Study On Development And Retrograde Degeneration Of DA Neurons In Zebrafish

ObjectiveParkinson’s disease(PD) is a common neurodegenerative disease which mainly affects aged adults. DA neuron loss in the subtantia nigra compacta of midbrain, and subsequently lacking of dopamine in striatum body are regarded as the main causes of PD symptoms[1]. Recently, researches on PD have demonstrated that oxidative stress,dysfunction of mitochondrion,ubiquitin-proteasome system,and lysosome dysfunction leanding to α-synuclein abnormally accumulation may be key factor of DA neurons degeneration and death.[2-3].Previous studies have done a series of experiments to protect dopamine neurons, and found inhibiting DA neurons degeneration can not prevent PD desease from progressing.[4] We come to focus on dysfunction in axon and microtubule.Our research in vivo observed position, distribution of dopaminergic neurons in zebrafish by using Vmat2 transgenic zebrafish and TH antibody staining method to construct an retrograde degeneration zebrafish model and to study DA neurons degeneration and death mechanism. MethodsFirstly, the develoment of DA neurons in the zebrafish CNS were observed in real time pattern with the combining of TH antibody staining and Vmat2: GFP transgenice zebrafish. Continuous real-time observation of zebrafish embryonic development were performed at timepoint 24h、30h、48h、54h、72h、4d、5d. Secondly, the DA neuron death and the axon degeneration were observed in real time patter after different concentration of MPTP were administrated to zebrafish. Meanwhile, the m RNA level of PD related genes were detected via quantitative PCR method. Results1. Based on the observation of TH antibody staining and Vmat2:GFP zebrafish, the DC2 neuron group, DC4 neuron group are dopaminergic neurons, and DC1、DC3、DC5and DC6 groups may not be dopaminergic neurons.It’s mainly dapaminergic neurons in pretectum and telencephalon area,,conversely in locus and raphe nuclei are noradrenergic neurons,not DA nerons.2. we have revealed that zebrafish brain(forebrain,midbrain,hindbrain) DA neurons development process. Fertilized after 24 hours,the earlist neurons observed are DC1/DC2 DA neurons in ventral diencephalon; 48 hpf,all neuron groups in ventral diencephalon complately developed,DC1~7 neuron groups、locus and raphe nuclei can be observed;3dpf,axon can be observed,axon of DC2 groups project to telencephalon;Nervous system of zebrafish developed complately in 4day. Our research in vivo observed continous growth of dopaminergic neurons by using Vmat2 transgenic zebrafish;We also found the number of DA neurons in TPp area showed firstly increasing and then decreased during developing process.Furthermore,the position of neurons have moved from area of origin to final positon.3. We constructed dynamic observing PD model in vivo by Vmat2:GFP transgenic zebrafish,to observe degeneration process of neuron and axon under confocal microscrope. After different concentration of MPTP exerted to zebrafish, the fluorescence intensity of DA neurons were obviously decreased,as well as behavior and molecular changed. especially in 200μM and 400μM group.4. Firstly observing diencephalon ventral TPp area exist axons projecting to subpilium in vivo with Vmat2:GFP transgenic zebrafish; Under high concentration of MPTP, The DA neuron axons death, prior to the neuron cells body,in TPp area.We also screened several PD related genes expression level. After different concentration of MPTP exerted to zebrafish embryo. Through q PCR analysis, the expreession level of PD related gene Pink1, LRRk2, Parkin, DJ-1 were downregulated under 100 u M MPTP administration at 100μM concentration before DA neurons morphology changed. Conclusion1.Through WT zebrafish TH staining and Vmat2:GFP transgenic zebrafish observation in vivo, we found that the DC2 and DC4 neuron groups are mainly dopaminergic neurons. Besides, pretectum etectum and telencephalon area are mainly consisted of DA neurons.we revised and supplement DA neurons positon and distribution in zebrafish brain.2. Study on DA nervous system successive growth and development for the first time. we have revealed that zebrafish brain(forebrain,midbrain,hindbrain) DA neurons development process. Fertilized after 24 hours,the earlist neurons observed are DC1/DC2 DA neurons in ventral diencephalon; 48 hpf,all neuron groups in ventral diencephalon complately developed,DC1~7 neuron groups、locus and raphe nuclei can be observed;3dpf,axon can be observed,axon of DC2 groups project to telencephalon;Nervous system of zebrafish developed complately in 4day. Our research in vivo observed continous growth of dopaminergic neurons by using Vmat2 transgenic zebrafish;We also found the number of DA neurons in TPp area showed firstly increasing and then decreased during developing process.Furthermore,the position of neurons have moved from area of origin to final positon.3. In vivo Vmat2:GFP transgenic zebrafish,It’s conveniently to observe DA neurons developing successively, the number of DA neurons in TPp area showed firstly increasing and then decreased during developing process.Furthermore,the position of neurons have moved from area of origin to final positon.Study on DA neurons development and distribution of Vmat2:GFP transgenic zebrafish have laid the foundation of building zebrafish axon restrograde degenerstion model in vivo with real time observation next step.4. We constructed successfully dynamic observing axon restrograde degenerstion PD model in vivo with Vmat2:GFP transgenic zebrafish,to observe degeneration process of neuron and axon,and have found after different concentration of MPTP exerted to zebrafish, the fluorescence intensity of DA neurons were obviously decreased,as well as behavior and molecular change, especially in 200 u M and 400 u M group.5. Firstly observing diencephalon ventral TPp area exist axons projecting to subpilium in vivo with Vmat2:GFP transgenic zebrafish; Through q PCR analysis, the expreession level of PD related gene Pink1, LRRk2, Parkin and DJ-1 have changed.at 100μM concentration before DA neurons morphology changed,especially Pink1 and DJ-1. In addition, DA neuron axon death in TPp area disappeared before neuron cell bodies after administration fo high MPTP concentration, which verified our previous hypothesis that the axon death is prior to the cell body death in PD pathogenesis.It revealed that the PD related genes Pink1, LRRk2, Parkin, and DJ-1 affect axon restrograde degeneration.