The Protective Effect Of Prion Protein On Dopamine Neurons

Prion protein（PrP）is a highly conserved glycoprotein,consisting of 254 amino acids encoded by the Prnp gene and is anchored to the surface of cell membrane by the glycoscophosphosphalyl inositol（GPI）structure.PrP is widely expressed in tissues and organs of animals,especially in the central nervous system.Misfolded PrP forms a pathogenic conformer of prion protein(PrP^Sc)and causes prion diseases leading to the neurodegenerative syndromes.Although conformational changes and pathogenic characters of PrP have been extensively studied,little is known about its physiological functions.Parkinson’s disease（PD）is the second most common neurodegenerative disease.The main pathological feature of PD is the loss of dopaminergic neurons in the nigro-striatal region of the brain,which results in insufficiency of dopamine transmitters.PD patients have a relatively hyperactive acetylcholine system and develop slow movements and resting tremors during the course of disease.In this study,we used both the MPTP mouse model of PD and the dopaminergic cell-line SH-SY5Y to explore the possible protective functions of PrP in dopaminergic neurons in vitro and in vivo.We first construced the PrP KO mice and confirmed that no PrP expression in these mice by PCR and WB,respectively.Moreover,no significant changes in cell morphology and quantity in the striatum region were observed in PrP KO mice.Then we injected MPTP（30mg/kg）intraperitoneally once per day for five days to WT and KO mice,which resulted in the expected pathology in MPTP-treated mice.Results of the open-field and rotarod tests showed that the total distance,average speed and stick retention time of the MPTP-treated mice were less than those of the saline-treated mice.The average speed of the MPTP-treated WT mice（WT+MPTPs）was significantly higher than that of the MPTP-treated KO mice（KO+MPTPs）in open field test,indicating a protective role of PrP against MPTP-induced neurotoxicity.Results from IHC and WB analyses showed that the middle brain of WT+MPTPs had higher amount of TH（tyrosine hydroxylase）and contained more TH positive cells than KO+MPTPs.In addition,the density of TH and DAT（dopamine transporter）staining in the striatum of WT+MPTPs was also significantly higher than that of KO+MPTPs.Since TH and DAT are two dopaminergic-specific protein markers involved in dopamine synthesis and transport,respectively,these results suggest a significant protective function of PrP in MPTP-induced toxicity in dopaminergic neurons.Finally,we used a dopaminergic cell line to construct both transient and stable PrP knockdown cell-lines（Tcs and Scs）by RNAi technology.The results of MTT and WB analyses showed that two MPP⁺-treated PrP knockdown cell-lines had a lower survival rate and less TH than MPP⁺-treated WT cells.In addition,MPP⁺-treated Tcs had more severe mitochondrial damage,a higher expression level of caspase-3 protein and Bax protein,and a lower expression level of Bcl-2 than MPP⁺treated WT cells,suggesting that PrP protects dopaminergic neurons via Bax-Bcl-2-caspase-3 signaling pathway to reduce the toxic effect of MPTP/MPP⁺on the neurons.Our finding provides a new insight in the medical treatment of Parkinson’s disease.