1. The periciliary membrane complex, Tubby family of proteins and ciliary trafficking of GPCR Tubby is the founding member of the Tubby-like family of proteins. The naturally occurring tubby mutation in mice causes retinitis pigmentosa, hearing loss and obesity. We show that the tubby is required for the correct localization of a select group of membrane receptors to the distal portion of primary and sensory cilia. By using tubby and tubby like protein 1 as baits, we have identified interacting proteins ankrd54 and an unknown protein LOC69239. Preliminary studies indicate that these proteins are likely to function in ciliary trafficking and we have engineered knockout mouse lines that carry mutations in these genes. Phenotype analysis is underway. We further hypothesize that phosphatidyl inoside bisphosphate (PIP2) is likely to be a critical signaling molecule for ciliary trafficking and experiments are underway to delineate the mechanism of this transport process. 2. Macf1 is required for microtubule and actin interactions during ciliated sensory cell maturation and function. The cytoskeleton, present in all eukaryotic cells, is comprised of microtubules, actin filaments and intermediate filaments, and forms important structures such as cilia and lamellipodia. It plays vital roles during cell division and intracellular transport. Spectraplakins are cytoskeletal cross-linkers, which associate with F-actin, microtubules and intermediate filaments to integrate cytoskeletal networks. In vertebrates, in addition to their role as cytoskeletal cross-linkers, spectraplakins have also been shown to play a role in intracellular trafficking. Microtubule actin cross linking factor 1 (MACF1/ACF7), one of only two spectraplakin proteins found in mammalian systems, is a 586 kDa protein, highly conserved across species. Previous work has shown that Macf1 plays a role in integrating coordinated microtubule and actin dynamics along cell borders (focal adhesions/adherens junctions). In mouse, Macf1 was found to be the third most abundant protein peptide in photoreceptor sensory cilium proteome and the Drosophila homolog shot, is to thought act as an actin-MT cross-linker during photoreceptor morphogenesis. We identified that Macf1 interacts with centrosomal/basal body protein Mkks and therefore may contribute to ciliogenesis. We generated conditional knock out mice, in which Macf1 had been deleted in developing ciliated sensory tissues (retina, cochlea, lateral ventricles) and Macf1 antibodies against its actin-binding domain. Immunohistochemistry suggests that Macf1 is widely expressed in developing retina, being concentrated at the apical border of the neuroepithelium. Loss of Macf1 in the developing retina results in a complete loss of visual function due to severely disrupted retinal lamination primarily affecting photoreceptors. Loss of apical basal polarity of the outer nuclear layer is seen as early as P5, which is preceded by ectopic ciliary vesicles and loss of basal body migration and/or docking at the apical membrane. Integrity of junctional markers (adherens junctions and tight junctions) is not compromised but polarity components RhoA and Par3 are differentially expressed. In developing cochlea sensory neurons, Macf1 expression is highly enriched at the base of the kinocilium. Mutant cochlea sensory neurons display mild polarity defects yet the cochlea length and tissue organization is not disrupted, suggesting that basal body migration is one of the underlying defects. Development of ciliated ventricular neuroepithelia is dependent on the correct polarization of primary cilia. Loss of Macf1 severely hinders the development of the lateral ventricle. These findings suggest a critical role for Macf1 in developing sensory neurons, and highlight possible novel functions for microtubule and actin interactions in ciliary related processes. These insights provide the cellular basis for clinical phenotypes associated with ciliopathies. We are currently investigating whether Macf1 also plays a separate role in mature photoreceptors at the point of actin-microtubule network connection for proteins en route to the outer segments via the connecting cilia. 3. A mouse model for spontaneous CNV Ppp2r5δis a regulatory subunit of the protein phosphatase 2A (PP2A) a trimeric holoenzyme that functions to reverse the action of kinases in most signaling cascades. It is implicated in the negative control of cell growth and division. Ppp2r5δbelongs to the B'/B56/PR61/PPP2R5 family of regulatory subunits which have been shown to negatively regulate Wnt signaling. Expression of Ppp2r5δis thought to be localized predominantly in the RPE. When we look at ocular tissue from Ppp2r5δ-/- mice we see sectorial photoreceptor degeneration accompanied with choroidal neovascularization in older animals. This is closely associated with thickened/multilayered RPE tissue and loss of Bruchs membrane integrity seen prior to onset of CNV in younger animals. We hypothesize that primary defects originate from un-curtailed β-catenin dependent Wnt signaling in the RPE. For the first months the defect is subclinical until the RPE begins to de-differentiate possibly undergoing epithelial to mesenchyme transition. When the RPE is not able to maintain the underlying pathology, these epithelial cells lose their apical and basolateral polarity, break their intercellular tight junctions, and degrade basement membrane extracellular matrix components to become migratory mesenchymal cells. With the integrity of the RPE compromised, choroidal blood vessels are able to migrate through the Bruchs membrane (requiring expression of VEGF) and permeate the outer retina Ultimately resulting in sectorial photoreceptor loss. We are interested in further exploring the molecular mechanism underlying the pathogenesis of this mouse mutant to see if the observed CNV is a direct effect of dysreglated WNT in the RPE . CNV is one of the hall marks of AMD, one of the most common degenerative eye disorders, yet so far only few animal models have been identified. Wnt signaling pathway is activated in the retinas and retinal pigment epithelia of animal models of age-related macular degeneration (AMD) and diabetic retinopathy (DR), yet non of the animal models that have been described have a primary defect related to Wnt signaling or the RPE. The mechanism underlying current animal models identified focus on the Bruch's membrane and inflammatory responses (cytokines/VEGF). This mouse could possibly be a new model for CNV and consequently for AMD. 4. Planar cell polarity (PCP) and retinal development Our interest in this area began when studies carried out at the NNRL showed that loss of the rod transcription factor NRL caused mis-regulation of Prickle homolog 1 and 2 (Pk1 and Pk2), which are putative core PCP components based on extensive studies in Drosophila. PCP refers to the polarization of a field of cells within the plane of the cell sheet. PCP is critical in driving normal tissue axis extension during development but the roles the PCP plays in neural tissues such as the retina is less clear. Our recent work shows that Prickle 1 (Pk1), a previously classified core planar cell polarity (PCP) component in Drosophila, also plays a role in tissue-axis extension and organization. Systemically we have identified a multitude of developmental defect that gave new insights into the molecular mechanism of Prickle function. A manuscript describing this study is under review. In the neural retina, we have identified preliminarily cellular defects in both the inner and outer retina. We are making intense effort towards elucidating the molecular pathways that normally operate involving prickle1 and the pathogenic mechanisms.

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National Eye Institute (NEI)
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