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

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

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EXPLORING ALTERNATIVE ROLES OF VISUAL ARRESTIN 1 IN
PHOTORECEPTOR SYNAPTIC REGULATION AND DECIPHERING THE
MOLECULAR PATHWAY OF RETINAL DEGENERATION USING MOUSE
KNOCKOUT TECHNOLOGY

by

Shun-Ping Huang

_______________________________

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETICS, MOLECULAR and CELLULAR BIOLOGY)

August 2010

Copyright 2010 Shun-Ping Huang

Dedication
This thesis is dedicated to my grandparents and my parents whose love and
devotion in life made it possible for me to pursue my graduate degree, to my little
auntie for believing in me, teaching me always follow my dream and being there
when I needed her and to my sisters for supporting and encouraging me to be a
good scientist.
ii

Acknowledgements

I wish to express my deep and heartfelt gratitude to my mentor Dr. Cheryl M.
Craft, who is the most important person to guide me along my path. I am
especially indebted for her invaluable comments and unfailing patience,
professionalism and wisdom. She has provided a wonderful environment to
explore many exciting questions. Her guidance and encouragement has enabled
me to complete my dissertation work successfully. My thesis committee, Dr. David
R. Hinton, Dr. Austin K. Mircheff, Dr. Alapakkam P. Sampath and Dr. Chien-Ping
Ko, have always been ready to provide helpful suggestions and constructive
criticism. Their input in my thesis work helped get much of material published
During my time in the Mary D. Allen Laboratory for Vision Research, many
people within the Craft lab have provided guidance and shared their experiences.
I thank Mr. Bruce Brown for responsible animal handling and expert retinal
surgery. I am very grateful to Freddi Zuniga and Rosanne Yetemian for their
friendship and support, and both Mrs. Gerda Goette and. Mrs. Gloria Arcieniega
who have been extremely helpful throughout my time in the lab.
iii

iv

Thanks to my grandparents, my parents, uncles, aunts and my sisters for
being the best family I could ever hope for. They have always given me their love
and support throughout my life. I would like to take this opportunity to express my
deepest thanks to Dr. Shui-Mei Lee, Dr. Jorn-Hon Liu, Dr. Ten-Nan Lin and Dr. J.
Mark Petrash for their support and firm confidence in my ability and helped me to
persevere in academic pursuit.

Table of Contents

Dedication ii
Acknowledgements iii
List of Tables viii
List of Figures ix
Abbreviations xi
Abstract xiii
Chapter 1: Introduction 1
1.1 The phototransduction cascade 1
1.2 The arrestin superfamily 6
1.3 Photoreceptor ribbon synapse 12
1.4 N-ethylmaleimide sensitive factor 14
1.5 ARR1 in Oguchi disease and retinitis pigmentosa 18
1.6 Summary 20

Chapter 2: Methods and Experiments 22
2.1 Animals 22
2.2 Antibody pull-down assays and co-immunoprecipitation 22
2.3 Isolation of proteins for mass spectrometry analysis 24
2.4 Plasmid construction 25
2.5 GST pull-down and in vitro binding assay 26
2.6 Quantitative RT-PCR 27
2.7 Immunoblot analysis 30
2.8 Immunohistochemistry 31
2.9 Cell culture and Transfection 32
2.10 NSF ATPase activity assay 33
2.11 NSF disassembly activity assay 33
2.12 Evaluation of exocytosis rate using FM1-43 staining 34
v

2.13 Electroretinography (ERG) 35
2.14 TUNEL analysis of apoptosis 36
2.15 Affymetrix
TM
GeneChip Microarray hybridization and Ingenuity
Pathway Analysis (IPA)

37
2.16 Statistical analysis 39

Chapter 3: Arr1 acts as a modulator for NSF in photoreceptor
synaptic regulation

40
3.1 Introduction 40
3.2 Results 42
3.2.1 NSF is an interacting partner for Arr1 in the
photoreceptor synapse

42
3.2.2 The Arr1-NSF complex formation is enhanced by ATP 50
3.2.3 Arr1 increases NSF ATPase activity and NSF-driven
SNARE complex disassembly

54
3.2.4 Arr1 deletion markedly reduces the expression level of
synapse-enriched proteins

59
3.2.5 Arr1 deletion strongly suppresses the synaptic activity
in the photoreceptor synapse

63
3.3 Discussion 68

Chapter 4: Gene analysis profiles of light-independent photoreceptor
loss in visual arrestin 1 knockout mice

75
4.1 Introduction 75
4.2 Results 77
4.2.1 Age-related light-independent photoreceptor loss in
Arr1
-/-
mice

77
4.2.2 Verification of Affymetrix exon array data 81
4.2.3 Biological functional groups and pathway analysis 83
4.2.4 Induction of early complement pathway components in
Arr1
-/-
retina

88
4.2.5 Early expression of Annexin A1 in Arr1
-/-
retina 90
4.2.6 Activation of oncostatin M and JAK/STAT3 signaling in
Arr1
-/-
retina

94
4.2.7 Highly induction of Edn2 and Serpin A3n transcript in
Arr1
-/-
retina

95
4.2.8 Activation of reactive gliosis in Arr1
-/-
mice 97
4.3 Discussion 101

vi

Chapter 5: Conclusions 107
Bibliography 111
Appendices:
Appendix A. Up-regulated transcripts in Arr1
-/-
retina 129
Appendix B. Down-regulated transcripts in Arr1
-/-
retina 142

vii

LIST OF TABLES

Table 2.1 Quantitative RT-PCR primer pair sequences 37
Table 4.1 Selective up-regulated transcripts in Arr1-/- retinas
compared with WT

82
Table 4.2 Comparison of top biological pathways identified as
statistically significant between Arr1
-/-
and WT

83
Table 4.3 Comparison of Top Associated Network Functions Identified
by IPA Analysis between Arr1
-/-
and

WT

86

viii

LIST OF FIGURES

Figure 1.1 Phototransduction cascades in rod photoreceptor 4
Figure 1.2 Light-dependent translocation of Arr1 in the
photoreceptor
8
Figure 1.3 Photoreceptor ribbon synapse 13
Figure 1.4 Schematic representation of the role of NSF in vesicle
transport

16
Figure 3.1 Retinal homogenates were immunoprecipitated with
anti-Arr1 MAb D9F2 and bands were identified by mass
spectrometry

43
Figure 3.2 NSF was identified by mass spectrometry 44
Figure 3.3 NSF co-immunoprecipitated with Arr1 in mouse retina 46
Figure 3.4 Cone Arrestin did not interact with NSF 47
Figure 3.5 Immunohistochemical fluorescent labeling of NSF and
Arr1 in the WT mouse retinas

49
Figure 3.6 Functional analysis of the interaction between Arr1 and
NSF

51
Figure 3.7 Association of Arr1 with NSF in COS-7 Cells 53
Figure 3.8 Arr1-NSF complex formation is ATP-dependent 55
Figure 3.9 NSF ATPase activity assay 56
Figure 3.10 SNARE complex disassembly assay 58
Figure 3.11 Quantitative RT-PCR of NSF, vGLUT1, VAMP2 and
EAAT5 in the retinas

61
ix

Figure 3.12 Immunoblots analysis of NSF, vGLUT1, EAAT5,
VAMP2, SNAP-25

63
Figure 3.13 Synaptic uptake of FM1-43 revealed a decrease of
synaptic activity in the Arr1
-/-
mouse retina

65
Figure 3.14 Figure 3.14 ERG analysis of WT, Arr1
-/-
, Arr4
-/-
, and
mCAR-H
arr1-/-
mice

67
Figure 4.1 TUNEL staining of the dark-adapted WT and Arr1
-/-

retina at different time points

79
Figure 4.2 Maximum b-wave amplitude of photopic ERG
responses in WT and Arr1
-/-
mice

80
Figure 4.3 Comparison of top canonical pathways identified by IPA
analysis between Arr1
-/-
and WT

85
Figure 4.4 Schematic representation of the hypothetical network
for photoreceptor degeneration in Arr1
-/-
mice

87
Figure 4.5 Induction of early complement pathway components in
Arr1
-/-
retina

89
Figure 4.6 Confirmation of differential transcription expression of
serping1, C3, C3ar1 and C4b using quantitative
RT-PCR (qPCR)

91
Figure 4.7 Early induction of Anxa1 in Arr1
-/-
retina with
photoreceptor degeneration

93
Figure 4.8 Activation of oncostatin M receptor and STAT3 signaling
in Arr1
-/-
retina

95
Figure 4.9 High induction of Edn2 and Serpina3n transcript in
Arr1
-/-
retina

97
Figure 4.10 A Activation of Muller glial cells in Arr1
-/-
retina 99
Figure 4.10 B Activation of Muller glial cells in Arr1
-/-
retina 100

x

Abbreviations

ANOVA: Analysis of variance
Arr1: Visual Arrestin 1
Arr4: Cone Arrestin
ATP: Adenosine triphosphatae
cDNA: Cyclic deoxyribonucleic acid
EAAT5: Excitatory amino acid transporter 5
ERG: Electroretinography
GC: Guanylyl cyclases
GCAP: Guanylyl cyclase activating proteins
GFAP: Glial fibrillary acidic protein
GPCR: G-protein-coupled receptor
GRK1: G protein-coupled receptor kinase 1
JAK: Janus kinase
NSF: N-ethylmaleimide sensitive factor
PDE: Phosphodiesterase enzyme
xi

SNAP-25: soluble N-ethylmaleimide sensitive factor attachment proteins 25
SNARE: soluble N-ethylmaleimide sensitive factor attachment proteins receptors
STAT3: signal transducer and activator of transcription 3
VAMP2: Vesicle associated membrane protein 2
vGLUT1: Vesicular glutamate transporter 1
xii

Abstract

In the G-protein-coupled receptor (GPCR) phototransduction cascade,
visual Arrestin1 (Arr1) binds to and deactivates phosphorylated light-activated
opsins, a process that is critical for effective recovery and normal vision. In this
dissertation study, we discovered a novel synaptic interaction between Arr1 and
N-ethylmaleimide sensitive factor (NSF) that is enhanced in a dark environment
when photoreceptors are depolarized and the rate of exocytosis is elevated. In the
photoreceptor synapse, NSF functions to sustain a higher rate of exocytosis, in
addition to the compensatory endocytosis to retrieve and to recycle vesicle
membrane and synaptic proteins. Not only does Arr1 bind to the junction of NSF
N-terminal and first ATPase domains in an ATP-dependent manner, but Arr1 also
enhances both NSF ATPase and NSF disassembly activities. In mouse retinas
with no Arr1 expression, the expression levels of NSF and other
synapse-enriched genes are markedly reduced and lead to a substantial
decrease in the exocytosis rate. This study demonstrates a vital modulatory role
of Arr1 in the photoreceptor synapse and provides key insights into the potential
xiii

molecular mechanisms of inherited retinal diseases, such as Oguchi disease and
Arr1-associated retinitis pigmentosa.
Arr1
-/-
mice develop a light-dependent retinal degeneration and a
light-independent cone dystrophy. We observed increased terminal
deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), activation of
phosphorylated signal transducer and activator of transcription 3 (pSTAT3) in the
Janus kinase (JAK)-STAT3 pathway, reactive gliosis, and cone dystrophy in Arr1
-/-

mice. To further explore the molecular pathways leading to Arr1
-/-
-related
light-independent cone dystrophy, we compared controls and Arr1
-/-
with
Affymetrix exon array analysis and observed in Arr1
-/-
up-regulated retinal
transcripts including serping1, C4b, C3 and C3a receptor 1 (C3ar1), annexin
A1(anxa1), oncostatin M receptor, STAT3, endothelin2, and glial fibrillary acidic
protein (GFAP). Top canonical pathways reveal several potential pathways
involved in this cone dystrophy phenotype including complement system
activation, acute phase response signaling, oncostatin M signaling and
JAK3-STAT3 activation. Our data support Arr1 is crucial for regulating an
xiv

xv

endogenous defense mechanism to ensure survival and normal function of cone
photoreceptors.

Chapter 1. Introduction

1.1 The phototransduction cascade
There are two types of photoreceptors in the mammalian retina, rods and
cones. Rods contain the light-sensitive visual pigment rhodopsin; the prototypical
G-protein coupled receptor (GPCR) that mediates the visual response in dim light.
Cones mediate vision at higher light intensities and depending upon the species,
comprise two or more classes, each containing a visual pigment with a distinctive
absorption spectrum.

Cascade Activation: Vision begins when absorbed photons are converted by the
G-protein-coupled phototransduction cascade into neural signals that are
processed within the retina and transmitted by the optic nerves to the visual
centers of the brain. Phototransduction in rod outer segments is initiated when the
photoreceptor protein rhodopsin captures a photon of light converting the 11-cis
retinal chromophore to all-trans retinal. This reaction leads to a conformational
change in rhodopsin to its activated form, metarhodopsin II (R*) (Yau, 1994;Baylor,
1

1996;Jindrova, 1998;Shichida and Imai, 1998). R* interacts with a heterotrimeric
G protein transducin (Gαβγ), causing the exchange of bound GDP for GTP and
the subsequent dissociation of the complex into Gα-GTP and Gβγ, which binds to
phosducin (Figure 1.1 Middle). G α* in turn activates the phosphodiesterase
enzyme (PDE*) and the resulting decrease in cGMP leads to the closure of
cGMP-gated channels in the plasma membrane of rod outer segments to the
influx of Na
+
and Ca
2+
ions and a hyperpolarization of the rod photoreceptors.
Hyperpolarization in the rod terminal diminishes the influx of Ca
2+
into the synaptic
terminal and decreases the release of synaptic transmitter into the synapse
connecting to bipolar cell.
Cascade inactivation: A single R* can activate sequentially hundreds of
transducin molecules. Active cGMP-PDE hydrolyzes large numbers of cGMP
molecules. Thus, the signal amplification provides high sensitivity but requires
mechanisms to rapidly shut down the light-activated receptor by the combined
effect of rhodopsin kinase (G protein-coupled receptor kinase 1, GRK1) followed
by Arr1 (Figure 1.1 Bottom), which prevents further activation of transducin.
2

P-R*-Arr1 is, in turn, attacked by retinol dehydrogenase to reduce the all-trans
retinal in R* to all-trans retinol, which is released from the complex. This reaction
causes a release of arrestin from phosphorhodopsin (P-R), which is slowly
dephosphorylated by a protein phosphatase 2A complex (PP2A) (Brown et al.,
2002) and regenerated to rhodopsin by binding with newly synthesized
11-cis-retinal. PDE*/Gα* turns itself off by the intrinsic GTPase activity of Gα*,
which is substantially enhanced by the cooperative action of RGS9-1 and Gβ5-L
(He et al., 1998;Makino et al., 1999). Intracellular cGMP is re-synthesized by
retinal guanylyl cyclases (GC), whose activities are accelerated by guanylyl
cyclase activating proteins (GCAPs) in a Ca2+-dependent manner. The increase
in cGMP levels results in a reopening of cGMP-gated channels and a return of the
photoreceptor cell to its dark, depolarized state (Figure 1.1 Top).
3

Figure 1.1 Phototransduction cascades in rod photoreceptor. (Modified from Burn ME
et al., Neuron, 2005, 48(3)387-401 with permission)

Dysfunctional Signal Termination Leads to Photoreceptor Death: Numerous
genes encoding specific proteins participate in the phototransduction cascade
and have been shown to be responsible for retinal degeneration, including genes
of transcription factors, genes involved in photoreceptor metabolism and
structural support (Travis, 1997;Lev, 2001). Termination of the phototransduction
cascade is critical for cell survival. Defects in rod phototransduction can lead to
continuous signaling equivalent to that produced by light. Constitutive activation of
4

phototransduction is thought to ultimately lead to photoreceptor cell death (Fain
and Lisman, 1993). Photoreceptor degeneration by this proposed mechanism has
been studied with both genetic mutants and prolonged light exposure. Continual
activation of transducin by opsin unbound to chromophore in RPE65
-/-
mice,
which is defective in producing the 11-cis-retinal chromophore (Grimm et al.,
2000;Wenzel et al., 2001;Woodruff et al., 2003), and mutants in rhodopsin
interfere with arrestin binding and leads to photoreceptor cell death (Fain and
Lisman, 1993). Morphological assessments in Arr1
-/-
retinas after light exposure
effectively demonstrate the loss of rod photoreceptor cells (Chen et al., 1999). In
null mutants of transducin, the G-protein necessary to propagate the rhodopsin
receptor signal, photoreceptor cell death was not observed (Calvert et al., 2000).
These studies indicate that the retinal degeneration results from constitutive
activation of phototransduction cascade. Restoration of proper phototransduction
termination will lead to recovery of photoreceptor. Understanding the molecular
mechanism of photoreceptor cell death may help to identify similar mechanisms in
retinitis pigmentosa (Lisman and Fain, 1995), age related macular degeneration,
and other retinal dystrophies.
5

1.2 Arrestin Superfamily
There are four distinct members in the arrestin superfamily expressed in
vertebrates (Craft and Whitmore, 1995). Visual Arrestin 1 (Arr1) is expressed in
both rod and cone photoreceptors, while Arrestin 4 (Arr4, cone or X-arrestin) is
expressed in cone photoreceptors (Nikonov et al., 2008). Both visual arrestins are
also highly expressed in pinealocytes (Craft et al., 1994). β-arrestin 1 and
β-arrestin 2 are ubiquitously expressed and mediate the desensitization of
β-adrenergic receptors and many other GPCRs (Lefkowitz et al., 1992).

a. Visual Arrestin 1 (rod arrestin or S-antigen)
Visual Arrestin1 (Arr1) has been studied extensively during recent years and
has differential mRNA and protein isoforms regulated by light and circadian
rhythms (Craft et al., 1990). Further work has demonstrated Arr1’s function, which
is to quench light-activated, transducin-mediated phototransduction in rod
photoreceptors by specific binding to the phosphorylated rhodopsin (Kuhn,
1984;Gurevich and Benovic, 1992;Gurevich and Benovic, 1993;Gurevich et al.,
1994;Gurevich and Benovic, 1995;Gurevich et al., 1995;Krupnick et al., 1997).
6

One principal binding region of arrestin with phosphorylated rhodopsin is
contained within the regions of residues 109-130 (number in bovine arrestin).
Arrestin has also been found to participate in a molecular pathway for
light-induced photoreceptor apoptosis in Drosophila through the formation of
stable rhodopsin-arrestin complexes that are recruited to the cytoplasmic
compartment through clathrin-dependent endocytosis (Alloway and Dolph,
1999;Alloway et al., 2000;Kiselev et al., 2000;Miller and Lefkowitz, 2001).
In dark-adapted photoreceptors, Arr1 localizes to the inner segment,
perinuclear region, and synaptic terminals (Figure 1.2), whereas light exposure
induces translocation of Arr1 to the outer segments and contributes to
light-adaptation (Broekhuyse and Winkens, 1985;Philp et al., 1987;Whelan and
McGinnis, 1988;Arshavsky, 2003;Brown et al., 2010a). The light-driven
translocation of rod arrestin is unaffected in several mouse models in which
rhodopsin phosphorylation is lacking. Also, transducin signaling is not required for
the light-induced redistribution of rod arrestin (Mendez et al., 2003;Zhang et al.,
2003). However, rhodopsin activation is required to initiate the movement or rod
arrestin translocation because, in RPE65
-/-
mice that are deficient in rhodopsin, no
7

light-dependent arrestin translocation was observed (Mendez et al., 2003). These
results suggest the presence of another light-dependent pathway to trigger
translocation in rod photoreceptor cell.

