Proteomics Analysis Of Mutant Autophagy Genes PINK1 And WDR45 During Neurodegeneration

Autophagy is a process in which cells use lysosomes to degrade damaged organelles and molecules.The main function is to eliminate intracellular aggregated proteins and damaged organelles,and to recover building blocks such as amino acids and fatty acids for macromolecules.Abnormal autophagy can lead to intracellular dysfunction and induce a range of diseases.Neurons are highly differentiated cells.During ageing process stress conditions such as ROS cause accumulation of abnormally aggregated proteins in the cell.If the autophagy function is impaired,these aggregated proteins cannot be cleared in time,which may lead to neuronal death,resulting in neurodegeneration.At present,the majority of research on autophagy and neurodegenerative diseases focus on the initiation of autophagy,the isolation of substrates and the mechanism of lysosome degradation.It is not entirely clear how autophagy regulates the apoptosis of neurons.Quantitative proteomics and phosphoproteomics based on liquid chromatographyhigh resolution mass spectrometry(LC-MS/MS)enable large-scale identification and quantification of proteins and phosphorylation sites,promoting our systematic understanding of cell biology such as cell cycle,receptor signaling,DNA damage and repair,signal transduction and synaptic remodeling.In this study,two mouse models with autophagic gene knocking out were used to comprehensively analyze the proteomics and phosphoproteomics of cultured neurons and brain tissues,with the hope to elucidate the mechanism of neuronal apoptosis caused by impairment of autophagy.PINK1 mutations that disrupt its kinase activity cause autosomal recessive early onset Parkinson's disease(PD).Although research in recent years has elucidated a PINK1-Parkin pathway of mitophagy activation that requires PINK1 kinase activity,mitophagy-independent functions of PINK1 and their possible roles in PD pathogenesis have been proposed.Using an unbiased quantitative mass spectrometry approach to analyze the phosphoproteome in primary neurons from wild type and Pink1 knockout mice after mitochondrial depolarization,we uncovered PINK1-regulated phosphorylation sites,which involve coordinated activation of multiple signaling pathways that control cellular response to stress.We identified the pro-apoptotic protein BAD as a potential mitochondrial substrate of PINK1 both in vitro and in vivo,and found that cells more susceptible to apoptosis induced by mitochondria damage can be rescued by phosphorylation mimic BAD.Our results suggest that PINK1 kinase activity is important for pro-apoptotic protein function in regulation of cell death.Mutations in the autophagy gene WDR45 cause ?-propeller protein-associated neurodegeneration(BPAN);however the molecular and cellular mechanism of the disease process is largely unknown.We generated constitutive Wdr45 knockout(KO)mouse that displayed cognitive impairments,abnormal synaptic transmission and lesions in several brain regions.Immunohistochemistry analysis shows loss of neurons in prefrontal cortex and basal ganglion in aged mice,and increased apoptosis in prefrontal cortex,recapitulating a hallmark of neurodegeneration.Quantitative proteomic analysis shows accumulation of endoplasmic reticulum(ER)proteins in KO mouse.At the cellular level,our data suggest that a defect in autophagy due to Wdr45 deficiency results in increased ER stress and impaired ER quality control.The unfolded protein response(UPR)is elevated through IRE1? or PERK pathway,and eventually leads to neuronal apoptosis.Suppression of ER stress,or activation of autophagy through mTOR inhibition alleviates cell death.Thus,the loss of Wdr45 cripples macroautophagy machinery in neurons and leads to impairment in organelle autophagy,which provides a mechanistic understanding of cause of BPAN and potential therapeutic strategy to treat this genetic disorder.