RIP1/RIP3/MLKL Mediates Dopaminergic Neuron Necroptosis In A Mouse Model Of Parkinson Disease

Objective:Parkinson’s disease（PD）is the second most common neurodegenerative disease and the most common movement disorder.At its core,PD is characterized as a neurodegenerative disease with progressive loss of dopaminergic neurons in the substantia nigra pars compacta（SNpc）and a movement disorder caused by resultant dopamine deficiency within the striatum.Necroptosis is a programmed,controlled necrosis mediated by the RIP1/RIP3/MLKL pathway.Recent studies have found that necroptosis was involved in the pathogenesis of a variety of neurodegenerative diseases.However,the role of necroptosis in PD remains unclear.This study was aimed to investigate the role of RIP1/RIP3/MLKL-mediated necroptosis on the development of PD and identify its potential mechanism.Methods:In this study,C57BL/6 mouse（8-10w,male）was used as experimental animals,and an acute PD model was induced by intraperitoneal injection of MPTP.Pretreatment with Necrostatin-1（a specific inhibitor of RIP1 protein kinase）or knockout of RIP3/MLKL gene was used to block necroptosis pathway.Combined with immunohistochemical staining,high performance liquid chromatography,western blot,double immunofluorescence labeling,TUNEL detection and q RT-PCR,we investigated:1.the activation of RIP1/RIP3/MLKL mediated necroptosis in SNpc in the MPTP-induced mouse PD model.2.the neuroprotective effect of Necrostatin-1 via affecting the loss of dopaminergic neurons in SNpc and the decrease of dopamine in striatum through regulating the RIP1/RIP3/MLKL mediated necroptosis in SNpc in the MPTP-induced mouse PD model.3.the effect of RIP3/MLKL gene knockout in the number of dopaminergic neurons in SNpc and the content of dopamine in striatum and the mechanism being associated with necroptosis,rather than apotosis.4.the effect of RIP3/MLKL gene knockout in neuroinflammatory response through gene intervention on the activation of RIP1/RIP3/MLKL mediated necroptosis in the midbrain tissue in the MPTP-induced mouse PD model.Results:On the first day,the fourth day,the seventh day and the 21^stday after MPTP treatment,western blot showed that the expression of RIP3 in the midbrain increased significantly and reached the peak on the seventh day.This trend was positively correlated with the number of dopaminergic neurons in SNpc and the amount of striatal dopamine content.Double immunofluorescence staining showed that the protein of RIP1/RIP3/MLKL was highly expressed and co-localized in TH-positive dopaminergic neurons on day 7 after MPTP injection,and western blot revealed that phosphorylation of RIP3 at Thr231/Ser232 and phosphorylation of MLKL at Ser345 were also significantly elevated compared to the control group after MPTP treatment.Pretreated with necrostatin-1 could down-regulate the activation of RIP1/RIP3/MLKL-mediated necroptosis and partially reduce the loss of dopaminergic neurons in SNpc and the decrease of dopamine in striatum.Knockout of RIP3/MLKL revealed protective effect on brain injury in a MPTP mouse PD model,and the protective mechanism was attributed to the inhibition of dopaminergic neuronal necroptosis other than cellular apoptosis.Moreover,inflammation was involved in the pathogenesis of PD,and up-regulating inflammatory cytokine in MPTP-treated mice was partially inhibited by deletion of RIP3 or MLKL gene.Conclusions:This study demonstrates that RIP1/RIP3/MLKL-mediated necroptosis is involved in the pathogenesis of MPTP-induced PD.Pretreated with Necrostatin-1 or knockout of RIP3/MLKL gene to block necroptosis pathway dramatically ameliorated PD through increasing the level of dopamine and rescuing the loss of dopaminergic neurons,indicating that Necrostatin-1 has neuroprotective effect on brain injury in PD.Additionally,inhibition of dopaminergic neuronal necroptosis by deletion of RIP3/MLKL gene can also partially alleviate the inflammatory response in a mouse PD model,and the reduction of inflammatory response may in turn ameliorate neuronal necroptosis to some extent.Therefore,regulating dopaminergic neuronal necroptosis may be a new potential therapeutic target for PD.