Figure 1.2 Light-dependent translocation of Arr1 in the photoreceptor. (Modified from
SlepakVZ and Hurley JB, IUBMB Life. 2008 Jan; 60(1):2-9) Reprint with permission.

A truncated form (44kDa) of arrestin, called p44, was isolated from fresh
bovine rod outer segments and binds unphosphorylated, phototoactivated
rhodopsin (Palczewski et al., 1994;Smith et al., 1994;Smith, 1996;Pulvermuller et
al., 1997). This p44 isoform of arrestin undergoes dramatic translocation to the
lipid rafts of the membrane upon illumination (Nair et al., 2002). Under dim light
8

conditions, virtually all arrestin is in the rod inner segment and the splice variant
p44 (Arr(1-370A) is the stop protein responsible for receptor
deactivation(Schroder et al., 2002). The p44 not only interacts with the
phosphorylated active receptor but also with inactive phosphorylated rhodopsin or
opsin in membranes or solution. Because of the latter interaction, p44 is not
soluble but is membrane-bound in the dark. Upon photoexcitation, p44 switches
form inactive to active prephosphorylated and phosphorylated rhodopsin faster
than arrestin and starts to quench transducin activation on a subsecond time
scale. This mechanism provides a new aspect of receptor shutoff in the single
photon operating range of the rod cell (Schroder et al., 2002).
Based on its 3.3 Å X-ray crystal structure, arrestin from bovine rod outer
segments comprises two domains of anti-parallel β-sheets connected through a
hinge region and one short a-helix on the back of the amino terminal fold (Granzin
et al., 1998). The resolution of the crystal structure of bovine rod arrestin to 2.8 Å
(Hirsch et al., 1999) placed into perspective the functional domains and the
significance of specific residues. The salient feature of the structure is a bipartite
molecule with an unusual polar core, which is partially stabilized by an extended
9

carboxyl-terminal tail that locks the molecule into an inactive state. Intrusion of the
phosphate moiety on the C-terminal end of the rhodopsin receptor into polar core
region of arrestin disrupts the electrostatic interactions and leads to a
rearrangement in the structure of arrestin into an active state enabling receptor
binding. In the crystal state, rod arrestin exhibits a tetrameric arrangement,
wherein an asymmetric dimer with an extensive interface between
conformationally different subunits is related to a second asymmetric dimer by a
local two-fold rotation axis. The self-association of rod arrestin may provide a
mechanism for regulation of arrestin activity (Schubert et al., 1999).

b. β-arrestin1 and β-arrestin2
The non-visual arrestins, β-arrestin1 and β-arrestin2 are ubiquitous proteins
predominantly localized in neuronal tissues and spleen. Originally discovered as
proteins that mediate the desensitization of β-adrenergic receptors and many
other GPCRs, β-arrestins also function as GPCR signal transducer with an
ever-increasing list of additional functions. Moreover, β-arrestin 1 also directs
receptors to coated pits for internalization and switches signaling to alternative
10

pathways (Gagnon et al., 1998;Orsini and Benovic, 1998;Lefkowitz and Shenoy,
2005) such as the mitogen-activated protein kinase (MAPKs) ERK1 and 2 (Luttrell
et al., 1999;Miller et al., 2000;Defea et al., 2000;Luttrell and Lefkowitz, 2002), the
c-Jun amino terminal kinase (JNK3)(McDonald et al., 2000) and the tyrosine
kinase c-Src.

c. Cone arrestin (Arr4)
Cone arrestin (Arr4), distinct from Arr1, was initially discovered from cone
photoreceptors and a subset of pinealocytes using a targeted predicted arrestin
functional anchor domain screening strategy (Craft et al., 1994). Craft and her
collaborators (Zhu et al., 2003) demonstrated that light-dependent
phosphorylation of both cone S- and M-opsins occurred in mouse retina and in the
absence of Grk1 neither phosphorylation of cone opsins nor Arr4 binding was
detectable. Similar to Arr1, Arr4 also showed light-dependent but
phosphorylation-independent translocation to cone outer segment in mouse retina
(Zhu et al., 2002).

11

Later, it was shown that both Arr1 and Arr4 function in arresting the activity
of either S-opsin or M-opsin for a fully normal recovery (Nikonov et al., 2008)

1.3 Photoreceptor ribbon synapse
Photoreceptor ribbon synapse is a specialized structure that allows the
photoreceptor to sustain the continuous release of vesicles and sense and
transmit sensory stimuli over a broad range of stimulus intensities.
Photoreceptors are tonically depolarized in the dark with membrane potentials of
-35 to -45mV that allow graded release of the neurotransmitter, L-glutamate. The
continuous cycles of exocytosis and endocytosis require structural and functional
specializations. Photoreceptor synaptic ribbons are large presynaptic structures
associated with active zones and constitute a plate-like structure (Figure 1.3) of
large surface area that extends from the site of neurotransmitter release into the
presynaptic cytoplasm. A large number of regularly aligned synaptic vesicles are
tethered to the presynaptic vesicle release sites where voltage-dependent
calcium channels are concentrated (Rao-Mirotznik et al., 1995;von Gersdorff,
2001).
12

Photoreceptor synaptic ribbons are considered to dynamically tune the
synaptic vesicles cycle in adjustable, efficient and precise machinery.

Figure 1.3 Photoreceptor Ribbon Synapse. (Modified from Dieck ST, Cell and Tissue
Research, 2006, 326(2) Reprinted with permission

The small terminals of the rod photoreceptor synapses contain a single
active zone and a single, large planar synaptic ribbon to which a total of ~770
synaptic vesicles bind. The rod synaptic ribbon is approximate 0.4 μm in height
and bends around four deeply invaginating postsynaptic elements, dendrites of
bipolar cells and processes of horizontal cells, at an active zone length of ~ 2 μm.
13

Cone photoreceptor synapses are usually larger than the rod terminals and
contain more ribbons than rod (usually 10-12 ribbons per terminal; in some
species, up to 50) with shorter active zones (~1 μm long, 0.2 μm high) contacted
by invaginating postsynaptic elements (Sterling and Matthews, 2005). The light
responses of cones are faster than those of rods and synaptic transmission from
cones is also faster than transmission from rods (tom and Brandstatter, 2006).
The total ribbon surface and the pool of ribbon-associated synaptic vesicles in
cone synapses is considerably larger than in rod synapses, which is likely a
consequence of higher synaptic demands in cone photoreceptors (Heidelberger
et al., 2005;Sterling and Matthews, 2005;Thoreson, 2007). Because all of the
visual information available to downstream retinal neurons must first pass through
the photoreceptor synapse, synaptic transmission from photoreceptors has a
considerable impact on visual perception.

1.4 N-ethylmaleimide sensitive factor
Vesicle traffic and fusion are essential, not only for cellular homeostasis but
also for neuronal signal transmission across the synaptic junction of nerves, cell
14

growth, and membrane repair. Exocytotic release of neurotransmitter from
synaptic vesicles is an exquisitely regulated form of intracellular membrane fusion.
The basic fusion process is mediated by vesicle (v)-soluble N-ethylmaleimide
sensitive factor attachment proteins receptors (SNAP receptors; SNAREs) on the
secretory vesicle with their cognate target (t)-SNAREs on the target membrane
(Sollner et al., 1993), which assemble in trans-configuration into four-helix bundle
complexes, bringing the membranes into close proximity (Jahn and Scheller,
2006;Rizo and Rosenmund, 2008;Martens and McMahon, 2008), which then
directly or indirectly leads to fusion. After membrane fusion, all SNAREs
constituting one complex are anchored in a relaxed cis-configuration to one
membrane. Disassembly of these SNAREs complexes for subsequent vesicle
transport and recycling is achieved by the concerted action of α-SNAP and NSF.
N-ethylmaleimide sensitive factor (NSF) is a homo-hexameric member of
the ATPase associated with various cellular activities protein (AAA) family, broadly
required for intracellular membrane fusion. NSF functions as a SNAP receptor
(SNARE) chaperone that binds through soluble NSF attachment proteins
(SNAPs), to SNARE complexes and utilizes the energy of ATP hydrolysis to
15

dissociate SNARE complexes after membrane fusion thus facilitating SNAREs
recycling (Figure 1.4).

Figure 1.4 Schematic representation of the role of NSF in vesicle transport. Vesicles
dock and fuse with the target membrane, releasing their cargo. α-SNAP and NSF bind to
SNARE complexes to form the 20 S complex. Upon hydrolysis of ATP, the individual
components of the SNARE complex are released. (Modified from May AP et al., J Biol
Chem. 2001 Jun22; 176(25):21991-4)
Each NSF protomer contain an N-terminal domain (NSF-N) and two
AAA-domains; a catalytic NSF-D1 and a structural NSF-D2: NSF-N (residue
1-205) is required for SNAP-SNARE binding; NSF-D1 (residue 206-477) accounts
for the majority of the ATP hydrolysis; NSF-D2 (residue 478-744) is required for
16

hexamerization (Tagaya et al., 1993;Nagiec et al., 1995). Within the N-terminal
subdomain of both NSF-D1 and NSF-D2, there is a highly conserved region
called Second Region of Homology (SRH), which is highly conserved in AAA
proteins (Hanson and Whiteheart, 2005). By using detailed mutagenesis analysis,
Zhao and collaborators (Zhao et al., 2010) showed that a positively-charged
surface on NSF-N, bounded by R67 and K105, and the conserved central pore
motifs in NSF-D1 (Y296 and G298) are involved in SNAP-SNARE binding but not
basal ATP hydrolysis. Sensor 1 is at the N-terminus of the SRH and is important
for basal ATPase activity and nucleotide binding. At its C-terminus are two
arginine residues termed Arginine Fingers, which are critical for ATP hydrolysis by
NSF hexamer. Sensor 2 comes from C-terminal helical subdomain and plays a
role in ATP- and SNAP-dependent SNARE complex binding and disassembly.
NSF binds to SNARE complexes via its adaptor protein, a-SNAP, only in the
presence of ATP (Nagiec et al., 1995). The intrinsic ATPase activity of NSF is very
low (Tagaya et al., 1993). Binding to immobilized a-SNAP stimulates the ATPase
activity (Morgan et al., 1994) and maximal stimulation of ATPase activity is

17

achieved when both α-SNAP and SNARE complexes are included (Matveeva and
Whiteheart, 1998).
While the major function of NSF is involved in vesicle transport and
recycling, it does seem to interact with other proteins such as the AMPA receptor
subunit (Osten et al., 1998), β-arrestin1(McDonald et al., 1999), GluR2
(Nishimune et al., 1998) and β2-AR (Cong et al., 2001) and is thought to affect
their trafficking pattern. More evidences suggest that NSF may be regulated by
transient post-translational modifications such as phosphorylation and
nitrosylation. These modifications are generally offered as an ideal mechanism for
reversible regulation of membrane trafficking.

1.5 ARR1 in Oguchi disease and retinitis pigmentosa
Oguchi disease, first reported by Oguchi in 1907 (Oguchi, 1907), is a rare
autosomal recessive form of congenital stationary night blindness, which is
characterized by abnormally slow dark adaptation after light exposure and diffuse
yellow or grey discoloration of the fundus which disappears after prolonged dark
adaptation (Mizuo-Nakamura phenomenon)(Mizuo, 1913), along with
18

characteristic electroretinographic (ERG) abnormalities. Patients generally have
normal day time visual acuity, visual fields, and color vision.

Full-field ERGs show
the absence of rod b waves after 30 minutes of dark adaptation, and nearly
normal a waves and extremely reduced b waves (negative ERG results) in
standard combined responses. In contrast, the single-flash cone and 30-Hz flicker
responses are within normal limits. The regeneration of rhodopsin is reported to
be approximately normal in patients with Oguchi disease but the time-course of
dark adaptation is some 8- to 10-fold slower than in normal subjects.
Oguchi disease presents with genetic heterogeneity. In the Japanese and
European population, several cases have been reported in which the causative
mutations are either in the ARR1 gene, or in the G-protein coupled receptor
kinase 1(GRK1) gene and both are responsible for deactivation of photoactivated
rhodopsin in the phototransduction cascade and for dark adaptation. Some
reports provide evidence that the ARR1 null mutation is causally related not only
to Oguchi disease but also to autosomal recessive retinitis pigmentosa
(Nakazawa et al., 1998;Nakamachi et al., 1998;Isashiki et al., 1999), which is

19

characterized as progressive night blindness, narrowing of the visual field,
reduced central vision and complete vision loss in later life.

1.6 Summary
In this thesis, we concentrated on investigating the alternative roles and
functions of Arr1 in the photoreceptor synapse and understanding the
pathological molecular mechanisms of light-independent retinal degeneration in
visual Arr1 knockout mice by employing a combination of genetic,
electrophysiological, and biochemical techniques.
In Chapter 3, we demonstrate a protein-protein interaction between Arr1 and
N-ethylmaleimide sensitive factor (NSF), an ATPase with diverse cellular activities,
which is critical for soluble NSF attachment protein receptor (SNARE) complex
disassembly in membrane trafficking events, including neurotransmitter release.
Arr1 co-immunoprecipitates with NSF and deletion analysis maps the relevant
binding site to the junction of two functional domains of NSF. We also provide
evidence supporting the interaction of Arr1 with NSF to modulate its ATPase
activity and to drive disassembly of the SNARE complex. Finally, we observe
20

that synaptic vesicle recycling in photoreceptors is dramatically decreased and
analysis of the photopic electroretinogram (ERG) b-wave also demonstrates a
light adaptation defect in Arr1
-/-
mice (Brown et al., 2010a). These cumulative
findings demonstrate that normal photoreceptor synaptic function involves the
ability of Arr1 to regulate and to enhance the dark-associated activity of NSF such
that photoreceptor synaptic vesicle demand is met efficiently in these specialized
sensory cells.
In Chapter 4, the investigation is focused on the findings that the retinas of
Arr1
-/-
mice show progressive cone dystrophy independent of light exposure in our
previous study (Brown et al., 2010a) . This degeneration is particularly evident
beyond 2 months of age. Evaluation of differential gene and protein expression
between WT and Arr1
-/-
retina is one approach used to identify pathogenic
pathways involved in this cone dystrophy.
21

Chapter 2. Methods and Experiments

2.1 Animals
Mice were dark-reared in the USC Vivarium following the appropriate
established guidelines of the National Institutes of Health and adapted by the
Institutional Animal Care and Use Committee of the University of Southern
California. Breeding pairs of the Arrestin1 (Arr1
-/-
) knockout mice were
generously provided by Dr. Jeannie Chen (USC) (Chen et al., 1999;Burns et al.,
2006). Colony control (C57Bl/6J and SVJ129 [WT] mixed genetic background)
mice were created by breeding heterozygote littermates of the Arr1
+/-
Arr4
+/-

knockout mice. Their offspring were verified by genotyping for both Arr1
+/+
and
Arr4
+/+
and used as breeders (WT)(Nikonov et al., 2008).

2.2 Antibody pull-down assays and co-immunoprecipitation
To verify the potential physiological interaction of Arr1 with NSF, antibody
pull-down assays and co-immunoprecipitation experiments were carried out as
described (Zhu et al., 2003). The reactions were performed in both light and
22

dark (infrared or dim red light) conditions. Briefly, retinas from WT mice were
homogenized in lysis buffer (50mM Tris-HCl pH 8.0, 1% Triton X-100, 150mM
NaCl with 1X proteinase inhibitor cocktail (Roche, Indianapolis, IN), sonicated on
ice for 30 sec, incubated at 4°C with gentle shaking for 30 min, and centrifuged at
13000g for 15 min. Equal volumes of supernatant were precleared for 1 hr
incubation with 50μl of a 50% slurry of protein-G agarose (KPL, Inc). After
centrifugation and the Arr1 in the supernatant was immunoprecipitated using 10μl
of mouse monoclonal antibody (MAb) D9F2 specific for Arr1 (AA
361-369/PEDPDTAKE). The tubes were incubated at 4°C overnight and then
incubated with 50μl protein-G agarose at 4°C for 2 hrs. The anti-rabbit mouse
cone arrestin (mCar-LUMIj) polyclonal antibodies (PAb)(Zhu et al., 2002) was
used as a nonspecific antibody control. The pellets were washed five times with
the lysis buffer containing no proteinase inhibitor. The immunoprecipitated
proteins were eluted after boiling for 5 min in SDS-PAGE sample buffer and
subjected to 10% SDS-PAGE followed by transfer to polyvinylidene difluoride
membrane (PVDF) membrane. Immunoblot analysis was performed using a PAb
specific for NSF (1:10,000, Upstate, 07-364), and NSF was visualized using the
23

enhanced luminol-based chemiluminescent (ECL) system (Amersham
Biosciences). To confirm equal loading of Arr1, the PVDF membrane was stripped
and reprobed to visualize Arr1 using an anti-rabbit PAb C10C10 (1:10,000, AA
293-301/RERRGIALD), which was developed and characterized in our lab. This
antibody is specific for Arr1 and is similar to the results with MAb D9F2.

2.3 Isolation of proteins for mass spectrometry analysis
The identification of interacting partners for Arr1 was performed by liquid
chromatography-tandem mass spectrometry at the USC School of Pharmacy
Proteomics Core Facility. Proteins were prepared and analyzed by mass
spectrometry using a method similar to that described in Gallaher TK et
al.(Gallaher et al., 2006). Briefly, the Arr1 interacting proteins were separated by
electrophoresis on 10% SDS-PAGE and the proteins were visualized by
Coomassie blue stain. Excised bands were destained and dehydrated and then
digested with trypsin at 37°C overnight. The supernatant was collected and
analyzed by liquid chromatography- tandem mass spectrometry. Protein
identification was carried out with the MS/MS search software Mascot 1.9 (Matrix
24

Science) with confirmatory or complementary analyses with TurboSequest as
implemented in the Bioworks Browser 3.2, build 41 (ThermoFinnegan).

2.4 Plasmid construction.
Mouse cDNA encoding NSF was amplified by PCR from Image Clone:
4506351(Open Biosystems, Huntsville, AL) with oligonucleotide primers sense
+NSF-f76 5’-ATGGCGGGCCGGACTATGCA-3’ and antisense -NSF-r2310
5’-TCAATCAAAGTCCAGGGGAC-3’ (NM_008740 mouse NSF complete coding
sequence) was subcloned into the PtrcHis-TOPO vector (Invitrogen, Carlsbad,
CA). Mouse Arr1, vGLUT1, α-SNAP, VAMP-2 cDNAs were PCR amplified with
specific 5’-sense and 3’-anti-sense primers and subcloned into the PtrcHis-TOPO
vector after PCR amplification from cDNA from total mRNA isolated from mouse
retina. The cDNA encoding the mouse NSF and Syntaxin 4 were subcloned as
fragments into the multiple cloning site using restriction endonucleases for
EcoRI-XhoI into the pGEX-4T1 vector (GE Healthcare, Piscataway, NJ). The
sequences of all constructs were confirmed by DNA sequencing. Recombinant

25

fusion proteins were purified on columns using the Histidine (His) tags with the
Bio-Rad Profinia purification system as recommended by the manufacturer.

2.5 GST pull-down and in vitro binding assay
To define the functional domain in NSF that interacts with Arr1, His
6
-tagged,
NSF-truncated segments of varying lengths (AA residues 1-744, 251-744,
197-744, and 1-205^478-744) and GST-tagged NSF1-250 and NSF1-197 were
constructed. GST-Arr1 proteins (3μg) were immobilized on glutathione-agarose
beads in 25 mM HEPES-KOH (pH 7.4), 200 mM KCl, 1% Triton X-100, 10%
glycerol and 1 mM DTT (buffer A) and then incubated with His
6
-NSF1-744,
251-744, 197-744 or 1-205^478-744 at 4°C for 1hr. GST-NSF1-250 or
GST-NSF1-197 proteins (3μg) were also immobilized on glutathione-agarose
beads in buffer A and then incubated with His
6
-Arr1 at 4°C for 1h. After six washes
in buffer A plus 2 mM ATP, 8 mM MgCl
2
(buffer B), bound proteins were eluted
with 20mM glutathione and detected by immunoblot analysis.
To evaluate the influence of the ATPase state of NSF on its direct interaction
with Arr1, GST-tagged Arr1[AA 1-403] (3 μg), or truncated Arr1[AA 1-191],
26

Arr1[AA 1-370] were immobilized on glutathione-agarose beads in buffer A.
Beads were washed twice with buffer B, or 2mM ATP- γ-S and 8mM MgCl
2
in the
presence of 1% BSA and incubated with 3μg His
6
-tagged NSF at 4°C for 1hr.
After four washes in buffer B without BSA, bound proteins were eluted with 20 mM
glutathione and detected by immunoblot analysis as described above. To
determine the effect of the Arr1 binding to NSF-ATPase activity, the same
procedure was performed in the presence of 8mM MgCl
2
, 10mMEDTA and 2mM
ATP or ATP-γ-S. Densitometric analysis was conducted using the ImageQuant TL

software (Amersham Biosciences).

2.6 Quantitative RT-PCR
Total RNA was prepared from individual frozen retinas using Trizol reagent
(Invitrogen, Carlsbad, CA). The cDNA made from 0.5μg total retina RNA was
prepared using a reverse transcription system from Invitrogen with oligo(dT)
20
.
Each quantitative RT-PCR reaction was set up in a final volume of 25μl containing
12.5μl SYBR Green from Superarray (Frederick, MD). Reactions were done in
triplicate on 96-well plates and quantified (LightCycler 480 Real-Time PCR
27

System; Roche). Data analysis was performed using the Light-Cycler Software
Version LCS480 1.2.0. The housekeeping gene, mouse
glyceraldehyde-3-phosphate dehydrogenase (mGAPDH), was used as the
reference to normalize the expression levels of the NSF, VAMP2, EAAT5 and
vGLUT1 transcripts. Values for retinas from light-adapted WT mice were set to 1.

28

Table 2.1 Quantitative RT-PCR primer pair sequences, sense/forward (+/f) and
antisense/reverse (-/r) (gene name, accession number; nucleotide numbers):
Gene Name Primer Sequence
Accession number;
Nucleotide numbers
+mGAPDH-f148 5’-ACCCCTTCATTGACCTCAACTACATGG-3’ NM_008084; 148-174
-mGAPDH-r303 5’-ATTTGATGTTAGTGGGGTCTCGCTCCT-3’ NM_008084; 277-303
+qNSF-f2158 5’-GCTCAGCAAGTCAAAGGGAA-3’ NM_008740; 2158-2177
-qNSF-r2248 5’-GGTACTCAGGATCCATCTGC-3’ NM_008740; 2229-2248
+qVAMP2-f213 5’-GTGGATGAGGTGGTGGACAT-3’ NM_009497; 213-232
-qVAMP2-r348 5’-GCTTGGCTGCACTTGTTTCAA-3’ NM_009497; 328-348
+vGLUT1-f1532 5’-GTGCAATGACCAAGCACAAG-3’ NM_182993; 1532-1551
-vGLUT1-r1601 5’-TAGTGCACCAGGGAGGCTAT-3’ NM_182993; 1582-1601
+EAAT5-f1250 5’-GCTCTGCTCATTGCGTTG-3’ NM_146255; 1250-1267
-EAAT5-r1317 5’-AGCAGGCACTTGAAGGTGAT-3’ NM_146255; 1298-1317

29

2.7 Immunoblot Analysis
Protein extracts from dark-adapted and light-adapted WT or Arr1
-/-
mouse
retina were prepared using NP-40 lysis buffer (50mM Tris-HCl pH7.6, 150 mM
NaCl, 1% NP-40 and 1mM EDTA). The protein concentrations were determined
using the bicinchoninic acid (BCA) protein assay kit (Pierce). 20μg of extracts
were separated on 12% SDS PAGE and transferred to PVDF membrane. After
one hr blocking with 5% nonfat milk, the membrane were incubated in different
primary antibody (anti-NSF PAb (1:2500; Abcam), Anti-EAAT5 PAb (1:5000; a gift
from Dr. David Pow (University of Queensland, Queensland, Australia)
anti-vGLUT1 MAb (1:2000; Millipore), anti-VAMP2 MAb (1:5000; Synaptic
System), anti-SNAP-25 PAb (1:3000; Abcam) at 4°C overnight. After washing, the
blots were then incubated with appropriate anti- horseradish peroxidase
conjugated secondary antibodies (1:10000; Bio-Rad) at room temperature for 1 hr.
The proteins on the membranes were detected using the ECL
chemiluminescence system (Amersham Biosciences). The blots were also probed
with antibody for glyceraldehydes-3-phophate dehydrogenase (GAPDH) as an
internal loading control. Densitometric analysis was conducted using the
30

ImageQuant TL

software (Amersham Biosciences).

Each experiment was
repeated three times with independent retinal samples from different animals.

2.8 Immunohistochemistry.
To further verify the potential physiological relevance of the interaction
between Arr1 and NSF, we performed indirect fluorescent dual localization
immunoreactive staining, as described (Zhu et al., 2003). Briefly, the eyes were
enucleated under infrared or light conditions, the cornea was removed, and the
eyes were immediately immersed in 4% (w/v) paraformaldehyde (PFA) in 0.1M
PBS for 2 hrs at room temperature. Eyes were rinsed in PBS, pH7.4 and
cryoprotected in 30% sucrose-PBS solution at 4°C overnight, and then embedded
in ornithine carbamyl transferase (OCT; Tissue-Tek, Elkhart, IN). Sections (7μm)
of the retina were cut through the optic nerve with a cryostat, and retina sections
were washed in 0.1M PBS, blocked in blocking buffer (1% BSA, 1% NGS, 1%
Triton X-100 in 1XPBS), and incubated with mouse MAb D9F2 (1:20,000) for Arr1
and anti-rabbit PAb (1:2,500) for NSF at 4°C overnight. To visualize binding of the
primary antibodies, sections were incubated in secondary antibody conjugated to
31

Alexa Fluor 488 or 568, respectively (1:500, Invitrogen) and TOPO-3 (1:2500,
Invitrogen) nuclear staining for 1 hr at room temperature. Samples stained without
either or both of the primary antibodies were included as controls to ensure the
dual-staining pattern results were reliable. The sections were visualized and
photographed with a Zeiss confocal laser-scanning microscope (Carl Zeiss, Inc).

2.9 Cell culture and transfection.
COS-7 cells were cultured in Dulbecco’s modified Eagle’s medium
(Invitrogen) supplemented with 10% fetal bovine serum and antibiotics and
maintained at 37°C in a humidified atmosphere with 5% CO
2
as previously
described
49
. The plasmids of interest were transfected into COS-7 cells using
Fugene Transfection Reagent according to the manufacturer’s instruction (Roche).
COS-7 cells were co-transfected with plasmids encoding empty vector,
pcDNA4/HisMax-Arr1, pcDNA3.1/GFP-NSF or pcDNA4/HisMax-Arr1 plus
pcDNA3.1/GFP-NSF for 48 hrs. Cell were washed in PBS and fixed in freshly
prepared 4% paraformaldehyde (PFA). Fixed cells were permeabilized and
blocked with 0.1% Trixon-X100 and 2.5% normal goat serum in PBS for 30 min at
32

room temperature and then incubated with primary and secondary antibodies
diluted in the same buffer for 1 hr at room temperature. After three washes with
PBS, the cells were mounted and viewed under a Zeiss confocal laser-scanning
microscope.

2.10 NSF ATPase activity assay.
The basal ATPase activity of NSF was measured by a colorimetric assay
(Huynh et al., 2004). Recombinant NSF (0.2μg/μl) was pretreated with 10mM
N-ethylmaleimide (NEM), a NSF inhibitor, as a negative control or with increasing
concentrations of recombinant Arr1 protein for 10 min at 37°C. ATPase reaction
buffer (25mM Tris-HCl at pH 9.0, 100mM KCl, 0.65mM β-mercaptoethanol, 2mM
MgCl
2
and 10% glycerol, 10mM ATP) was added to the mixture. The release of
inorganic phosphate was measured by adding Biomol Green (Biomol, Plymouth
Meeting, PA) and the absorbance at 620 nm was determined on a Benchmark
Plus microplate reader (Bio-Rad, Los Angles, CA). Corrections were made for
minor ATPase contaminants, nonenzymatic hydrolysis of ATP and preexisting

33

phosphate in protein samples by subtracting the NEM-treated control values from
those obtained at 37 °C.

2.11 NSF disassembly activity assay.
The disassembly activity of NSF was measured by a co-precipitation assay
as described previously (Matsushita et al., 2003). Recombinant His
6
-NSF
(0.1μg/μl) was pretreated with buffer or with increasing concentration of
recombinant Arr1 protein for 10 min at 37°C. Immobilized GST-Syntaxin 4
(0.1μg/μl) on glutathione-agarose beads was incubated with recombinant
His
6
-α-SNAP (0.1μg/μl) and SNARE polypeptide (0.1μg/μl each of VAMP-2 and
SNAP-25) at 4 °C for 1hr. The beads were then washed three times with
binding/wash buffer (4mM HEPES, pH 7.4, 0.1M NaCl, 1mM EDTA, 3.5mM CaCl
2
,
3.5mM MgCl
2
, and 0.5% Triton X-100). The mixture of NSF with increasing
concentration of Arr1 was added to the beads and then incubated in binding/wash
buffer with 2.5 mM ATP/5mM MgCl
2
for 30 min at 4°C with rotation. The beads
were washed with binding/wash buffer six times, mixed with SDS-PAGE sample

34

buffer at 60 °C for 3 min, resolved on 12% SDS-PAGE, transferred to PVDF, and
then analyzed on immunoblots.

2.12 Evaluation of exocytosis rate using FM1- 43 staining.
The procedure for activity-dependent staining with FM1-43 was performed
as described previously (Miller et al., 2001;Caicedo et al., 2005). The eyecup
preparations from WT and Arr1
-/-
mice were immersed in mammalian Ringer’s
solution. The eyecups were superfused with depolarizing Ringer’s solution
containing 10μM fixable FM1-43 (Invitrogen) with or without 25mM K
+
for 30 min.
After incubation, preparations were washed 3 times with Ringer’s solution
containing 5mM Co
2+
for 5 min to minimize dye release through
calcium-dependent exocytosis and to eliminate background staining. Eyecups
were fixed in 4% PFA for 1 hr, cryoprotected in 30% sucrose, and cut on a cryostat.
FM1-43 staining was visualized and photographed with a Zeiss confocal
laser-scanning microscope.

35

2.13 Electroretinography (ERG)
Photopic, cone-driven ERG responses were recorded from mice in the
presence of a steady white background light to suppress the rod response, as
previously described (Brown et al., 2010a). In our study, the background light was
turned on, and after 1 min of light adaptation, a single maximum intensity flash
was delivered and averaged every 2 min until 15 min of recording. The amplitudes
from at least 6 mice in each group were averaged and two-way ANOVA with
Bonferroni post-tests were performed on the data at each time point during
recording.

2.14 TUNEL analysis of apoptosis
The observed cone photoreceptor dystrophy was evident by 2 months;
therefore, in order to verify cone apoptosis prior to outer nuclear layer (ONL)
thinning, eyes were enucleated from three dark-adapted (24 hrs) WT and Arr1
-/-

mice at postnatal day (p) 22, p30, p45 and p60 under infrared. Eye cups were
fixed and embedded as previously described (Zhu et al., 2002). Three adjacent
sections cut through the optic nerve along the vertical meridian were used from
36

each eye. Apoptotic cells were visualized by means of the terminal
deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay
using the DeadEnd
TM
Fluorometric TUNEL System (Promega), following the
manufacturer’s instructions (Li et al., 2003). After labeling, the slides were
mounted with Vectashield Mounting Medium for fluorescence with DAPI nuclear
stain (Vector Laboratories). Apoptotic cells were quantified by counting TUNEL
positive cells from the entire section, and the average number of apoptotic cells
per section was calculated from 3 sections of both eyes of 3 mice from each
genotype and each time course. Statistical analysis was performed using the
one-way ANOVA and results are expressed as means ± standard error of the
mean (SEM).

2.15 Affymetrix
TM
GeneChip Microarray hybridization and Ingenuity Pathway
Analysis (IPA)
Transcriptional variability when apoptosis was documented and prior to
severe morphological changes was examined in retinas from 1 month old
dark-adapted WT and Arr1
-/-
mice. Retinas at this age also have a similar ONL
37

thickness, which allowed for comparable mRNA levels between the two strains.
Total RNA from three animals at each condition was prepared from using Trizol
reagent (Invitrogen, Carlsbad, CA). RNA purity and concentration was determined
using spectrophotometry A
260
/A
280
ratios. Affymetrix
TM
Genechip Mouse Exon 1.0
ST Arrays (Affymetrix, Inc. Santa Clara CA) were used for hybridization. This
array is comprised of more than 750,000 unique oligonucleotide features
constituting more than 266,260 genes. Labeling, hybridization and scanning of the
exon arrays were performed in the Children’s Hospital Los Angeles genome core
facility according to Affymetrix protocols. Experiments were performed in triplicate
for statistical and biological relevance. The original Affymetrix raw intensity files
were imported into Partek Genomic Suite (Partek, Inc.) and pre-processed using
the software implemented GCRMA background correction (Goto et al., 1991) and
quantile normalization algorithms(Bolstad et al., 2003). The log2 transformed data
was then subjected to a 2-way mixed-model factorial ANOVA analysis and the list
of differentially expressed genes between the two genotypes was subsequently
generated (p<0.05 coupled with fold changes >2 or <2).Transcripts with
statistically significant differences and annotated function were categorized using
38

Ingenuity Pathway Analysis (IPA) (Ingenuity Systems). Statistical significance of
canonical pathways was conducting using Fisher’s Exact Test with a p value
threshold of 0.05. Ratios are the number of molecules identified from the data set
per pathway. Scores for top networks were based on the hypergeometric
distribution and calculated with the right tailed Fisher’s Exact Test by IPA.

2.16 Statistical analysis
Analysis of statistical significance was determined using two-tailed Student’s
t test and one-way ANOVA followed by Bonferroni’s multiple comparison test.
Data are presented as means ± standard error of the mean (SEM). In all cases,
p<0.05 denotes statistical significance.
39

Chapter 3. Arr1 Acts as a Modulator for NSF in Photoreceptor
Synaptic Regulation

3.1 Introduction
Arrestins play established roles in the regulation of G-protein coupled
receptor (GPCR) signaling by specifically binding to agonist-activated,
phosphorylated receptors, terminating further G-protein activation. There are four
distinct members in the arrestin superfamily expressed in vertebrates (Craft and
Whitmore, 1995). Arr1 is expressed in both rod and cone photoreceptors, while
Arrestin 4 (Arr4, cone or X-arrestin) is expressed in cone photoreceptors (Nikonov
et al., 2008). Both visual arrestins are also highly expressed in pinealocytes (Craft
et al., 1994), while β-arrestin1 and β-arrestin2 are ubiquitously expressed and
mediate the desensitization of β-adrenergic receptors and many other GPCRs
(Lefkowitz et al., 1992). Moreover, β-arrestin1 also directs receptors to coated pits
for internalization and switches signaling to alternative pathways (Orsini and
Benovic, 1998;Gagnon et al., 1998;Lefkowitz and Shenoy, 2005).

40

Previous work, which includes gene knockouts of mouse Arr1,
demonstrated its function in modulating the phototransduction shutoff and
recovery in the rod and cone photoreceptor outer segment by binding to
light-activated, phosphorylated opsins and quenching the GPCR
phototransduction cascade (Wilden et al., 1986;Xu et al., 1997;Nikonov et al.,
2008). Furthermore, mutations in the human gene encoding ARR1 lead to either
an inherited recessive form of stationary night blindness known as Oguchi
disease that is associated with abnormal electroretinograms (ERGs) of reduced
a-wave amplitudes and absent scotopic b-waves (Fuchs et al., 1995;Nakazawa et
al., 1997), or retinitis pigmentosa (Nakamachi et al., 1998) that leads to retinal
degeneration.
Subcellular localization of signaling molecules is vital for their biological
function. Constitutively shuttling of signaling proteins and the redistribution of
their interactive partners between subcellular compartments are important for the
modulation of their activity. With light exposure, Arr1 is translocated to the
outer segments in rods and cones and also contributes to light-adaptation; while
in dark-adapted photoreceptors, high concentrations of Arr1 localizes to the inner
41

segment, perinuclear region, and synaptic terminals (Broekhuyse and Winkens,
1985;Philp et al., 1987;Whelan and McGinnis, 1988;Arshavsky, 2003;Brown et al.,
2010a). Based on the photoreceptor synaptic localization in the dark of Arr1 and
alternative functions of the β-arrestins in neuronal synapses, we examined
potential binding partners of Arr1.

3.2 Results
3.2.1 se
nvestigate potential physiologically relevant photoreceptor partners of
Arr1,
NSF (Figure 3.2).
NSF is an interacting partner for Arr1 in the photoreceptor synap
To i
we performed a pull-down immunological assay from colony control wild
type (WT) mouse retina homogenates, using a mouse monoclonal antibody (MAb
D9F2) against Arr1, followed by SDS-PAGE separation and analysis. Among the
major bands identified by mass spectrometry, one band was identified as
N-ethylmaleimide sensitive factor (NSF) (NP_032766) (Figure 3.1). Protein
database search using Mascot analysis (Matrix Science) revealed ~22% tryptic
peptide coverage with a total Mascot score of 610 of a protein corresponding to
42

Figure 3.1 Retinal homogenates were immunoprecipitated with anti-Arr1 MAb D9F2 and
bands were identified by mass spectrometry. The immunoprecipitated proteins were
separated by 10% SDS-PAGE and visualized by Coomassie blue stain. The anti-m se
phosducin (PhD) MAb 1D6 was used as a nonspecific antibody control. Excised bands were
ou
then analyzed by mass spectrometry. Several proteins were identified: IgG dimer, Acrylpeptide
hydrolase (NP_666338), phosphofructokinase (NP_032852), NSF (NP_032766), Heat shock
protein 5 (NP_071705), Heat shock protein 9 (NP_034611), Heat shock protein 8
(NP_112442), albumin (NP_033784), Arr1, ATP synthase, H+ transporting, mitochondrial F1
complex, alpha subunit (NP_031531).
43

Figure 3.2 NSF was identified by mass spectrometry. The protein database search
using Mascot analysis revealed ~22% tryptic peptide coverage with a total Mascot
score of 610, corresponding to mouse N-ethylmaleimide sensitive factor. The identified
matched peptide sequences are shown in bold.
NSF is an ATPase that is an essential component of various membrane
fusions including the exocytosis of synaptic vesicles (Kawasaki et al., 1998;Tolar
and Pallanck, 1998;Singh et al., 2004). To verify the interaction between Arr1 and
NSF, we performed additional co-immunoprecipitation assays from retinal
homogenates from WT and Arr1 knockout (Arr1
-/-
). Immunoblot analysis with a
44

rabbit anti-NSF polyclonal antibody (PAb) identified a single band in WT and
Arr1
-/-
retinal homogenates (Figure 3.3(a)), while the anti-Arr1 MAb identified a 48
kDa band corresponding to Arr1 only in the WT, but not in the Arr1
-/-
retinal
homogenates (Figure 3.3(b)). A protein of approximately 76 kDa, corresponding to
NSF, was co-immunoprecipitated with Arr1 MAb from the WT but not from the
Arr1
-/-
retina homogenates (Figure 3.3(c)), confirming the specific protein-protein
interaction of Arr1 with NSF. The interaction between Arr1 and NSF is greater in
dark-adapted (DA) retinas compared to light-adapted (LA) retinas (Figure 3.3(d)
and (e)). We also observed that NSF was not co-immunoprecipitated with
anti-rabbit cone arrestin Pab in WT retinal homogenates (Figure 3.4).

45

Figure 3.3 NSF co-immunoprecipitated with Arr1 in mouse retina. Arr1 was
immunoprecipitated from the homogenates of 10 mouse retinas using the Arr1 monoclonal
antibody (MAb D9F2). NSF and Arr1 proteins are shown in the initial retinal homogenates
(a and b). NSF co-immunoprecipitated with Arr1 in the WT retinal homogenates (c). The
retinal homogenates from Arr1
-/-
mice were used as a negative control (c).
Immunoprecipitated proteins were isolated from mouse retinas under either light- or
dark-adapted conditions, analyzed by SDS-PAGE, transferred to PVDF membranes and
detected by ECL using anti-rabbit NSF(d) or anti-mouse Arr1(e) antibodies. IP:
immunoprecipitation; IB: immunoblot analysis
46

Figure 3.4 Cone Arrestin does not interact with NSF. The co-immunoprecipitation studies
using anti-rabbit mouse cone arrestin-Luminaire juniors (mCar-LUMIj) PAb in the mouse
retinal homogenate. NSF and Arr4 proteins were shown in the initial retinal homogenates.
NSF was not co-immunoprecipitated with mCar-LUMIj PAb in WT retinal homogenates. The
retinal homogenates from Arr4
-/-
mice were used as negative control.
To be a relevant interacting partner for Arr1, NSF must co-localize with Arr1
in the appropriate subcellular compartments within the retina. In this study,
retinas from WT mice were either light- or dark-adapted; their retinas were
prepared for immunohistochemistry and sections were examined with a confocal
microscope. The retinal sections were incubated with Arr1 MAb and NSF PAb that
were used in the co-immunoprecipitation experiments, followed with the
appropriate secondary antibodies, Alexa Fluor 568 goat anti-mouse IgG antibody
47

and Alexa Fluor 488 goat anti-rabbit IgG antibody, respectively. In the DA retinas,
the majority of immunoreactive Arr1 was localized to the inner segments,
perinuclear region with a small fraction in the synaptic terminals (Figure 3.5(a)). In
the LA retinas, the majority of Arr1 translocated to the outer segments (Figure
3.5(b)). NSF showed some perinuclear labeling in the inner nuclear layer and
intensive staining in the outer plexiform layer (OPL) and inner plexiform layer (IPL)
in retinas from both LA and DA mice. Dual immunohistochemical labels showed
that Arr1 and NSF extensively co-localized in the photoreceptor terminals (OPL;
Figure 3.5(c)). In the DA retinas, Arr1 and NSF co-localized in the junction
between the outer segment and inner segment; however, in the LA retinas, Arr1
immunoreactive label was limited and co-localized with NSF only in the OPL
(Figure 3.5(d)). These observations further confirm the potential physiological
interactions between Arr1 and NSF in vivo. Their intense dual expression
pattern at the specialized ribbon synapse of photoreceptors in the dark condition
suggests that Arr1 and NSF may be critical partners for modulating
neurotransmitter transmission. Alternatively, Arr1 may be acting at the synapse as
a co-chaperone for NSF in the SNARE complex to regulate exocytosis.
48

Figure.3.5 Immunohistochemical fluorescent labeling of NSF and Arr1 in the WT mouse
retinas. Adult WT mouse retina frozen sections were triple labeled fluorescently with the
anti-mouse Arr1 MAb D9F2 (red), anti-rabbit NSF PAb (green), and appropriate secondary
antibodies and TO-PRO3 for the nuclei (blue). The immunoreactive staining pattern of NSF is
mainly in the OPL and IPL in dark-adapted (DA) (2a) or light-adapted (LA) (2b) retinas. The Arr1
MAb immunoreactive label is predominantly in the inner segment, perinuclear area and a
fraction in the photoreceptor terminal in DA retinas, whereas the Arr1 MAb immunoreactivity is
translocated to the outer segment in LA retinas. The Arr1 MAb immunoreactivity is extensively
dual localized with NSF immunological staining in the OPL in DA retinas (2c) and only limited
dual staining in the OPL in LA retina (2d). DA: dark-adapted; LA: light-adapted; OS, outer
segment; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner
nuclear layer; IPL, inner plexiform layer; RGC, retinal ganglion cell layer. Scale bar, 50μm in a
and b; 10μm in c and d.

49

3.2.2 The Arr1-NSF complex formation is enhanced by ATP
Each NSF monomer has three functional domains (Whiteheart et al.,
1994;Nagiec et al., 1995): an N-terminal domain (N, amino acid [AA] 1-205) that is
required for the binding to α-SNAP and SNARE proteins; and two homologous
ATP-binding domains, D1 (AA 206-477), in which the ATP hydrolytic activity is
associated with NSF-driven SNARE complex disassembly, and D2 (AA 478-744),
which is responsible for hexamerization. In order to define the potential
functional domain in NSF that interacts with Arr1, His6-tagged or Glutathione
S-transferase (GST)-tagged NSF-truncated segments of varying lengths were
constructed and a GST pull-down assay was performed. Our results indicate that
only the NSF fragments that included AA 197-250, which is in the junction of the N
and D1 domains (Figure 3.6(i)), interact with Arr1.
We further mapped the regions of Arr1 that interact with NSF using in vitro
GST pull-down assays. GST-Arr1 (AA 1-191), GST-Arr1 (AA 1-370), GST-Arr1
(AA 1-403) and GST control proteins were immobilized on glutathione-agarose
beads and incubated with recombinant His
6
-NSF. His
6
-NSF bound specifically to

50

Figure 3.6 Functional analysis of the interaction between Arr1 and NSF. (i) Mapping the
binding site of Arr1 on NSF. NSF has 3 functional domains: N domain is for SNARE protein
complex binding, D1 domain is for fusion complex disassembly, and D2 domain is for NSF
homo-hexamer formation. To define the region in NSF that interacts with Arr1, His
6
-tagged
truncated segments of NSF with varying lengths (AA residues 1-744, 197-744, 1-477, and
1-205^478-744) and GST-tagged NSF (AA 1-250) and NSF (AA197-250) were constructed and
GST pull-down assay was performed. Bound proteins were detected by immunoblot analysis
with anti-mouse His-tag MAb and anti-mouse Arr1 MAb D9F2. Arr1 only bound NSF
fragments that included the AA 197-250, which was located in the junction of the N and D1
domains. (ii) Defining the binding site of NSF on Arr1. GST pulldown assay demonstrated a
direct interaction between the N-terminal domain of Arr1 and NSF. GST alone or GST-Arr1(AA
1-191), GST-Arr1(AA 1-370) and GST-Arr1(AA 1-403) coupled to beads were incubated with
purified His
6
-tagged NSF in the binding buffer containing 2mM ATP + 8mM MgCl
2
or 2mM
ATP-γ-S + 8mM MgCl
2
.

Bound NSF was detected by immunoblot analysis using anti-rabbit NSF
antibody.
51

each of the GST-Arr1 recombinant proteins but not the GST control (Figure 3.6
(ii)). These data demonstrate that the amino-terminal domain (AA 1-191) of Arr1
interacts with NSF. Others have shown the association of NSF with other
binding partners exhibited an ATP dependence (Banerjee et al., 1996;Nishimune
et al., 1998). In the presence of ATP-γ-s/MgCl
2
(non-hydrolyzable ATP) or
ATP/ethylenediamine tetra-acetic acid (EDTA) (Figure 3.7), the interaction of each
GST-Arr1 recombinant construct and NSF showed enhanced binding compared
with the ATP/MgCl
2
(hydrolyzable ATP). These results also confirm that the Arr1
binding to NSF is enhanced by ATP.

52

Fig.3.7 Arr1-NSF complex formation is ATP-dependent. GST-hybrid Arr1 proteins (3mg)
were immobilized on glutathione-agarose beads and then incubated with His
6
-tagged NSF
protein (3mg) and buffers containing 2mM ATP + 8mM MgCl
2
, 2mM ATP+ 8mM
MgCl
2
+EDTA, 2mM ATP-g-S + 8mM MgCl
2
or only 8mM MgCl
2
. Bound proteins were
eluted with 20mM glutathione and detected by immunoblot analysis. The amount of NSF
bound was normalized to the amount of Arr1 immunoprecipitated. Results represent the
means ± standard error of the mean (s.e.m.) for three independent experiments.**p<0.01,
***p<0.001.
Interactions between Arr1 and NSF were examined in tissue cultured
COS7-cells that were transiently co-transfected with plasmids encoding either
empty vector, pcDNA4-HisMax-Arr1 and pcDNA3.1-GFP-NSF (AA 1-744), or
53

pcDNA3.1-GFP- NSF (AA 197-250) for 48 hrs. As shown in Figure 3.8, both
GFP-NSF (AA 1-744) (a) and GFP-NSF (AA 197-250) (d) showed a cytoplasmic
diffuse fluorescent pattern. HisMax-Arr1 was localized to the cytosol and
perinuclear region. The GFP empty vector (g) showed diffuse staining throughout
the cell, including the nucleus. The GFP-NSF (AA 1-744) and GFP-NSF (AA
197-250) extensively co-localized with HisMax-Arr1 (b, e, His-Arr1; c, f, Merge),
whereas the GFP empty vector had no dual staining with HisMax-Arr1 in the
COS7-cells (h, HisMax-Arr1; i, Merge).

3.2.3 lex Arr1 increases NSF ATPase activity and NSF-driven SNARE comp
disassembly
In order to examine the effect of Arr1 binding on NSF ATPase activity,
which is critical for NSF function, increasing concentrations of recombinant
His
6
-Arr1 were added to 10μg of recombinant His
6
-NSF, and the ATPase activity
of the NSF was measured by a colorimetric assay. Arr1 significantly enhanced
NSF ATPase activity in a dose-dependent manner (Figure 3.9).
54

Figure 3.8 Association of Arr1 with NSF in COS-7 Cells. COS-7 cells co-expressing
pcDNA4-HisMax-Arr1 and pcDNA3.1-GFP- NSF(AA 1-744) or NSF(AA 197-250) were
processed for immunofluorescence and analyzed by confocal microscopy for the extent of
colocalization of pcDNA4-HisMax-Arr1 (b, e, h, red (Anti-mouse His-tag MAb); c, f, i,
Merge) with pcDNA3.1-GFP-NSF(AA 1-744) (a, green (GFP); c, Merge), NSF(AA
197-250) (d, green (GFP); f, Merge) and empty vector (g, green (GFP); i, Merge).
55

Figure 3.9 NSF ATPase activity assay. The ATPase activity of NSF was measured
with a colorimetric assay. Recombinant NSF (0.2μg/μl) was pretreated with 10mM
N-ethylmaleimide (NEM), a NSF inhibitor, as a negative control or with increasing
concentrations of recombinant Arr1 protein for 10 min at 37°C. The release of inorganic
phosphate was measured by adding Biomol Green (Biomol, Plymouth Meeting, PA) and
reading the absorbance at 620 nm on a Benchmark Plus, microplate reader (Bio-Rad,
Los Angeles, CA). Arr1 significantly increases the NSF ATPase activity in a
dose-dependent manner. Corrections were made by subtracting the NEM-treated
control values. Results of the ATPase assay represent the means ± s.e.m. for four
independent experiments. **p<0.01, ***p<0.001.

56

We next explored the effect of Arr1 on NSF disassembly activity. NSF has
been shown to bind stably to SNARE complex molecules using α-SNAP as an
adaptor that locks it in the ATP state (Barnard et al., 1997;Muller et al., 1999).
Hydrolysis of ATP enables NSF to separate from and disassemble the SNARE
complex. Accordingly, we examined the effect of Arr1 on NSF disassembly of
purified, recombinant SNARE molecules. The recombinant His
6
-NSF proteins
were pretreated with increasing concentrations of recombinant Arr1. The
GST-tagged-Syntaxin 4 proteins were immobilized to glutathione-agarose beads
and then incubated with soluble N-ethylmaleimide sensitive factor attachment
protein (α-SNAP) and SNARE polypeptides, including vesicle associated
membrane protein 2 (VAMP-2) and SNAP-25 in binding buffer. The mixture of
NSF, Arr1 and SNARE complex was precipitated with beads and the precipitated
proteins were separated by SDS-PAGE and analyzed on immunoblots with
specific antibodies to NSF, Syntaxin-4, SNAP-25 and VAMP2. These data verified
that Arr1 enhanced NSF disassembly activity in a dose-dependent manner
(Figure 3.10).

57

Figure 3.10 SNARE complex disassembly assay. (a) Assembly status of SNARE
complexes. GST-Syntaxin 4 was immobilized on glutathione-agarose beads and
incubated with His
6
-tagged VAMP2 and SNAP-25 with (Lane2) or without (Lane 1) NSF
and α-SNAP for 30 min at 4°C. Proteins precipitated with beads were separated by 12%
SDS-PAGE and visualized by Coomassie blue staining. (b) Arr1 enhanced NSF
disassembly activity in a dose-dependent manner. The NSF disassembly assay was
performed by pretreating recombinant His
6
-tagged NSF with increasing concentration of
Arr1 and then mixing with immobilized GST-Syntaxin4 with His
6
-tagged-α-SNAP, VAMP2,
or SNAP-25. Proteins precipitated with glutathione-agarose beads were transferred to
PVDF and incubated with (from top to bottom) anti- rabbit NSF PAb, anti-mouse Syntaxin
4 MAb, anti-rabbit SNAP-25 PAb and anti- mouse VAMP2 MAb and appropriate
secondary antibodies. SNARE complexes of ~250 kDa were detected with all the
antibodies listed above.
58

3.2.4 Arr1 deletion markedly reduces the expression level of
synapse-enriched proteins
Unlike conventional synaptic terminals that release neurotransmitters
episodically in response to action potentials, the photoreceptor ribbon synapses
are depolarized in the dark, resulting in maintained activation of voltage-gated
calcium channels, continual Ca
2+
influx, and higher rates of exocytosis required
for tonic release of the neurotransmitter glutamate. Increasing light intensity
induces a graded hyperpolarization that turns off these events and suppresses
glutamate release (Morgans, 2000;Heidelberger et al., 2005;von Gersdorff, 2001).
Many presynaptic proteins regulate synaptic vesicle exocytosis and
neurotransmitter release. These proteins are differentially distributed among the
different synapses of the mouse retina. For example, glutamate transporters, such
as vesicular glutamate transporter 1 (vGLUT1), which refills the synaptic vesicles
with glutamate, is essential for transmission of visual signaling from
photoreceptors to second- and third-order neurons(Johnson et al., 2007).
Excitatory amino acid transporter 5 (EAAT5), which is expressed in synaptic
terminals of photoreceptors and rod bipolar cells, plays a predominant role in
59

re-uptake of glutamate from the synaptic cleft to ensure reliable synaptic
transmission (Pow and Barnett, 2000;Wersinger et al., 2006). Also, a synaptic
vesicle protein, VAMP2, which is the predominant isoform in the mouse retina, is
an integral membrane protein associated with forming the fusion core complex
required for docking and fusing of synaptic vesicles at the synaptic active
zone(Sherry et al., 2003). To investigate if the presence of Arr1 alters the
expression levels of NSF and the synapse-enriched encoded genes such as
vGLUT1, EAAT5 and VAMP2, we performed quantitative real time polymerase
chain reaction technology (RT-PCR) to compare the mRNA expression level of
these genes in WT and Arr1
-/-
retinas under different lighting conditions.
Comparing DA and LA WT retinas, the mRNA levels of NSF, vGLUT1, EAAT5 and
VAMP2 were significantly higher in the dark (Figure 3.11).

60

Fig 3.11. Quantitative RT-PCR of NSF, vGLUT1, VAMP2 and EAAT5 in the retinas.
Expression levels of NSF, VAMP2, EAAT5 and vGLUT1 in WT and Arr1
-/-
mouse retina were
measured by quantitative RT-PCR. Each column represents the average of 3 amplification
reactions (mean ± s.e.m.), performed on a cDNA sample reverse transcribed from total RNA
prepared from ten pooled retinas using Trizol reagent and transcribed into cDNA with oligo
(dT)
20
using the Superscript III system (Invitrogen). Values for light-adapted WT retinas were
set to 1. The NSF, vGLUT1, VAMP2, and EAAT5 mRNA levels were significantly higher in
the dark-adapted (D) WT retinas compared with light-adapted (L) WT retinas. In the light,
significantly lower expression levels of vGLUT1 and EAAT5 were observed in the Arr1
-/-

retinas compared with WT retinas. In the dark, NSF, vGLUT1, VAMP2 and EAAT5 mRNA
levels were markedly decreased in the Arr1
-/-
retinas compared with the WT retinas.*p<0.05,
**p<0.01, ***p<0.001, mean ± s.e.m.

61

In the light, there was no significant difference in the transcriptional level of
NSF and VAMP2, but the expression levels of vGLUT1 and EAAT5 were lower
than in Arr1
-/-
retinas than the WT retinas. Difference in expression levels
between WT and Arr1
-/-
retinas is light dependent. In the dark, the mRNA levels
of NSF, vGLUT1, VAMP2 and EAAT5 were markedly decreased in the Arr1
-/-

retinas compared with the WT retinas. The protein expression level of NSF,
vGLUT1, VAMP2 and EAAT5 in WT and Arr1
-/-
retinas under different lighting
conditions correlated to the transcription level of these genes (Figure 3.12). For
SNAP-25, the protein expression level was lower in the DA Arr1
-/-
retinas
compared with DA WT retinas and there was no significantly difference between
LA WT and Arr1
-/-
retinas.
62

Figure 3.12 Immunoblots analysis of NSF, vGLUT1, EAAT5, VAMP2, SNAP-25.
Mouse mGAPDH was used as internal control. In the bar graph, the expression level of
these proteins is expressed as a ratio to GAPDH expression. Results represent the
means ± s.e.m. for three independent experiments
3.2.5 Arr1 deletion strongly suppresses the synaptic activity in the
photoreceptor synapse
To further test whether photoreceptor synapses are dysfunctional in vivo in
the Arr1
-/-
retinas, we performed experiments that were based on the widely used
63

fluorescent dye, FM1-43, which can selectively label structures and living cells
that are undergoing exocytosis and endocytosis. In WT retinas depolarized with
25mM KCl, FM1-43 was avidly sequestered into the presynaptic terminals in the
OPL and IPL (Figure 3.13 (a)), whereas in the depolarized Arr1
-/-
retinas FM1-43
staining in the OPL and IPL was strongly reduced (Figure 3.13 (b)). In the
non-depolarized WT (Figure 3.13 (c)) and Arr1
-/-
(Figure 3.13 (d)) retinas, the
FM1-43 uptake was diminished in both OPL and IPL. These results verify that
when Arr1 expression is absent in the retina, the synaptic activity is dramatically
reduced in the photoreceptors.

64

Figure 3.13 Synaptic uptake of FM1-43 revealed a decrease of synaptic activity in the
Arr1
-/-
mouse retina. Enhanced FM1-43 uptake in the outer (OPL) and inner plexiform
layers (IPL) was observed in depolarized (25mM K
+
) WT retina (a), compared to
depolarized Arr1
-/-
retina (b). FM1-43 intensity was also significantly reduced in the OPL
and IPL in non-depolarized WT (c) and Arr1
-/-
(d) retinas. Scale bars =50 μm.

65

In our studies, unlike Arr1, we observed that Arr4 does not interact or
modulate NSF (Figure 3.4), which suggests a divergence of visual Arr function in
the cone photoreceptor. In cones, at least one visual Arrestin is essential for
normal photoreceptor recovery (Nikonov et al., 2008).
The averaged b-wave amplitudes recorded from the WT, Arr1
-/-
and Arr4
-/-

every 2 min from 1 to 15 min during light adaptation are graphed in Figure. 3.14
(a). The photopic b-wave amplitudes for these three genotypes are similar at 1
min. Only WT and Arr4
-/-
mice demonstrated equivalent light adapting increases in
the b-wave, reaching maximum amplitudes at 9 min. In contrast, Arr1
-/-
mice
showed no b-wave amplitude increase over the 15 min of light adaptation.
Similar results were observed with twenty multiple intensity flashes to further
saturate rhodopsin (Brown et al., 2010a). In order to further delineate the
functional differences between Arr1 and Arr4 in synaptic regulation from their
established function in phototransduction inactivation, we also tested transgenic
mice, mCAR-H
arr1-/-
, where Arr4 expression is driven by the rhodopsin promoter
and is over-expressed in the rod photoreceptors with normal expression in the
cones of Arr1
-/-
mice. Previously, mCAR-H
arr1-/-
mice were observed to restore
66

partial recovery of rod function and reduced levels of light dependent rod
degeneration associated with the loss of Arr1 (Chan et al., 2007). In Figure 3.14
(b), the 15 min of light adaptation showed no appreciable increase in amplitudes
of b-wave responses in the mCAR-H
arr1-/-
, similar to the observed phenotype in
the Arr1
-/-
.

Figure 3.14 ERG analysis of WT, Arr1
-/-
, Arr4
-/-
, and mCAR-H
arr1-/-
mice. Average
photopic b-wave amplitudes ( μV) recorded every 2 min during 15 min of light
adaptation of the (a) WT, Arr1
-/-
, Arr4
-/-
, and (b) mCAR-H
arr1-/-
mice. Two-way ANOVA
with Bonferroni post-tests used for statistical comparisons with WT (*p<0.05, **p<0.01,
***p<0.001).

67

3.3 Discussion
In this study, we demonstrate the alternative structural and potential
functional interactions between visual Arr1 and NSF in the photoreceptor synapse
using a combination of biochemical, cellular, and molecular biological techniques.
The crystal structures of Arrestin family members, including Arr1, Arr4 and
β-arrestin1, are remarkably similar (Wilden et al., 1997;Hirsch et al., 1999;Han et
al., 2001;Sutton et al., 2005). In a previous in vitro yeast two-hybrid study,
β-arrestin1 was shown to interact in vitro with NSF and it bound to the identical
region (AA 197-250 of NSF) as observed for Arr1 binding. The β-arrestin1-NSF
complex formation was also ATP-dependent (McDonald et al., 1999). Over
expression of NSF rescued the dominant negative effect of a β-arrestin1
phosphorylation mutant (S412D) by restoring normal sequestration of the GPCR,
the β2-adrenergic receptor (AR). It is important to note that other members of the
Arrestin family do not possess the Ser412 residue and therefore are not subject to
the same regulation in receptor internalization as β-arrestin1. In our work, we
show that the Arr1 N-terminal motif (AA 1-191) contains a key domain involved in
activation-recognition and phosphorylation-specific binding of the Arr1 (Gurevich
68

and Benovic, 1992;Gurevich and Benovic, 1993;Gurevich et al., 1994) for
interaction with NSF, but no evidence documents that this region of β-arrestin1
interacts with NSF. We also determined that cone Arr4, which is also expressed
in cone pedicles with Arr1

(Nikonov et al., 2008), did not interact with NSF (Figure
3.4). In previous studies, the interaction of β-arrestin1 with NSF directs the
clathrin-mediated receptor internalization. In our experiments, Arr1 binds directly
to the junction of the NSF N and D1 domains, which are required for SNARE
complex binding and ATP hydrolysis, to modulate the process of exocytosis.
Unlike conventional synaptic terminals that release neurotransmitter
episodically in response to action potentials, the photoreceptor ribbon synapses,
which are specialized structures at active zones in the rod spherules and cone
pedicles, are depolarized in the dark, resulting in maintained activation of
voltage-gated calcium channels, continual Ca
2+
influx, and higher rates of
exocytosis required for tonic neurotransmitter (L-glutamate) release (Morgans,
2000; von Gersdorff, 2001; Heidelberger et al., 2005). Increasing light intensity
induces a graded hyperpolarization that turns off these events and suppresses
neurotransmitter release. This higher rate of exocytosis in the photoreceptor
69

synapses must be balanced by compensatory endocytosis to retrieve vesicle
membrane and vesicle proteins incorporated into the plasma membrane during
fusion.
Our study demonstrates that Arr1 interacts with NSF to enhance its ATPase
activity and to also stimulate its ability to disassemble the SNARE complex. These
functions are crucial for NSF in regulation of vesicular transport and synaptic
transmission. Moreover, Arr1 deletion markedly reduces the expression level of
NSF and synapse-enriched proteins, including vGLUT1, EAAT5, VAMP2 and
SNAP-25. SNAP-25 and VAMP2 are the major components for SNARE complex
formation (Sogaard et al., 1994;Roth and Burgoyne, 1994). The vGLUT1
regulates the glutamate sequestered into synaptic vesicles in photoreceptor
terminals (Johnson et al., 2007), and EAAT5 is involved in synaptic uptake of
glutamate into photoreceptor to ensure reliable synaptic transmission (Wersinger
et al., 2006). Dynamic exocytosis, vesicle replenishment and glutamate removal
are principally responsible for regulating the kinetics of synaptic transmission in
the photoreceptors (Thoreson, 2007).

70

We observe that a loss of Arr1 gene expression in the retina of Arr1
-/-
mice is
also associated with a reduced level of mRNAs and proteins involved in these
processes, and synaptic activities are dramatically suppressed in the
photoreceptors of these mice. On the basis of previous studies (Mendez et al.,
2003;Smith et al., 2006;Brown et al., 2010a) and our current findings, we suggest
that Arr1 serves a dual functional role in distinct subcellular compartments in the
photoreceptor. Bright light exposure induces Arr1 translocation to the
photoreceptor outer segment for termination and recovery of the light response by
binding to light-activated, G-protein receptor kinase1 dependent phosphorylated
rhodopsin (Wilden et al., 1986; Xu et al., 1997). In the dark, Arr1 is highly
expressed and maintained in all mouse photoreceptor synapses where it interacts
with NSF for modulation of sustained synaptic vesicles release to fulfill the need to
accurately encode subtle membrane potential changes leading to specific
adaptations of the synaptic machinery employed in the photoreceptor synapses.
Mutations in the ARR1 gene lead to Oguchi disease (Fuchs et al., 1995;
Nakazawa et al., 1997) and retinitis pigmentosa (Nakamachi et al., 1998). Oguchi
disease is a rare recessive form of congenital stationary night blindness (CSNB)
71

characterized by distinctive golden-brown discoloration of the fundus that
disappears after prolonged dark adaptation. The typical ERG findings in patients
with Oguchi disease show a negative configuration with subnormal to normal
a-waves and nearly absent b-waves, similar to those in complete CSNB. After
longer dark adaptation (2-4 hrs), the mixed rod-cone ERG shows increases in
both a- and b-waves. This recovery of light sensitivity was thought to be related
to the time course of rhodopsin regeneration (Dryja, 2000). From another
perspective, our study suggests an alternative explanation with Arr1 involvement.
The b-wave reflects the depolarizing response of bipolar cells and the absence of
a b-wave suggests that the synaptic transmission between photoreceptors and
depolarizing bipolar cells may be defective. In our experiments, we observe that
Arr1 enhances NSF ATPase activity and also facilitates SNARE complex
disassembly to maintain tonic neurotransmitter release. We also observe an
abnormal photopic b-wave phenotype associated with loss of light adaptation in
the Arr1
-/-
mice (Figure 3.14a and (Brown et al., 2010)). Increased light
adaptation gradually changes the synaptic signal transfer from photoreceptor
synapse to the second order neuron. We propose that this phenotype reflects a
72

defect of intercellular feedback mechanisms dependent on synaptic transmission
(Van Epps et al., 2001) downstream of the light-activated, phosphorylated GPCR
recovery observed with Arr1 function. Mouse rod photoreceptors only express
Arr1, while the cone photoreceptors express both Arr1 and Arr4. When recording
electrophysiological signals from single mice cones with either one or both visual
Arrestins knocked out, at least one visual Arrestin is required for normal cone
inactivation (Nikonov et al., 2008). In other studies, Arr4 was shown to partially
substitute for Arr1 in Arr1
-/-
rod photoreceptors in transgenic mCAR-H
arr1-/-
retina
(Chan et al., 2007), which targeted the expression of Arr4 to rod photoreceptors
on an Arr1 null

background. In Figure 3.14b, we showed the light-adaptation
b-wave curve in mCAR-H
arr1-/-
retina is almost identical to the Arr1
-/-
. This suggests
that even though Arr4 can partially substitute for the function of Arr1 in rod
phototransduction inactivation, it cannot substitute for the function of Arr1 in
photoreceptor synaptic regulation since Arr4 does not interact with NSF (Figure
3.4). Therefore, the ERG b-wave abnormality in patients with Oguchi disease,
who lack normal expression of ARR1 may result from the disruption in synaptic
transmission in the photoreceptor terminals. Photoreceptors can properly respond
73

to the change in light stimuli depending on precise control of neurotransmitter
release. Reliable photoreceptor synaptic transmission requires accurate detection
of the changes in glutamate concentration in the synaptic cleft by the postsynaptic
cells. We propose that without Arr1 acting as a modulator for NSF, the
photoreceptor synapse cannot maintain the normal graded exocytosis rate to
continuously adjust the release of glutamate to optimize the signal transfer to the
postsynaptic horizontal and bipolar cells.

74

Chapter 4. Gene Expression Profiles of Light-independent
Photoreceptor Loss in Visual Arrestin 1 Knockout Mice

4.1 Introduction
The retina is a highly specialized extension of the central nervous system,
which has evolved to carry out phototransduction and initial visual signal
processing. Retinal degenerations characterized by photoreceptor cell death,
such as retinitis pigmentosa and age-related macular degeneration, are the
leading causes of blindness in developed countries. In most cases, photoreceptor
cell death is the result of the long-term exposure to environmental, inflammatory
and genetic insults. Retinal cell apoptotic process has been implicated as a
common pathway leading to retinal degeneration. Elucidating the molecular
mechanisms that lead to photoreceptor cell death and identifying endogenous
rescue pathways for photoreceptor survival are crucial steps to enhance our
potential for developing new therapeutics.
Vision is initiated when photons are absorbed by visual pigments, triggering
the conversion of chromophore 11-cis-retinal to the all-trans isomer, resulting in
75

activation of the phototransduction cascade. The lifetime of the activated pigment
is limited by a two-step process: active rhodopsin is first phosphorylated by
rhodopsin kinase (GRK1) and then becomes completely inactivated when visual
Arr1 binds (Arshavsky, 2002). It has been showed that the absence of Arr1 (Chen
et al., 1999) sensitizes the photoreceptors of mutant mice to the induction of cell
death by light. Constitutive activation of the phototransduction cascade is
presumably responsible for initiating the subsequent process that lead to
photoreceptor loss (Fain and Lisman, 1999). Furthermore, mutations in the
human gene encoding ARR1 lead to either an inherited recessive form of
stationary night blindness known as Oguchi disease (Fuchs et al.,
1995;Nakazawa et al., 1997), or retinitis pigmentosa (Nakamachi et al., 1998) that
leads to retinal degeneration.
Craft and coworkers identified a progressive cone dystrophy independent of
light exposure in the retinas of Arr1
-/-
mice (Brown et al., 2010a). This
degeneration is particularly evident beyond 2 months of age. It suggested that
constitutive activation of phototransduction is not the mechanism of Arr1
-/-
retinal
degeneration as traditionally thought. Evaluation of differential gene and protein
76

expression between WT and Arr1
-/-
retina is one approach used to identify
pathogenic pathways involved in this cone dystrophy. To resolve this question, it is
necessary to show that altered expression of the target gene exists by using exon
array analysis to identify differentially expressed genes as meaningful cone
dystrophy targets. In the present study, we found that early complement pathway
components, such as serpineg1, C4b, C3, C3ar1, were differentially expressed in
Arr1
-/-
retina compared with WT retina. We further showed that activation of
oncostatin M signaling and JAK-STAT signaling and high induction of endothelin 2
transcripts in the Arr1
-/-
retina. In addition, we observed overexpression of GFAP,
a characteristic marker of reactive gliosis, in Arr1
-/-
retina. Thus our results
demonstrate modifications in gene regulation are associated with
light-independent mechanisms for retinal degeneration driven by the lack of Arr1.

4.2 Results
4.2.1 Age-related light-independent photoreceptor loss in Arr1
-/-
mice
The time course of cell death was assessed by TUNEL analysis (Figure 4.1).
At p22, TUNEL staining in the outer nuclear layer (ONL) in WT and Arr1
-/-
retina
77

(data not shown) was not statistically different in Arr1
-/-
retina compared with WT.
From p30 to p60, the Arr1
-/-
mouse retina has significantly more apoptotic cells
than the WT retina (p<0.001). In Arr1
-/-
mice, there is statistically significantly
greater number of apoptotic cells at p30 and p45 when compared to p22 (p<0.01)
and p60 (p<0.05). These results suggest that the cone photoreceptors of these
mice die through apoptosis and the degeneration is exacerbated following
ablation of Arr1.

78

Figure 4.1 TUNEL staining of the dark-adapted WT and Arr1
-/-
retina at different time
points. TUNEL staining was conducted on sections from p30 to p60 (a). Shown are images of
the middle superior region of the retina. TUNEL-positive apoptotic cells (green) were counted
under a fluorescence microscope. (b) The total number of TUNEL-positive cells from 3
sections of each mouse was recorded, and the average count (mean ± SEM) was calculated
from 3 mice of the same genotype. *P<0.05; **P<0.01; ***P<0.001.
Electroretinography (ERG) was performed to compare visual function
in Arr1
-/-
mice and age-matched WT controls. ERG analyses of retinal physiology
and cone function show that the b-wave amplitudes of photopic ERG responses
decreases in Arr1
-/-
mice in an age-dependent manner, with a 44% reduction in
79

b-wave amplitude by p60 (Figure 4.2). Importantly, the average b-wave
amplitudes of Arr1
-/-
mice are rapidly decreased 28% in p30 compared with p22.
This phenomenon is compatible with the TUNEL staining pattern in Arr1
-/-
retina
(Figure 4.1). The ERG results indicate a defect in cone function and suggest that
the degeneration of cone photoreceptors in the Arr1
-/-
retina is related to
increasing age.

Figure 4.2 Maximum b-wave amplitude of photopic ERG responses in WT and Arr1
-/-

mice. Plot of b-wave amplitudes of photopic ERG responses averaged from mice 22-60
days of age. At p22 days the b-waves in all genotypes were higher than at later ages. Also,
there is a greater disparity in the mean b-wave amplitude of the Arr1
-/-
compared to WT at
p30-60 compared to p22. *P<0.05; **P<0.01; ***P<0.001.

80

4.2.2 Verification of Affymetrix exon array data
To identify potential genetic modifiers and cellular pathways involved in the
cone dystrophy in the Arr1
-/-
mouse, we performed exon array analysis in triplicate
and analyzed using Partek Genomic Suite and Ingenuity Pathway Analysis. The
data reveal statistically significant differences (p value ≤ 0.05) and average fold
changes (AFC) ≥ 2.0 of 211 mapped genes using a two-way ANOVA analysis. In
the Arr1
-/-
retina compared with WT retina, over 266,260 transcripts with signals
above background, only 14 showed more than a fivefold and 167 more than a
twofold increase with a p value less than 0.05. Only 33 transcripts showed more
than a twofold decrease with a p value less than 0.05 (see Appendix A and B). For
a subset of these transcripts (Table 4.1), we have more precisely assessed
changes in transcription level, protein expression level and anatomic localization
between WT and Arr1
-/-
retina.

81

Table 4.1. Selective up-regulated transcripts in Arr1
-/-
retinas compared with WT
Gene Assignment
Gene
Symbol
Fold
Change
(Arr1
-/-
vs.
WT)
p-value
(Arr1
-/-
vs.
WT)
complement component 4B (Childo
blood group)
C4b 6.76275 3.83E-07
complement component 3a receptor 1 C3ar1 4.39176 5.03E-05
serine (or cysteine) peptidase inhibitor,
clade G, memb
Serping1 4.22181 9.05E-07
complement component 1, q
subcomponent, beta polypeptide
C1qb 3.70483 1.99E-06
complement component 1, q
subcomponent, alpha polypeptide
C1qa 3.46567 9.14E-07
complement component 1, q
subcomponent, C chain
C1qc 2.75508 6.00E-05
complement component 3 C3 2.22494 2.79E-07
endothelin 2 Edn2 11.7733 1.85E-08
glial fibrillary acidic protein Gfap 6.86561 1.60E-06
serine (or cysteine) peptidase inhibitor,
clade A, mem
Serpina3n 6.35121 2.56E-06
oncostatin M receptor Osmr 3.73712 1.15E-05
annexin A1 Anxa1 3.6083 1.98E-06
suppressor of cytokine signaling 3 Socs3 3.44784 3.53E-05
signal transducer and activator of
transcription 3
Stat3 2.91539 1.60E-06
Janus kinase 3 Jak3 2.20732 1.54E-06

82

4.2.3 Biological functional groups and pathway analysis
Transcripts were categorized using Ingenuity Pathway Analysis according to
biological function with respect to diseases and disorders, molecular and cellular
functions, and physiological system development and function. Of these pathways,
inflammatory response, cell-to-cell signaling and interaction, and hematological
system development and function were ranked as the top categories according to
p value and the number of molecules categorized (Table 4.2).
Table 4.2 Comparison of Top Biological Pathways Identified as Statistically Significant
between Arr1
-/-
and WT

83

The top canonical pathway identified was the Complement System, which
has been implicated in the pathogenesis of age-related macular degeneration
(Souied et al., 2005;McGeer et al., 2005;Klein et al., 2005;Edwards et al.,
2005;Haines et al., 2005;McKay et al., 2010). Other key canonical pathways
associated with the Arr1
-/-
retinal dystrophy model include Acute Phase Response
Signaling, Oncostatin M Signaling and Jak/Stat Signaling (Figure 4.3).
84

Figure 4.3 Comparison of Top Canonical Pathways Identified by IPA Analysis between
Arr1
-/-
and WT. Canonical pathways are presented on the x axis, and the statistical
significance of each pathway with a threshold of 0.05 is presented on the y axis. Top
canonical pathways above the threshold value are presented. The orange line displays the
ratio of up-regulated transcripts in the data set to the number of genes in each pathway;
revealing complement system to have the largest ratio, while Acute Phase Response
Signaling, Oncostatin M Signaling and Jak/Stat Signaling among others, is above threshold
for statistical significance.
85

The top networks identified with their respective number of focus molecules
are presented in Table 4.3. Of these identified networks, the Inflammatory
Disease, Inflammatory Response network was the most relevant to our model
system due to its known association with some retinal degenerative disease
models.

Table 4.3 Comparison of Top Associated Network Functions Identified by IPA
Analysis between Arr1
-/-
and

WT. Networks are scored based on the number of
Network Eligible Molecules they contain. Networks with higher scores have a lower
probability of finding the observed number of Network Eligible Molecules in a given
network by random chance. Scores are numerical values used to rank networks
according to their degree of relevance to the Network Eligible Molecules in the IPA
dataset. The Inflammatory Disease, Inflammatory Response Network was determined to
be the most relevant to the Arr1
-/-
phenotype.

86

The up-regulated transcripts from the key canonical pathways and networks
were organized into a schematic diagram and are presented in Figure 4.4.

Figure 4.4. Schematic representation of the hypothetical network for photoreceptor
degeneration in Arr1
-/-
mice. Microarray data set analysis was used to generate a
hypothetical network based on up-regulated transcripts. Dotted lines represent indirect
protein relationships, whereas solid lines represent direct relationships; including protein-
protein interactions, phosphorylation, activation, and ligand receptor binding. Molecules in
red represent significantly up-regulated transcripts, green represent significantly
down-regulated transcripts and uncolored molecules were not differentially expressed but
are important components of the network.
87

4.2.4
tivation of the complement cascade and chronic local inflammation has
been
Induction of early complement pathway components in Arr1
-/-
retina
Ac
implicated in the pathogenesis of age-related macular degeneration (AMD).
A number of complement system proteins, complement activators and
complement regulatory protein were identified in drusen, the hallmarker
extracellular deposit associated with early stage AMD (Anderson et al.,
2002;Crabb et al., 2002;Anderson et al., 2004;Johnson et al., 2001;Johnson et al.,
2000;Johnson and Anderson, 2004;Mullins et al., 1997;Mullins et al., 2000;Mullins
et al., 2001). In our exon array analysis data, we found seven transcripts in
complement system were upregulated in dark-adapted Arr1
-/-
retina at one month
(Table1; Figure 4.5). Serping1, Complement C4b (C4b), Complement C3 (C3),
and Complement C3a receptor 1 (C3ar1) were selected for confirmation by
qRT-PCR to identify specific retinal responses to cone dystrophy in Arr1-/- retina.
At p30 or p60, the transcription level of the Serping1, C4b, C3 and C3ar1
was significantly up-regulated in Arr1
-/-
retina compared with WT retina either in
dark or light-adapted condition (Figure 4.6). We also found that Sperping1, C4b,
C3, and C3ar1 mRNA level was slightly increased with age in WT retina. In Arr1
-/-
,
88

the transcription level of Serping1, C4b, C3 and C3ar1 was exaggeratedly
induced at p60 than the level at p30.

Figure 4.5 Induction of early complement pathway components in Arr1
-/-
retina.
Molecules in red represent significantly up-regulated transcripts in the complement pathways.
C1q, serping1, C3, C3ar1 and C4b which are the early complement pathway components
were up-regulated in Arr1
-/-
retina compared with WT retina at p30 based on the exon array
analysis.
89

Figure 4.6. Confirmation of differential transcription expression of serping1, C3,
C3ar1 and C4b using quantitative RT-PCR (qPCR). Expression levels of serping1, C3,
C3ar1 and C4 in WT and Arr1
-/-
mouse retina were measured by quantitative RT-PCR.
Each column represents the average of 3 amplification reactions (mean ± sem). Values for
dark-adapted WT retinas at p30 were set to 1. GAPDH served as references. *p<0.05;
**p<0.01; ***p<0.001. (+/+, WT; -/-, Arr1
-/-
)
4.2.5 Early expression of Annexin A1 in Arr1
-/-
retina
Annexin A1, one of the transcripts induced in the Arr1
-/-
retina, encodes a 37
kDa calcium and phospholipid binding protein that is a strong inhibitor of
90

glucocorticoid-induced eicosanoid synthesis and phospholipase A2. It has been
demonstrated that Anxa1 mediates the anti-inflammatory actions of glucocorticoid
in many experimental models (Podgorski et al., 1992; Yang et al., 1997, 2004).
Furthermore, overexpression of Anxa1 has been shown to induce apoptosis and
involve in the phagocytosis of apoptotic cells.
In order to confirm the expression level and to define subcellular localization
of Anxa1 in the retina, we performed quantitative RT-PCR, immunoblot analysis
and immunohistochemical localization in WT and Arr1
-/-
mice. The transcription
level of Anxa1 was significantly enhanced in the Arr1
-/-
retina compared with WT
retina independent of lighting conditions and age (Figure 4.7(A)). In Arr1
-/-
retina,
the Anxa1 mRNA level was significantly higher at p30 than the level at p60.
At p30 or p60, Anxa1 was below the limit of detection by immunoblot
analysis (Figure 4.7(B)) independent of lightning condition in WT retina. Anxa1
can be detected in Arr1
-/-
retina and the protein expression level at p30 was higher
than the level at p60 which was correlated to the pattern of transcription level.
At p30, Anxa1 immunoreactivity was below the limit of detection in WT retina
(Figure 4.7C(a)). At p60, weak Anxa1 immunostaining was present in the bottom
91

of the ganglion cell layer and nerve fiber layer in WT retina (Figure 4.7C (b)). In
Arr1
-/-
retina, Anxa1 staining was observed in inner segment (IS), outer plexiform
layer and some staining in the inner nuclear layer and ganglion cell layer at p30
(Figure 4.7C (c)). At p60, the Anxa1 immunostaining was present weakly in the
OPL but extensively in the nerve fiber layer (Figure 4.7(d)). The immunoreactivity
in the inner segment was undetectable. This staining pattern was different from
what we observed at p30.

92

Figure 4.7 Early induction of Anxa1 in Arr1
-/-
retina with photoreceptor
degeneration. Anxa1 mRNA were analyzed by quantitative qPCR (A) and immunoblot (B)
in dark-adapted and light-adapted WT and Arr1
-/-
retina at p30 and p60. Transcription
levels were expressed relative to levels in p30 dark-adapted WT retina which were set to
1. GAPDH served as references. Shown are means ± S.E.M of n=3. *p<0.05; **p<0.01;
***p<0.001. (+/+, WT; -/-, Arr1
-/-
) (C) p30 and p60 dark-adapted WT and Arr1
-/-
mouse
retina frozen sections were double labeled fluorescently with the anti-rabbit Anxa1 PAb
(green) and appropriate secondary antibodies and TO-PRO3 for the nuclei (blue). OS,
outer segment; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer;
INL, inner nuclear layer; IPL, inner plexiform layer; GC, retinal ganglion cell layer. Scale
bar, 50µm.

93

4.2.6
phosphorylation has been commonly
d STAT3 was
signific
Activation of oncostatin M and JAK/STAT3 signaling in Arr1
-/-
retina
Up-regulated STAT3 expression and
observed during the degenerative processes in the retina (Samardzija et al.,
2006;Ueki et al., 2008). Similarly, the induction of oncostatin M receptor has been
shown in the light-damaged RPE and retina (Rattner et al., 2008).
In this study, we found the transcription level of OSMR an
antly enhanced in the Arr1
-/-
retina compared with WT retina independent
of lighting conditions and age (Figure 4.8 Top). In Arr1
-/-
retina, the STAT3 mRNA
level was significantly higher at p60 than the level at p30. Light can significantly
induce STAT3 transcripts expression at p30 and also enhance OSMR mRNA level
at p60 in Arr1
-/-
retina. The protein expression level of OSMR and STAT3 in WT
and Arr1
-/-
retinas (Figure 4.8 bottom) under different lighting conditions and age
correlated to the pattern of transcription level. Importantly, phosphorylated STAT3
(pSTAT3) protein was only observed in Arr1
-/-
retina. In WT retina, pSTAT3 was
below the limit of detection by immunoblotting.
94

Figure 4.8 Activation of oncostatin M receptor and STAT3 signaling in Arr1
-/-
retina. OSMR and STAT3 mRNAs were analyzed by quantitative qPCR and
immunoblot analysis in dark-adapted and light-adapted WT and Arr1
-/-
retina at p30
and p60. Transcription levels were expressed relative to levels in p30 dark-adapted
WT retina which were set to 1. GAPDH served as references. Shown are means ±
S.E.M of n=3. *p<0.05; **p<0.01; ***p<0.001. (+/+, WT; -/-, Arr1
-/-
)
4.2.7
revealed an increased expression of genes related to
an a
High induction of Edn2 and Serpina3n transcript in Arr1
-/-
retina
Exon array analysis
cute phase response at p30 in the dark-adapted Arr1
-/-
retina (Table 4.1).
These gene included Edn2 and Serpina3n that are indicators of retinal stress and
95

inflammatory responses.
At p30 or p60, the transcription level of the Edn2 and Serpina3n was
significantly up-regulated in Arr1
-/-
retina compared with WT retina either in dark or
light-adapted condition (Figure 4.9). We also found that Edn2 mRNA level was
persistently elevated to in Arr1
-/-
retina at p60. In Arr1
-/-
retina, the transcription
level of Serpina3n was exaggeratedly induced up at p60 than the level at p30.
However, there is no significantly difference in endothelin receptor B (Ednrb)
mRNA level between WT and Arr1
-/-
retina at p30 or p60. Light can slightly induce
Ednrb transcription level in WT and Arr1
-/-
retina at p30.

96

Figure 4.9 High induction of Edn2 and Serpina3n transcript in Arr1
-/-
retina. Edn2, Ednrb
and Serpina3n mRNAs were analyzed by qPCR in dark-adapted and light-adapted WT and
Arr1
-/-
retina at p30 and p60. Transcription levels were expressed relative to levels in p30
dark-adapted WT retina which were set to 1. GAPDH served as references. *p<0.05;
**p<0.01; ***p<0.001. (+/+, WT; -/-, Arr1
-/-
)
4.2.8 Activation of reactive gliosis in Arr1
-/-
mice
During neuronal degeneration, Müller cells are well known to undergo
reactive gliosis characterized by the upregulation of intermediate filaments
(Bringmann A, et al., 2006,2010).This was clearly demonstrated
byimmunostaining against GFAP, a sensitive marker for retinal gliosis in response
to photoreceptor degeneration (Hong et al., 2000). At p30 or p60, the transcription
97

level of the GFAP is significantly up-regulated in Arr1
-/-
retina compared with WT
retina either in dark or light-adapted condition (Figure 4.10A top). We also found
that exposed to light for one hour can further enhance the GFAP mRNA level in
Arr1
-/-
retina. The transcription level of GFAP in Arr1
-/-
retina is significantly higher
at p60 than the level at p30. The protein expression level of GFAP in WT and
Arr1
-/-
retinas under different lighting conditions and age correlated to the pattern
of transcription level (Figure 4.10A bottom).

Figure 4.10 Activation of Müller glial cells in Arr1
-/-
retina. (A) GFAP mRNA was analyzed by
quantitative qPCR in dark-adapted and light-adapted WT and Arr1
-/-
retina at p30 and p60.
Transcription levels were expressed relative to levels in p30 dark-adapted WT retina which were
set to 1. GAPDH served as references. Shown are means ± S.E.M of n=3. *p<0.05; **p<0.01;
***p<0.001 (+/+, WT; -/-, Arr1
-/-
)
98

GFAP immunoreactivity is normally found only in the astrocytes in the
ganglion cell layer of the retina in the WT retina (Figure 4.10B (a-d)). A slight
upregulation of GFAP immunoreactivity was observed in the Arr1
-/-
retina at p30,
mainly in the inner plexiform layer (IPL, Figure 4.10B (e) arrows). At p45, GFAP is
stronger upregulated and extends to the top of the inner nuclear layer (INL, Figure
4.10B (f)). At p60, the intensive staining of GFAP further extends to the outer
plexiform layer of the photoreceptor and co-localization of GFAP and cone
arrestin was observed in the OPL (Figure 4.10B (g) arrows).

99

Figure 4.10 Activation of Müller glial cells in Arr1
-/-
retina (B) p30 and p60 dark-adapted
WT and Arr1
-/-
mouse retina frozen sections were double labeled fluorescently with the
anti-mouse GFAP MAb (green), anti-rabbit cone arrestin PAb (red) and TO-PRO3 for the
nuclei (blue). Wild type retina staining for GFAP shows the typical, mild signal restricted to the
GCL (a-d). Similar to wild-type retina, only mild GFAP immunostaining was seen in Arr1
-/-

retina at p22 (e). A slight upregulation of GFAP immunoreactivity in the IPL was observed in
the Arr1
-/-
retina at p30 ((f), arrows). At p45, GFAP staining extends to the top of the inner
nuclear layer (g). At p60, the intensive staining of GFAP further extends to the OPL and
co-localization of GFAP and cone arrestin was observed ((h), arrows). Scale bar, 50μm.

100

4.3 Discussion
In this study, we used an exon array analysis to investigate profile changes
in gene expression associated with retinal degeneration in Arr1
-/-
mice. We verify
these patterns in gene expression, we did quantitative real time (qRT)-PCR

to
assess if gene expression changes were evident at earlier

or later time points
when the retinal degeneration is initiated. As discussed in detail below, a subset of
the gene expression changes identified were

comparable to those identified in
previous microarray studies in retinal damage and retinal degeneration. This
suggests

that these gene expression changes may represent a shared
retinal

response to retinal degeneration that may enhance the development

of
disease.
Complement Pathway in Retinal Disease
The complement cascade is a pathway that is largely involved in the
recruitment and chemotaxis of inflammatory cells and direct induction of cell death
(Rus et al., 2005). Recent evidence suggests that complement pathways play
important roles in neuronal survival and even in normal neuronal development.
101

Activation of complement cascade has also potential initiating pathway involved in
promoting neuronal death.
In our study, several complement components were elevated in Arr1
-/-

retinas,

including Serping1, C3, C3ar1 and C4b, which were significantly
upregulated

at one month and further enhanced by two months (Figure 4).
Increased complement

gene expression has been reported in retinas from a
mouse photoreceptor

degeneration model and in Müller cells from a rat model

of
diabetic retinopathy (Gerhardinger et al., 2005). Abnormal activation of the
alternative pathway of complement has been implicated in common
neurodegeneration diseases including age-related macular degeneration.
Activation of the alternative pathway of complement in blood is under genetic
control and increases with age. Thus, uncontrolled activation of the expression of
complement genes

may be associated with retinal damage or disease. In our
study, we detected the expression of early pathway components was increased
with age and exaggeratedly induced in the Arr1
-/-
retina with cone dystrophy. It is
possible that the early

complement components contribute to cone dystrophy in
102

the Arr1
-/-
retina due to their opsonization

or chemoattractant activity results in
persistent inflammation to promote local tissue damage, although this remains to
be

investigated.
Induction of Oncostatin M signaling and Jak3/STAT3 signaling pathways in
retinal degeneration
Oncostatin M (OSM), a member of interleukin 6 (IL-6) family, forms two
types of heterodimeric signaling complex; gp130/leukemia inhibitory factor
receptor (LIFR) which can be activated by LIF or OSM and gp130/OSMR which is
activated by OSM only. Upon ligand binding, the gp130/OSMR receptor
activates Jak/STATs signaling pathway and phosphoryalted STAT proteins
translocate to the nucleus and alter transcriptional activity. Phosphorylation of the
STAT3 protein has been commonly observed during degenerative process in the
retina (Mechoulam and Pierce, 2005; Samardzija et al., 2006; Yang et al., 2007;
Ueki et al., 2008).In our study, we also showed the induction of OSMR and
pSTAT3 in the Arr1
-/-
retina. However, the up- and downstream signaling
cascades are not yet clear. A likely candidate up- or downstream is Jak3, which
103

was induced at p30 in the Arr1
-/-
retina based on the exon array data (Table 4.1).
Since Jak3 activity is mainly regulated on the gene expression level (Mangan et al.
2006), it is possible that Jak3 is part of an immune-related response induced by
retinal damage. This response may be responsible for the attraction of
immune-cells entering the degenerated retina (Zeng et al. 2005; Zhang et al.
2005).
Muller glial cells activation
Müller cells are connected to almost all the retinal neurons by their
specialized morphology, which is the foundation of normal retinal function
(Reichenbach et al., 1995; Newman 1996). Müller cells are involved in many
retinal physiological activities including glycometabolism, blood regulation,
neurotransmitter cycling, homeostasis and control of neuronal excitability
(Newman and Zahs 1998; Stevens et al., 2003; Bringmann et al., 2004). Müller
cell become reactivated in response to many retinal pathophysiological processes,
including light damage, retinal trauma, ischemia, retinal detachment, glaucoma,

104

diabetic retinopathy, inherited retinal degeneration and age-related macular
degeneration (Bringmann and Reichenbach, 2001; Bringmann et al., 2006, 2010).
In the murine retina, Edn2 is mainly expressed in photoreceptor cells (Joly,
et al., 2008), and the receptor for Edn2 is predominantly localized to astrocytes
and Müller cells (Ratterner and Nathans, 2005). In our study, we found the
transcription level of Edn2 was highly induced 20 fold up-regulation at p30 and
persistently enhanced at p60 in Arr1
-/-
retina. However, the Ednrb mRNA level was
not induced in Arr1
-/-
retina compared with WT but light can slightly increase the
Ednrb mRNA level in both Arr1
-/-
and WT retina at p30. Photoreceptor injury
from retinal degeneration and light damage induces the molecular signal
photoreceptor-derived Edn2 that stimulates the endothelin receptor B (Ednrb) in
Müller cells and results in Müller cell activation, as indicated by the increased
expression of GFAP and decrease in glutamine synthetase. Activated Müller cells
may have direct cytotoxic effect by the release of soluble factors such as
proinflammatory cytokines contributing to photoreceptor apoptosis and retinal
degeneration (de et al., 1994;de et al., 1997;Nakazawa et al., 2006;Nakazawa et
al., 2007). We found upregulation of GFAP in Arr1
-/-
retina initiating from p30
105

