Neuroprotective Effects Of PINK1 On A Mouse Model Of Tauopathy And The Related Mechanisms

Objective: To explore the effect of phosphatase and tensin homolog-induced kinase 1(PINK1)on Tau pathology,the resulting neurological tissue damage and cognitive impairment,as well as the possible mechanisms.Methods:(1)The establishment of experimental animal model: Recombinant adeno-associated virus(AAV)with target gene fragment MAPT(encoding human Tau protein)or PINK1(encoding human PINK1 protein)was constructed respectively,namely AAV-MAPT and AAV-PINK1.Then AAV-MAPT was stereotaxically injected into the hippocampal CA1 region of 2-month-old and wild-type C57 mice,to establish the mouse model with overexpression of human Tau(hTau)protein,which was recorded as hTau group.Simultaneous overexpression of hTau and PINK1 proteins in CA1 region was achieved by combined injection of AAV-MAPT and AAV-PINK1,which was recorded as hTau+PINK1 group.(2)The evaluation of cognitive function: Behavioral tests(novel object recognition and Morris water maze)were performed one month after the injection of virus,to evaluate the learning and memory ability of the mice.(3)The observation of nervous tissue in CA1 region: The morphology,structure,number and distribution of neurons in CA1 region were observed by Nissl’s staining and neuronal nuclei staining,and the level of apoptotic proteins was detected by Western Blots.The distribution and density of dendrites and dendritic spines in CA1 region were observed by Golgi staining.(4)The detection of Tau pathology: Western Blots and immunohistochemical staining were used to observe the accumulation and distribution of hTau protein in CA1 region.(5)Probing possible mechanisms of PINK1 affecting Tau pathology: The m RNA level of hTau was detected by q PCR.The expression of autophagy related proteins was detected through Western Blots,and the ubiquitination of Tau was assayed by Co-immunoprecipitation.Subsequently,mice were intraperitoneally injected with the autophagy-lysosome pathway inhibitor chloroquine or proteasome inhibitor MG132,to observe whether blocking the autophagy-lysosome pathway or proteasome could inhibit the effect of PINK1 on Tau protein level and its influence on nervous tissue damage and cognitive impairment.(6)Exploring the effects of PINK1 on mitochondria: The level of hTau protein in mitochondrial components of CA1 region was detected by Western Blots,and the effects of PINK1 on mitochondrial function were evaluated by assaying ATP,antioxidant enzyme and lipid oxidation levels of CA1 area.Morphological and structural changes of mitochondria were observed by electron microscopy.Results: 1.HTau mice showed learning and memory impairments,while the cognitive dysfunction was rescued in hTau+PINK1 mice.2.HTau mice showed neuronal injury,neuronal loss and excessive apoptosis,and synaptic damage in CA1 region,while up-regulation of PINK1 attenuated such phenotype.3.PINK1 overexpression distinctly reduced hTau protein(including total protein and phosphorylated protein)level in CA1 region.4.HTau mice showed accumulation of hTau in mitochondria,morphological and structural damage,and mitochondrial disorders,while PINK1 reduced the accumulation of hTau in mitochondria,alleviated mitochondrial morphology and structural damage,and improved mitochondrial dysfunction.5.PINK1 enhanced autophagy-lysosome pathway,and promoted the ubiquitination of Tau.Blocking the autophagy-lysosomal pathway with chloroquine could reverse the ameliorated effect of PINK1 on Tau pathology and inhibit the improvement effect of PINK1 on neuron loss,synaptic damage and cognitive impairment.While inhibition of proteasome by MG132 did not show the above reversal effects.Conclusions: Upregulation of PINK1 reduced the pathological accumulation of hTau in CA1 region,thereby alleviating neuron loss,synaptic damage,and cognitive impairments in hTau mice.This probably occurred through inducing hTau degradation via autophagylysosome pathway.Taken together,this study supported that PINK1 may be a promising therapeutic target for AD.