The Mechanism And Effects Of Endogenous Calcium Release Induced By 6-OHDA Via ER Stress On The Function And Survival Of DA Neurons

Background:Parkinson’s disease（PD）is the degenerative diseases of the nervous system.The main clinical symptoms are bradykinesia,tremor,stiffness and gait disorders,and sometimes accompanied by hyposmia,depression and constipation.The pathogenesis of PD are not clear.The pathological manifestation is the selective death of dopamine（DA）neurons in the Substantia Nigral compacta（SNc）,and the Lewy body（LB）in the residual neurons,.Parkinson’s disease has become one of the major problems that need to be addressed in the field of nervous system diseases.The dyshomeostasis of calcium caused by ER stress is an important molecular mechanism of selective loss of dopaminergic neurons.Inositol 1,4,5-triphosphate receptors（IP3Rs）and the ryanodine receptor（RyRs）,which are the predominant endogenous Ca²⁺ release channels,play a key role in regulating Ca²⁺ homeostasis.However,the role of these endogenous Ca²⁺ release channels in PD and its effect on the function and the survival of DA neurons are still unknown.Studies have shown that the dyshomeostasis of calcium in the cytoplasm induced by 6-OHDA can cause a series of changes in plasma channels of the dopaminergic neurons,but it is unclear for endogenous release of calcium and its effects on cell excitability,cells oxidative stress and cell survival.The importance of the two channels on endoplasmic reticulum in the endogenous release of calcium is still unknown.Objective:To investigate the effects and the mechanism of 6-OHDA on the endoplasmic reticulum stress,the release of Ca²⁺,the excitability of dopaminergic neurons,the oxidative stress of dopaminergic neurons and cell survival of dopaminergic neurons.And,try to provide some theoretical foundation t for the vulnerability of dopaminergic neurons in SNc region.Methods:1.The effects of 6-OHDA on endoplasmic reticulum stress and the release of endogenous calcium were measured by Western-Blot and calcium imaging.And the mechanism was studied via the use of the ER stress inhibitor and channel blocker;2.The effects of 6-OHDA-induced increase of intracellular Ca²⁺ concentration on the excitability of DA neuron were studied by whole-cell patch-clamp experiments,3.The effects of 6-OHDA-induced high calcium concentration in cytoplasm on the oxidative stress of DA neurons were investigated via ROS staining and mitochondrial staining;4.the effects of 6-OHDA-induced high calcium concentration in cytoplasm on the survival of dopaminergic were investigated by MTT and TUNEL staining.Results:1.Western-Blot experiments confirmed that 6-OHDA could induce endoplasmic reticulum stress in dopaminergic neurons.Calcium imaging showed that endoplasmic reticulum stress induced by 6-OHDA in dopaminergic neuronal could cause intracellular calcium concentration persistence and irreversible increase,and this effects may closely relate to the endoplasmic reticulum stress state and endoplasmic reticulum calcium channel RyR.2.Whole-cell patch-clamp experiments confirmed that sustained and irreversible increase induced by 6-OHDA on intracellular Ca²⁺ concentration in neurons affectes the excitability of dopaminergic neuron,and this effect was closely associated with endoplasmic reticulum stress state and endoplasmic calcium channels RyR.3.ROS experiments and mitochondrial staining confirmed that 6-OHDA-induced increase on intracellular Ca²⁺ concentration in neurons was involved in the induction of oxidative stress in neurons,and this effect was associated with endoplasmic reticulum stress and the RyR calcium channel on the endoplasmic reticulum.4.MTT assay and TUNEL staining confirmed that 6-OHDA-induced increase in intracellular Ca²⁺ concentration in neurons induced dopaminergic neuronal death,and this effect was closely related to endoplasmic reticulum stress status and RyR calcium channel in endoplasmic reticulum.Conclusion:1.6-OHDA can induce the endoplasmic reticulum stress in dopaminergic neuron,which leads to the increase of intracellular Ca²⁺ concentration in the cytoplasm,this effect is mainly related to the RYR calcium channel on the endoplasmic reticulum.2.6-OHDA-induced increase of intracellular Ca²⁺ concentration can reduce the excitability of dopaminergic neurons in SNc region.3.6-OHDA-induced increase of cytoplasmic calcium concentration can partly participate in the 6-OHDA-induced oxidative stress of dopaminergic neuronal.4.6-OHDA-induced increase of cytoplasmic calcium concentration can induce cytotoxic effects of dopaminergic neurons.