MANF (Mesencephalic Astrocyte-derived Neurotrophic Factor) Expression Protects Retinal Photoreceptor Neurons And Ganglion Cells

Retinitis pigmentosa(RP)is a group of genetic eye diseases that is characterized by progressive degeneration of the retina and loss of rob photoreceptor neurons gradually,then following by the demise of cone PNs,eventually resulting in blindness.RP has incidence of about 1 in every 3000 to 5000 people worldwide.There are about more than 40 thousands RP patients in China,who are suffered by low vision and heavy cost of treatment.More than 60 genes defects have been reported to be associated to RP.In general,30-40% of cases are inherited as an autosomal dominant,50-60% of cases are inherited as autosomal recessive,5-15% of cases are inherited as X-linked.Theoretically,genetic therapy is the best way to treat RP,but its effectiveness and safety need to be verified because too many genes are involved in RP etiology and high genetic heterogeneity of pathological RP.Hence,more treatments are focusing in medicines(including neurotrophic factors)which could slow down photoreceptor neurons degeneration.Recently,more and more reports suggest that endoplasmic reticulum(ER)stress may be an important common mechanism in RP.Whereas how Transcriptional factor C/EBP homologous protein(CHOP)plays a important role in the UPR and ER stress-induced apoptosis is undefined.Retinal ischemia-reperfusion(RIR)is a usually pathological process in many opthalmic clinical diseases.Lots of literature illuminated RIR is related to glaucoma,retinal and choroidal vessel occlusions,diabetic retinopathy,anterior ischemic optic neuropathy,retinopathy of prematurity,and traumatic optic neuropathy.Due to relatively ineffective therapy,it remains a common cause of visual impairment and blindness.As we know,the pathophysiological process of RIR involve in multiple processes,including reactive oxygen species formation,oxygen and glucose deprivation,ER-stress and apoptosis,finally cause lots of retinal ganglion cells(RGCs)apoptosis.More and more studies demonstrated ERS play a key role in RGCs apoptosis after RIR,but how it play a role in impacting this course is ambiguity.Mesencephalic astrocyte-derived neurotrophic factor(MANF,also known as ARMET)is a new neurotrophic factor extracted from a rat mesencephalic cell line 1,which is specific for dopaminergic neurons.It is located in central nervous system of mammal,and can selectively protect the activity of dopaminergic neurons.We have found that recombinant human MANF could protect photoreceptors in a retinal degeneration rat model carrying the S334 ter rhodopsin mutation previously.Now we will investigate if MANF could protect RGCs after RIR.Beside MANF is a neurotrophic factor,accumulating evidence suggests that MANF is an ER stress response protein and is able to protect cells against ER stress-induced cell death.The effects of MANF on the expression of 6 ER-stress markers were examined,including Bi P,IRE1α,PERK,ATF6,and CHOP,in S334 ter line 3(S334ter-3)rat retina,in order to illuminate the mechanism to make MANF protecting photoreceptors neurons and RGCs.Part Ⅰ : Mesencephalic astrocyte-derived neurotrophic factor(MANF)up-regulates CHOP and ATF6 protecting photoreceptor neurons in the rat retinaPurpose:We have found that recombinant human MANF could protects photoreceptors in a retinal degeneration rat model carrying the S334 ter rhodopsin mutation previously.In the partⅠ,we investigated the effects of MANF by examining the level of 5 ERS markers(including Bi P,IRE1α,PERK,ATF6,and CHOP)in S334 ter rat retina,in order to illuminate the signal of MANF protecting photoreceptor neurons induced by ERS.Methods:Recombinant human MANF was expressed in E.coli and purified.MANF(5 μg in 2μl)was intravitreally injected to the left eyes,and the right eyes were injected with 2μl PBS(phosphate buffered saline)as controls.The levels of ER stress markers Bi P,IRE1α,PERK,ATF6,and CHOP in different time(6h,12 h,24h,48 h,72h)were assessed by Western blot analysis in S334ter(line 3)rats and wild-type Sprague Dawley(SD)rats.The ER stress markers Bi P,IRE1α,PERK,ATF6,and