ARVO 2015 Annual Meeting Abstracts 131 Photoreceptor

ARVO 2015 Annual Meeting Abstracts

131 Photoreceptor development

Sunday, May 03, 2015 1:30 PM–3:15 PM

Exhibit Hall Poster Session

Program #/Board # Range:

Organizing Section:

Visual Neuroscience

436–453/A0149–A0166

Retinal Cell Biology

Contributing Section(s): Biochemistry/Molecular Biology, Retina,

Program Number: 436 Poster Board Number: A0149

Presentation Time: 1:30 PM–3:15 PM

Regulation of photoreceptor development by competitive activation of cell type-specific enhancers

Timothy J. Cherry, Milena Andzelm, David Harmin, Michael

Greenberg. Neurbiology, Harvard Medical School, Boston, MA .

Purpose: Photoreceptor development is driven by a network of transcription factors (TFs), however the cis-regulatory elements

(CREs) through which these factors regulate gene expression are not fully understood. We sought to comprehensively identify active

CREs in developing photoreceptors and to dissect the combinatorial mechanisms by which TFs regulate photoreceptor gene expression.

Such mechanisms may provide a roadmap for reprogramming of somatic cells to replace photoreceptors lost to disease.

Methods: We performed ChIP-Seq for markers of active chromatin and total RNA-Seq to assess enhancer RNA (eRNA) and gene expression in the developing mouse retina. These datasets were compared to DNAse I hypersensitivity (DHS) data (ENCODE) to identify candidate active CREs. Motif analysis was performed to identify TF binding motifs enriched at active CREs. ChIP-Seq analysis was then performed to determine binding of corresponding

TF proteins. ChIP-Seq of H3K27Ac and total RNA-Seq was performed in TF KO retinas to determine the contribution of these factors to CRE activity and gene expression. ChIP-Seq of MEF2D in

Crx KO retinas was performed to determine the requirement of CRX for competitive recruitment of MEF2D to photoreceptor specific

CREs.

Results: Our analysis identified approximately 20,000 DHS sites with high levels of both H3K27Ac and eRNA expression in the p11 mouse retina. These sites were enriched for DNA sequences corresponding to CRX, RORA/B and MEF2 consensus binding sites and demonstrated CRX and MEF2D protein occupancy. A subset of these of these sites demonstrated a significant reduction in H3K27Ac and eRNAs in CRX KO and Mef2d KO retinas respectively. 330

MEF2D binding sites were reduced >2x in CRX KO retinas, while

227 sites demonstrated an increase in MEF2D binding. Many of the latter sites demonstrated an increase in eRNAs and proximal gene expression.

Conclusions: These studies identify candidate active CREs in the developing retina and suggest mechanisms by which TFs regulate gene expression in photoreceptors. TFs bind to distal enhancers to regulate their activity and demonstrate a functional interdependence whereby MEF2D requires CRX for binding to and activation of certain photoreceptor enhancers. This suggests that an active competition for MEF2D recruitment to photoreceptor enhancers may be required for normal photoreceptor development.

Commercial Relationships:

Andzelm , None;

Timothy J. Cherry

David Harmin , None;

, None; Milena

Michael Greenberg , None



Integrated transcriptome and epigenome analysis of developing mouse rod photoreceptors reveals dynamic epigenetic control of lineage priming and developmental plasticity

Hyun-Jin Yang, Jung-Woong Kim, Vijender Chaitankar, Matthew

Brooks, Hong Hao, Anand Swaroop. Neurodegeneration and Repair,

National Eye Institute, Bethesda, MD .

Purpose: Functional maturation of rod photoreceptors takes place over an extended period of time and is associated with extensive changes in gene expression patterns. Despite a well-established role of NRL in controlling vast majority of rod genes, how precise temporal gene expression is established remains poorly understood.

To explore the possible contribution of epigenetic factors in temporal regulation of rod gene expression, we have undertaken integrated transcriptome and epigenome analysis of developing and mature mouse rod photoreceptors.

Methods: We isolated rod photoreceptors from Nrl p-GFP mouse retina at various developmental stages and generated rod-specific transcriptome, NRL targetome and epigenome profiles.

Results: Epigenetic architecture of newly post-mitotic rods was highly favorable for expression of cone genes but not of rod genes, while rod-photoreceptor specific epigenetic signature was fully established by adult stage. This change is consistent with transient cone gene expression detected in most immature rods and late onset of many rod genes. Notably, NRL recruitment to target genes was also delayed despite a constantly high level of Nrl expression throughout rod development. Our epigenome data also revealed genes specific for non-photoreceptor retinal neurons bearing both active and repressive epigenetic marks in immature rods, constituting a novel class of genes with bivalent chromatin marks. This bivalency was lost in mature rods.

Conclusions: plastic with their chromatin state primed for S-cone fate, consistent with our recent finding that most mouse rods are recruited from S cone lineage. Bivalent chromatin state identified in non-photoreceptor retinal genes likely reflects the developmental history transiently retained in newly post-mitotic rods as rods are one of the late born neurons from common pools of retinal progenitor cells. A subsequent massive reorganization of chromatin state takes place as rods mature, thereby allowing regulation of rod gene expression in a timely manner.


Kim , None;

Hong Hao

Support:

We conclude that immature rods remain epigenetically

Vijender Chaitankar

, None;

Hyun-Jin Yang

, None;

Anand Swaroop , None

, None; Jung-Woong

Matthew Brooks , None;

National Institutes of Health Intramural Research Program



The role of Lysine demethylase 1 in mouse rod photoreceptor development and maturation.

Evgenya Popova, Carolina Pinzon-Guzman, Anna Salzberg, Daniel

Hass, Samuel S. Zhang, Colin J. Barnstable. Penn State University

College of Medicine, Hershey, PA .

Purpose: The transition of dividing progenitors into terminally differentiated cells in the Central Nervous System requires large-scale repression of progenitor genes and activation of genes characteristic of each differentiated cell type. A key element in this reprogramming is the action of histone modifying enzymes inside complexes binding to promoters and enhancers of the genes. One of the wellcharacterized enzymes is lysine-specific demethylase 1(LSD1).

