MicroRNA-24-3p Regulates Dopaminergic Neurons Death In A MPTP-induced Parkinson's Disease Mouse Model By Targeting Pinkl

Parkinson's disease(PD)is the second most common neurodegenerative disorder which is characterized by a loss of dopaminergic neurons in the substantia nigra(SN)and the aggregation of a-synuclein(?Syn).The major clinical features include static tremor,movement delay,muscle rigidity and so on.Although the present studies suggest that oxidative stress,mitochondrial dysfunction and neuronal inflammation play an important role in the pathogenesis of PD,the exact aetiology has not been elucidated.Pinkl(PTEN induced putative kinase 1)is a PD-related gene,mutation of which leads to autosomal recessive early-onset Parkinson's disease.The Pink1 gene is located in the PARK6 locus and encodes a highly conserved putative serine and/or threonine kinase which consist of an N-terminal mitochondrial targeting motif and putative trans-membrane segment.Mounting evidence demonstrates that Pinkl plays an crucial role in maintaining mitochondrial homeostasis,promoting mitophagy,and protecting cells from apoptosis induced by oxidative stress.Therefore,improving the mitochondrial function by regulating the expression of Pinkl will be effective against PD.Although the neuroprotective effect of Pinkl has been extensively studied,little is known about the regulation mechanism of Pinkl at the post-transcriptional levels.MicroRNAs(miRNAs)are a class of endogenous non-coding small RNAs,which represse gene expression by directly binding to their target sequences in the 3'untranslated region(3'UTR)of mRNAs.Approximately,about one-third of functional genes are regulated by miRNAs which are getting more attention as critical regulators for many cellular processes such as cell proliferation and apoptosis,tumor formation and neurodegenerative diseases.Previous studies from postmortem brain analyses and animal model researches have suggested that miRNA dysfunction induces neurodegeneration.For example,the expression of miR-7 decreases dramatically in the brain of PD patients and is shown to be invovled in the regulation of a-synuclein.Together,microRNAs may be used as biomarkers for early diagnosis and become potential therapeutic targets for PD.MiR-24-3p is widely expresses among various tissues and cells of mammals,which primarily acts the role of regulating cell proliferation and apoptosis in several cancers.Intriguingly,recent researches reveal that miR-24-3p expression is upregulated in serum of patients affected by PD,indicating that miR-24-3p may function at a certain extent in the disease.However,the exact regulatory mechanism of miR-24-3p involved in PD remains speculated.In the present study,we found that the expression of Pinkl is essential in neuroprotection,and miR-24-3p,significantly upregulated in the midbrain of MPTP-treated mice as well as A53T mutant a-synuclein transgenic(A53Ttg/tg)mice compared with C57BL/6J mice,participates in the pathogenesis of PD and initiates neuron death by suppressing of Pinkl translation.We further demonstrated that transfection of miR-24-3p into SH-SY5Y cells exacerbates apoptosis after MPP+treatment while inhibition of miR-24-3p protects cells against MPP+-induced mitochondrial deficits and promotes clearance of damaged mitochondria,leading to decreased cytotoxicity and increased dopaminergic cells survival.Pink 1 interference RNA(si-Pinkl)as well as the autophagy inhibitor 3-MA,can partly reverse the neuroprotection of miR-24-3p inhibitor.Finally,our datas suggest that stereotactically injected miR-24-3p antagomir into the SNc of A53Ttg/tg mice rescued the loss of dopaminergic neurons induced by sub-acute MPTP administration.In summary,We demonstrated