Retinitispigmentosa(RP)causesirreversibleblindnessinindividualsofallages.Itaffects~1in3,500people worldwide.WhileRPisinitiallycharacterizedbynightblindnessandperipheralvisionloss,mostRPpatients losetheircentralvisionandbecomelegallyblindbytheageof40.Thereisnotreatmenttosloworstop visionloss.Mislocalizationofthelight-absorbingproteinrhodopsinintherodsisacommonhallmarkshared bymanyanimalmodelsofRP.Thelong-termobjectiveofthisapplicationistodissectthemolecularpathway thatunderliesthesortinganddeliveryoftherhodopsinanddetermineitsdiseaserelevance.Thisinformation willacceleratethediscoveryofnewtreatmentsforRPandotherretinaldegenerativediseases.Inthe mammalianrods,whicharehighlycompartmentalized,rhodopsinissynthesizedinthebiosynthetic organellesconfinedtotheinnersegment.Rhodopsinisthenvectoriallydeliveredtoandconcentratedinthe outersegment(OS).Whilerhodopsin?straffickingthroughtheendoplasmicreticulum-Golgipathwayhas beeninvestigated,theimportanceoftheendosomeinrhodopsin?sOStargetingisunclear.Inmanyothercell types,theendosomes ervesasakeysortingstationforproteinsatthecrossroadsofmultipleintracellular traffickingpathways.Prolongedendosomalaccumulationofflyrhodopsinhasbeenshowntoleadtolight- dependentretinaldegeneration.Ourpreliminaryresultsshowedthatthenewly-synthesizedrhodopsin transitsthroughtheendosomalcompartmentsinmouserodsinvivo.TheOStargetingsignalofthe rhodopsinbindstoanearlyendosome-specificprotein,SARA.SARAdeficiencyinmouserodsnotonly causesrhodopsinmislocalizationbutalsoseveralothercellulardefectsintheendolysosomalsystem.Inthis application,wewilltestthecentralhypothesisthatinmammalianrodsthetrans-endosomalpathwaycritically regulatesthefidelityandtheefficiencyoftheOStargetingofrhodopsin.First,wewilladdresswhetherthe RPmutantrhodopsinsareretainedintheendosomesabnormallyduringtheirtransittotheOS,andwhether thisdefectperturbsthehomeostasisofotherendomembranes(Aim1).Wewillgeneratemultiple, complementarymousemodelstoexaminetheroleofthetrans-endosomalpathwayinthemorphogenesisof theOSanditsrhodopsinexpression(Aim2).Wewillalsoprofiletherodproteinsthattransitthroughthe endosomalsystemandcharacterizetheirinteractionwithkeyendosomaltraffickingregulators(Aim3).We willachievetheseaimsbyapplyingstate-of-the-arttechniquessuchasrod-specificinduciblegene expressionandgenedeletion,super-resolutionconfocalmicroscopy,correlativelight-electronmicroscopy, and3Dscanningelectronmicroscopy.Byprovidingmechanisticinsightsonphotoreceptorproteintrafficking andOSbiogenesis,thisresearchwillcontributetothedevelopmentofnewtherapiesforRPandrelated diseases.
There is currently no treatment to slow or stop vision loss in patients with RP in which rhodopsin mislocalizaiton a common defect. The overall goal of this proposal is to demonstrate the critical importance of trans-endosomal pathway in regulating the high-fidelity sorting of rhodopsin and to identify novel endosomal trafficking regulators. Success in confirming our model will open new doors to rationale therapies that broadly treat RP and other retinal degenerative diseases.