LSD1 demethylates mono- and dimethylated H3K4 and H3K9. The purpose of this study was to understand how LSD1 may be involved

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to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org

.

ARVO 2015 Annual Meeting Abstracts in regulating the epigenetic status of genes during retina development and the role of this status in controlling rod photoreceptor maturation.

Methods: We compared pharmacological inhibition and conditional knockouts of this enzyme in developing mouse retina. Animal use was in accordance with ARVO/IACUC guidelines. Whole retina explants were isolated from C57BL/6j mice pups at postnatal day

1 and were cultured individually in media as a control or with

LSD1inhibitors for 24h, 4 days or 8 days. Illumina MouseRef8 v2.0

Expression BeadChip (BD-202-0202) was processed starting with

500ng of template and using standard Illumina Total Prep protocol.

Results: LSD1 is expressed in retinal postmitotic cells at the peak of rod photoreceptor differentiation. Pharmacological inhibition of

LSD1 in retinal explants cultured from PN1 to PN8 had three major effects. It prevented normal decrease in expression of transcriptional repressors and genes associated with proliferation and progenitor specification; it blocked rod photoreceptor development and it increased expression of genes associated with other retinal cell types, including cone photoreceptors. We generated two Cre-loxPmediated conditional knockout mice. Mice with LSD1 floxed alles

(LSD1 fl/fl) were crossed with Crx-Cre or Rho-Cre mice to create LSD1 knockouts in retina at two developing windows to determine LSD1 role in photoreceptor development and maturation. The results of this genetic approach will be compared with the pharmacological studies.

Conclusions: Our results indicate that LSD1 activity is essential both for the cessation of progenitor programs and for the correct execution of terminal differentiation programs.

Commercial Relationships: Evgenya Popova , None; Carolina

Pinzon-Guzman , None; Anna Salzberg , None; Daniel Hass , None;

Samuel S. Zhang , None; Colin J. Barnstable , None

Support: Macula Vision Research Foundation



Efficient retina formation requires suppression of both Activin and BMP signaling pathways in pluripotent cells

4 , Syafiq Abd Wahab 5 , Andrea Kimberly A. Wong 1, 3 , Michael Trembley

S. Viczian 2, 3 . 1 Neuroscience & Physiology, SUNY Upstate Medical

University, Syracuse, NY;

University, Syracuse, NY;

2

3

Ophthalmology, SUNY Upstate Medical

The Center for Vision Research, SUNY

Eye Institute, Syracuse, NY; 4 Pharmacology & Physiology, Aab

Cardiovascular Research Institute, University of Rochester School of

Medicine and Dentistry, Rochester, NY; 5 Molecular Biology, Weill

Cornell Graduate School of Medical Sciences, New York, NY .

Purpose: Retina formation requires the correct spatiotemporal patterning of key regulatory factors. While it is known that repression of several signaling pathways lead to specification of retinal fates in vivo, we have found that treatment of pluripotent cells with only Noggin, a known BMP antagonist, can direct cells to become functional retinal cells. The aim of this study is to determine if

Noggin affects intracellular signaling pathways other than BMP to efficiently direct this conversion.

Methods: We treated pluripotent Xenopus laevis animal caps with chemical inhibitors and dominant negative components of the BMP and Activin signaling pathways. Their effect on retina formation was determined using the Animal Cap Transplant (ACT) assay, in which treated pluripotent cells were transplanted into the eye field of sibling embryos at neural plate stage, after endogenous neural induction has already occurred. Signaling activity at the point of transplantation was determined by Western blot and semi-quantitative PCR (RT-

PCR) to measure downstream protein and gene target expression.

Results: Overexpressing Noggin in pluripotent cells resulted in a concentration-dependent suppression of both Smad1 and Smad2 phosphorylation, which act downstream of BMP and Activin receptors, respectively. This caused a decrease in downstream transcriptional ability, reflected by the reduced expression of both the endothelial marker, xk81 , and the mesodermal marker, xbra .

Expression of dominant negative BMP and Activin receptors or

R-Smads revealed that retinal specification was increased when both pathways were inhibited simultaneously. Similar results were observed when the chemical inhibitors dorsomorphin and SB431542 were used to inhibit Smad1 and Smad2 phosphorylation, respectively.

Conclusions: Thus, the dual inhibition of BMP and Activin pathways promotes retinal specification in Xenopus tissue. This suggests that Noggin extrinsically modulates intercellular BMP and Activin signaling in order to efficiently specify retinal cell fate. Future studies could translate these findings to a mammalian culture assay, in order to efficiently produce retinal cells in culture.

Commercial Relationships: Kimberly A. Wong , None; Michael

Trembley , None; Syafiq Abd Wahab , None; Andrea S. Viczian ,

None

Support: NEI Grant EY019517, Research to Prevent Blindness

Unrestricted Award, and the Lions of District 20-Y1



Control of maintenance and regeneration of planarian eyes by ovo

Samuel D. Cross, Benjamin J. Gilles, Lori A. Bachman, Alan D.

Marmorstein, Lihua Y. Marmorstein. Ophthalmology, Mayo Clinic,

Rochester, MN .

Purpose: More than 2 million traumatic eye injuries occur in the U.S. each year with bilateral blindness occurring at a rate as high as 75 per

100,000 cases. The planarian Schmidtea mediterranea has the ability to regenerate its eyes. In this study we sought to determine whether eye regeneration in S. mediterranea could occur in the absence of the wound healing response typically associated with regeneration in many organisms.

Methods: A previous study (Lapan & Reddien, Cell Rep. 2(2):294-

307,2012) demonstrated that maintenance of eyes in S. mediterranea requires the gene ovo . 240 S. mediterranea were treated with ovo

RNAi or control ( unc-22 ) RNAi by feeding liver paste containing dsRNA. Following loss of eyes, ovo RNAi treatment was halted and replaced with control RNAi treatment. Quantitative real-time PCR was used to monitor changes in ovo expression during ovo RNAi knock-down and following the switch to control RNAi. Fluorescent in situ hybridization was used to localize ovo expression in control and ovo RNAi treated worms. Eye functionality was monitored via a phototaxis assay.

