Study On The Effect And Mechanism Of DHDDS On Retinal Photoreceptor

Objective:Retinitis pigmentosa(RP)is the mostcommon inherited retinal degeneration,leading vision loss and blindness.RP refers to a large group of highlyheterogeneous retinal degenerative disorders characterizedby early rod photoreceptor dysfunction followedby progressive rod and cone photoreceptor dysfunctionand photoreceptor death.The disease has several patterns of inheritance.Anew K42E mutation in DHDDS(dehydrodolichol diphosphate synthase)has recently identified asthe cause of retinitis pigmentosa(RP)phenotype in a single family with non-syndromic recessive RP.To furtherstudy dehydrodolichol diphosphate synthase in the retina,we genetically ablated DHDDS globally and conditionally in mouse.The phenotype of retina was analyzed when glycosylation of photoreceptor was blocked to elucidate whether dysfunction of glycosylation is the mechanism of RP.Methods:1.Mouse embryonic stem(ES)cells containing the DHDDStmla allele,trapping cassette "SA-βgeo-pA"(splice acceptor-beta-geopolyA)and thus creating a constitutive null mutation,were injected into blastocysts and 5 chimeric mice were born.Crossing the chimeric mice with wild type mice produced two F1 mice carrying the DHDDStmla allele.Intercrossing produced more DHDDStmla/+.2.The DHDDSfl/fl mice are been crossed with mice expressing Crerecombinase under the control ofLMOP(long-mouse opsin promoter)and Chx10 to create DHDDS conditionalknock-out mice in which DHDDS is ablated only in mature photoreceptors and retinal neurocyte development respectively.3.Dark adapted electroretinogram(ERG)was performed observe the functional changes.Eyes were collected at different endpoint and semi-thin plastic sections were cut to display the entire retina.4.Conditional knockout of DHDDS was achieved by subretinal injection of AAV-Rho-Cre(adeno-associated virus carring Cre recombinase driven by a 500bp muring rhodopsin)to DHDDSfl/fl mice in which exon 4 was floxed.Immunoflueorescence assay detected the locations of Cre and GFP(green fluorescent protein)after injection 1 month.5.TM was dissolved in DMSO and final concentrations of 25ng/μl and 50ng/μl.Adult Balb/c mice were injected intravitreally in 2μl.After injection 1,2,4weeks,OCT,ERG and semi-thin plastic sections were taken.Using western blotting technique to detect the changes of rhodopsin in photoreceptors,for forther investigate the mechanisms of glycosylation in RP.Results:1.Morphological showed normal development of photoreceptors and functionalanalysis indicates normal ERGs from DHDDStmla/+ mice.No such well structured embryos were found in DHDDS-/-mice at E7 as DHDDS-/-miceand no homozygous DHDDStmla(DHDDS-/-)mice were obtained even afterextensive crossingDHDDZStmla/+ mice.The absence of homozygous DHDDStmla may suggest that DHDDS-/-is lethal.2.Abnormal structures and loss of photoreceptors is evident when the DHDDSfl/fl mice were crossed with the LMOP-Cre and Chx10 Cre.The ERG showed abnormal a wave and b wave amplitude compared with control group.3.Photoreceptors in the AAV-Rho-GFP injected eyes appeared having normal structures and fuctions.In the AAV-Rho-Cre injected eyes showed the loss of photoreceptors and ERG shows the dysfunction of photoreceptors.4.By 4weeks after TM injection,significant loss of photoreceptor occurred in the retina both 50ng and 100ng TM group.ERG shows the dysfunction of photoreceptors.Western Blotting showed the rhodopsin deglycosylated by TM.Conclusion:DHDDS plays a vital role in early life state and embryo development.The dysfunctions of DHDDS,leading the incomplete glycosylation,could cause RP.Loss of photoreceptor was evident when protein of retina dycglycosylaed by TM blocking.Photoreceptors are vulnerable to glycosylation deficiency and the glycosylation pathway is one of potential mechanisms for RP.