Mechanisms Of Tyrosine Hydroxylase Expression And Neurotoxicity Regulated By Parkinson's Disease-related Genes

Part ?.DJ-1 regulates tyrosine hydroxylase expression through CaMKK?/CaMKIV/CREB1 pathwayAim:Decreased expression of tyrosine hydroxylase(TH)in the brain is a hallmark of Parkinson's disease(PD)pathogenesis.Deletion or mutations of the DJ-1 gene results in early onset of familial PD.The purpose of this study is to explore the molecular mechanism of PD-associated protein DJ-1 in TH expression regulation in this section.Methods:N2a cells were transfected with control or Dj-1 siRNAs to construct DJ-1 deficiency cells.we measured the mRNA and protein levels of several key enzymes that are involved in the production and release of DA by quantitative RT-PCR(qRT-PCR)and Western Blot.TH protein levels in substantia nigra of Dj-1 KO mice and the littermate controls were detected using Western Blot.To further identify whether DJ-1 regulates TH transcription,we constructed a 2 kb murine TH promoter fragment into PGL3-Basic vector(wild type and mutants).In order to explain whether the regulation of TH at the transcriptional level,we overexpress DJ-1 in HEK293 cells and measure the activity of wild type or mutant TH promoter.The levels of upstream related proteins of TH expression in Dj-1 deficiency cells were detected by Western Blot,and further validated in Dj-1 KO mice.The interaction between proteins was investigated by immunoprecipitation assay and proximity ligation assay(PLA),and then the co-localization of the two proteins was investigated by cellular immunofluorescence.Results:In the substantia nigra of Dj-1 knockdown cells and knockout mice,we found that the expression levels of TH,but not other DA anabolism-related enzymes,were significantly lower than those of the control group.Further studies revealed that the decrease in TH expression induced by DJ-1 deficiency is mediated by its transcription factor cAMP-response element binding protein 1(CREB1)acitity,whereas silencing of Crebl abolishes the change in TH expression caused by DJ-1 deficiency.It was further found that silencing Dj-1 inhibits CREB1 activity by inhibiting the phosphorylation level of upstream kinase Ca2+/calmodulin-dependent protein kinase ?(CaMKIV)Thr196/200 of CREB1.Finally,we found that DJ-1 regulates the transcriptional expression of TH by directly binding Ca2+/calmodulin-dependent protein kinase kinase ?(CaMKK?)to regulate CaMKIV/CREB1 pathway activity,whereas DJ-1 deficiency leads to CaMKK?/CaMK[V/CREB1 pathway activity.Decreased resulting in decreased TH expression.Conclusion:Our results elucidate a novel target and a new pathway for the regulation of TH expression by the Parkinson,s disease-associated protein DJ-1,which regulates the expression of TH by directly binding CaMKKp to regulate the activity of CaMKIV/CREB1 pathway.Decreased activity of CaMKKp/CaMKIV/CREB1 pathway and down-regulation of TH expression caused by DJ-1 deficiency may be one of the underlying mechanisms that are involved in the pathogenesis of PD.Part ?.The mechanism of neurotoxicity induced by Parkinson's disease-related gene NUS1 deficiencyAim:NUS1 was identified as a new Parkinson's disease risk gene in 2018,and there is a relatively lack of research on the role of NUS1 gene in the central nervous system.This study attends to explore the role and mechanism of loss of NUS1 in neurotoxicity at the cellular and molecular levels.Methods:Firstly,mouse primary cortical neurons were extracted and cultured in vitro.The Nus1-deficient cell model was constructed by knocking down the expression of the Nusl gene by siRNAs transfection using RNAiMax in primary cortical neurons and Neuro-2a(N2a)cell lines.qRT.PCR was performed to detect mRNA levels of endoplasmic reticulum stress-related genes in the model.Western blotting was used to verify the expression level of endoplasmic reticulum(ER)stress-related proteins in the model.In order to investigate whether the expression of silencing Nus1 gene can activate the apoptotic pathway,the mRNA and protein levels of apoptosis and apoptosis-related genes in NUS1 deficiency model were detected by lactate dehydrogenase(LDH)activity detection kit,qRT-PCR and Western blotting.The cells were treated with the related signaling pathway inhibitors such as 2-Aminoethoxydiphenyl borate(2-APB),Integrated Stress Response Inhibitor(ISRLB)or ER stress inducer thapsigargin(TG)to further observe the mRNA and protein changes of NUS1 on ER stress-related genes.Results:We found that knockdown of Nus1 in primary cortical neurons specifically activates ER stress through protein kinase R-like ER kinase(PERK)signaling and induces ER stress-related protein activating transcription factor 4(ATF4)and C/EBP homologous protein(CHOP)expression by qRT-PCR and Western blotting.The PERK inhibitor ISRIB significantly inhibited the activation of ER stress-related proteins induced by Nus1 deficiency.In addition,silencing of Nus1 activates the endoplasmic reticulum stress-mediated apoptotic signaling pathway,including increased expression of p53 and Bax as well as Cleaved-caspase3 activation,whereas ISRIB treatment of primary cortical neurons significantly inhibits Nus1 deficiency-induced neuronal apoptosis.In addition,we also found that silencing Nus1 disrupts the endoplasmic reticulum calcium homeostasis,and pretreatment with calcium chelating agent 2-APB partly inhibits the activation of Cleaved-caspase3 induced by Nus1 deficiency,suggesting that calcium homeostasis may be involved in Nus1 deficiency mediated ER stress and apoptosis.Conclusion:Nus1 silince significantly induced ER stress by activating the PERK pathway to activate ER stress-related protein expression and induce ER stress-mediated neuronal apoptosis,whereas inhibition of PERK significantly inhibited Nus1 deficiency-induced ER stress-related protein activation and neuronal apoptosis.