(Figure 8b (f)),consistent with reports of glial

activation in both human tissue and
animal models of retina damage and degeneration, which indicated degenerative
changes in Arr1
-/-
were already underway starting at p30. The significant increase
of GFAP protein seen in Arr1
-/-
retina at p60 (Figure 8b (h)) supports the data
showing proliferation of Müller cells and their activation in response to the
progression of retinal degeneration.
106

Chapter 5. Conclusions

These studies have demonstrated that the interaction of Arr1 and NSF is
ATP-dependent, and the N-terminal domain of Arr1 interacts with the N and D1
junctional domains of NSF. The Arr1-NSF interactions are greater in the
photoreceptor synaptic terminal in the dark. Furthermore, Arr1 enhances the
NSF ATPase activity and increases the NSF disassembly activities, which are
critical for NSF functions in sustaining a higher rate of exocytosis in the
photoreceptor synapses and the compensatory endocytosis to retrieve vesicle
membrane and vesicle proteins for vesicle recycling. Deletion of Arr1 expression
also leads to reduced levels of mRNA expression of NSF, vGLUT1, EAAT5 and
VAMP2, which have the potential to markedly depress the exocytosis rate in the
dark-adapted retinas. These data provide strong in vitro results and for the first
time, in vivo evidence that, in photoreceptors demonstrate the Arr1 and NSF
interaction is necessary for the maintenance of normal vision.
This dissertation reveals an alternative functional role of Arr1 in
photoreceptor synapses by interacting as a modulator for NSF to regulate
107

synaptic transmission and provides further insights into potential mechanisms of
inherited retinal diseases. Continued study of Arr1
-/-
mouse model retinas will
provide useful information regarding the early pathophysiology of
light-independent photoreceptor loss that might be useful in the design of
therapeutic measures to delay or decrease retinal degeneration.
While exploring the functional significance of Arr1 expression in cone
photoreceptors, we observed a light-independent cone dystrophy and completed
a(Brown et al., 2010b) microarray analysis of Arr1
-/-
mouse eyes undergoing
retinal degeneration. In this study, we identified a set of genes that are
preferentially expressed in the photoreceptor cells. It also revealed increased
expression of an overlapping set of stress response genes that respond to the
early stages of retinal degeneration in several mouse models of the disorder. This
discovery will lead to additional studies aiming to determine the primary cause of
altered Annexin a1 expression and potential effect of the activation of Oncostatin
M signaling and the early complement pathways components involved in this
light-independent cone dystrophy. Determining the factors that initiate these

108

processes and ways to control these processes may significantly improve the
rehabilitation of the degenerating retina.
Inherited retinal degeneration affects about 1:2000 of the United States
population and they are the most important cause of visual loss in the young and
account for a large proportion of blindness in adult life. These disorders may be
inherited in any one of the recognized patterns, and they fall within a spectrum
ranging from retinitis pigmentosa (RP) to macular dystrophies, which are often the
consequence of genetic defects in proteins within the phototransduction cascade.
Age-related macular degeneration is the leading cause of irreversible visual
impairment and blindness worldwide in the elderly population. Elucidating the
molecular signals involved in retina health and disease is crucial to understand
the mechanisms that underlie retinal development and death, and also may
enhance our potential for developing new therapeutics. Since the retina is an
integral part of the central nervous system, many of the characteristics of cell
death in the retina and in photoreceptor in particular may be relevant for neuronal
cell death in general. Our various strains of visual transduction targeted knockout

109

mice are effective experimental models to help us investigate and elucidate the
etiology associated with the pathogenesis of the visual disorders.
The goal of this dissertation is to accelerate our ability to gain insight into the
alternative roles of the Arr1 in the photoreceptor cells and enable us to
understand more precisely the mechanisms underlying night blindness
associated with clinically diagnosed Oguchi disease or other Arr1-associated
forms of retinitis pigmentosa. Gaining an understanding of the molecular
pathways underlying the neuroprotective roles of these complement factors, we
identified signaling proteins that may be involved in the retinal degeneration in
visual Arrestin 1 knockout mice model. Further studies will determine what the
regulatory switches are that maintain the balance between neurodegeneration
and neuroprotection and will be important avenues of investigation towards the
development of novel therapies for preventing retinal degeneration.

110

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128

Appendix A. Up-regulated transcripts in Arr1
-/-
retina
Gene_assignment Gene Symbol
Fold-Chang
e (Arr1
-/-
vs.
WT)
p-value
(Arr1
-/-
vs.
WT)
NM_013532 // Lilrb4 // leukocyte
immunoglobulin-like receptor, subfamily
B, memb
Lilrb4 14.5854 8.87E-06
NM_007902 // Edn2 // endothelin 2 // 4 D2.2
// 13615 /// ENSMUST00000030384 // E
Edn2 11.7733 1.85E-08
NM_008331 // Ifit1 // interferon-induced
protein with tetratricopeptide repeats
Ifit1 10.0723 0.000279054
NM_001131020 // Gfap // glial fibrillary
acidic protein // 11 D|11 62.0 cM // 14
Gfap 6.86561 1.60E-06
NM_198657 // EG381438 // predicted gene,
EG381438 // 3 B|3 // 381438 /// ENSMUST
EG381438 6.78452 1.46E-06
NM_009780 // C4b // complement
component 4B (Childo blood group) // 17
B1|17 18.
C4b 6.76275 3.83E-07
NM_010260 // Gbp2 // guanylate binding
protein 2 // 3 H1|3 67.4 cM // 14469 ///
Gbp2 6.43829 1.69E-07
NM_008655 // Gadd45b // growth arrest
and DNA-damage-inducible 45 beta // 10
C1|
Gadd45b 6.39688 1.21E-06
NM_009252 // Serpina3n // serine (or
cysteine) peptidase inhibitor, clade A,
mem
Serpina3n 6.35121 2.56E-06
NM_011313 // S100a6 // S100 calcium
binding protein A6 (calcyclin) // 3 F1-F2|3
S100a6 5.95383 8.40E-07
NM_133664 // Lad1 // ladinin // 1 E4 //
16763 /// ENSMUST00000038760 // Lad1 //
Lad1 5.58255 3.01E-08
NM_009099 // Trim30 // tripartite
motif-containing 30 // 7 E3|7 50.4 cM //
20128
Trim30 5.24396 1.01E-06
129

NM_010745 // Ly86 // lymphocyte antigen
86 // 13 A3.3 // 17084 /// ENSMUST000000
Ly86 5.16919 2.43E-06
NM_008134 // Glycam1 // glycosylation
dependent cell adhesion molecule 1 // 15
F
Glycam1 4.93135 2.97E-06
NM_144761 // Crygb // crystallin, gamma B
// 1 C2|1 32.0 cM // 12965 /// ENSMUST
Crygb 4.84826 0.00045938
NM_175628 // A2m //
alpha-2-macroglobulin // 6 F1|6 61.7 cM //
232345 /// ENSMUS
A2m 4.7437 9.57E-06
NM_025378 // Ifitm3 // interferon induced
transmembrane protein 3 // 7 F5 // 661
Ifitm3 4.5846 1.43E-05
NM_009890 // Ch25h // cholesterol
25-hydroxylase // 19 C1 // 12642 ///
ENSMUST00
Ch25h 4.52628 2.22E-05
NM_011337 // Ccl3 // chemokine (C-C
motif) ligand 3 // 11 C|11 47.59 cM // 20302
Ccl3 4.49413 5.99E-07
ENSMUST00000068009 //
OTTMUSG00000007392 // predicted gene,
OTTMUSG00000007392 /
OTTMUSG0000
0007392
4.41399 6.65E-07
NM_009779 // C3ar1 // complement
component 3a receptor 1 // 6 F1 // 12267 ///
EN
C3ar1 4.39176 5.03E-05
NM_021274 // Cxcl10 // chemokine (C-X-C
motif) ligand 10 // 5 E2|5 53.0 cM // 15
Cxcl10 4.37737 2.14E-07
NM_007695 // Chi3l1 // chitinase 3-like 1 //
1 E4|1 72.3 cM // 12654 /// ENSMUST
Chi3l1 4.29729 1.62E-08
NM_008919 // Ppyr1 // pancreatic
polypeptide receptor 1 // 14 B|14 10.5 cM //
19
Ppyr1 4.22706 1.52E-05
NM_009776 // Serping1 // serine (or
cysteine) peptidase inhibitor, clade G,
memb
Serping1 4.22181 9.05E-07
130

NM_019949 // Ube2l6 //
ubiquitin-conjugating enzyme E2L 6 // 2
E1 // 56791 /// E
Ube2l6 4.22046 2.71E-05
NM_007774 // Cryga // crystallin, gamma A
// 1 C2|1 32.0 cM // 12964 /// ENSMUST
Cryga 4.09507 1.63E-06
NM_010516 // Cyr61 // cysteine rich
protein 61 // 3 H2|3 72.9 cM // 16007 /// EN
Cyr61 4.0659 6.97E-06
NM_001130174 // Tnnt2 // troponin T2,
cardiac // 1 E4|1 60.0 cM // 21956 /// NM_
Tnnt2 4.06071 1.08E-05
NM_008880 // Plscr2 // phospholipid
scramblase 2 // 9 E3.3 // 18828 ///
ENSMUST0
Plscr2 4.05746 1.29E-05
NM_001044384 // Timp1 // tissue inhibitor
of metalloproteinase 1 // X A1.3|X 6.2
Timp1 3.99689 8.00E-06
NM_008491 // Lcn2 // lipocalin 2 // 2 A3|2
27.0 cM // 16819 /// ENSMUST000000507
Lcn2 3.86923 1.87E-05
NM_010380 // H2-D1 // histocompatibility
2, D region locus 1 // 17 B1|17 19.09 c
H2-D1 3.82132 1.04E-06
NM_008006 // Fgf2 // fibroblast growth
factor 2 // 3 A2-B|3 19.3 cM // 14173 ///

Fgf2 3.78348 3.84E-06
NM_008788 // Pcolce // procollagen
C-endopeptidase enhancer protein // 5
G2|5 78
Pcolce 3.77625 7.70E-06
NM_145825 // Cetn4 // centrin 4 // 3 B //
207175 /// ENSMUST00000102955 // Cetn4

Cetn4 3.75602 0.0069332
NM_011019 // Osmr // oncostatin M
receptor // 15 A1|15 4.6 cM // 18414 ///
ENSMU
Osmr 3.73712 1.15E-05
NM_009777 // C1qb // complement
component 1, q subcomponent, beta
polypeptide //
C1qb 3.70483 1.99E-06
131

NM_133738 // Antxr2 // anthrax toxin
receptor 2 // 5 E3 // 71914 ///
ENSMUST0000
Antxr2 3.67495 1.07E-07
NM_007498 // Atf3 // activating
transcription factor 3 // 1 H6|1 103.2 cM //
119
Atf3 3.65014 3.34E-06
NM_010730 // Anxa1 // annexin A1 // 19
B|19 18.0 cM // 16952 ///
ENSMUST00000025
Anxa1 3.6083 1.98E-06
NM_203507 // Rwdd4a // RWD domain
containing 4A // 8 B1.1 // 192174 ///
ENSMUST0
Rwdd4a 3.58136 3.76E-07
NM_183166 // EG233164 // predicted gene,
EG233164 // 7 B3|7 // 233164 /// NM_177

EG233164 3.55732 0.000886574
NM_021281 // Ctss // cathepsin S // 3 F2.1|3
42.7 cM // 13040 /// ENSMUST0000001
Ctss 3.48656 5.90E-06
NM_030707 // Fcrls // Fc receptor-like S,
scavenger receptor // 3 F1 // 80891 //
Fcrls 3.47865 3.38E-05
NM_033601 // Bcl3 // B-cell
leukemia/lymphoma 3 // 7 A3|7 6.5 cM //
12051 /// EN
Bcl3 3.47458 2.49E-07
NM_007572 // C1qa // complement
component 1, q subcomponent, alpha
polypeptide /
C1qa 3.46567 9.14E-07
NM_153561 // Nudt6 // nudix (nucleoside
diphosphate linked moiety X)-type motif
Nudt6 3.46213 9.89E-07
NM_007707 // Socs3 // suppressor of
cytokine signaling 3 // 11 E2 // 12702 /// E
Socs3 3.44784 3.53E-05
NM_008630 // Mt2 // metallothionein 2 // 8
C5|8 45.0 cM // 17750 /// ENSMUST0000
Mt2 3.40369 3.64E-07
NM_008416 // Junb // Jun-B oncogene // 8
C2-D1|8 38.6 cM // 16477 /// ENSMUST000
Junb 3.36986 4.73E-07
132

NM_013706 // Cd52 // CD52 antigen // 4
D3|4 73.5 cM // 23833 ///
ENSMUST00000000
Cd52 3.35928 1.35E-05
NM_028838 // Lrrc2 // leucine rich repeat
containing 2 // 9 F2|9 70.4 cM // 7424
Lrrc2 3.35017 9.81E-08
NM_011173 // Pros1 // protein S (alpha) //
16 C1.3 // 19128 /// ENSMUST000000236
Pros1 3.34819 5.86E-08
NM_018734 // Gbp3 // guanylate binding
protein 3 // 3 H1 // 55932 /// ENSMUST000
Gbp3 3.31058 9.66E-06
NM_001159417 // Irf9 // interferon
regulatory factor 9 // 14 C3|14 21.5 cM // 16
Irf9 3.29625 7.67E-07
ENSMUST00000037953 // 2810032G03Rik
// RIKEN cDNA 2810032G03 gene // 12 A1.1
2810032G03Rik3.24455 2.41E-06
NM_007651 // Cd53 // CD53 antigen // 3
F2.3|3 50.5 cM // 12508 ///
ENSMUST000000
Cd53 3.22857 1.39E-06
NM_026331 // Slc25a37 // solute carrier
family 25, member 37 // 14 D2 // 67712 /
Slc25a37 3.10538 5.37E-06
NM_012028 // St6galnac5 // ST6
(alpha-N-acetyl-neuraminyl-2,3-beta-galac
tosyl-1,
St6galnac5 3.06348 8.48E-07
NM_007447 // Ang // angiogenin,
ribonuclease, RNase A family, 5 // 14
B-C1|14 18
Ang 3.0479 4.06E-07
NM_001001892 // H2-K1 //
histocompatibility 2, K1, K region // 17
B1|17 18.44 cM
H2-K1 3.02205 0.000330982
NM_007697 // Chl1 // cell adhesion
molecule with homology to L1CAM // --- //
126
Chl1 2.96231 3.76E-07
NM_028595 // Ms4a6c //
membrane-spanning 4-domains,
subfamily A, member 6C // 19
Ms4a6c 2.94023 2.63E-05
133

NM_011331 // Ccl12 // chemokine (C-C
motif) ligand 12 // 11 C|11 47.0 cM // 2029
Ccl12 2.92294 9.38E-07
NM_213659 // Stat3 // signal transducer
and activator of transcription 3 // 11 D
Stat3 2.91539 1.60E-06
NM_001039530 // Parp14 // poly
(ADP-ribose) polymerase family, member
14 // 16 B
Parp14 2.91198 3.16E-07
NM_054098 // Steap4 // STEAP family
member 4 // 5 A1 // 117167 ///
ENSMUST000001
Steap4 2.90753 6.12E-05
NM_207648 // H2-Q6 // histocompatibility
2, Q region locus 6 // 17 B1|17 19.18 c
H2-Q6 2.90472 0.00102213
NM_011662 // Tyrobp // TYRO protein
tyrosine kinase binding protein // 7 B|7 10.
Tyrobp 2.89264 8.20E-05
NM_013602 // Mt1 // metallothionein 1 // 8
C5|8 45.0 cM // 17748 /// ENSMUST0000

Mt1 2.87723 2.05E-05
NM_007568 // Btc // betacellulin, epidermal
growth factor family member // 5 E2|
Btc 2.86978 8.93E-06
NM_053109 // Clec2d // C-type lectin
domain family 2, member d // 6 F3 // 93694
Clec2d 2.85688 9.50E-05
NM_153601 // Lgsn // lengsin, lens protein
with glutamine synthetase domain // 1
Lgsn 2.85614 0.00129525
NM_024184 // Asf1b // ASF1 anti-silencing
function 1 homolog B (S. cerevisiae) /
Asf1b 2.84161 5.54E-07
NM_199146 // AI451617 // expressed
sequence AI451617 // 7 E3 // 209387 ///
NM_00

AI451617 2.79736 2.52E-05
NM_008137 // Gna14 // guanine nucleotide
binding protein, alpha 14 // 19 A-B|19
Gna14 2.77255 1.61E-06
NM_008326 // Irgm1 // immunity-related
GTPase family M member 1 // 11 B1.2 // 15
Irgm1 2.7627 1.70E-06
134

NM_007574 // C1qc // complement
component 1, q subcomponent, C chain //
4 D3|4 6
C1qc 2.75508 6.00E-05
NM_011854 // Oasl2 // 2'-5' oligoadenylate
synthetase-like 2 // 5 F // 23962 ///
Oasl2 2.74045 1.32E-06
NM_001082573 // Crygc // crystallin,
gamma C // 1 C2|1 32.0 cM // 12966 ///
NM_0
Crygc 2.70895 0.000146321
NM_026473 // Tubb6 // tubulin, beta 6 // 18
E1 // 67951 /// ENSMUST00000001513 /
Tubb6 2.69868 1.66E-05
NM_018773 // Skap2 // src family
associated phosphoprotein 2 // 6 B3 //
54353 //
Skap2 2.69632 6.01E-06
NM_001143689 // H2-gs10 // MHC class I
like protein GS10 // 17 B1|17 // 436493 /
H2-gs10 2.69483 1.99E-05
NM_023386 // Rtp4 // receptor transporter
protein 4 // 16 B1 // 67775 /// ENSMUS
Rtp4 2.69326 3.89E-05
NM_010397 // H2-T22 // histocompatibility
2, T region locus 22 // 17 B1|17 19.74
H2-T22 2.69312 0.000429645
NM_175485 // Prtg // protogenin homolog
(Gallus gallus) // 9 D // 235472 /// ENS
Prtg 2.69305 1.17E-06
NM_009763 // Bst1 // bone marrow stromal
cell antigen 1 // 5 B3|5 25.0 cM // 121
Bst1 2.69156 1.40E-06
NM_001083929 // Gpx3 // glutathione
peroxidase 3 // 11 B3-B5 // 14778 ///
NM_008
Gpx3 2.65257 0.00443777
NM_007801 // Ctsh // cathepsin H // 9
E3.1|9 50.0 cM // 13036 ///
ENSMUST0000003
Ctsh 2.64311 4.59E-06
NM_019472 // Myo10 // myosin X // 15 C|15
9.2 cM // 17909 /// ENSMUST00000110457
Myo10 2.61044 1.42E-05
NM_010234 // Fos // FBJ osteosarcoma
oncogene // 12 D2|12 40.0 cM // 14281 /// E
Fos 2.58506 2.97E-07
135