Results: In the ovo RNAi treated group, the eyes were observed to gradually shrink until they were completely absent. 100% of the planarians receiving ovo RNAi lost both eyes within 120 days of the onset of treatment. Functional loss of eyes was validated using a phototaxis assay. Ovo RNAi treated planarians were unable to regenerate eyes in response to wounding. Upon switching from ovo

RNAi to control RNAi, eyes becamevisible as small pigmented spots in the head within 25 days. The eyes slowly developed, gaining pigmented cells first and more slowly developing the non-pigmented photoreceptors.

Conclusions: S. mediterranea have the ability to generate whole eyes in the absence of a wound healing response. This ability requires expression of ovo . Understanding how S. mediterranea control eye maintenance and regeneration could help to provide clues to genetic programs necessary to coax human stem cells to differentiate into various eye tissues.



.



J. Gilles

None;

, None;

Support:

Lori A. Bachman

Lihua Y. Marmorstein

NIH Grant EY013847 (to LYM), EY013160 (to ADM),

Prevent Blindness.

Samuel D. Cross

, None;

, None

, None; Benjamin

Alan D. Marmorstein ,

R01EY021153 (to ADM), and an unrestricted grant from Research to



Characterization of the zebrafish cone cytoskeleton using a fluorescent tubulin

Tylor Lewis, Peter J. Volberding, Joseph C. Besharse. Cell

Biology, Neurobiology & Anatomy, Medical College of Wisconsin,

Milwaukee, WI .

Purpose: In explanted zebrafish retinas, cone photoreceptors frequently exhibit microtubule-containing neuritic processes at the distal axoneme that extend beyond the outer segment (OS) and axonemes frequently exhibit branch points within the OS

(Bader, et al., 2012. Vision Research). In order to better understand microtubule organization in cone photoreceptors, we have used confocal microscopy and transmission electron microscopy (TEM) on developing and adult zebrafish that express fluorescent tubulin specifically in cone photoreceptors.

Methods: A line of zebrafish was generated that express tdEOS, a photoconvertible fluorophore, tagged to α -tubulin (tdEOS-tubulin) under control of the transducinα cone promoter (TaCP), allowing the cone cytoskeleton to be visualized. Cytoskeletal structure was analyzed with confocal microscopy on histological cryosections of larval and adult eyes, combined with confocal microscopy of ex vivo cultures of isolated photoreceptors and intact retinal slices.

In conjunction, TEM was performed on adult zebrafish to analyze ultrastructure of putative microtubule-based processes.

Results: In isolated preparations, a splayed structure adjacent to the

OS contained microtubules, and was characteristic of the accessory

OS described in other teleosts. Myoid elongation, a well-defined retinomotor movement, was observed in isolated photoreceptors and, as expected, was regulated by addition of dopamine. Interestingly, cultured cones and retinal slices formed neuritic projections containing microtubules that emanated from both the cone inner segment (IS) and OS that also appeared to be regulated by dopamine.

TEM of intact retina revealed microtubule-based structures associated cone photoreceptors that are embedded within the retinal pigmented epithelium (RPE).

Conclusions: A variety of unique characteristics of the cone cytoskeleton were observed using tdEOS-tubulin, including the accessory OS and myoid elongation regulated by dopamine, characteristic of retinomotor movements. More unconventionally, putative neuritic extensions that were also regulated by dopamine were observed in culture. Evidence in TEM suggests that there are microtubule-based processes associated with cones in vivo that interact with the RPE. Further characterization of the structure and biological function of these microtubule-based processes is ongoing.

Commercial Relationships: Tylor Lewis , None; Peter J.

Volberding , None; Joseph C. Besharse , None

Support: NEI R01 EY03222, T32 EY014537



A Human Cone Precursor Program Underlying a Proliferative

Response to RB Loss

Hardeep P. Singh 1 , David Cobrinik 2, 3 . 1 The Vision Center, Division of Ophthalmology, Children’s Hospital Los Angeles, Los Angeles,

CA; 2 The Vision Center, Division of Ophthalmology and the Saban

Research Institute, Children’s Hospital Los Angeles, Los Angeles,

CA; 3 USC Eye Institute, Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA .

Purpose: Inactivating RB1 mutations are thought to collaborate with retinal cell type-specific circuitry to initiate retinoblastoma tumorigenesis. We have earlier shown that human cone precursors proliferate in response to RB loss, dependent upon intrinsically high levels of MDM2 and MYCN and low levels of p27 as compared to other retinal cell types, and can develop into retinoblastoma tumors.

In contrast, mouse cone precursors fail to proliferate in response to

RB loss, and lack prominent MDM2, yet have not been evaluated for other components implicated in retinoblastoma development.

The goals of this study are to 1) define the spatio-temporal sequence of appearance of human cone precursor features implicated in retinoblastoma development, 2) determine whether these features appear in mouse cone precursors, and 3) test whether human-specific features such as high-level MDM2 expression can sensitize mouse cones to the loss of RB function.

Methods: Human fetal week (Fwk) 15 and Fwk 21 retinas and mouse post-natal day (P) 6, P10, and P20 retinas were used for immunostaining. Transgenic Red-Green-opsin Promoter (RGP)-

MDM2 mice were mated with RGP-Cre and Rb1 l/l mice to generate mice predicted to have cone-directed RB loss and MDM2 expression.

Results: In developing human retinas, RB, MDM2, and MYCN were not detected in RXR γ + cone precursors in the periphery but were prominent in central maturing cone precursors with expression initiating peripheral to the boundary of cone arrestin (ARR3) expression. In developing mouse retinas Rb and Mdm2 were barely detectable at any ages, while Mycn was detected at P10 but not at

P20. RGP-MDM2 mice expressed high levels of MDM2 in cone cells, beginning at P8. No abnormal proliferation was detected at P8,

P10, P15, or P20 in RGP-MDM2, RGP-Cre, Rb1 l/l mice. No tumors were observed in more than 40 RGP-MDM2, RGP-Cre, Rb1 l/l mice.

