Brian D. Perkins - US grants

DESCRIPTION: The proposed experiments are designed to utilize a vertebrate genetic system capable of identifying dominant mutations affecting the zebrafish retina, with particular emphasis on mutations involved in rhodopsin trafficking. The long-term objectives of the proposed research include the identification of genes associated with blindness caused by age-related retinal degeneration and the characterization of the cellular and molecular mechanisms underlying retinal degeneration. Mutations causing dominantly inherited night blindness will be isolated by a behavioral assay and confirmed by histological and physiological methods. Proper rhodopsin localization and transport will be evaluated by breeding mutant zebrafish with transgenic zebrafish that express a transgene encoding a rhodopsin-green fluorescent protein (GFP) fusion protein and examining the progeny by confocal fluorescent microscopy. Additional characterization of other transport systems within the photoreceptors of mutant fish will elucidate possible interactions between cellular transport mechanisms. Finally, identification of the genes responsible for these mutations will be achieved by positional cloning strategies. The proposed work will provide insight to the mechanisms underlying photoreceptor cell death that ultimately lead to retinal degeneration and blindness associated with aging.

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the molecular basis of cilia formation and maintenance in vertebrate photoreceptor cells. In vertebrates, the assembly and maintenance of photoreceptor outer segments begins with the formation of a connecting cilium. The connecting cilium contains a microtubule- based axoneme that is anchored to the apical inner segment by a basal body. Cilia formation begins with the docking of basal bodies at the apical surface of the inner segment. Genetic mutations disrupting the assembly, structure, or function of basal bodies and/or cilia result in a spectrum of diseases known as ciliopathies. These multisyndromic disorders often present with retinal degeneration, kidney disease, mental retardation, and polydactyly. In the current application, we will utilize loss-of-function strategies in zebrafish to investigate the mechanisms controlling basal body localization. In Specific Aim 1, we will test the hypothesis that the dynein/dynactin complex regulates the apical transport of basal bodies preceding cilia formation by examining zebrafish mutants in dynein and the p150 and p50 subunits of dynactin. In Specific Aim 2, we provide preliminary evidence that basal bodies show a highly polarized arrangement within the adult zebrafish retina. We will directly test the hypothesis that the PCP pathway regulates this patterning and is essential for photoreceptor survival. In Specific Aim 3, we will examine zebrafish carrying null mutations in the Joubert Syndrome gene arl13b for retinal phenotypes. Proposed experiments will also test the requirement of the GTPase domain and a ciliary-targeting sequence RVxPx for Arl13b function. We will also test arl13b for functional interactions with Bardet-Biedl Syndrome (BBS) genes, and components of the PCP pathway. These interactions will identify potential second-site modifiers that enhance expression of photoreceptor phenotypes. The results of these studies will reveal novel mechanisms required for basal body placement prior to cilia formation and to identify novel genetic interactions that influence hereditary blindness.

Project Summary/Abstract The synapses of vertebrate photoreceptors and sensory hair cells release neurotransmitter through specialized structures known as synaptic ribbons. In a screen for insertional mutations affecting visual function in zebrafish, we identified the zebrafish wrb mutant, which exhibited photoreceptor degeneration and hearing loss. The mutation disrupts the wrb gene, which encodes a novel 170 amino acid protein with no known function. In preliminary studies, we found that wrb mutants exhibited mislocalization of ribbon synapse components, reduced numbers of docked ribbons at photoreceptor synapses, and significantly reduced ERG responses. These data strongly suggest that Wrb could be integrally involved in ribbon synapse assembly and/or function. To validate this hypothesis, we will study the role of Wrb in zebrafish synaptic function through a combination of histological and molecular approaches. In Aim 1, we will identify the subcellular localization of Wrb using transgenic approaches, we will determine if Wrb affects ribbon assembly during development, how loss of Wrb affects dendritic morphology of postsynaptic cells, and whether Wrb blocks synaptic vesicle release or membrane endocytosis. In Aim 2, we will identify binding partners of Wrb using a transgenic approach to purify tagged Wrb complexes and subsequent analysis by mass spectroscopy. These studies will provide insights into the role of Wrb, a novel protein that may be a critical component to ribbon synapse structure and function. The identity of Wrb binding partners and the subsequent characterization of Wrb in an in vivo system will open new avenues of research on synaptic architecture and possibly provide insight into candidate genes for blindness/deafness disorders.

Project Summary Mutations in CEP290 result a number of genetic diseases termed ciliopathies, which manifest with a variety of clinical symptoms, including retinal degeneration. While it is well-established that photoreceptor survival requires Cep290 function, the causes of photoreceptor death remain largely unknown. In vertebrate photoreceptors, Cep290 localizes to the connecting cilium, which is analogous to the transition zone of primary cilia. Work from cell culture and invertebrates suggest that Cep290 organizes the assembly of protein complexes that form a ?ciliary gate? within the transition zone. However, whether loss of Cep290 impacts such a ciliary gate have not been demonstrated in photoreceptors. Furthermore, the highly variable nature of CEP290-associated disease phenotypes cannot be explained by traditional genotype-phenotype correlations. Two models have been proposed to explain this variability. One possibility is that second-site genetic modifiers enhance disease severity in some patients. The second possibility is that exons harboring nonsense mutations and that also begin and end in the same reading frame can be preferentially skipped. In such a case the resulting mRNA transcript eludes nonsense-mediated decay and can produce a near-full-length protein. Disease severity therefore correlates with the total amount of full- length and near-full-length protein produced. This proposal seeks to address fundamental questions related to photoreceptor cell biology and the role of Cep290 in photoreceptor degeneration. In Aim 1, we will utilize two distinct zebrafish cep290 mutants to determine if defects in ciliary gating play a role in degeneration. In Aim 2, we will determine if other genes associated with Joubert Syndrome, namely arl13b, ahi1 or cc2d2a act as genetic modifiers to cep290-associated retinal degeneration in zebrafish. Finally, in Aim 3, we will take fibroblasts from patients with CEP290 mutations and generate human induced pluripotent stem cells (hiPSCs) and subsequently differentiated into 3D retinal cups. These hiPSC-derived retinal cups (hiPSC-DRCs) will be used determine if basal exon skipping occurs in from humans carrying CEP290 mutations and whether total protein levels correlate with disease severity. In addition, how disease-causing mutations lead to alterations in cilia architecture and protein trafficking will be investigated. These experiments will establish the molecular and genetic mechanisms that determine the severity of disease progression. Understanding the basis for phenotypic variability will provide much-needed clarification on the mechanisms of pathogenesis and lead to better treatment of ciliary disease.

Abstract Functional Vision Module The Functional Vision Module will provide the technologies required to interrogate visual function in experimental animals to the investigators involved in this P30 Core Grant for Vision Research. This module will provide state of the art assessment of visual function in mice and zebrafish. Included are visual electrophysiological assays such as the electroretinogram (ERG) and the visual evoked potential (VEP); and visual behavior assays such as the pupillary light reflex (PLR), the optokinetic reflex (OKR), optomotor reflex (OMR) and threshold measurement. Collectively, these assays interrogate visual function from light detection in the photoreceptors to signal integration and interpretation within the brain. Access to this core module will support NEI R01 funded investigators, help newly recruited investigators obtain data for NEI R01 grant submissions, foster collaborative interactions between RO1 funded investigators and newly recruited investigators, attract investigators from other departments to research on the visual system and be a valuable departmental resource in attracting additional vision scientists to our program as we expand our research faculty.

Inherited retinal degenerations (IRDs) result in the progressive and permanent death of neurons. In recent years, however, our knowledge of endogenous stem cells in the retina has opened the possibility of stimulating regeneration of lost neurons in patients suffering from IRDs. Leber's Congenital Amaurosis (LCA), Bardet-Biedl Syndrome, and Retinitis Pigmentosa are among the most common form of IRDs. Mutations in CEP290 are one of the most common causes of LCA, while mutations in BBS2 contribute to BBS and mutations in the EYS gene cause RP. We demonstrate that zebrafish with mutations in the either cep290, bbs2, or eys genes undergo a progressive cone degeneration with evidence of rod dysfunction. Unlike mammals, zebrafish have the innate ability to regenerate retinal neurons when they are damaged or lost. In response to retinal injury, zebrafish exhibit a robust capability of regenerating lost neurons, including photoreceptors. Retinal damage causes release of growth factors and inflammatory cytokines that trigger Müller glia to divide and generate multipotent retinal progenitor cells that regenerate lost neurons. Central to this process is a reprogramming event that involves activation of Stat3. While robust regeneration occurs following acute injury, evidence from the literature and preliminary data indicate that regeneration does not occur in zebrafish with inherited forms of retinal degeneration, such as the cep290-/-, bbs2-/-, or eys-/- mutants. These observations suggest that Müller glia respond differently to acute vs. inherited forms of retinal injury and that regeneration is only triggered when the degree of retinal injury crosses a ?damage threshold? within a temporal window. In zebrafish degeneration mutants there is widespread proliferation of rod progenitor cells. However, Müller glia, which are the source of cone progenitors, fail to proliferate. Using what is known about mechanisms involved in regeneration after acute retinal injury in zebrafish, we will test components of these signaling pathways to determine if they are activated in Müller cells of these photoreceptor degeneration mutants. In particular, we will focus on pathways that converge to activate Stat3. Using RNAseq on purified Muller glia from these degeneration mutants, we will investigate how degeneration and inflammation alter the transcriptome of Muller glia. Finally, we will use pharmacological and genetic approaches to suppress immune cell activity and understand the role of microglia and macrophages on degeneration and regeneration. Understanding the mechanisms that underpin retinal regeneration in multiple zebrafish disease models will generate novel hypotheses that can ultimately be translated into humans with retinal degenerative diseases.