Study On The Mechanism Of Dopaminergic Amacrine Cells Regulating Melanopsin Expression In The Retinal Degenerative Rats

Retinitis pigmentosa (RP) is a heritage and occoecatio ophthalmopathy，which belongsto degenerative diseases of photoreceptor cells and pigment epithelium layer (RPE). TheRPE is invaded firstly because RPE lacks of phagocytosis of disc membrane ablated fromacromere and metabolic waste. Subsequently, retinal photoreceptor cells are in a state ofprogressing degeneration so that the patients lose their eyesight. So far，the phthogenesis isnot well known. However, people noted that the blind humans retaining eyeballs in the latestage of retinal degeneration could still perceive the change of circadia. Recent studiesconfirm existing a third class of photoreceptor Melanopsin-expressing retinal ganglion cells(mRGCs) or intrinsically photosensitive retinal ganglion cells(ipRGCs).Over the past150years, the cones and rods were thought to be the sole mammalianretinal photoreceptor. However, the discovery of the photopigment Melanopsin andMelanopsin-containing RGCs (mRGCs) represents a brand-new and exciting direction inthe field of visual field. Melanopsin belongs to the G protein-coupled receptor (GPCR)superfamily, and is made of a single peptide including heptahelical transmembraneconstructure, and Lys336in the seventh helix links retinaldehyde. The light stimulus canchange the construction of retinaldehyde linking melanoposin and initiate the light signaltransduction. Melanopsin-expressing photostable HEK-293suggested the photosensitivepeak is at480nm, that is to say Melanopsin expressing is sensitive to blue light. Althoughthe Melanopsin is much less sensitive to light and has far less spatial resolution, mRGCsnot only can receive the input information from rob and cone cells, but also can receive theinput information from the inner retina. More inportantly, mRGCs can receive theinformation transduction of DA ACs and have the unique ability to project to brain areas bythe retinohypothalamic tract (RHT) and communicate directly with the cerebrumsuprachiasmaticus nucleus(SCN). So it is important for how to remain or boost the function of mRGCs after the defuntionalization of photoreceptors in the late stage of retinaldegeneration. Unfortunately, Melanopsin presents lower expression levels in many acuteand chronic retinal diseases, and the molecular mechanisms underlying Melanopsinexpression are not yet really understood till now.MicroRNAs play important roles during development. Most importantly, the link ofmicroRNA biology to a diverse set of cellular processes, ranging from proliferation,apoptosis and malignant transformation to neuronal development is emerging. So thisresearch is based on some hypothesises as following: in the early stage of retinaldegeneration in RCS rats, overexpression of miR-133b in DA ACs downregulates theexpression of an important transcription factor, pitx3, depresses the expression of TH andD2receptor, restrains the development, maturation and function of DA ACs, thus makes theDA ACs injury. The injured DA ACs apparently decrease the synthesis and release of DA,can not form the normal effective synapse connection with mRGCs, and finally decreasethe Melanopsin expression.In this research, we employed classical pigmentary degeneration of retina RoyalCollege of Surgeon (RCS) rats as animal models to investigate the underlying molecularmechanism regulating Melanopsin expression using a panel of microRNA. The researchconsists of detecting the expression of Melanopsin in the retinal degeneration progress byWestern blot and qRT-PCR assays; detecting the morphological characteristic and fuction ofDA ACs and mRGCs by immunohistostaining, qRT-PCR and injecting D2receptoractivator by cavum vitreum; detecting miR-133b expression and location during retinaldegeneration by microRNA qRT-PCR, in situ hybridization and immunohistostaining;Moreover, detecting whether Melanopsin expression was restored after delivery ofmiR-133b/RNAi.The results and conclusions are as following.Part One Expression level of Melanopsin progrssively decreased during theretinal degeneration in RCS ratsSome studies showed the number of RGCs has greatly decreased in the retinaldegeneration progress compared to the control rats, but the distribution and the number ofmRGCs did not change during the postnatal development, even in the late stage of retinaldegeneration. So, we hope to know the level of Melanopsin expression during the retinal degeneration.Our studies suggested:①M elanopsin expressiondecreased in both mRNAand protein levels. At the initial stage of retinal degeneration, P15, Melanopsin expressiondid not change, but the mRNA showed a little more than the control rats mRNA, Perhapsthis is a temporary increasing due to the light stimulus after the RCS rats open the eyespostnatal two weeks. On P30, Melanopsin expression in the levels of protein and mRNAdecreased compared to the control rats. At the middle or late stage of retinal degeneration,P60and P90, Melanopsin expression greatly decreased and almost reached the lower limitof the detection method.②M elanopsin wasdistributed as same as the RGCs with pituitaryadenylate cyclase activation peptide(PACAP). That is to say Melanopsin located in the RHTRGCs with PACAP, so the expression level of PACAP mRNA could indirectly reflect thenumber of mRGCs. Our findings showed that PACAP mRNAs presented a little more thanthat in the control group at the early stage of degeneration, P15and P30, and no changeswere observed at the middle or late stage of degeneration, P60and P90. Meanwhile, themRNA of Thy1.1, a marker of RGCs, progressively decreased during retinal degenerationin RCS rats, that is to say it increased on P15, but decreased on P30, P60and P90. Theseresults are the same as the prophase research of counting mRGCs and RGCs. In conclusion,decreased Melanopsin expression during the retinal degeneration is not due to thedecreasing of mRGCs number, may be due to the signal transduction barrier regulatingMelanopsin expression.Part two Dopamine(DA)secreted by dopaminergic amacrine cells(DA ACs)is theimportant factor of regulating Melanopsin expressionSome researches has revealed that DA is one of the important neurotransmitter in theretina, it can modulate Melanopsin expression level by dopamine receptor Ⅱ(D2). DAismainly secreted by amacrine cells, as same as the important neurotransmitter and modulatorin the retinal information processing progress. As a rate-limiting enzyme of DA synthesis,Tyrosine Hydroxylase(TH) can catalyze tyrosine to form DA in the cytoplasma. Thus, TH isthought to be an important modulator of DA synthesisin the retina. TH is main distributedin the retinal dopaminergic amacrine cells(DA ACs or A18), so TH is the reliable marker ofDA Acs. DA receptors include D1receptor and D2receptor. D1receptor can activateadenylate cyclase, but D2receptor can restrain adynelate cyclase. D2receptor includespostsynaptic receptor and autoreceptor. D2postsynaptic receptor and D2autoreceptor located in the retinal photoreceptor and pigment epithelium can both feedback regulatedopamine synthesis and release from dopaminergic neurons. Some researches showed thatDA ACs and mRGCs dendrites colocalized in the S1sublaminae of the retinal innerplexiform layer(IPL), DA ACs could direct the mRGCs dendrites layering, and provide thespecial instruction for mRGCs selectively synapse connection. So, we hope to studywhether the decreased Melanopsin expression during retinal degeneration correlated withthe decreased TH and D2receptor or the decreased synthesis and release of DA, we alsowant to know how the DA ACs and mRGCs change during retinal degeneration.Our findings were as follows:①On the one hand, based on the immunofluorescencestaining assay by using calretinin,a marker of amacrine cells, we found inner plexiformlayer (IPL) was unordered in RCS rats retina, especially the structure of S1sublaminae wasdisordered. Meanwhile, DA Acs located in the most inner layer of INL, its dendrites mainlylocated in the S1sublaminae of IPL. These suggested that the dendrites of DA Acs wereinjured during the retinal degeneration. On the other hand, based on theimmunofluorescence staining assay against Melanopsin, a marker of mRGCs, we found thestructure of mRGCs dendrities disordered in IPL, positive signals apparently weaken, evendisapperaed in IPL and INL in RCS rats’ retinas. These show mRGCs are also injuriedduring retinal degeneration. Thus, there may be no normal effective synapse connectionbetween mRGCs and DA Acs.②TH and D2receptor mRNA level apparently decreasedduring the retinal degeneration. Immunofluorescence staining assay against D2showed D2receptors mainly expressed in INL beneath the inner limiting membrane and ganglion cellsin the ganglion cell layer (GCL), and also presented on axons in OPL (outer plexiform layer)and IPL. But D2receptors in all four layers above are slightly reduced.③Melanopsinexpression levels were recovered or even increased after D2receptors were activated by D2receptor activator R()-propylnorapomorphine hydrochloride(NPA) in the cavum vitreum.These findings show: mRGCs can still express Melanopsin during the retinal degeneration,but decreased Melanopsin expression may due to the defuctionalization of DA ACs.Part Three Overexpressed microRNA133b and downregulated transcriptionfactor, pitx3, control Melanopsin expression.MicroRNAs play important roles during development. Most importantly, the link ofmicroRNA biology to a diverse set of cellular processes, ranging from proliferation, apoptosis and malignant transformation to neuronal development is emerging. Recentreaearch suggested that the negative feedback loop between miR-133b and pitx3couldregulate the development, maturation and function of dopaminergic neurons in themesocephalon. So, we hope to clarify whether there is the negative feedback loop betweenmiR-133b and pitx3in the retina, whether the overexpression of miR-133b lead to thenonfunction of DA ACs during degeneration, how to rescue the function of DA ACs andrecover Melanopsin expression.Our findings were: microRNA, miR-133b, specifically expressed in retinal DA ACs,markedly increased expression at the initial stage, P15, during the retinal degeneration inRCS rats, as well as miR-133b and pitx3colocalized in DA ACs. The overexpression ofmiR-133b downregulated the expression level of pitx3, an important transcription factor.These studies suggeste that there is the negative feedback loop between miR-133b andpitx3in the retina. The overexpression of miR-133b in DA ACs downregulates pitx3expression, and suppresses development, maturation and function of DA ACs. The lowlevel expression of pitx3decreases TH and D2receptor expression and dopamineproduction. On the one hand, injured DA ACs can not form the effective synapseconnection with mRGCs. On the other hand, decreased DA finally leads to reducedMelanopsin expression. Upregulation of pitx3and TH in DA ACs recovers the synthesisand release of DA by delivery of miR-133b/RNAi, which finally result in recoveredMelanopsin expression.In summary:1.Clarifying the mechanisms of regulating Melanopsin expression on miRNAexpression levels. Decreased Melanopsin expression is not due to the number loss ofmRGCs, but due to the overexpression of miR-133b and decreased pitx3expression whichleads to defunctionalization of DA ACs, impaired DA/D2receptor interaction during retinaldegeneration, and finally results in decreased Melanopsin expression.2.Decreased mRNA and protein levels of TH and D2receptor are main reasons fordecreased Melanopsin expression in RCS rats retina. These suggeste that DA is theimportant factor regulating nonvisual imaging system. Furthermore, a lot of dopaminereceptors family molecules are distributed on the surface of photoreceptors of visualimaging system. So, DA may be also the important factor of regulating visual imaging system. These results indirectly reflect that there may be the direct connection betweennonvisual imaging system and visual imaging system.3. TH, D2receptor and Melanopsin expression levels were increased by delivery ofmiR-133b/RNAi, which suggestes the ability of Melanopsin expression of mRGCs is notinjured and Melanopsin expression can be recovered by rescuing the function of DA ACsduring the retinal degeneration. These findings may provide the theoretical foundation forthe clinical treatment of RP at late stage.