The Gene PINK1Protects Neurons From Ischemic Damage Via Regulating Mitochondrial Fission/Fusion Pathways

Recent studies have shown that mitochondrial fission fusion pathway plays an important role in ischemic neuronal damage. Our previous studies also showed that ischemic stimulation in rat hippocampal neurons mitochondria obvious transition rupture phenomenon. The PINK1genes that cause familial Parkinson’s disease are more reported in the literature involved in mitochondrial fission and fusion process. In mammalian cells, PINK1(PTEN-induced putative kinase1) gene knockout can be induced mitochondrial fission and fragmention. To explore the role of PINK1in ischemic neuronal injury, and its impact on mitochondria1function in neurons after ischemia. We prepared oxygen glucose deprivation (OGD) injury neuron model, and over-expression of wild-type PINK1or its mutant L347p and W437x of lentiviral expression system. And PINK1-shRNA lentivirus-mediated inhibition of the expression of the endogenous Pinkl. The results show that, PINK1improve significantly reduced OGD-induced cell death, to improve after OGD mitochondria1morphology and function after OGD to reduce the attenuation of the mitochondrial membrane potential and energy metabolism. Its protective effect may be PINK1suppressed OGD-induced mitochondrial fission protein Drpl translocation from the cytoplasm to the mitochondria within the cells, and thus restore the balance of mitochondrial fission and fusion. Studies suggest that, PINK1can inhibit ischemic injury induced mitochondrial division, so as to improve mitochondrial function and to reduce the role of ischemic neuronal death. Mitochondrial fission fusion pathway intervention may become ischemic injury in neuroprotection research targets.Part I The PINK1protective effect of ischemic injury of neurons Aim:To study whether the PINK1gene has a preotective effect of neurons after oxygen glucose deprivation injury.Methods:We cultured the primary neurons of rat, building a wild-type PINK1Ientivirus mutations PINK1and PINK1-shRNA expression vector. The expression efficiency is confirmed by the Real-time PCR. The cell LDH release was measured to study the protective effect of PINK1neurons after hypoxic-ischemic injury.Results:Successfully cultured primary neurons, build a variety of PINK1expression vector and to verify the efficiency of expression. Overexpression of wild-type PINK1neurons OGD injury has a protective effect and the mutant PINK1loss of this role, PINK1knock down aggravate OGD injury.Conclusions:PINK1has a protective effect of OGD-induced nerve injury.Part II The effects of PINK1of mitochondrial morphology and function of neurons in ischemic injuryAim:To investigate role of PINK1for neurons mitochondrial morphology and function after OGD(Oxygen glucose deprivation) injuryMethod:Primary cultured neurons, neurons PINK1wild-type and mutant overexpression and knock. By Mito-DsRed mark mitochondria measurement of the degree of mitochondrial fragmentation after OGD. After the processing of in the OGD through the determination of ATP as well as the evaluation of the mitochondrial membrane potential mitochondrial damageResults:Compared with the control group, the OGD hypoxia reoxygenation-induced neuronal damage, over-expression of wild-type PINK1can reduce mitochondrial damage and inhibition of mitochondrial fragmentation, mutant and knockout PINK1can increase the mitochondrial mitochondrial damage and fragmentation.Conclusion:PINK1gene can alleviate the mitochondrial damage and mitochondrial fragmention after OGD injury. Part III The PINK1protects neurons from ischemic damage through the mitochondrial fission/fusion pathwaysAim:To investigate neuroprotective mechanism of mitochondrial fission fusion of PINK1after OGD hypoxia reoxygenation-induced neuronal injury.Method:Primary cultured neurons, neurons PINK1wild-type and mutant overexpression and knock. Cultured SH-SY-5Y cell lines, split after OGD mitochondria and Western Blot Determination of mitochondrial fission fusion protein content. To research the Drp1protein translocation by immunostaining studies.Results:Compared with the control group,, OGD hypoxia reoxygenation-induced neuronal damage, over-expression of wild-type PINK1can inhibit Drpl pack slurry translocation to the mitochondria, mitochondrial Drpl decreased. Knockout Pinkl can exacerbate pack slurry Drpl to the mitochondrial translocation, increased mitochondrial Drp1content.Conclusion:PINK1can play a role to improve the mitochondrial function and neuroprotection by inhibiting the Drp1translocation from cytoplasm to the mitochondrial and reducing the the fragmentation of the mitochondrial.Conclusion1. PINK gene has a protective effect for neurons after ischemic injury. This protective effect related mitochondrial function of the neurons and the kinase domain activity in cytoplasm of PENK1.2. After OGD injury, the mitochondrial take more fragmentation, due to the increasing of Drpl in mitochondrial. PINK1inhibition the mitochondrial fragmentation by reducing mitochondrial Drpl after OGD thus play a neuroprotective effect.