Phosphorylation Of XBP1s By CDK5 Mediates The Activation Of IRE1?/XBP1 Pathway In Parkinson's Disease

Background:Parkinson's disease(PD)is a common age-related neurodegenerative disorder that is characterized by loss of dopaminergic neurons in the substantia nigra pars compacta(SNpc),with the presence of cytoplasmic inclusion bodies Within the neurons,known as Lewy bodies(LBs).The mechanisms behind the neuronal death in PD are currently focused on unfolded or misfolded proteins aggregation,mitochondrial dysfunction and oxidative stress.Recently growing evidence suggests that above pathological changesis closely related to the development and progression of endoplasmic reticulum stress(ER stress).Intracellular gene mutations,oxidative stress and other factors may destroy the balance of the ER homeostasis and the oxidizing environment of endoplasmic reticulum lumen,leading to ER stress due to unfolded or misfolded proteins aggregation in the ER.The UPR is activated and mediated by three main signaling branches,including protein kinase RNA-like ER kinase(PERK),inositol-requiring enzyme 1(IRE1)and activating transcription factor 6(ATF6).The most conserved UPR signaling branch is initiated by IRE1.Dimerization of IRE1 and its autophosphorylation activate its endoribonuclease activity to catalyse the unconventional splicing of the mRNA encoding the transcription factor X-box binding protein 1(XBP1).This event excises a 26-nucleotide intron that shifts the coding reading frame of the mRNA.Spliced XBP1(XBPls)is a stable and active transcription factor that controls a subset of UPR target genes.Studies in animal models have revealed that XBPls has important functions in liver lipogenesis,inflammation,and energy metabolism.A study using cell culture and mouse model of PD has reported that overexpression of XBP1scan protect MPP+ and MPTP-induced cell from death.However,the mechanism of IREla/XBP1 pathway in PD is not fully understood.Cyclindependent kinase 5(CDK5)is a proline-dependent serine/threonine protein kinase that highly expressed in the nervous system.CDK5 is aberrantly activated in neurodegenerative diseases,and involves in the regulation of neuronal death by phosphorylating various protein molecules.Based on the above background,the present study,the specific mechanism of IREla/XBPl pathway is explored at cell and animal levelsof PD,and further investigates the molecular mechanism of that activated CDK5 induces neuronal degenerationby regulating XBP1s.Methods and Results:The IREla/XBP1 pathway is activated in the cell models of PD.In primary cortical neurons treated by MPP+ at different times,the expression levels of XBP1s mRNA were increased with the increase of processing time from the results of-RT-qPCR.The phospho-IRE1?(pIRE1?)and the expression levels of XBP1s protein are also increased from the results of Western blot.Those results suggest that IRE1?/XBP1 pathway is activated in MPP+-treated cortical neurons.The IREla/XBP1 pathway is activated in MPTP mouse model of PD.Male mice with weight 25-30g were randomly divided into two groups,the experimental groups were performed intraperitoneal injection with 25 mg/kg dose of MPTP,and the control groups were injected volume of saline.SNpc tissues were isolated and detected by RT-qPCR and Western blot.The results showed that expression levels of XBP1s mRNA and the expression levels of XBP1s protein in experimental groups were significantly higher than the control groups.The pIRE1awas also increased in experimental groups.Those results indicate that IRE1?/XBP1 pathway was also activated in MPTP drug-induced model of PD.The IRE1?a/XBP1 pathway is activated in the substantia nigra tissue of transgenic animal models of PD.The transgenic mice(Th-SNCA*A30P*A53T)(TG)and normal littermates mice(wild type,WT)with age of 2 months and 15 months were selected to separate the SNpc tissues.The mRNA and protein expression levels of XBP1s in 15 months TG mice were significantly higher than WT mice from the results of RT-qPCR and Western blot test.The pIRE1? was also increased in 15 months TG mice.Those results strongly suggested that the IRE1?/XBP1 pathway was activated in TG animal model of PD.The nuclear translocation of XBPls occurs as a CDK5-dependent manner in PD.Western blot results showed that in primary cortical neurons treated by MPP+ at different times,the expression levels of XBP1s were significantly increased in neuronal nuclei lysate with the extension of treatment time.Roscovitine,a CDK5 inhibitor,was added in neuronal cells 30min before treated by MPP+.and the nuclear translocation of XBP1s significantly reduced.The above results showed that nuclear translocation of XBP1s may be associated with CDK5.The nuclear translocation of XBP1s is not dependent on p38MAPK in PD.The result that XBP1s was phosphorylated by p38 MAPK(p-p38)and promoted its nuclear translocation has been reported in previous research.To identify whether the nuclear translocation of XBP1s was related to p38 or not.p-p38 expression levels were detected by Western blot.The result showed that p-p38 was barely detected in MPP+-treated neurons.SB203580,a p38 inhibitor,was added in neuronal cells 30min before MPP+-treated,and the nuclear translocation of XBP1s did not affected.The increased nuclear translocation of XBP1s was independent of p-p38 in MPP+-treated neurons.CDK5 mediates the phosphorylation of XBPls.In the total lysate of MPP+-treated neurons,the phosphorylation of XBPls was analyzed by immunoprecipitation and immunoblot assay.The results from Phos S/TP antibody testing found that the phosphorylation of XBP1s in MPP+-treatment neurons was significantly increased.The phosphorylation of XBPls in the cortex tissue lysate of' CDK5 knockout mice was also significantly reduced compared to littermate WT mice.In addition,the interaction between XBPls and CDK5 was verified in vitro and in vivo by co-immunoprecipitation methods.Those results indicated that the phosphorylation of XBP1s was increased and related to CDK5 in PD.CDK5 phosphorylates XBPls on its Ser61 site.XBP1s Ser61 site was a potential phosphorylated site of CDK5,which detected by bioinformatics software GPS3.0,Scansite and DNAMAN7.11 peptides containing XBPls Ser61 site were synthesized in vitro and as a substrate reacted with CDK5/p25 kinase in vitro.The result from mass spectrometry showed that CDK5 could phosphorylate XBP1s Ser61 sites in vitro.Wild-type GST-XBP1-WT and mutated GST-XBP1s-S61 A protein induced in vitrowas used as the substrate respectively to react with CDK5/p25 kinase in vitro,the results detected by Phos S/TP and Phos-tag SDS-PAGE gels found that GST-XBPls-WT could be phosphorylated by CDK5.Synthesized penetrating peptide Myr-RLTHLSPEEKA containing XBP1s Ser61 in vitro was added in MPP+-treated neurons,and protective effect was founded in neuronal death caused by MPP+.The results further showed that CDK5 may cause neurons death by phosphorylation of XBP1s Ser61 sites in PD.Phosphorylation of XBPls Ser61 enhances its nuclear translocation.Primary cultured cortical neurons transfected with wild-type plasmid(pEGFP-XBPls-WT)and the phospho-mimetic mutant of XBP1s(pEGFP-XBP1s-S61D)with green fluorescent protein(GFP)tag respectively,were observed by fluorescence microscopy after 24h,revealed that XBP1s-WT protein was expressed in the cytoplasm and nucleus,while XBP1s-S61D expressed mainly in the nucleus.HEK293 cells transfected with HA-CDK5/Myc-p35.pEGFP-XBPls-WT or pEGFP-XBPls-S61 A plasmid were detected by GFP antibody,the results showed that the nuclear translocation of XBP1s-WT was significantly increased in the case of overexpression of CDK5/p35.In contrast,the nuclear translocatioin of XBP1s-S61A was significantly reduced.Those results suggested that phosphorylation of XBP1s Ser61 sites promoted its nuclear translocation.Phosphorylated XBPls as a transcription factor that induce multiple genes expression.Phosphorylation of XBP1s Ser61 sites promoted XBP1s into the nucleus and regulated downstream gene expression as a transcription factor.HEK293 cells were transfected with pEGFP-XBP 1 s-S61D and control vector plasmids.The downstream genes regulated by phospho-XBP1s were detected by RNA-Seq.96 differentially expressed genes were screened according to fold change greater than 2.0 times and P<0.01.91 upregulated genes and 5 downregulated genes were found in overexpressed XBP1s-S61D groups compared with the vector groups.GO analysis and KEGG pathway about the differentially expressed genes found that multiple genes associated with metabolic processes,apoptosis and non-coding RNAs were upregulated in XBP1s-S61D groups.Conclusions:This study found that the IRE1?/XBP1 pathway is activated in PD.and nuclear translocation of XBP1s occurs as a CDK5 manners but not p-p38.Activated CDK5 interacts with XBPls and enhances its nuclear translocation by phosphorylation its Ser61 site during the development of PD.The nuclear translocation of phosphomimetic XBPls leads to up-regulation of the target genes associated with metabolic processes,apoptosis and non-coding RNAs.which may be involved in neuronal death in PD.This study revealsa novel role for XBP1s for the pathogenesis of PD and is worthy of further investigation in the context of new therapeutic strategy for PD.