sci U S A 97(20): 11020‐11025.

Strettoi, E., V. Pignatelli, et al. (2003). "Remodeling of second‐order neurons in the retina of
rd/rd mutant mice." Vision Res 43(8): 867‐877.

Strettoi, E., V. Porciatti, et al. (2002). "Morphological and functional abnormalities in the inner
retina of the rd/rd mouse." J Neurosci 22(13): 5492‐5504.

Tabas, I. and D. Ron (2011). "Integrating the mechanisms of apoptosis induced by endoplasmic
reticulum stress." Nat Cell Biol 13(3): 184‐190.

Takano, Y., H. Ohguro, et al. (2004). "Study of drug effects of calcium channel blockers on retinal
degeneration of rd mouse." Biochem Biophys Res Commun 313(4): 1015‐1022.

Tanito, M., H. Masutani, et al. (2002). "Cytoprotective effect of thioredoxin against retinal photic
injury in mice." Invest Ophthalmol Vis Sci 43(4): 1162‐1167.

Tanito, M., A. Nishiyama, et al. (2002). "Change of redox status and modulation by thiol
replenishment in retinal photooxidative damage." Invest Ophthalmol Vis Sci 43(7): 2392‐
2400.

Tosi, J., R. J. Davis, et al. (2011). "shRNA knockdown of guanylate cyclase 2e or cyclic nucleotide
gated channel alpha 1 increases photoreceptor survival in a cGMP phosphodiesterase
mouse model of retinitis pigmentosa." J Cell Mol Med 15(8): 1778‐1787.

109

Tosi, J., J. Sancho‐Pelluz, et al. (2011). "Lentivirus‐mediated expression of cDNA and shRNA slows
degeneration in retinitis pigmentosa." Exp Biol Med (Maywood) 236(10): 1211‐1217.

Travers, K. J., C. K. Patil, et al. (2000). "Functional and genomic analyses reveal an essential
coordination between the unfolded protein response and ER‐associated degradation."
Cell 101(3): 249‐258.

Travis, G. H. (1998). "Mechanisms of cell death in the inherited retinal degenerations." Am J
Hum Genet 62(3): 503‐508.

Tsang, S. H., P. Gouras, et al. (1996). "Retinal degeneration in mice lacking the gamma subunit of
the rod cGMP phosphodiesterase." Science 272(5264): 1026‐1029.

Turner, B. J. and J. D. Atkin (2006). "ER stress and UPR in familial amyotrophic lateral sclerosis."
Curr Mol Med 6(1): 79‐86.

van Soest, S., A. Westerveld, et al. (1999). "Retinitis pigmentosa: defined from a molecular point
of view." Surv Ophthalmol 43(4): 321‐334.

Walter, P. and D. Ron (2011). "The unfolded protein response: from stress pathway to
homeostatic regulation." Science 334(6059): 1081‐1086.

Wei, J. Y., E. D. Cohen, et al. (1998). "Substituted cGMP analogs can act as selective agonists of
the rod photoreceptor cGMP‐gated cation channel." J Mol Neurosci 10(1): 53‐64.

Wei, J. Y., E. D. Cohen, et al. (1996). "Identification of competitive antagonists of the rod
photoreceptor cGMP‐gated cation channel: beta‐phenyl‐1,N2‐etheno‐substituted cGMP
analogues as probes of the cGMP‐binding site." Biochemistry 35(51): 16815‐16823.

Wenzel, A., C. Grimm, et al. (2000). "c‐fos controls the "private pathway" of light‐induced
apoptosis of retinal photoreceptors." J Neurosci 20(1): 81‐88.

Wenzel, A., C. Grimm, et al. (2005). "Molecular mechanisms of light‐induced photoreceptor
apoptosis and neuroprotection for retinal degeneration." Prog Retin Eye Res 24(2): 275‐
306.

Wenzel, A., C. Grimm, et al. (2005). "Molecular mechanisms of light‐induced photoreceptor
apoptosis and neuroprotection for retinal degeneration." Progress in retinal and eye
research 24(2): 275‐306.

110

Wenzel, A., C. E. Reme, et al. (2001). "The Rpe65 Leu450Met variation increases retinal
resistance against light‐induced degeneration by slowing rhodopsin regeneration." J
Neurosci 21(1): 53‐58.

Xu, J., R. L. Dodd, et al. (1997). "Prolonged photoresponses in transgenic mouse rods lacking
arrestin." Nature 389(6650): 505‐509.

Yamamoto, S., K. C. Sippel, et al. (1997). "Defects in the rhodopsin kinase gene in the Oguchi
form of stationary night blindness." Nat Genet 15(2): 175‐178.

Yang, J. H., S. F. Basinger, et al. (2003). "Blue light‐induced generation of reactive oxygen species
in photoreceptor ellipsoids requires mitochondrial electron transport." Invest
Ophthalmol Vis Sci 44(3): 1312‐1319.

