1. Replacement gene therapy for TULP1 (RP14/LCA15) Gene defects in TULP1 cause retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) in humans. A knockout mouse model lacking Tulp1 (TULP1/) develops rapid photoreceptor degeneration with severe mislocalization of rod and cone opsins. Following our previous work with a mouse replacement gene, we evaluated whether AAV-mediated human gene replacement therapy in TULP1/ model could improve photoreceptor survival and recovery of rhodopsin and cone opsin in outer segments of rods and cones respectively. The retina-specific variant of human TULP1 proved unclonable from retinal cDNA, hence we used a synthetic fragment to replace over 400 bp of the human gene that has a high GC content and is always mutated when propagated through E. coli hosts. We found substantial rescue of the disease phenotype as a result of transgene expression. We propose that the vector and the synthetic construct design used in this study could serve as a prototype for a human clinical trial (manuscript in preparation). 2. Tubby is required for trafficking G protein-coupled receptors to cilia 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 (in press). 3. Planar cell polarity (PCP), primary cilia and neurobiology of the mammalian retina 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. In the neural retina, we have identified starburst amacrine cells as the major site of Pk1 expression. This maybe functionally related to the elaboration of dendritic arbors that these cells develop and the direction-selective wiring with RGCs. We are currently collaborating with Wei Li at NEI and Jimmy Zhou at Yale to perform imaging and functional studies to decipher the role of PK1 in this cell population. We are also beginning to explore a possible role for PCP in photoreceptor development and function. 4: Elastic fiber homeostasis and pseudoexfoliation glaucoma. The etiology of pseudoexfoliation glaucoma is not clear, but available evidence suggests a link to a defect in elastic fiber maintenance. Using a line of LOXL1 mutant mice we have previously generated, we conducted two sets of studies to characterize the phenotypic changes in the anterior segment as well as the posterior portion of the eye. a) Anterior segment defects We have identified blood-aqueous barrier breakdown and cataract as major anterior segment defects in mouse eyes due to loss of LOXL1. A similar breakdown in the blood-aqueous barrier has been well documented in humans with pseudoexfoliation leading to changes in the composition of the aqueous. The Loxl1 mutant also develops early cataract, which is also a common complication of human exfoliation syndrome. Interestingly the lens defect in mice is confined to the area in close apposition to the posterior iris with sparing of the pupillary area. In human patients with pseudoexfoliation, iris fluorescein angiography and histopathologic studies have shown the iris vasculature to be the site of blood-aqueous barrier breakdown. In mice, a large area of the anterior lens surface is virtually in contact with the posterior iris epithelial lining. This area of the lens, therefore, will be exposed to the highest concentration of any abnormal substances released from the iris. We propose that in both human exfoliation patients and in Loxl1 mutant mice, the lens defect is secondary to the compromised blood-aqueous barrier. The Loxl1 null mice do not exhibit spontaneous accumulation of exfoliative materials characteristic of human exfoliation syndrome. Our findings are consistent with current genetic data suggesting that additional genetic and/or environment factors aside from genetic variation in LOXL1 are necessary for disease development. The risk alleles of LOXL1 may affect the way LOXL1 gene is controlled rather than affecting LOXL1 expression or function constitutively. This work was done in collaboration with Dr. Janey Wiggs (Harvard Medical School;manuscript in preparation). b) Susceptibility to ocular hypertension at the optic nerve exit (mouse equivalent of lamina cribrosa. The anterior chamber defects does not represent the entire spectrum of changes occurring in pseudoexfoliation. Abundant elastic tissues, and hence LOXL1, are also found in the ciliary body, trabecular meshwork, Bruchs membrane and the point where optic nerve exit the globe corresponding to the lamina cribrosa of human eye (our unpublished observation). A defect in elastic fiber homeostasis will likely impact the structure and function of those tissues as well. Mechanical weakness or changes in the compliance and resilience of the connective tissue at the lamina cribrosa could make the optic nerve more vulnerable to elevation of intraocular pressure . Our studies in mice confirm that the optic nerve exit is weakened in the loxl1 mutant mice. Upon induction of high IOP by injection of microbeads into the anterior chamber, LOXL1 deficient mice show increased vulnerability to elevated IOP, as evidenced by increased axial length of the eye, pronounced optic nerve excavation and a greater loss of retinal ganglion cell axons. These changes are associated with increased degradation of elastic tissues. These data suggest that LOXL1 insufficiency affects the tensile strength and resilience in the connective tissues surrounding the optic nerve exit. Though not sufficient to cause pseudoexfoliation glaucoma on its own, functional insufficiency of LOLX1 is one aspect of the pathogenic mechanism conferred by the LOXL1 risk allele in humans.
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