CHOP were detected by Western blotting in different postnatal time(PD6-20)in S334ter(line 3)rat.After MANF was injected into vitreous of S334ter-3 rat,the morphology of retina was observed and compared with that of SD rat.Results:We first examined the expression levels of Bi P,IRE1α,PERK,ATF6,and CHOP in the retinas of the S334ter(line 3)rats during rapid photoreceptor degeneration from postnatal day(PD)10 to PD 20.Among the 5 markers,no marker showed obvious fluctuation.Surprisingly,MANF treatment induced a dramatic increase in ATF6 by 6hr post injection(injected at PD9).The increase in ATF6 then rapidly declined to control level by 12 hr post injection.In addition,a significant increase in CHOP was detected at 6 hr,peaked at 12 hr,and lasted to 48 hr post injection.No significant changes were seen in the other 3 proteins(Bi P,IRE1α,PERK,).Similar increases in ATF6 and CHOP were observed in wild-type SD rats after MANF injection.Conclusions:No significant changes in ER stress marker were found during the rapid photoreceptor degeneration in the S334ter-3 rats,suggesting that ER stress does not play a key role in photoreceptor degenerations in this model.Intravitreal injection of MANF significantly induces increasing levels of ATF6 and CHOP to protect photoreceptor neurons.Part Ⅱ :AAV-mediated MANF expression protects retinal ganglion cells in a retinal ischemia-reperfusion mouse modelPurpose:Previously we found that recombinant human MANF plays a protective role in degeneration of photoreceptors in a S334-ter rat model.In the partⅡ,we investigates potential neuroprotective effects of MANF on retinal ganglion cells(RGCs)using a C57BL/6J mouse retinal ischemia–reperfusion(IR)model.Methods:A viral vector AAV-MANF was used to deliver the MANF gene to retinal cells.The expression of MANF was driven by a CMV promoter.A control viral vector,AAV-GFP,was constructed by replacing the ORF of MANF with the ORF of GFP.Adult C57B/6J mice(male,3 months old)were used in all experiments.Animals were divided into 3 groups: RIR(retinal ischemia reperfusion,RIR)only(n=5),RIR+AAV-GFP(n=3),and RIR+AAV-MANF(n=5).Three microliters of AAV-GFP(2x1013 vg/ml)or AAV-MANF(2x1013 vg/ml)were injected intravitreally into each eye 3 weeks prior to RIR.The other animals were divided intro 4 groups: RIR only(n=5),RIR+MANF(n=5),RIR+PBS(n=5),RIR+dry needle(n=5).The expression levels of MANF were assessed by Western blotting.The ischemia mouse modle was made as below: A 33-gauge needle was inserted into the anterior chamber for 45 min in the left eye,which connected to a reservoir of saline that hung on to keep the intraocular pressure(IOP)to 120 mm Hg.By raising the reservoir to a given height relative to the eye,the desired IOP was achieved.The right eye was untreated as a control.Retinas were harvested 7 days after IR,stained with Neu N,and flat-mounted.Neu N positive cells in the retinas from both eyes were counted,and cell survival rate in the RIR retinas was calculated and compared between groups by percentile,the percentile came from the mean cell density of treated eye divide that of the control fellow eye.The location of MANF was analyzed by Immunofluorescence staining.Results:Seven days after RIR,the remaining of RGCs in the control ischemic retinas without injection was 25±1%,indicating about 75% cell loss.A similar survival rate(21±3%)was found in retinas injected with AAV-GFP.In AAV-MANF treated retinas,however,the survival rate was 78±8%,remarkably higher than both two control groups(P<0.001 compared with each control group).The survival rate in MANF treated retinas is similar with that of PBS treated retinas((P>0.05).Western blotting analysis also showed the expression of MANF protein in the retina and vitreous in the AAV-MANF treated eyes were significantly increased.AAV-MANF induced MANF expression is mainly seen in Muller cells,including the cell bodies,fibers,and endfeet,and in the ganglion cells.Conclusions:Our results demonstrated that AAV-mediated MANF expression significantly protects against RGC loss.MANF thus could be considered as a clinical neuroprotective agent for retinal degenerative disorders.