Yang, L. P., L. M. Wu, et al. (2008). "Endoplasmic reticulum stress is activated in light‐induced
retinal degeneration." J Neurosci Res 86(4): 910‐919.

Yang, L. P., L. M. Wu, et al. (2008). "Endoplasmic reticulum stress is activated in light‐induced
retinal degeneration." Journal of neuroscience research 86(4): 910‐919.

Yang, L. P., L. M. Wu, et al. (2007). "Activation of endoplasmic reticulum stress in degenerating
photoreceptors of the rd1 mouse." Invest Ophthalmol Vis Sci 48(11): 5191‐5198.

Yau, K. W. (1994). "Phototransduction mechanism in retinal rods and cones. The Friedenwald
Lecture." Invest Ophthalmol Vis Sci 35(1): 9‐32.

Yau, K. W. and D. A. Baylor (1989). "Cyclic GMP‐activated conductance of retinal photoreceptor
cells." Annu Rev Neurosci 12: 289‐327.

Yau, K. W. and K. Nakatani (1984). "Cation selectivity of light‐sensitive conductance in retinal
rods." Nature 309(5966): 352‐354.

Yip, H. K. and K. F. So (2000). "Axonal regeneration of retinal ganglion cells: effect of trophic
factors." Prog Retin Eye Res 19(5): 559‐575.

Zhong, Y., J. Li, et al. (2012). "Activation of endoplasmic reticulum stress by hyperglycemia is
essential for Muller cell‐derived inflammatory cytokine production in diabetes."
Diabetes 61(2): 492‐504.

Abstract (if available)

Abstract Retinitis pigmentosa (RP) is a set of hereditary retinal diseases characterized by progressive retinal degeneration and eventual photoreceptor cell death, affecting 1 in 4000 for a total of about 15 million people in the world. There is currently no treatment for this debilitating retinal degenerative disorder. To develop clinical treatments, two fundamental questions need to be answered: first, what are the underlying mechanisms

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Elements of photoreceptor homeostasis: investigating phenotypic manifestations and susceptibility to photoreceptor degeneration in genetic knockout models for retinal disease

PDF

Exploring alternative roles of visual arrestin 1 in photoreceptor synaptic regulation and deciphering the molecular pathway of retinal degeneration using mouse knockout technology

PDF

Identification of novel protein-protein interactions and functional analysis in the mouse photoreceptor of the hypothetical protein FLJ33282-Als2cr4 and the small Rho GTPase, Rnd2

PDF

Visual arrestin interactions with clathrin adaptor AP-2 regulate photoreceptor survival

PDF

Characterization of human embryonic stem cell derived retinal pigment epithelial cells for age-related macular degeneration

PDF

Essential role of the carboxyl-terminus for proper rhodopsin trafficking and "enlightenment" to the pathway(s) causing retinal degeneration in a mouse model expressing a truncated rhodopsin mutant

PDF

An investigation of dark adaptation: the role of metabolism and alternative rod pathways in shaping visual sensitivity following bright light

PDF

Cone arrestin 4 contributes to vision, cone health, and desensitization of the dopamine receptor D4

PDF

Understanding the degenerate retina's response to electrical stimulation: an in vitro approach

PDF

Before they were amyloid: understanding the toxicity of disease-associated monomers and oligomers prior to their aggregation

PDF

Protein phosphatase 2A and annexin A5: modulators of cellular functions

PDF

Utilizing zebrafish and mouse models to uncover the underlying genetics of human craniofacial anomalies

PDF

Genetic and molecular analysis of a rhythmic behavior in C. elegans: how neuropeptide signaling conveys temporal information

PDF

LIGHT-ing up prostate cancer: remodeling the tumor microenvironment and enhancing therapeutic vaccine efficacy through forced LIGHT expression in prostate cancer

PDF

The role of HES/HEY transcriptional repressors in specification and maintenance of cell fate in the mouse organ of Corti

PDF

The mechanisms of somatic cell reprogramming

PDF

An analysis of disease-free survival and overall survival in inflammatory breast cancer

PDF

New understanding of rhodopsin in retinal degeneration and high gain phosphorylation

PDF

Application of genetic association methods in mice to understand phenotypes with a complex etiology

PDF

RPE secretome for the treatment of retinal degeneration in the RCS rat

Asset Metadata

Creator Wang, Tian (author)

Core Title Mechanisms of retinal degeneration caused by genetic and environmental factors

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publication Date 04/23/2015

Defense Date 03/18/2013

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag OAI-PMH Harvest,Retinal degeneration

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Sampath, Alapakkam P. (committee chair), Chen, Jeannie (committee member), Crump, Gage D. (committee member)

Creator Email tianwang@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-242739

Unique identifier UC11287957

Identifier etd-WangTian-1583.pdf (filename),usctheses-c3-242739 (legacy record id)

Legacy Identifier etd-WangTian-1583.pdf

Dmrecord 242739

Document Type Dissertation

Rights Wang, Tian

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA