1. PDZPH1, a newly identified protein in photoreceptor function and disease. Intracellular trafficking to photoreceptor outer segment and to the synaptic terminal is a polarized process that involves transport of proteins synthesized and processed in the biosynthetic inner segment. It is known that several trafficking routes exist and that outer segment bound proteins appear to rely on utilize these routes to different extent. Impairment of protein trafficking linked to mutations in the genes coding for Tubby and Whirlin causes early and late onset retinal degeneration, respectively. PDZPH1, a novel protein was discovered in our lab through yeast two hybrid screens by virtue of its interaction with tubby and whirlin. We report here that PDZPH1 is highly and specifically expressed in photoreceptors and conserved across vertebrate species from human to zebrafish. PDZPH1 protein is enriched at photoreceptor apical inner segments in endocytic trafficking organelles. CRISPR/Cas9 mediated disruption of PDZPH1 in mice leads to strongly reduced cyclic nucleotide gated (CNG) channels and potassium-dependent sodium/calcium exchanger (NCKX) trafficking to the plasma membrane of photoreceptor outer segments, and impaired photoreceptor function. Moreover, aberrant accumulation of early endosomes, strong reduction in lysosomes, altered CNG/NCKX transport at endosomal compartments and vacuolar structures in the photoreceptor inner segment are found. The amplitudes of electroretinograms (ERG) originating from both rods and cones under regular inter stimulus intervals (ISI) are comparable between mutant and control mice. However, significant reduction in amplitudes occurred in the knockout mice when the ISI were shortened between flashes. Deficits in visual acuity were seen at higher spatial frequencies as tested using optomotor responses, whereas the responses were normal at lower spatial frequencies. CRISPR/Cas9 mediated disruption of PDZPH1 in zebra fish showed early visual defects (6dpf) as determined by strongly reduced optokinetic responses. We conclude that PDZPH1 functions in endosomal transport of distinct subset of outer segment cargoes that include NCKX (Na+/Ca2+-K+ exchangers) and CNG channels. Our data indicate that CNG channels and NCKX are critically dependent on the endocytic routes for trafficking to the outer segments. We propose a model whereby reduced NCKX at the outer segment plasma membrane leads to a slowed calcium response kinetics, which in turn, slows photoreceptor light response recovery (slower response kinetics). This visual dysfunction is similar to patients with bradyopsia, caused by mutations in RGS9 or R9AP, who manifest profoundly reduced dynamic visual acuity and an inability to navigate through common daily tasks. A manuscript is in preparation (Vetrivel Sengottuvel et al.) 2. Stem cell studies.
We aim to make stem cell-derived photoreceptors that are well differentiated as indicated by the elaboration of outer segments. Such a culture system would be much better suited for retinal disease modeling, drug discoveries and provide donor tissues for transplantation. Given the dependence of photoreceptors on RPE, one of the critical components that is missing in current 3D retinal culture systems is a functioning RPE layer. Therefore, our long term goal is to develop a photoreceptor-RPE co-culture system. In the first phase of the research, we have used an embryonic stem (ES) cell line (H9) to differentiate stem cells into RPE and after 60 days of induction we have been able to confirm successful differentiation of the ES cells into RPE cells. We have tested and compared RPE cells grown on conventional Trans-well system and on electrospun polymer support, which better simulates the physical properties of the Brunchs membrane, and have demonstrated the suitability of the latter as a substrate for growing and differentiating RPE from stem cells. Our differentiated RPE cells correctly display RPE markers, express typical proteins of mature RPE cells, have polarity of mature RPE cells, and display distinct RPE morphology such as: forming necessary tight junctions between the cells required for epithelial monolayer and hexagonal shape. This shows reliability and success of our protocol and allows a baseline comparison of differentiation of Induced Pluripotent Stem Cells (iPSCs). In the second phase which has recently begun, we are generating stem cell derived human and mouse retinal progenitor cells and testing different setups to achieve insulated culturing system where RPE and photoreceptor progenitors are in close juxtaposition with the correct polarity orientation and at the same time the media conditions on the RPE and photoreceptor side can be varied independently with no leakage (short circuiting) from the peripheries. Taking an reductionist approach, we are also testing liposome-mediated delivery of chromophore to retinal organoids, exposure to RPE apically and basally derived, conditioned media in an attempt to identify factors that facilitate outer segment formation (In progress. Dawn Landis in our Section in close collaboration with Dr. Swaroops Stem Cell group). 