that miR-24-3p inhibitior exerted its neuroprotective role through upregulating the expression of Pinkl and enhancement of the clearance of damaged mitochondria in PD.AIM:To explore the effects and mechanisms of miR-24-3p in MPTP-induced Parkinson's disease model.METHODS:(1)The expression of Pinkl and miR-24-3p in the midbrain of C57BL/6J mice were detected by Western Blot(WB)and RT-PCR(Real-time Polymerase Chain Reaction)respectively after sub-acute MPTP administration.Besides,the Pinkl as well as miR-24-3p levels in human neuroglioma cells SH-SY5Y was detected in response to MPP+(0?100?M?200?M?400?M).(2)We utilized the 10-month-old Pink1 knockout(Pink1-/-)rat and evaluated the TH positive cells in SNc by Immunohistochemistry(IHC).(3)We first screened some miRNAs by RT-PCR analysis,which were considered to have potential binding sites on Pinkl mRNA 3'UTR and filtered out the most likely candidate(miR-24-3p).MiR-24-3p mimic and miR-24-3p inhibitor were synthesized and the levels of Pink1 in SH-SY5Y cells were semi-quantified by WB after transfection of miR-24-3p mimic/miR-24-3p inhibitor.The WT-3'UTR and MT-3'UTR of human Pinkl mRNA were constructed andl luciferase activity assay was performed using the Dual-Luciferase Reporter Assay System(Promega)according to the manufacturer's instructions.(4)SH-SY5Y cells were transfected with miR-24-3p mimic/miR-24-3p inhibitor(100nM)followed by stimulation with MPP+(400?M).Cell viability were measured by CCK-8 and LDH,cell apoptosis were assessed by using Hoechst 33342 staining and flow cytometry.WB was used to measure the protein levels of Bax,Bcl-2 and Caspase-9.(5)miR-24-3p inhibitor and GFP-LC3 plasmid were co-transfected into SH-SY5Y cells,MitoTracker Deep Red was taken to label mitochondria.The colocalization of mitochondria and autophagosomes was observed by fluorescence microscope.The expression of Parkin both in cytoplasm and mitochondria were detected by WB.(6)SH-SY5Y cells were transfected with miR-24-3p inhibitor(100nM)followed by stimulation with MPP+(400?M).Mitochondrial-specific ROS levels as well as mitochondrial membrane integrity in SH-SY5Y cells were measured by MitoSox and JC-1 staining respectively.A combination of MitoTracker Green with MitoTracker Red was used to quantitate mitochondrial damage after cells were treated with 3-MA.(7)miR-24-3p inhibitor(100nM)and si-Pinkl(100nM)were co-transfected into SH-SY5Y cells followed by stimulation with MPP+(400?M).Cell viability were measured by CCK-8 and LDH,cell apoptosis were assessed by flow cytometry.(8)The expression of Pinkl and miR-24-3p in the midbrain of A53Ttg/tg mice were detected by WB and RT-PCR respectively.Cy3-labled miR-24-3p antagomir was stereotactically injected into the SNc of A53Ttg/tg mice that treated with sub-acute MPTP challenge.Survival neurons were measured by Nissl Staining and immunohistochemistry was taken for TH expression.Western Blot was used to analyze the expression of Bax,Bcl-2 and Cleaved-caspase-9.(9)Immunofluorescence was taken for TH and a-synuclein in SNc of A53Ttg/tg mice after stereotaxic injection of miR-24-3p antagomir.(10)The levels of LC3 and p62 were analyzed by Western Blot and immunofluorescence was taken for TH and LC3 in SNc of A53Ttg/tg mice after stereotaxic injection of miR-24-3p antagomir.RESULTS:(1)The expression of Pinkl deceased after MPTP administration.C57BL/6J mice showed a lower Pinkl level in the midbrain after sub-acute MPTP administration and the expression of Pinkl in human neuroglioma cells SH-SY5Y was detected to be downregulated in response to MPP+.(2)Pink1 knockout rat showed a lower number of TH+ neurons in SNc.We utilized the 10-month-old Pink1 knockout(Pink1-/-)rats and evaluated the TH positive cells in SNc.Immunohistochemical staining revealed that Pink1-/-induced a 43%loss of TH-positive cells in SNc compared with WT rat.