Conclusions: In human cone precursors, expression of RB, MDM2, and MYCN are first detected after the onset of RXR γ and before

ARR3, and further increase during cone precursor maturation.

Maturing mouse cone precursors fail to induce high-level Rb, Mdm2, or Mycn. Cone-directed MDM2 overexpression is not sufficient to sensitize RB deficient mouse cones to retinoblastoma. Additional factors specific to human cones such as high MYCN may be needed for cone precursor proliferation and retinoblastoma development in response to Rb loss.

Commercial Relationships: Hardeep P. Singh , None; David

Cobrinik , None

Support: NIH grant R01 CA137124, The Larry and Celia Moh

Foundation, Research to Prevent Blindness



Photoreceptor Synaptic Vesicle Cycling in tulp1-/ Mice

Gayle J. Pauer 1 , Glenn Lobo 1 , Adrian Au 1 , Stephanie A. Hagstrom 1,

2 . 1 Cole Eye Institute i-31, Cleveland Clinic, Cleveland, OH;

2 Ohthalmology, Cleveland Clinic Lerner College of Medicine of Case

Western Reserve University, Cleveland, OH .

Purpose: Tulp1 is a retina-specific protein that localizes to the inner segment (IS), connecting cilium, perikarya and synaptic terminal



.


(ST) of photoreceptor cells. We hypothesize that Tulp1 functions in vesicular trafficking in both the IS and ST. To directly analyze vesicle cycling, we compared tulp1-/ versus wild-type (wt) STs using styryl dye (FM1-43X) photo-conversion followed by electron microscopy

(EM).

Methods: Eyes were harvested from P16 dark-adapted mice under infrared light. Photoreceptor STs were labeled in the dark by loading 45 uM FM1-43X styryl dye to the intact retina in normal saline containing 1.5 mM Ca 2+ . Loading was followed by washing in a Ca 2+ -free solution containing 1 mM EGTA and 1 mM

Advasep-7. Retinal slices were assessed for styryl dye uptake and imaged using confocal microscopy. To observe vesicle number and location at the ribbon synapse, retinas were fixed in a 2.5% glutaraldehyde/4% paraformaldehyde solution and incubated in 1 mg/ ml diaminobenzidine (DAB). Photo-conversion of DAB to form an electron-dense reaction product was achieved focusing high power

488 nm light onto the tissue with confocal microscopy. Following photo-conversion, the retinal section was prepared and imaged using

EM.

Results: In both wt and tulp1-/ retinal sections, we observed strong

FM1-43X styryl dye uptake and labeling of photoreceptor inner and outer segments and cell bodies. While wt photoreceptor STs showed strong FM1-43X styryl dye uptake, tulp1-/ photoreceptor STs were significantly reduced of styryl dye staining. EM images of wt retinal sections revealed photoreceptor STs to be packed with small electron dense vesicles that were tightly tethered to the ribbon. In contrast,

EM images of tulp1-/ retinal sections showed much fewer electron dense vesicles at the STs, most of which did not tether to the synaptic ribbons.

Conclusions: Our data suggests that tulp1 is required for vesicle cycling in the STs of photoreceptor cells. In addition, in the absence of tulp1, occur correctly, suggesting a possible explanation for poor synaptic transmission observed in


None; Adrian Au

Support: vesicle docking on the ribbon synapse does not appear to

, None; tulp1-/- mice.

Gayle J. Pauer , None;

Stephanie A. Hagstrom

NH Grant EY16072, RPB

Glenn Lobo

, None

,



Surveying the electrostatic binding potential of rod photoreceptor membranes and its role in setting peripheral membrane protein distribution

Nycole A. Maza 3, 2 , Mehdi Najafi

Calvert 3, 2 . 1

1, 2 , Ivayla Geneva 1, 2 , Peter D.

Ophthalmology, SUNY Upstate Medical University,

Syracuse, NY; 2 SUNY Eye Institute, Syracuse, NY; 3 Ophthalmology

& Neuroscience/Physiology, SUNY Upstate Medical University,

Syracuse, NY .

Purpose: The mechanisms underlying signal-dependent translocation of key phototransduction proteins in the rod photoreceptor are not understood. We propose a systematic study of peripheral membrane proteins and their interaction with cell structures and membranes, in live photoreceptors, in order to determine the roles of electrostatic charge and posttranslational lipidation on peripheral membrane protein localization and distribution in dark- and light- adapted rods.

Methods: Live cell electrostatic membrane probes were created by attaching genetic sequences with lipid transferase motifs and amino acid linkers bearing systematically varied degrees of charge to E/

PAGFP. Probes in which the lipidation motif is mutated such that no lipid modification occurs were used to assess the impact of charge alone, whereas charge neutral linkers with lipid transferase motifs reported the impact of lipidation alone. The sequence for each probe was inserted behind the XOP promoter, and introduced to Xenopus sperm nuclei in vitro using restriction enzyme mediated integration.

Retinas were harvested, and the distribution patterns and mobilities of each probe were assessed in rods using live cell confocal microscopy and multiphoton fluorescence relaxation after photoactivation

(mpFRAPa). Five tadpoles were required to reach statistical significance for each probe.

Results: Addition of a myristoylated, poly-basic targeting sequence to the N-terminus of EGFP results in a concentration distribution that differs from both EGFP alone as well as the Myr-EGFP construct. Since the inner segment is less crowded than the outer segment, soluble EGFP fluorescence in the inner segment is approximately twice as bright as that of the outer segment. However, the concentration distribution of a myristoylated EGFP probe with a +8 linker is localized primarily to the outer segment, with a small amount of fluorescence found in the ellipsoid region of the inner segment.

Conclusions:

None;

Support:

Our results indicate that positive charge, in addition to post-translational lipidation, is important for outer segment localization of peripheral membrane proteins. This study suggests that electrostatic charge and post-translational lipidation are both intimately involved in the distribution and localization of photoreceptor proteins.


Ivayla Geneva , None;

Nycole A. Maza

Peter D. Calvert

NIH Grant EY018421

, None;

, None

Mehdi Najafi



Rhodopsin organization in retinal membranes of heterozygous rhodopsin knockout mice assessed by atomic force microscopy

Tatini Rakshit, Paul S. Park. Ophthalmology, Case Western Reserve

University, Cleveland, OH .

Purpose: Mutations in rhodopsin are a leading cause of autosomal dominant retinitis pigmentosa (adRP). A majority of mutations in rhodopsin causing adRP result in misfolded receptor, thereby reducing the level of properly folded receptor available for incorporation into disc membranes of rod outer segments (ROS).

Heterozygous rhodopsin knockout (Rho+/-) mice provide a suitable model to study the effects of reduced rhodopsin expression levels.

Atomic force microcopy (AFM) was performed on retinal samples from Rho+/- mice to investigate the impact of reduced rhodopsin expression on ROS disc membrane structure.

Methods: ROS disc membranes were isolated from the eyes of wild-

, type (WT) and Rho+/- mice. Disc membranes were imaged by AFM.

AFM revealed the organization of rhodopsin into nanodomains. The dimensions of disc membranes and rhodopsin nanodomains were determined and analyzed.

Results: The size of ROS disc membranes in Rho+/- mice is smaller than those in WT mice. Like rhodopsin in WT mice, rhodopsin forms nanodomains in disc membranes of Rho+/- mice. The spatial density of rhodopsin in 4-week-old Rho+/- mice was less than that of rhodopsin in 6-week-old Rho+/- mice. No difference in spatial density of rhodopsin was observed in samples from 4- and 6-weekold WT mice. The spatial density of rhodopsin in disc membranes of 6-week-old Rho+/- mice were similar to that found in disc membranes of WT mice. The average surface area of nanodomains was the same for all mice studied.

Conclusions: Reduced rhodopsin expression reduces the size of ROS discs. Initially, a reduction in the expression of rhodopsin decreases the density of rhodopsin in disc membranes. However, as mice age the reduced level of rhodopsin expression is overcome to achieve similar rhodopsin densities as that observed in WT mice. Thus, there appears to be a mechanism in place to achieve a constant density



.

ARVO 2015 Annual Meeting Abstracts of rhodopsin in disc membranes. The surface area of rhodopsin nanodomains is unaffected by the reduced level of rhodopsin expression.


None

Support:

Tatini Rakshit

R01EY021731, P30EY011373

, None; Paul S. Park mice reveals that the effect of GAP overexpression on TCS depends on the level of background illumination. GAP overexpression resulted in: 1) increased TCS in response to intermediate (3 - 15 Hz) but not to high temporal

,



Over-expression of the GAP complex in rods increases temporal contrast sensitivity of mice determined with an operant behavior assay

Yumiko Umino 1, 2 , Eduardo C. Solessio 1, 2 . 1 Ophthalmology, SUNY

Upstate Medical University, Syracuse, NY; 2 SUNY Eye Institute,

Syracuse, NY .

Purpose: Past work has assigned the photoreceptor frequency response as one mechanism that limits Temporal Contrast Sensitivity

(TCS); however, this concept has not been demonstrated empirically.

Our overall hypothesis is that under mesopic conditions, when rods integrate multiple photon events, rod photoresponse recovery kinetics limits rod-driven temporal contrast sensitivity. Here we apply an operant behavioral assay to measure TCS in mouse while removing the confounding factors inherent to the optomotor assay (see our

ARVO abstract, 2013).

Methods: We applied a novel operant behavioral assay to R9AP95 transgenic mice, which display accelerated rod photo-response recovery kinetics due to the over-expression of transducin GTPase activating protein (GAP) complex selectively in rods (Krispel et al, 2006). We selectively isolated rod-mediated vision in mesopic conditions by determining TCS functions for R9AP95 mice in animals (GNAT2 cpfl3 ) that have no cone responses due to a missense mutation in the cone-specific transducin α -subunit gene (Gnat2).

We measured TCS as a function of temporal frequency (1.5-48 Hz) in response to full-field flicker. Flicker ERGs were determined to measure retinal sensitivity.

Results: Comparison of operant responses in R9AP95(+)::GNAT2 vs R9AP95(-)::GNAT2 cpfl3 frequencies (20 - 42 Hz) at low mesopic lights (20 R*/rod/s), when rods integrate photons; 2) no change in TCS at scotopic lights (0.2

R*/rod/s), when rods count photons. The flicker ERG sensitivities were in line with visual sensitivities but their dynamic range was

Pang et al; 2010). Circuit constraints may limit responses to high temporal frequencies.

Commercial Relationships: Yumiko Umino , None; Eduardo C.

Solessio , None cpfl3 limited to temporal frequencies <24 Hz.

Conclusions: Our data indicate that photoreceptor response kinetics constrain operation of rod pathways under specific illumination conditions and temporal frequencies. Candidate mechanisms include shorter integration times in R9AP95 rods (Krispel et al; 2006), lower sensitivity to background lights and extended response range in

‘fast’ rods (Chen et al; 2010), and/ or transmission of enhanced OFF signals along selective (eg, tertiary) rod pathways (Li et al; 2010,

Support: Research to Prevent Blindness, Lions Club of Central New

York, SUNY/RF Research Collaboration Award



Light-adapted electroretinograms in Cx36 knockout mice

Rose Pasquale 1, 2 , Yumiko Umino 3, 2 , Eduardo C. Solessio 3, 2 .

1 Neuroscience, SUNY Upstate Medical University, Syracuse, NY;

2 Center for Vision Research and SUNY Eye Institute, Syracuse, NY;

3 Ophthalmology, SUNY Upstate Medical University, Syracuse, NY .

Purpose: Rod signals spread to cone pathways via Connexin36

(Cx36) gap junctions in the inner and outer retinas. The conductance of gap junctions and their degree of coupling in the outer retina is decreased by light adaptation, potentially controlling the contribution of rod secondary pathways to the ERG. Although the contribution of

Cx36-dependent pathways to dark-adapted ERG responses are well documented, their contribution in the light-adapted retina is not well understood. Our goal was to test the hypothesis that, by decreasing neuronal coupling, light-adaptation decreases the contribution of

Cx36-dependent pathways to the ERG. In such case, we expect that ERGs of light-adapted control and Cx36 knockout mice match closely.

Methods: We measured light-adapted flash ERGs in wildtype vs

Cx36 -/ mice to assess the activity of the retina and light-adapted flicker ERGs to infer contributions of separate frequency dependent pathways to the retinal response. To isolate rod signals, we tested

ERGs in GNAT2 cpfl3 cone mutant and Cx36 -/::GNAT2 cpfl3 double mutant mice. Immunohistochemical analysis and confocal images were used to assess retinal integrity. The optomotor behavioral assay was used to determine visual function.

Results: In the dark and low flash intensities (<3 R*/rod), ERG responses of all three mutant lines match closely with wildtype as expected ( Abd-El-Barr et al , 2009). At mid and high flash intensities

(>10 R*/rod), b-wave amplitudes of Cx36 -/-

-/-

are 80% of wildtype

::GNAT2 cpfl3 values while those of GNAT2 cpfl3 and Cx36 mice are reduced to 60%. Light-adapted flicker ERGs of wildtype and

GNAT2 cpfl3 mice match closely in dim and intermediate background lights (<10000R*/rod/s) while those of Cx36 -/::GNAT2 cpfl3 -/- and Cx36 mice exhibit a frequency-dependent reduction in response at intermediate lights (1 to 10000 R*/rod/s). Confocal images show no difference in Cx36 expression in GNAT2 to controls. Optomotor responses of Cx36 -/- cpfl3 mice compared mice show reduced sensitivity in dim and intermediate lights consistent with night blindness.

Conclusions: Our comparison of light-adapted ERG responses in WT, Cx36 -/, GNAT2 cpfl3 and Cx36 -/::GNAT2 cpfl3 mice indicates functional Cx36-dependent pathways in background lights producing up to ~10000R*/rod/s. We do not discard uncoupling effects of light adaptation at higher light intensities. Potential retinal remodeling effects of the knockouts are also considered.

Commercial Relationships: Rose Pasquale , None; Yumiko Umino ,

None; Eduardo C. Solessio , None

Support: Research to Prevent Blindness, Lions CLub of Central New

York, SUNY/RF Research Collaboration Award



Retinal Function in P23H rats assessed by full-field ERGRetinal

Function in P23H rats assessed by full-field ERG

Tobias Peters 1 , Krunoslav Stingl 1 , Torsten Strasser 1 , Vinicius

M. Castro 1 , Doaa Akl 1 , Seong-Woo Kim 1 , Eberhart Zrenner 2 .

1 Ophthalmology, Institute for Ophthalmic Research, Tübingen,

Germany; 2 Werner Reichardt Centre for Integrative Neuroscience

(CIN) and Centre for Ophthalmology, University of Tübingen,

Tübingen, Germany .



.


Purpose: Longitudinal study in a rod-dominated animal model with slow retinal degeneration.

Methods: Ten P23H rats were evaluated at different time points after birth starting from postnatal day 40 (PN40). ERG protocol included scotopic and photopic responses to single flashes followed by flicker series in light adapted state with frequencies of 5, 10, 15, 20 and 30

Hz.

The ERG was recorded and stored for offline analysis with an

Espion® system (Diagnosys LLC, Littleton, MA). The recorded signal was filtered on-line with a band-pass filter from 0,625 Hz to

300 Hz. Eyes were stimulated using a Ganzfeld stimulator (Color

Dome; Diagnosys LLC, Littleton, MA).

Results: ERG amplitudes and implicit times show different courses of decay of rod and cone functionality. Dim single flash responses indicate that rod function is already abolished between PN150 and

PN200. In contrast we observed reduced but maintained cone specific responses up to PN300. In the cone flicker response at all frequencies we consistently find some surprising signs of transient recovery of the function between PN150 and PN250. Different decay rates of cone and rod function allowed to model these results by a functional inhibition of rod pathways onto cone function. This model was tested in the flicker series and indicates that even relatively small remnants of rod function can substantially inhibit the cone response.

Conclusions: Evaluation of flicker series in retinal degeneration can offer new insights in terms of functional interaction of rods and cones during the course of retinal degeneration. Here we present first promising results in a small number of P23H rats that may trigger further similar investigations also in other animal models with retinal degeneration.

Commercial Relationships: Tobias Peters , None; Krunoslav

Stingl , None; Torsten Strasser , None; Vinicius M. Castro , None;

Doaa Akl , None; Seong-Woo Kim , None; Eberhart Zrenner , None

Support: EU grant 246180 PANOPTES; Egon Schuhmacher Stiftung

Program Number:

Presentation Time:

Temporal contrast sensitivities elicited by the four photoreceptor classes using the silent substitution paradigm in retinitis pigmentosa

Cord R. Huchzermeyer, Julia Auer, Jan J. Kremers.

Ophthalmology, University Erlangen-Nürnberg (FAU), Erlangen,

Germany

Purpose: To measure psychophysical temporal contrast sensitivities

(TCS) elicited by the four photoreceptor classes (L-, M-, S-cones and rods) in the pericentral retina of patients with retinitis pigmentosa

(RP) using the silent substitution paradigm.

Methods:

.

1:30 PM–3:15 PM

Eleven patients with RP (3 AD, 1 AR, 1 X-linked, 2 Usher,

4 sporadic) were examined. Their data were compared with those from 18 normal subjects for the cone sensitivities and from 7 subjects for the rod sensitivities.

Stimuli were created using an 8-channel LED stimulator with a

2° diameter central circular field and a 13° outer diameter annular surround field (each with 4 independent primaries). Sine-wave modulated stimuli were presented in the white surround field (mean luminance 2.7 log phot Td) while subjects fixated the darker steady central field.

449 Poster Board Number: A0162

Department of

For each photoreceptor class, isolating stimuli were created using the silent substitution paradigm based on the 10° cone fundamentals and the scotopic luminosity curve. For each condition, the critical flicker fusion frequency (CFF) was determined at the maximal available contrast. TCS was measured at 1, 2, 4, 6, 8, 10, 12, 16, 20, and 28

Hz, but only up to the frequency just above the CFF. Thresholds were determined using a randomly-interleaved double staircase algorithm.

Sensitivity was defined as 1/(contrast at threshold).

Results: Measurements were feasible in 10 patients. One patient with

X-chromosomal RP also displayed a deuteranomalous defect, so that data from this patient were excluded from further analysis.

On average, sensitivities of the RP patients were lower at all frequencies and for all photoreceptor classes. Differences between patients and normal subjects were statistically significant at high frequencies for L-cones, M-cones and rods and at all frequencies for

S-cones. Sensitivities showed significant overlap for L-cones and

M-cones. In our population, the best diagnostic criteria were the rod sensitivities at 10Hz and at 12Hz (area under the ROC curve of 1).

Conclusions: Psychophysical measurements of TCS elicited by the different photoreceptor classes is feasible in patients with RP using our protocol. As expected, rod sensitivities were best suited to separate RP patients from normal subjects. In the future, our paradigm may allow more accurate genotype-phenotype correlations and offer advantages in the monitoring of RP progression.


Auer , None;

Support:

Jan J. Kremers

Cord R. Huchzermeyer

, None

, None;

ELAN fund University of Erlangen-Nürnberg,

Julia

Interdisciplinary Center for Clinical Research Erlangen (IZKF), Pro

Retina Foundation



Regulation of the tandemly duplicated LWS opsin genes during development and regeneration in zebrafish

Diana M. Mitchell, Timothy McGinn, Ruth Frey, Deborah L.

Stenkamp. Biological Sciences, University of Idaho, Moscow, ID .

Purpose: Retinoic Acid (RA) regulates photoreceptor fate, differentiation, and survival. Our microarray analysis revealed upregulation of LWS-1 in embryonic zebrafish eyes treated with RA during photoreceptor differentiation. LWS-1 is the upstream member of the tandemly duplicated LWS array also containing LWS-2, orthologous to the human L/M array. Normally, LWS-1 is expressed in red-sensitive cones located mainly in ventral retina, but not until larval stages. Adult retinas show specific spatial patterns of LWS-1 and LWS-2 cones, but how these patterns are achieved is not known.

Understanding how the LWS array functions during development and regeneration is important for achieving long-term goals that aim to regenerate proper photoreceptor subtype ratios and high acuity color vision in damaged retinas.

Methods: We used the transgenic line LWS-PAC(H) reporting LWS-

1 :GFP and LWS-2 :RFP expression to examine individual cones of RA treated and control retinas by confocal microscopy and spatial pattern analysis. To determine if LWS cones recapitulate spatial patterns following regeneration, we examined LWS-1 and LWS-2 expression by in situ hybridization in regenerated adult retinas following ouabain damage that destroys all retinal neurons.

Results: Confocal imaging of LWS-PAC(H) retinas revealed that RA treatment results in ventrally located LWS-1 expressing cones, and a large proportion of these cones co-express GFP and RFP, indicating that RA induced a switch from LWS-2 to LWS-1 in individual cones.

Spatial pattern analysis of cone mosaics indicated that LWS-1 cones present in RA-treated eyes did not disrupt the LWS-2 cone mosaic, providing further evidence that LWS-1 cones following RA treatment are post-mitotic LWS-2 cones that switched opsin expression. We find that cones expressing LWS-1 and LWS-2 in regenerated retinas do not recapitulate the topographic distribution seen in undamaged retinas.

Conclusions: Our results suggest that RA signaling may act as a molecular toggle regulating the LWS gene array to promote the



.

ARVO 2015 Annual Meeting Abstracts proper patterning of LWS-1 and LWS-2 cones during development.

However, the patterning mechanism(s) regulating the

McGinn , None; Ruth Frey , None;

LWS array during development is not similarly engaged during regeneration.

This is the first indication that global topographic patterning cues during regeneration do not function in the same manner as during development.

Commercial Relationships: Diana M. Mitchell , None; Timothy

Deborah L. Stenkamp , None



The bHLH transcription factor NeuroD governs injury-induced photoreceptor regeneration through Delta-Notch signaling

Scott M. Taylor 1 , Ryan Thummel 2 , Peter F. Hitchcock 1 . 1 Ophthal &

Visual Sciences, University of Michigan, Ann Arbor, MI; 2 Anatomy and Cell Biology, Wayne State University, Detroit, MI .

Purpose: In humans, photoreceptor death causes permanent visual impairment, whereas, in zebrafish, photoreceptor death stimulates a regenerative response that restores photoreceptors and visual function. Identifying the mechanisms that govern photoreceptor regeneration in zebrafish can provide critical information for developing regenerative therapies that restore photoreceptors in humans. In embryonic zebrafish, the bHLH transcription factor

NeuroD governs photoreceptor genesis through Delta-Notch signaling (Taylor et al., under review), but the role of NeuroD during photoreceptor regeneration is unknown. The purpose of this study was to test the role of NeuroD in adult photoreceptor regeneration and identify mechanisms through which NeuroD functions.

Methods: Following light-induced photoreceptor ablation, NeuroD was knocked down by electroporating ATG-targeted morpholinos into the retina. Standard control morpholinos were used as controls.

5-Ethynyl-2’-Deoxyuridine (EdU) was injected IP at 3 days post lesion (dpl), and 5-Bromo-2’-Deoxyuridine (BrdU) was administered by immersion at 5-6 dpl to label dividing cells and determine the percentage of progenitors remaining in the cell cycle at 6 dpl. In-situ hybridization was used to label regenerated rods and cones at 6 and

7 dpl and to evaluate the expression of Notch pathway molecules.

Finally, following NeuroD knockdown, the gamma secretase inhibitor

DAPT was used to inhibit Notch signaling.

Results: NeuroD knockdown resulted in an increased proportion of

EdU+ cells co-labeled with BrdU at 6 dpl, indicating that NeuroD is required for injury-induced progenitors to exit the cell cycle.

Correlated with this, there were significantly fewer regenerated photoreceptors in experimental retinas compared to controls, indicating that NeuroD is required for photoreceptor regeneration.

NeuroD knockdown caused up-regulation of notch1a , deltaA and deltaD , as well as the Notch targets her4 and ascl1a , suggesting that NeuroD transcriptionally represses Notch signaling. Following

NeuroD knockdown, inhibition of Notch signaling with the Gamma secretase inhibitor DAPT rescued the defects in cell cycle exit and photoreceptor regeneration.

Conclusions: These data are interpreted to show that following photoreceptor ablation, NeuroD governs progenitor cell cycle exit and photoreceptor regeneration by negatively regulating Delta-Notch signaling.


Thummel

Support:

, None;

Scott M. Taylor

Peter F. Hitchcock

NIH Grant EY07060

, None

, None; Ryan



Transcriptional changes in photoreceptor development genes during regeneration after cone ablation in zebrafish

A P. Oel, Michele DuVal, W T. Allison. Biological Sciences,

University of Alberta, Edmonton, AB, Canada .

Purpose: Genes controlling differentiation of developing photoreceptors have been identified in mouse and zebrafish; however, regulation of the teleost ability to robustly regenerate lost photoreceptors remains uncharacterized. We hypothesize that this regeneration recapitulates development; here, we test whether developmentally important genes are transcriptionally active during the regeneration period of post-injury neural retinas isolated from adult zebrafish.

Methods: We induced regeneration in adult zebrafish in two ways: intense light exposure causing widespread photoreceptor death, and conditional genetic ablation of either UV or blue light-sensitive cones. Conditional genetic ablation of cones occurs when the prodrug metronidazole (MTZ) is added to tank water of transgenic fish expressing bacterial nitroreductase (NTR) in either UV or blue cones; cells expressing NTR convert MTZ into a DNA-crosslinking agent, causing cell-autonomous apoptosis. After injury by light exposure or MTZ treatment, we isolated the total RNA from neural retinas, and examined the transcriptional activity of several photoreceptor differentiation genes over a time-course by quantitative PCR analysis.

Results: Through light-based ablation we have found changes in expression among rx1, nr2e3, nrl, and thr β 2. Compared to uninjured controls, light blasted retinas show a 2-fold reduction in rx1 transcripts, starting 12hours post ablation (hpa) and lasting until the end of the experiment (96hpa). Expression of nr2e3 is reduced 4-fold and nrl nearly 2-fold over the same period, whereas thr β 2 trends toward increased expression. Transgenic fish exposed to MTZ respond as predicted, showing a loss of NTR-expressing photoreceptors within 15 hours and regenerating new NTRexpressing photoreceptors within a week of recovery after transient treatment with MTZ, with qPCR studies pending.

Conclusions:

W T. Allison

We examined gene activity during regeneration after either widespread or targeted photoreceptor death. This allows us to evaluate how closely regeneration recapitulates development; to identify genes critical to the regeneration of cone photoreceptors; and finally, to use transgenic tools to modify the endogenous retinal stem cell population, to modulate the outcome of regeneration.

This has implications for the design of stem cell-based treatments for blindness, where the restoration of cone-mediated sight is a paramount goal.


, None

A P. Oel , None; Michele DuVal , None;



Identifying targeting motifs necessary for the differential localization of Na + /K + -ATPase isoforms in photoreceptors

Joseph G. Laird, Yuan Pan, David Yamaguchi, Sheila A. Baker.

Biochemistry, University of Iowa, Carver College of Medicine, Iowa

City, IA .

Purpose: Na+/K+-ATPase (NKA) is the sodium pump used by all cells to establish and maintain electrochemical gradients across cellular membranes. It can alternatively function in signaling as a receptor for cardiotonic steroids or participate in maintaining cellcell adhesion. Ways to accommodate the unique requirements of different cell types include the utilization of different NKA isoforms or altering the subcellular localization of NKA. In photoreceptors,

NKA composed of the α 3 subunit is located in the plasma membrane



.

ARVO 2015 Annual Meeting Abstracts of the inner segment. But in sperm, NKA is composed of the

α 4 subunit and localizes to the flagella. We observed that upon expression in photoreceptors NKA α 4 localized to the analogous ciliary compartment, the outer segment. Our goal in this study was to take advantage of that observation to dissect the differential targeting information encoded within each of these NKA isoforms.

Methods: A series of GFP-tagged NKA constructs were cloned.

These included a series of α 3/ α 4 chimeras, deletion mutants and point mutants of each isoform. The proteins were expressed in

Xenopus laevis rods by generating transgenic tadpoles. Retinas from stage 43-46 animals were immunostained with anti-GFP antibodies and imaged with confocal microscopy.

Results: NKA α 3 and α 4 are most divergent in their cytoplasmic, unstructured N-termini. Chimeras with this region exchanged resulted in changing the localization of α 3 from the inner segment plasma membrane to the outer segment and vice versa. Analysis of a series of paired chimeric constructs narrowed the region containing the motif responsible for this differential targeting to fourteen amino acids.

Within this region we identified a valine and proline necessary for outer segment localization.

Conclusions: This study allowed us to uncover a targeting motif within a flagellar NKA isoform with similarity to the targeting motif of rhodopsin. We speculate that lack of this targeting motif in NKA

α 3 contributes to its inner segment localization where competition for energy resources between NKA, phototransduction in the outer segment, and the synaptic vesicle cycle in the synapse can be minimized.

Commercial Relationships: Joseph G. Laird , None; Yuan Pan ,

None; David Yamaguchi , None; Sheila A. Baker , None



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ARVO 2015 Annual Meeting Abstracts 131 Photoreceptor

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