3. We have continued our study on protein post-translational modification in the cilia of photoreceptors and made significant new discoveries. The complex patterns of protein glutamylation contribute to the tubulin code that confers versatility to microtubule functions. Glutamylation is a form of post-translational modification initiated by the ligation of a single glutamate to the -carboxyl group of gene-encoded glutamate residues on target proteins known as monoglutamylation, and elongation of the branched peptide chains then generates polyglutamylation structures. Tubulin tyrosine ligase-like (TTLL) proteins are related enzymes that either initiate or elongate the glutamate side chains. Deglutamylases catalyzing the reverse reaction are members of the cytosolic carboxypeptidase (CCP) family, which similarly have preferential activities either to shorten long glutamate chains or remove the branching point glutamate. We show that cytosolic carboxypeptidase 5 (CCP5), the only CCP known to remove the branch point glutamate, is uniquely required for photoreceptor function and viability. In mice lacking CCP5, monoglutamylated tubulins are increased at the photoreceptor connecting cilia and axonemal microtubules but polyglutamylated tubulins remain unchanged. Photoreceptors degenerate soon after weaning with cones impacted more severely than rods. Prior to cell death there is marked ectopic accumulation of cone and rod opsins in the cell body. While increased tubulin monoglutamylation is seen systemically, mutant mice appear healthy other than retinal dystrophy and male infertility. These results are consistent with CCP5 being a critical deglutamulase responsible for removing the branch point glutamate in vivo, and support CCP5 mutations as a cause of human retinitis pigmentosa. The unexpected finding that an overt disease phenotype is restricted to retinal photoreceptors may be underscored by the exceptionally active transciliary trafficking of membrane receptors, and hence a greater sensitivity to changes in the tubulin code, relative to CNS neurons in general (Xun Sun et al., manuscript in preparation).

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Veleri, Shobi; Nellissery, Jacob; Mishra, Bibhudatta et al. (2017) REEP6 mediates trafficking of a subset of Clathrin-coated vesicles and is critical for rod photoreceptor function and survival. Hum Mol Genet 26:2218-2230
Fan, Jianguo; Jia, Li; Li, Yan et al. (2017) Maturation arrest in early postnatal sensory receptors by deletion of the miR-183/96/182 cluster in mouse. Proc Natl Acad Sci U S A 114:E4271-E4280
Yu, Wenhan; Mookherjee, Suddhasil; Chaitankar, Vijender et al. (2017) Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice. Nat Commun 8:14716
Shimada, Hiroko; Lu, Quanlong; Insinna-Kettenhofen, Christine et al. (2017) In Vitro Modeling Using Ciliopathy-Patient-Derived Cells Reveals Distinct Cilia Dysfunctions Caused by CEP290 Mutations. Cell Rep 20:384-396
Pawlyk, B S; Bulgakov, O V; Sun, X et al. (2016) Photoreceptor rescue by an abbreviated human RPGR gene in a murine model of X-linked retinitis pigmentosa. Gene Ther 23:196-204
Sun, Xun; Park, James H; Gumerson, Jessica et al. (2016) Loss of RPGR glutamylation underlies the pathogenic mechanism of retinal dystrophy caused by TTLL5 mutations. Proc Natl Acad Sci U S A 113:E2925-34
May-Simera, Helen L; Gumerson, Jessica D; Gao, Chun et al. (2016) Loss of MACF1 Abolishes Ciliogenesis and Disrupts Apicobasal Polarity Establishment in the Retina. Cell Rep 17:1399-1413
Liu, Chunqiao; Widen, Sonya A; Williamson, Kathleen A et al. (2016) A secreted WNT-ligand-binding domain of FZD5 generated by a frameshift mutation causes autosomal dominant coloboma. Hum Mol Genet 25:1382-91
Yadav, Sharda Prasad; Sharma, Neel Kamal; Liu, Chunqiao et al. (2016) Centrosomal protein CP110 controls maturation of the mother centriole during cilia biogenesis. Development 143:1491-501
Li, Tiansen (2015) Leber congenital amaurosis caused by mutations in RPGRIP1. Cold Spring Harb Perspect Med 5:

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