Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

THE HAGSTROMLABORATORYSENIOR TECHNOLOGISTGayle T. Pauer, B.S.POSTDOCTORAL FELLOWTULP1 Under Scrutiny forRole in Early OnsetProgressive Retinal DegenerationRetinalQuansheng Xi, Ph.D.degeneration encompasses a myriadof genetically and phenotypically heterogeneous diseases. The majority of these blindingSTUDENTdisorders are inherited. The retina’s light-sensitiveAndrea Crabbphotoreceptor cells degenerate due to defects inmany different genes required for the normalphysiology of these cells. The broad long-term goalsof my laboratory are to identify novel genes causingretinal degeneration in patients and to define theunderlying pathogenic mechanisms responsible forphotoreceptor degeneration.In collaboration with ophthalmologists in ourdepartment, a program has been designed to recruitlarge numbers of patients with a variety of types ofretinal degeneration. DNA samples from hundredsof patients are deposited into a centralized database.My laboratory has implemented highthroughputmutation screeningtechniques, allowing us to efficientlyanalyze many genes for mutations inthe DNA of these patients. Our goalis to identify genetic alterations andcorrelate these results with theclinical phenotype of the patient.Recently, we identifiedmutations in a novel gene, TULP1, inpatients with an autosomal recessiveform of retinitis pigmentosa, a groupof progressive retinal degenerationsleading to blindness. TULP1 is amember of a conserved family offour proteins of unknown functionnamed tubby-like proteins (TULPs).We have begun to explore theStephanie A. Hagstrom, Ph.D.physiologic properties of TULP1 inthe retina by analyzing the tissuedistribution of the protein in normalHagstrom, S.A., North, M.A., Nishina, P.A., Berson, E.L., and T.P. Dryja (1998) Recessive mutations inthe gene encoding the tubby-like protein TULP1 in patients with retinitis pigmentosa. Nat. Genet. 18:174-176.Hagstrom, S.A., and T.P. Dryja (1999) Mitotic recombination map of 13cen-13q14 derived from an investigationof loss of heterozygosity in retinoblastomas. Proc. Natl. Acad. Sci. USA 96:2952-2957.Hagstrom, S.A., Duyao, M., North, M.A., and T. Li (1999) Retinal degeneration in tulp1-/- mice: accumulationof extracellular vesicles in the interphotoreceptor space. Invest. Ophthalmol. Vis. Sci. 40:2795-2802.Hagstrom, S.A., Neitz, M., and J. Neitz (2000) Cone pigment gene expression in individual photoreceptorsand the chromatic topography of the retina. J. Optical Soc. Am. A 17:527-537.Hagstrom, S.A., Adamian, M., Scimeca, M., Pawlyk, B.S., Yue, G., and T. Li (2001) A role for the tubby-likeprotein 1 in rhodopsin transport. Invest. Ophthalmol. Vis. Sci. 42:1955-1962.Xi, Q., Pauer, G.J.T., West, K.A., Crabb, J.W., and S.A. Hagstrom (2003) Retinal degenerations causedby mutations in TULP1. In Anderson, R.E., Lavail, M.M., and J. Hollyfield, eds. New Insights into RetinalDegenerative Deseases. Kluwer Academic/Plenum.mice and the photoreceptor disease phenotype intulp1-knockout mice. We generated antibodiesagainst Tulp1 and determined that it is aphotoreceptor-specific protein. It is predominantlylocalized in two specialized compartmentsof photoreceptors, named the inner segment andthe connecting cilium. In addition, we generatedtulp1-knockout mice and determined that theydevelop an early-onset, progressive photoreceptordegeneration that parallels that seen in patientswith TULP1 mutations. Based on the localizationof Tulp1 and the features of the retinalphenotype in knockout mice, we hypothesize thatTULP1 is involved in the transport of proteinssynthesized in the inner segment compartment toits final location in the outer segment compartmentof photoreceptor cells.The steps and proteins involved in thetransport of proteins to the outer segments ofphotoreceptors is an essential but not wellunderstoodaspect of photoreceptor cell biology.The outer segment is connected to the innersegment via a narrow compartment, the connectingcilium. Outer segment-bound proteins aresynthesized in the inner segment and must beefficiently transported to their target location.The molecular basis of this transport systemremains unclear. Our localization of Tulp1 andanalysis of knockout mice suggest that Tulp1 maybe involved in this transport process.We are currently testing this hypothesis viaseveral approaches. We are generating an in vitrosystem in which to study the subcellular localizationand properties of wild-type and mutantforms of Tulp1. We are also searching forinteracting proteins by using yeast two-hybrid andimmunoprecipitation analysis. We are continuingto characterize the physiologicproperties of Tulp1 in vivo bystudying the knockout mice.We anticipate that results fromthese experiments will provideinsights into the pathogenicmechanism causing retinitispigmentosa and help us assignfunction(s) to a novelphotoreceptor-specific protein.As we continue tosearch and catalogue novelgenes causing retinal degeneration,the ultimate goal of ourresearch is to provide thefoundation for future studiesaimed at evaluating therapeuticmodalities that might slow,stop, or reverse the course ofretinal degeneration.164