The Role Of TRPC In Mice Modle Of Parkinson’s Disease Induced By MPTP And The Underlying Mechanism

Background:Parkinson’s Disease(PD)is a degenerative neurological disease with dyskinesia,muscle rigidity,and resting tremor as the main clinical symptoms.The main pathological changes are degeneration,necrosis of dopaminergic neurons(DA neurons)in the substantia nigra pars compacta,and formation of Lewy Bodies in DA neurons.The disease has been identified by WHO as one of the most common,difficult and complex neurological disorders.However,the pathogenesis of PD is still not clear.Therefore,it is particularly important to study the pathogenesis of PD.Previous studies have confirmed that endoplasmic reticulum(ER)stress is involved in the pathophysiological processes of PD.ER plays an important role in maintaining intracellular Ca2+ homeostasis.Calcium is a very important messenger in cells and intracellular calcium homeostasis can maintain and regulate cell functions.Disruption of Ca2+ homeostasis is fundamental events which lead to neuronal loss in PD.It is commonly believed that the voltage-gated Ca2+channel play an important role in maintaining intracellular Ca2+ homeostasis.Recent studies demonstrate that transient receptor potential(TRPCs)are also involved in the Ca2+ homeostasis in DA neurons.TRPC is a subfamily of TRP super family.There are currently seven types of TRPC(TRPC1~7)which are non-selective cation channel and able to gate Ca2+ and Na+ influx.TRPC is highly expressed in the nervous system.Recent reports show that individual TRPC is involved in the development of PD.Calcium influx through TRPC Channels causes calcium overload leading to the occurrence of PD.However,their effects on PD are controversial.Here we hypothesize that all seven-TRPC deletion in rodent model will clearly demonstrate the functionl role of TRPC in PD pathogenesis.This article focuses on the TRPC channels and studies its effect on Parkinson’s Disease.Objective:The purpose of this study was to investigate the role of TRPC gene knockout on apoptosis of DA neurons in PD and its mechanism by constructing an animal model of Parkinson’s disease with intraperitoneal injection of MPTP in WT and TRPC KO mice.By primary culture of neural cells in cerebral cortex of WT fetal mice to establish a cell model of PD with MPP+ stimulation.Methods and Results: 1.The Gene identification of TRPC knockout miceTo determine whether TRPC KO mice were successfully prepared,TRPCs from WT and TRPC KO mice were tested.RT-PCR results showed that TRPC m RNA was detected in brain tissue of WT mice but not in that of the TRPC KO mice.The results above suggest that TRPC KO mice are successfully prepared.2.Preparation and Identification of PD Mice ModelIn order to construct an animal model of PD,we used MPTP to intraperitoneally inject the WT and TRPC KO mice.Behavioral test was used to determine whether the PD model were successfully prepared.The results showed that the duration of crawling on the fatigue rotarod in the MPTP-treated PD model group was significantly shorter than that of the Sham group.In many cases,the motor coordination ability of the mice has decreased significantly.This result shows that the mice PD model were successfully prepared.3.TRPC Knockout Attenuates Damage and Apoptosis of Dopaminergic Neurons in PD ModelTo investigate the effect of TRPC knockout on PD,the number of TH positive cells in the region of SNc was detected by immunofluorescence.The results showed that the number of TH positive neurons in SNc of the PD model group was significantly lower than that in the Sham group;however the number of TH positive neurons in SNc of the MPTP-KO group was significantly higher than that of the MPTP-WT group.The results above indicate that TRPC KO has a protective effect on PD.4.TRPC Knockout Reduces the Damage and Apoptosis of Dopaminergic Neurons in the PD Model by Activating the PI3K/AKT/GSK3β PathwayTo investigate the mechanism of TRPC knockout in the development of PD,Western blotting was used to detect the expression of the related proteins.Result showed that the expression of TH was increased in TRPC KO mice,while the ratio of BAX/Bcl-2 and the expression of caspase-3 was decreased.These results suggest that TRPC knockout resist the apoptosis of DA neurons induced by MPP+ or MPTP and it has a protective effect on PD.What’s more,the expression of p-AKT,and p-GSK3β were increased in TRPC KO mice.The underlying mechanism may be that TRPC knockout activated the PI3K-AKT-GSK3β survival signaling pathway thus inhibited the conduction of apoptotic signal.Conclusions:These results above indicate that TRPC channels are involved in the occurrence of PD.TRPC knockout can reduce the apoptosis of DA neurons,thus alleviating the symptoms of PD and has a protective effect on PD.