(3)MiR-24-3p repressed the expression of Pinkl.Several miRNA-target prediction algorithms(TargetScan,mirDIP,miRcode)were used and miR-24-3p,miR-29b,miR-30b and miR-142-5p was predicted to have putative binding sites in the 3'UTR of Pinkl.Among several miRNAs,miR-24-3p levels in the midbrain of C57BL/6J mice were significantly upregulated on sub-acute MPTP administration.Moreover,the expression of miR-24-3p in SH-SY5Y cells in the presence of MPP+was significantly upregulated.MiR-24-3p overexpression led to a decrease in the Pink1 protein level(P<0.01)while inhibiting miR-24-3p resulted in a upregulation of Pinkl expression(P<0.01).The dual luciferase reporter assay revealed that the WT 3'-UTR of Pinkl exhibited a low translation level in the presence of miR-24-3p,whereas once the 3'-UTR was mutated,the miR-24-3p-mediated suppression of R-Luc reporter activity was abolished(P<0.01).(4)MiR-24-3p inihbitor rescued cytotoxicity and apoptosis induced byMPP+.Knockdown of miR-24-3p attenuated the decrease of cell viability induced by MPP+(P<0.05).Similar results were obtained with the LDH assay(P<0.05).Moreover,inhibition of miR-24-3p prevented activation of apoptosis pathway,transfection of si-Pink 1 abrogated its neuroprotection.On the contrary,miR-24-3p mimic aggravated SH-SY5Y cells death and apoptosis after MPP+ treatment.(5)MiR-24-3p inhibitior promoted mitophagy and the clearance of damaged mitochondria.Inhibition of miR-24-3p increased colocalization between mitochondria and of LC3-labelled autophagosomes and promoted translocation of Parkin from cytoplasm to mitochondria.Blockage of miR-24-3p decreased mitochondrial ROS accumulation induced by MPP+(P<0.01)and maintained the integrity of mitochondrial membrane potential(P<0.01).Pretreatment of autophagy inhibitor 3-MA could partly abolish the protective effect of miR-24-3p inhibitor on mitochondria and cell viability.(6)MiR-24-3p antagomir alleviated neuronal injury in SNc of A53Ttg/tg mice induced by MPTP administration.MiR-24-3p levels were upregulated(P<0.05)and the expression of Pinkl decreased(P<0.05)in the midbrain of A53Ttg/tg mice compared with WT mice.Stereotactically injection of miR-24-3p antagomir into the SNc of A53Ttg/tg mice prevented Cresyl violet positive cells reducion,rescued the loss of TH+ neuron number in the SNc after MPTP administration and inhibited activation of apoptotic pathway.Futhermore,anti-miR-24-3p significantly downregulated midbrain a-synuclein expression of A53Ttg/tg mice.(7)MiR-24-3p antagomir promoted autophagy of dopaminergic neurons in the midbrain of A53Ttg/tg mice.The expression of LC3 was markedly increased while p62 levels decreasd after stereotaxic injection of miR-24-3p antagomir.Immunofluorescence results showed that miR-24-3p antagomir increases colocalization between TH and of LC3 in SNc of A53Ttg/tg mice.CONCLUSION:(1)MiR-24-3p regulated the expression of Pinkl.(2)Inhibition of miR-24-3p alleviated dopaminergic neurons death and apoptosis induced by MPTP/MPP+ through promoting the clearance of dysfunctional mitochondria.The major contributions of the present work lie in:(1)This study demonstrats that miR-24-3p regulates the expression of Pinkl and enables us to take a new look at the important role of Pinkl in cell injury induced by MPTP/MPP+ from the post-transcriptional perspective.(2)We elucidate that miR-24-3p inhibitior exerts its neuroprotective role through upregulating the expression of Pinkl and promoting the clearance of damaged mitochondria,providing an insight into the potential of miR-24-3p/Pinkl/mitophagy axis in opening up novel therapeutic avenues for PD.