NM_001098271 // Tmem176a //
transmembrane protein 176A // 6 B2.3 //
66058 /// NM
Tmem176a 2.53579 1.15E-07
NM_133859 // Olfml3 // olfactomedin-like 3
// 3 F2.2 // 99543 /// ENSMUST0000002
Olfml3 2.52157 8.14E-06
NM_007776 // Crygd // crystallin, gamma D
// 1 C2|1 32.0 cM // 12967 /// NM_0270

Crygd 2.48904 5.42E-06
NM_011609 // Tnfrsf1a // tumor necrosis
factor receptor superfamily, member 1a /
Tnfrsf1a 2.48013 3.56E-06
NM_009735 // B2m // beta-2 microglobulin
// 2 F1-F3|2 69.0 cM // 12010 /// ENSMU
B2m 2.44473 7.67E-07
NM_013561 // Htr3a // 5-hydroxytryptamine
(serotonin) receptor 3A // 9 A5.3 // 1
Htr3a 2.42899 8.83E-06
NM_013904 // Hey2 //
hairy/enhancer-of-split related with YRPW
motif 2 // 10 A4
Hey2 2.40353 6.36E-08
NM_027030 // Dcps // decapping enzyme,
scavenger // 9 A4 // 69305 /// ENSMUST000
Dcps 2.37934 1.54E-06
NM_020008 // Clec7a // C-type lectin
domain family 7, member a // --- // 56644 /
Clec7a 2.37725 4.02E-06
NM_009621 // Adamts1 // a disintegrin-like
and metallopeptidase (reprolysin type
Adamts1 2.37708 2.44E-07
NM_009744 // Bcl6 // B-cell
leukemia/lymphoma 6 // 16 B1|16 13.9 cM
// 12053 ///
Bcl6 2.374 1.79E-05
NM_001081215 // Ddx60 // DEAD
(Asp-Glu-Ala-Asp) box polypeptide 60 // 8
B3.1 //
Ddx60 2.37311 0.000161978
NM_025374 // Glo1 // glyoxalase 1 // 17
A3.3|17 16.0 cM // 109801 /// NM_0011135
Glo1 2.36375 8.84E-06
NM_018738 // Igtp // interferon gamma
induced GTPase // 11 B1.3|11 32.0 cM // 16
Igtp 2.36116 7.94E-06
136

NM_001081079 // Ogfrl1 // opioid growth
factor receptor-like 1 // 1 A5 // 70155
Ogfrl1 2.34292 0.000385182
NM_172767 // Vwa5a // von Willebrand
factor A domain containing 5A // 9 B // 677
Vwa5a 2.34205 1.09E-07
NM_010329 // Pdpn // podoplanin // 4 E1 //
14726 /// ENSMUST00000030317 // Pdpn
Pdpn 2.3334 1.63E-06
NM_146042 // Rnf144b // ring finger
protein 144B // 13 A5 // 218215 ///
ENSMUST0
Rnf144b 2.3079 2.29E-05
L78788 // D17H6S56E-5 // DNA segment,
Chr 17, human D6S56E 5 // 17 B1|17 19.0
cM
D17H6S56E-5 2.2884 0.000203444
NM_008329 // Ifi204 // interferon activated
gene 204 // 1 H3|1 95.2 cM // 15951
Ifi204 2.27448 1.34E-05
NM_001082976 // Tc2n // tandem C2
domains, nuclear // 12 F1 // 74413 ///
NM_0289
Tc2n 2.27276 0.000799495
NM_146068 // 2310008H04Rik // RIKEN
cDNA 2310008H04 gene // 16 A2 // 224008 /
2310008H04Rik2.27255 1.46E-06
NM_007470 // Apod // apolipoprotein D //
16 B2|16 21.2 cM // 11815 /// ENSMUST00
Apod 2.26185 3.59E-06
NM_009853 // Cd68 // CD68 antigen // 11
B3|11 39.0 cM // 12514 /// ENSMUST000000
Cd68 2.25927 2.70E-05
NM_011150 // Lgals3bp // lectin,
galactoside-binding, soluble, 3 binding
protein
Lgals3bp 2.24994 3.87E-05
NM_017372 // Lyz2 // lysozyme 2 // 10
D2|10 66.0 cM // 17105 /// NM_013590 // Ly

Lyz2 2.23702 0.000559194
NM_009778 // C3 // complement
component 3 // 17 E1-E3|17 34.3 cM //
12266 /// EN

C3 2.22494 2.79E-07
137

NM_024257 // Hdhd3 // haloacid
dehalogenase-like hydrolase domain
containing 3 /
Hdhd3 2.2205 3.46E-06
NM_016669 // Crym // crystallin, mu // 7
F2|7 55.0 cM // 12971 /// ENSMUST000000
Crym 2.21211 4.76E-07
NM_010708 // Lgals9 // lectin, galactose
binding, soluble 9 // 11 B5 // 16859 //
Lgals9 2.21185 0.000113914
NM_010589 // Jak3 // Janus kinase 3 // 8
B3.3|8 33.0 cM // 16453 /// NM_013564 /
Jak3 2.20732 1.54E-06
NM_008597 // Mgp // matrix Gla protein // 6
G1 // 17313 /// ENSMUST00000032342 /
Mgp 2.18928 7.71E-05
NM_023142 // Arpc1b // actin related
protein 2/3 complex, subunit 1B // 5 G2 //
Arpc1b 2.17346 0.000427271
NM_008013 // Fgl2 // fibrinogen-like
protein 2 // 5 A3|5 7.0 cM // 14190 /// ENS
Fgl2 2.16943 0.000183929
NM_008533 // Cd180 // CD180 antigen // 13
D1 // 17079 /// ENSMUST00000022124 //
Cd180 2.16595 0.000485736
NM_016675 // Cldn2 // claudin 2 // X F1 //
12738 /// ENSMUST00000054889 // Cldn2
Cldn2 2.16518 0.000703597
NM_016762 // Matn2 // matrilin 2 // 15 B3.3
// 17181 /// ENSMUST00000022947 // M
Matn2 2.16408 0.00323911
NM_007799 // Ctse // cathepsin E // 1 F|1
69.1 cM // 13034 /// ENSMUST0000007335
Ctse 2.14366 8.78E-06
NM_178911 // Pld4 // phospholipase D
family, member 4 // 12 F1 // 104759 /// ENS
Pld4 2.13702 0.000109007
NM_178598 // Tagln2 // transgelin 2 // 1
H3|1 94.2 cM // 21346 /// ENSMUST000001
Tagln2 2.12364 2.99E-07
NM_009263 // Spp1 // secreted
phosphoprotein 1 // 5 E5|5 56.0 cM // 20750
Spp1 2.12183 2.80E-05
NM_011990 // Slc7a11 // solute carrier
family 7 (cationic amino acid transporter
Slc7a11 2.12021 5.69E-05
NM_178677 // Sec22c // SEC22 vesicle
trafficking protein homolog C (S. cerevisia
Sec22c 2.11528 1.94E-05
138

NM_013489 // Cd84 // CD84 antigen // 1
H3|1 93.3 cM // 12523 ///
ENSMUST00000042
Cd84 2.10514 0.00042248
NM_033270 // E2f6 // E2F transcription
factor 6 // 12 A1.1|12 6.0 cM // 50496 //
E2f6 2.10016 2.77E-07
NM_008966 // Ptgfr // prostaglandin F
receptor // 3 H3|3 75.8 cM // 19220 /// EN
Ptgfr 2.09796 0.0015131
NM_054096 // Tirap // toll-interleukin 1
receptor (TIR) domain-containing adapto
Tirap 2.08642 0.000339179
NM_011698 // Lin7b // lin-7 homolog B (C.
elegans) // 7 B2 // 22342 /// ENSMUST0
Lin7b 2.08521 5.47E-05
NM_172689 // Ddx58 // DEAD
(Asp-Glu-Ala-Asp) box polypeptide 58 // 4
A5 // 23007
Ddx58 2.07142 1.89E-07
NM_031159 // Apobec1 // apolipoprotein B
mRNA editing enzyme, catalytic polypept
Apobec1 2.06932 0.000283372
NM_001111096 // Lyn // Yamaguchi
sarcoma viral (v-yes-1) oncogene
homolog // 4 A

Lyn 2.06409 2.49E-05
NM_008872 // Plat // plasminogen
activator, tissue // 8 A2|8 9.0 cM // 18791 ///
Plat 2.06108 6.60E-06
BC096647 // E230001N04Rik // RIKEN
cDNA E230001N04 gene // 17 A3.3 //
320187 ///
E230001N04Rik2.0607 0.000670383
NM_009982 // Ctsc // cathepsin C // 7
D3-E1.1 // 13032 /// ENSMUST00000032779
Ctsc 2.04745 7.68E-07
NM_009283 // Stat1 // signal transducer
and activator of transcription 1 // 1 C1
Stat1 2.04168 1.09E-06
NM_001082960 // Itgam // integrin alpha M
// 7 F4 // 16409 /// NM_008401 // Itga
Itgam 2.03952 1.95E-05
NM_007671 // Cdkn2c // cyclin-dependent
kinase inhibitor 2C (p18, inhibits CDK4)
Cdkn2c 2.03566 5.74E-05
139

AK173199 // Rnf213 // ring finger protein
213 // 11 E2 // 672511
Rnf213 2.03563 4.69E-05
NM_008525 // Alad // aminolevulinate,
delta-, dehydratase // 4 B3|4 30.6 cM // 1
Alad 2.03122 6.27E-05
NM_023065 // Ifi30 // interferon gamma
inducible protein 30 // 8 B3.3 // 65972 /
Ifi30 2.03069 4.76E-05
NM_007657 // Cd9 // CD9 antigen // 6 F3|6
58.0 cM // 12527 /// ENSMUST0000003249
Cd9 2.03023 2.89E-05
NM_030253 // Parp9 // poly (ADP-ribose)
polymerase family, member 9 // 16 B3 //
Parp9 2.0287 2.93E-05
NM_010442 // Hmox1 // heme oxygenase
(decycling) 1 // 8 C1|8 35.0 cM // 15368 //
Hmox1 2.02815 0.000695264
NM_001005858 // I830012O16Rik // RIKEN
cDNA I830012O16 gene // 19 C1 // 667370 /
I830012O16Rik2.026 0.000666494
NM_009851 // Cd44 // CD44 antigen // 2
E2|2 56.0 cM // 12505 /// NM_001039150 //
Cd44 2.02337 9.72E-06
NM_027787 // Rfx2 // regulatory factor X, 2
(influences HLA class II expression)
Rfx2 2.01545 6.13E-06
NM_011163 // Eif2ak2 // eukaryotic
translation initiation factor 2-alpha kinase
Eif2ak2 2.01402 1.63E-07
NM_010185 // Fcer1g // Fc receptor, IgE,
high affinity I, gamma polypeptide // 1
Fcer1g 2.01152 0.000731203
NM_009135 // Scn7a // sodium channel,
voltage-gated, type VII, alpha // 2 C1.3|2
Scn7a 2.00524 5.02E-08
NM_023579 // Ipo5 // importin 5 // 14 E5 //
70572 /// ENSMUST00000032898 // Ipo5
Ipo5 2.00386 4.00E-08
NM_029537 // Tmem98 // transmembrane
protein 98 // 11 B5 // 103743 ///
ENSMUST00
Tmem98 2.003 5.38E-05
--- 2.00267 0.000113136
NM_027334 // Mettl7a1 //
methyltransferase like 7A1 // 15 F3 //
70152 /// NM_199
Mettl7a1 2.00078 6.89E-06
140

NM_053083 // Loxl4 // lysyl oxidase-like 4 //
--- // 67573 /// BC068307 // Loxl4
Loxl4 2.0002 5.58E-05
141

Appendix B. Down-regulated transcripts in Arr1
-/-
retina
Gene_assignment Gene Symbol
Fold-Change
(Arr1
-/-
vs.
WT)
p-value
(Arr1
-/-
vs.
WT)
NM_009118 // Sag // retinal S-antigen // 1
D|1 53.6 cM // 20215 /// ENSMUST00000
Sag -170.545 6.14E-11
NM_001039137 // Scoc // short coiled-coil
protein // 8 C2 // 56367 /// NM_019708
Scoc -49.4912 8.03E-08
NM_183389 // Duxbl // double homeobox
B-like // 14 A3 // 278672 /// EU257807 //
Duxbl -5.61068 4.05E-07
ENSMUST00000059351 //
5930422O12Rik // RIKEN cDNA
5930422O12 gene // 8 A4 // 319
5930422O12R
ik
-3.07035 3.55E-05
NM_134161 // Fut10 // fucosyltransferase
10 // 8 A3 // 171167 /// NM_001012517 /
Fut10 -2.85333 6.84E-05
NM_172461 // Nek11 // NIMA (never in
mitosis gene a)-related expressed
kinase 11
Nek11 -2.84035 2.76E-07
NM_008230 // Hdc // histidine
decarboxylase // 2 E5-G // 15186 ///
ENSMUST000000
Hdc -2.69552 5.08E-07
NM_153804 // Plekhg3 // pleckstrin
homology domain containing, family G
(with Rh
Plekhg3 -2.61069 3.60E-06
NM_173384 // Sox30 // SRY-box
containing gene 30 // 11 B1.1 // 214105 ///
ENSMUS
Sox30 -2.4879 2.09E-05
NM_027770 // Col24a1 // collagen, type
XXIV, alpha 1 // 3 H3 // 71355 /// ENSMUS
Col24a1 -2.41141 7.48E-05
NM_177866 // Catsper4 // cation channel,
sperm associated 4 // 4 D3 // 329954 //

Catsper4 -2.39628 1.31E-05
142

NM_146256 // Hpdl //
4-hydroxyphenylpyruvate
dioxygenase-like // 4 D1 // 242642
Hpdl -2.3767 0.00382836
ENSMUST00000070315 //
OTTMUSG00000005065 // predicted
gene, OTTMUSG00000005065 /
OTTMUSG000
00005065
-2.36316 3.82E-06
AY344585 // ENSMUSG00000074792 //
predicted gene, ENSMUSG00000074792
// --- // 1
ENSMUSG00
000074792
-2.35548 0.000692286
NM_207268 // Ccdc87 // coiled-coil
domain containing 87 // 19 A // 399599 ///
EN
Ccdc87 -2.32506 5.80E-05
NM_027998 // Cldn23 // claudin 23 // 8
B1.1 // 71908 /// ENSMUST00000060128 //
C
Cldn23 -2.31784 2.24E-05
NM_031172 // Trim17 // tripartite
motif-containing 17 // 11 B1.2-B1.3 //
56631 /
Trim17 -2.28347 6.55E-06
NM_173419 // Dleu7 // deleted in
lymphocytic leukemia, 7 // 14 D1 //
239133 ///
Dleu7 -2.27399 8.76E-06
NM_011563 // Prdx2 // peroxiredoxin 2 // 8
C3|8 36.0 cM // 21672 /// ENSMUST0000
Prdx2 -2.2685 0.00101103
BC140980 // Gpr115 // G protein-coupled
receptor 115 // 17 B3 // 78249 /// BC089
Gpr115 -2.26031 1.82E-05
NM_008567 // Mcm6 // minichromosome
maintenance deficient 6 (MIS5 homolog,
S. po
Mcm6 -2.24251 8.58E-06
NM_001039209 // OTTMUSG00000010671
// predicted gene,
OTTMUSG00000010671 // 4 E1
OTTMUSG000
00010671
-2.23852 0.00414807
NM_019670 // Diap3 // diaphanous
homolog 3 (Drosophila) // 14 D3 // 56419 /
Diap3 -2.20759 2.23E-06
143

144

NM_016803 // Chst3 // carbohydrate
(chondroitin 6/keratan) sulfotransferase
3 //
Chst3 -2.18332 6.75E-07
BC099566 // 1700007K13Rik // RIKEN
cDNA 1700007K13 gene // 2 A3 // 69327 ///
AY9
1700007K13R
ik
-2.18223 0.000336189
NM_172864 // Wdr63 // WD repeat
domain 63 // 3 H2 // 242253 ///
ENSMUST000000610
Wdr63 -2.13611 3.95E-07
NM_029621 // Haus8 // 4HAUS
augmin-like complex, subunit 8 // 8 C1 //
76478 ///
Haus8 -2.13013 3.25E-06
NM_175214 // Kif27 // kinesin family
member 27 // 13 B1 // 75050 ///
ENSMUST0000
Kif27 -2.11912 1.04E-06
NM_011848 // Nek3 // NIMA (never in
mitosis gene a)-related expressed
kinase 3 /
Nek3 -2.11442 1.29E-05
NM_145484 // Zfp758 // zinc finger
protein 758 // 17 A3.3 // 224598 ///
BC021442
Zfp758 -2.04102 2.56E-05
NM_153804 // Plekhg3 // pleckstrin
homology domain containing, family G
(with Rh
Plekhg3 -2.02619 0.000832244
NM_018751 // Sult1c1 // sulfotransferase
family, cytosolic, 1C, member 1 // 17 C
Sult1c1 -2.00673 0.00261143

Abstract (if available)

Abstract In the G-protein-coupled receptor (GPCR) phototransduction cascade, visual Arrestin1 (Arr1) binds to and deactivates phosphorylated light-activated opsins, a process that is critical for effective recovery and normal vision. In this dissertation study, we discovered a novel synaptic interaction between Arr1 and N-ethylmaleimide sensitive factor (NSF) that is enhanced in a dark environment when photoreceptors are depolarized and the rate of exocytosis is elevated. In the photoreceptor synapse, NSF functions to sustain a higher rate of exocytosis, in addition to the compensatory endocytosis to retrieve and to recycle vesicle membrane and synaptic proteins. Not only does Arr1 bind to the junction of NSF N-terminal and first ATPase domains in an ATP-dependent manner, but Arr1 also enhances both NSF ATPase and NSF disassembly activities. In mouse retinas with no Arr1 expression, the expression levels of NSF and other synapse-enriched genes are markedly reduced and lead to a substantial decrease in the exocytosis rate. This study demonstrates a vital modulatory role of Arr1 in the photoreceptor synapse and provides key insights into the potential molecular mechanisms of inherited retinal diseases, such as Oguchi disease and Arr1-associated retinitis pigmentosa.

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Asset Metadata

Creator Huang, Shun-Ping (author)

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

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publication Date 08/05/2010

Defense Date 06/09/2010

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

Tag beta-arrestin,cone arrestin,FM1-43,N-ethylmaleimide sensitive factor,OAI-PMH Harvest,photoreceptor,S-antigen,SNARE,synapse,visual arrestin

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Craft, Cheryl M. (committee chair), Hinton, David R. (committee member), Ko, Chien-Ping (committee member), Mircheff, Austin K. (committee member), Sampath, Alapakkam P. (committee member)

Creator Email sphophdoc1688@gmail.com,supergenius999@hotmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3303

Unique identifier UC1314537

Identifier etd-Huang-3815 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-370201 (legacy record id),usctheses-m3303 (legacy record id)

Legacy Identifier etd-Huang-3815.pdf

Dmrecord 370201

Document Type Dissertation

Rights Huang, Shun-Ping

Type texts

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

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu