Mutations in WHRN lead to Usher syndrome type 2d or to non-syndromic hearing impairment. The WHRN-encoded gene product whirlin directly interacts with the intracellular regions of the other two Usher syndrome type 2-associated proteins, usherin and ADGRV1. In photoreceptor cells, this protein complex constitutes fibrous links between the periciliary membrane and the connecting cilium. However, the molecular mechanism(s) of retinal degeneration due to compromised formation and function of the USH2-associated protein complex remains elusive. To unravel this pathogenic mechanism, we isolated and characterized whirlin-associated protein complexes from zebrafish photoreceptor cells. We generated transgenic zebrafish that express Strep/FLAG-tagged Whrna, a zebrafish ortholog of human whirlin, under the control of a photoreceptor-specific promoter. Affinity purification of Strep/FLAG-tagged Whrna and associated proteins from adult transgenic zebrafish retinas followed by mass spectrometry identified 19 novel candidate associated proteins. Pull down experiments and dedicated yeast two-hybrid assays confirmed the association of Whrna with 7 of the co-purified proteins. Several of the co-purified proteins are part of the synaptic proteome, which indicates a role for whirlin in the photoreceptor synapse. Future studies will elucidate which of the newly identified protein-protein interactions contribute to the development of the retinal phenotype observed in USH2d patients. SIGNIFICANCE: Since protein-protein interactions identified using targeted in vitro studies do not always recapitulate interactions that are functionally relevant in vivo, we established a transgenic zebrafish line that stably expresses a Strep/FLAG-tagged ortholog of human whirlin (SF-Whrna) in photoreceptor cells. Affinity purification of in vivo-assembled SF-Whrna-associated protein complexes from retinal lysates followed by mass spectrometry, identified 19 novel candidate interaction partners, many of which are enriched in the synaptic proteome. Two human orthologs of the identified candidate interaction partners, FRMPD4 and Kir2.3, were validated as direct interaction partners of human whirlin using a yeast two-hybrid assay. The strong connection of whirlin with postsynaptic density proteins was not identified in previous in vitro protein-protein interaction assays, presumably due to the absence of a biologically relevant context. Isolation and identification of in vivo-assembled whirlin-associated protein complexes from the tissue of interest is therefore a powerful methodology to obtain novel insight into tissue specific protein-protein interactions and has the potential to improve significantly our understanding of the function of whirlin and the molecular pathogenesis underlying Usher syndrome type 2.

Antisense oligonucleotide (AON)-based splice modulation is the most widely used therapeutic approach to redirect precursor messenger RNA (pre-mRNA) splicing. To study the functional effect of human mutations affecting pre-mRNA splicing for which AON-based splice redirection would be a potential therapeutic option, humanized knock-in animal models are pivotal. A major limitation of using humanized animal models for this purpose is the reported poor recognition of human splice sites by the splicing machineries of other species. To overcome this problem, we provide a detailed guideline for the generation of functional humanized knock-in zebrafish models to assess the effect of mutation-induced aberrant splicing and subsequent AON-based splice modulation therapy .

Mutations in USH2A , encoding usherin, are the most common cause of syndromic and non-syndromic retinitis pigmentosa (RP). The two founder mutations in exon 13 (c.2299delG and c.2276G>T) collectively account for ~ 34% of USH2A -associated RP cases. Skipping of exon 13 from the USH2A transcript during pre-mRNA splicing presents a potential treatment modality in which the resulting transcript is predicted to encode a slightly shortened usherin protein. Morpholino-induced skipping of ush2a exon 13 in larvae of the previously published ush2a exon 13 zebrafish mutant resulted in the production of usherinΔexon13 and completely restored retinal function. RNA antisense oligonucleotides were investigated for their potential to specifically induce human USH2A exon 13 skipping. Lead candidate QR-421a induced dose-dependent exon 13 skipping in iPSC-derived photoreceptor precursors from a patient homozygous for the USH2A c.2299delG mutation. Intravitreal delivery of QR-421a in non-human primates showed that QR-421a penetrates the retinal outer nuclear layer and induces detectable levels of exon 13 skipping until at least 3 months post injection. In conclusion, QR-421a-induced exon skipping proves to be a highly promising treatment for RP caused by mutations in exon 13 of the USH2A gene.

Mutations in eyes shut homolog (EYS), a gene predominantly expressed in the photoreceptor cells of the retina, are among the most frequent causes of autosomal recessive (ar) retinitis pigmentosa (RP), a progressive retinal disorder. Due to the absence of EYS in several rodent species and its retina-specific expression, still little is known about the exact function of EYS and the pathogenic mechanism underlying EYS-associated RP. We characterized eys in zebrafish, by RT-PCR analysis on zebrafish eye-derived RNA, which led to the identification of a 8,715 nucleotide coding sequence that is divided over 46 exons. The transcript is predicted to encode a 2,905-aa protein that contains 39 EGF-like domains and five laminin A G-like domains, which overall shows 33% identity with human EYS. To study the function of EYS, we generated a stable eysrmc101/rmc101 mutant zebrafish model using CRISPR/Cas9 technology. The introduced lesion is predicted to result in premature termination of protein synthesis and lead to loss of Eys function. Immunohistochemistry on retinal sections revealed that Eys localizes at the region of the connecting cilium and that both rhodopsin and cone transducin are mislocalized in the absence of Eys. Electroretinogram recordings showed diminished b-wave amplitudes in eysrmc101/rmc101 zebrafish (5 dpf) compared to age- and strain-matched wild-type larvae. In addition, decreased locomotor activity in response to light stimuli was observed in eys mutant larvae. Altogether, our study shows that absence of Eys leads to a disorganized retinal architecture and causes visual dysfunction in zebrafish.

Antisense oligonucleotide (AON)-based splice modulation has been proven to hold great promise as a therapeutic strategy for a number of hereditary conditions. AONs are small modified single-stranded RNA or DNA molecules that are complementary to splice enhancer or silencer target sites. Upon pre-mRNA binding, AONs will prevent or stimulate binding of the spliceosome thereby modulating splicing events. AONs can be designed and applied for different genes and genetic disorders as the specificity depends on their nucleotide sequence. Here we provide a guideline for setting up AON-based splice-modulation experiments by describing a detailed protocol to design and evaluate AONs using a combination of in silico and in vitro analyses.

Mutations in USH2A are the most frequent cause of Usher syndrome and autosomal recessive nonsyndromic retinitis pigmentosa. To unravel the pathogenic mechanisms underlying USH2A-associated retinal degeneration and to evaluate future therapeutic strategies that could potentially halt the progression of this devastating disorder, an animal model is needed. The available Ush2a knock-out mouse model does not mimic the human phenotype, because it presents with only a mild and late-onset retinal degeneration. Using CRISPR/Cas9-technology, we introduced protein-truncating germline lesions into the zebrafish ush2a gene (ush2armc1: c.2337_2342delinsAC; p.Cys780GlnfsTer32 and ush2ab1245: c.15520_15523delinsTG; p.Ala5174fsTer). Homozygous mutants were viable and displayed no obvious morphological or developmental defects. Immunohistochemical analyses with antibodies recognizing the N- or C-terminal region of the ush2a-encoded protein, usherin, demonstrated complete absence of usherin in photoreceptors of ush2armc1, but presence of the ectodomain of usherin at the periciliary membrane of ush2ab1245-derived photoreceptors. Furthermore, defects of usherin led to a reduction in localization of USH2 complex members, whirlin and Adgrv1, at the photoreceptor periciliary membrane of both mutants. Significantly elevated levels of apoptotic photoreceptors could be observed in both mutants when kept under constant bright illumination for three days. Electroretinogram (ERG) recordings revealed a significant and similar decrease in both a- and b-wave amplitudes in ush2armc1 as well as ush2ab1245 larvae as compared to strain- and age-matched wild-type larvae. In conclusion, this study shows that mutant ush2a zebrafish models present with early-onset retinal dysfunction that is exacerbated by light exposure. These models provide a better understanding of the pathophysiology underlying USH2A-associated RP and a unique opportunity to evaluate future therapeutic strategies.

The frequent deep-intronic c.7595-2144A>G mutation in intron 40 of USH2A generates a high-quality splice donor site, resulting in the incorporation of a pseudoexon (PE40) into the mature transcript that is predicted to prematurely terminate usherin translation. Aberrant USH2A pre-mRNA splicing could be corrected in patient-derived fibroblasts using antisense oligonucleotides. With the aim to study the effect of the c.7595-2144A>G mutation and USH2A splice redirection on retinal function, a humanized zebrafish knockin model was generated, in which 670 basepairs of ush2a intron 40 were exchanged for 557 basepairs of the corresponding human sequence using an optimized CRISPR/Cas9-based protocol. However, in the retina of adult homozygous humanized zebrafish, only 7.4% +/- 3.9% of ush2a transcripts contained the human PE40 sequence and immunohistochemical analyses revealed no differences in the usherin expression and localization between the retina of humanized and wild-type zebrafish larvae. Nevertheless, we were able to partially correct aberrant ush2a splicing using a PE40-targeting antisense morpholino. Our results indicate a clear difference in splice-site recognition by the human and zebrafish splicing machinery. Therefore, we propose a protocol in which the effect of human splice-modulating mutations is studied in a zebrafish-specific cell-based splice assay before the generation of a humanized zebrafish knockin model.

Program Number: 2485 Presentation Time: 8:30 AM–8:45 AM Photoreceptor-specific transition zone (PSTZ), a novel sub-region of the connecting cilium (CC), is maintained by retinal ciliopathy protein SPATA7 Rachayata Dharmat1, 2, Aiden Eblimit2, Yumei Li2, Michael Robichaux3, Zhixian Zhang3, Feng He3, Antrix Jain3, Graeme Mardon1, Sung Yun Jung3, Theodore G. Wensel3, Rui Chen1, 2. 1Molecular and Human Genetics, Baylor college of medicine, Houston, TX; 2HGSC, Baylor College of Medicine, Houston, TX; 3Dept of Biochemistry, Baylor College of Medicine, Houston, TX. Purpose: A hallmark of the photoreceptor sensory cilium is the presence of a specialized structural homolog of transition zone called the connecting cilium (CC). Interestingly, certain transition zone genes, such as SPATA7, specifically impairs the function of the CC without affecting the transition zone of primary cilia when mutated. To determine how the CC is functionally distinct from the transition zone, we investigated the impact of the loss of a photoreceptorspecific ciliary protein to probe the differences between the CC and the transition zone using Spata7 KO mice as the model. Methods: To understand the function of SPATA7 at the CC, we performed IP-MS based proteomic profiling to identify SPATA7-interacting proteins. We next assessed the localization of interacting partners in the absence of Spata7 and Sdccag8 using immunohistofluorescence and confirmed it using super-resolution STORM microscopy on the photoreceptors of P15 Spata7 KO mice. Since we observed morphological defects, we further assessed structural alterations of the microtubules using cryo-tomography. Results: SPATA7 interacts with the RPGR and NPHP complex and localizes throughout the length of the CC. In the absence of SPATA7, its interacting proteins are specifically absent (or excluded) from the distal CC which we named the photoreceptor-specific transition zone (“PSTZ”). However, the localization of CC proteins in the proximal CC (pCC) remains unaffected. In contrast, this peculiar phenotype is not observed in the absence of a pCC protein, SDCCAG8. Functionally, the PSTZ complex is important for stabilization of the CC structure as its absence leads to destabilization of ciliary microtubules specifically in the distal CC. This destabilization is caused due to the absence of CEP290, from the PSTZ region, a component of Y-links that are essential for the integrity of microtubules. Conclusions: Our data displays a novel photoreceptor-specific sub-region in the distal CC, termed the PSTZ, which plays a critical role in the functioning of the CC. Cilia-related proteins at the PSTZ are essential for maintaining the integrity of the microtubule core thereby stabilizing the CC. Hence we propose that this unique PSTZ region makes the CC functionally and structurally distinct from the transition zone found in other primary cilia. Commercial Relationships: Rachayata Dharmat, None; Aiden Eblimit, None; Yumei Li, None; Michael Robichaux, None; Zhixian Zhang, None; Feng He, None; Antrix Jain, None; Graeme Mardon, None; Sung Yun Jung, None; Theodore G. Wensel, None; Rui Chen, None Support: Grants from National Eye Institute (R01EY022356) and Retinal Research Foundation to R.C Program Number: 2486 Presentation Time: 8:45 AM–9:00 AM Contribution of autophagy to Usher syndrome pathogenesis Erik de Vrieze1, Ralph Slijkerman1, Margo Dona1, Sanne Broekman1, 2, Lisette Hetterschijt1, Theo Peters1, Hannie Kremer1, 2, Erwin van Wijk1. 1Otorhinolaryngology, Radboudumc, Nijmegen, Netherlands; 2Human Genetics, Radboudumc, Nijmegen, New Caledonia. Purpose: Usher syndrome (USH) is the most common cause of hereditary deaf-blindness. USH patients are born with congenital hearing impairment, and suffer from progressive vision loss (retinitis pigmentosa, RP), a combination that puts them at risk for social isolation and loss of independence. Mutations in the USH2A gene are the most frequent cause of USH, explaining up to 50% of all cases. Mutations in USH2A can also result in non-syndromic RP. Currently, virtually nothing is known about the pathogenesis of USH2A-associated RP. Methods: We used complementary proteomics techniques to identify novel interaction partners of USH2A. Using CRISPR/Cas9-mediated genome editing, we generated an ush2a zebrafish knockout model, carrying a protein-truncating mutation in exon 13. Guided by our proteomic studies, we deep-phenotyped our ush2a-/zebrafish. Results: We identified and validated interactions between multiple subunits of the Cop9 signalosome (CSN) and members of the USH protein complex. CSN subunit 8 (COPS8) is a direct interactor of USH2A. CSN/COPS8 is well documented to play a role in two proteostatic pathways: ubiquitin proteasome system (UPS) and autophagy. Immunohistochemistry showed that Cops8 localization in the photoreceptor overlaps with Ush2a, allowing interactions in vivo. Phenotypic analyses of the ush2a-/retina showed increased levels of photoreceptor apoptosis, as well as an increase in autophagosomes. However, we did not find evidence of increased activity of the UPS system. We also identified mislocalization of rhodopsin-containing transport vesicles. Surprisingly, ER stress (protein-accumulation in the endoplasmic reticulum) appears diminished. Conclusions: Using zebrafish as a model organism, we have identified that elevated levels of autophagy might be the pathogenic mechanism underlying USH2A-associated retinal degeneration. Whether this is a direct consequence of misregulated autophagy (through CSN), or is a response to the mislocalized transport vesicles, remains to be established. Both prolonged elevation of autophagy and activation of mislocalized photopigments can lead to the observed apoptosis of photoreceptors. The pathways that we have identified explain the slow progressive nature of the retinal degeneration in USH2A patients. This brings us closer to understanding the pathogenesis of USH2A-associated RP, which is also important for the development of future therapies. Commercial Relationships: Erik de Vrieze; Ralph Slijkerman, None; Margo Dona, None; Sanne Broekman, None; Lisette Hetterschijt, None; Theo Peters, None; Hannie Kremer, None; Erwin van Wijk, None

Over the last decade, huge progress has been made in the understanding of the molecular mechanisms underlying inherited retinal dystrophy (IRD), as well as in the development and implementation of novel therapies, especially in the field of gene therapy. The use of mutant animal models, either naturally occurring or generated by genetic modification, have contributed greatly to our knowledge on IRD. Yet, these mutant animal models do not always mimic the retinal phenotype that is observed in humans with mutations in the orthologous gene, often due to species-specific characteristics of the retina, and/or diverse functions of the gene products in different species. In this manuscript, we compare general and ocular characteristics of a series of widely used vertebrate animal models, i.e. zebrafish, chicken, rodents, cats, dogs, sheep, pigs and monkeys, in terms of genetic architecture and sequence homology, methods to modify genomes, anatomy of the eye, and structural details of the retina. Furthermore, we present an overview of mutant vertebrate animal models that have been used to study or develop treatments for the various genetic subtypes of IRD, and correlate the suitability of these models to the specific characteristics of each animal. Herewith, we provide tools that will help to select the most suitable animal model for specific research questions on IRDs in the future, and thereby assist in an optimal use of animals and resources to further increase our understanding of inherited retinal dystrophies, and develop novel treatments for these disorders.

Ciliopathies are Mendelian disorders caused by dysfunction of cilia, ubiquitous organelles involved in fluid propulsion (motile cilia) or signal transduction (primary cilia). Retinal dystrophy is a common phenotypic characteristic of ciliopathies since photoreceptor outer segments are specialized primary cilia. These ciliary structures heavily rely on intracellular minus-end directed transport of cargo, mediated at least in part by the cytoplasmic dynein 1 motor complex, for their formation, maintenance and function. Ninein-like protein (NINL) is known to associate with this motor complex and is an important interaction partner of the ciliopathy-associated proteins lebercilin, USH2A and CC2D2A. Here, we scrutinize the function of NINL with combined proteomic and zebrafish in vivo approaches. We identify Double Zinc Ribbon and Ankyrin Repeat domains 1 (DZANK1) as a novel interaction partner of NINL and show that loss of Ninl, Dzank1 or both synergistically leads to dysmorphic photoreceptor outer segments, accumulation of trans-Golgi-derived vesicles and mislocalization of Rhodopsin and Ush2a in zebrafish. In addition, retrograde melanosome transport is severely impaired in zebrafish lacking Ninl or Dzank1. We further demonstrate that NINL and DZANK1 are essential for intracellular dynein-based transport by associating with complementary subunits of the cytoplasmic dynein 1 motor complex, thus shedding light on the structure and stoichiometry of this important motor complex. Altogether, our results support a model in which the NINL-DZANK1 protein module is involved in the proper assembly and folding of the cytoplasmic dynein 1 motor complex in photoreceptor cells, a process essential for outer segment formation and function.