| PD is one of the neurodegeneration diseases characterized by movement disorder induced by decreased dopamine release caused by loss of substantia nigra compacta dopaminergic neuron.PD patients show different symptoms at different stages of the disease.Before the motor*symptoms appear,patients suffer the non-motor symptoms like rem sleep disorder,depression,anxiety.hyposmia and constipation.It is considered as the early stage of PD when motor disorders such as tremor,bradykinesia and rigidity are accompanied with non-movement disorders like mild cognitive impairment,fatigue,daytime sleepiness and apathy.As the disease progresses.patients appear the symptoms such as axial deformation,movement disability,orthostatic hypertension and urinary tract symptoms,then rapidly develop into dementia,easy-falling,dysphagia posture and gait disorder symptoms and ultimately die.Clinical treatments of PD are based on increasing brain dopamine levels and enhancing the function of dopamine receptor.Unfortunately,there still have no methods to cure PD thoroughly.All the drugs are to slow the progress of PD and improve the quality of life.Clarifying the mechanisms inducing dopaminergic neuron death is indispensable for PD treatment.At present,neuroinflammation,mitochondrial dysfunction,oxidative stress,lysosome dysfunction and immune dysregulation are considered as the pathogenesis of PD;of which oxidative stress is reported to be the trigger factor.For oxidative stress can lead to oxidized dopamine accumulation followed by reduced glucocerebrosidase enzymatic activity,lysosomal dysfunction and a-synuclein accumulation.Thus the toxic cascade ultimately results in neuron death.Therefore,early intervention with antioxidants might be an effective way for PD treatmentDopamine receptor agonists directly bind to dopamine receptor and relieve movement disorders and fluctuation symptoms of younger PD patients in a dopaminergic neuron-independent manner.Besides,dopamine receptor agonists ropinirole and cabergoline were also reported to enhance brain antioxidant capacity by inducing GSH biosynthesis.However,the targets of dopamine receptor agonists and the molecular mechanisms by which they promoted GSH synthesis remain unknown.Previous studies on dopamine receptor mainly focused on neuronal dopamine receptor.With the discovery and further study of dopamine receptors in astrocytes,it is found that astrocytic dopamine receptors played an important role in regulating neuroinflammation,immune function,neurotransmitter and calcium homeostasis.Therefore,the first part of this paper aims to explore the target and specific mechanism of dopamine receptor agonist to promote the synthesis of GSH.Results showed that the activation of Drd2 in astrocytes could promote the synthesis of GSH,while Drdl did not affect the synthesis of GSH.However,activating neither Drdl nor Drd2 in neurons affected GSH synthesis.Drd2 activation triggered β-arrestin2 binding to PKM2 and promoting PKM2 dimerization.The dimeric PKM2 interacted with Nrf2 and facilitated its binding to the promoter of glutamate-cysteine ligase followed by increasing GSH synthesis.PKM2 is a rate-limiting enzyme in the final step of glycolysis,which can regulate cellular glucose metabolism.In addition to the classical function,dimeric PKM2 can also function as protein kinase and co-activator of transcription factors.The non-classical functions of PKM2 participate in cell physiological and pathological process via regulating inflammation,oxidative stress,cell proliferation and apoptosis.Clinically,long-term use of dopamine receptor agonists leads to poor efficacy and even aggravates PD symptoms.Long-time exposure to dopamine receptor agonists typically contributes to Drd2 receptor internalization,thus leads to the loss of target of dopamine receptor agonists and loses the treatment efficiency.Hence,finding small molecules that directly dimerize PKM2 may maintain astrocytic GSH biosynthesis in a Drd2-independent manner.After screening 863 natural products,we found that pyridoxine,a type of vitamin B6,could dimerize PKM2 and increase astrocytic GSH synthesis through Nrf2 activation.Moreover,in vivo experiments also showed that pyridoxine supplementation increased the striatum GSH and relieved nigral dopaminergic neuron loss in MPTP-induced subacute PD mice model.Combined the clarified protective effect of astrocytic PKM2 in the first two parts and the appeared PKM2 expression after MPP+stimuli in primary neurons detected by 2D-PAGE-MS.In the third part we further explored the role of neuronal PKM2 in PD.Results demonstrated that neuronal PKM2 upregulated as a self-protective mechanism protected neuron from MPP+/MPTP-induced damage.Moreover,exogenous overexpression PKM2 to amplify the protective effect of PKM2 could reduce the loss of nigral dopaminergic neuron in MPTP-induced subacute PD mouse model.Part I Inducing glutathione biosynthesis by astrocytic dopamine D2 receptor via PKM2-mediated Nrf2 transactivationObject:To explore the mechanism of glutathione biosynthesis conducted by dopamine receptor agonist.Methods:Primary astrocytes and primary neurons were cultured and stimulated with carbergoline alone or with SCH23390 and sulpiride combined with cabergoline.GSH colorimetric assay kits were used to measure GSH level.WT and Drd2-/-astrocytes were stimulated with quinpirole,quinelorane and bromocriptine and GSH colorimetric assay kits were used to detect GSH level.Drd2foxp/foxp and Drd2hGFAPcKO mice were intraperitoneally injected with quinpirole or saline and striatal GSH level was assayed using GSH colorimetric assay kits.Adult astrocytes of WT mice with or without quinpirole administration were separated using Anti-ACSA2 microbead kits for RNA sequencing.After being treated with quinpirole in astrocytes,qRT-PCR was used to analyze the mRNA level of Nrf2 targets;immunoblotting was used to detect the protein expression of Gclc and Gclm;ChIP was used to detect the interaction between Nrf2 and promotors of Gclc and Gclm.In quinpirole treated WT and Nrf2-/-astrocytes,immunoblotting was used to detect the protein expression of Gclc and Gclm and GSH colorimetric assay kits were used to measure GSH level Label-free quantitative proteomic sequencing was used to detect the protein that bound with Nrf2 in greater abundance after quinpirole stimuli.qRT-PCR and selective enzyme digestion were used to analyze the mRNA expression of PKM1 and PKM2 in astrocyte and neuron;immunoblotting was used to analyze the protein expression of PKM1 and PKM2 in astrocyte and neuron;TSA staining of brain slice was used to detect the co-localization of PKM1 or PKM2 with NeuN or GFAP.Lipofectin transfection with PKM2 siRNA was used to knockdown PKM2 in astrocyte followed by quinpirole treatment;qRT-PCR was used to analyze the mRNA level of Gclc and Gclm and the amount of anti-Nrf2 immunoprecipitated DNA with primers flanking the promoters in the Gclc and Gclm genomic regions;immunoblotting was used to detect the protein expression of Gclc and Gclm;GSH colorimetric assay kits were used to measure GSH level.Astrocytes were pre-treated with DASA followed by quinpirole stimuli.BN-PAGE was used to detect the dimeric and tetrameric PKM2;PLA and co-IP were used to analyze the co-localization of PKM2 and Nrf2;qRT-PCR was used to analyze the mRNA level of Gclc and Gclm and the amount of anti-Nrf2 immunoprecipitated DNA with primers flanking the promoters in the Gclc and Gclm genomic regions;immunoblotting was used to detect the protein expression of Gclc and Gclm;GSH colorimetric assay kits were used to measure GSH level.Adenovirus AV-PKM1 infection was used to over-express PKM1 in astrocyte followed by quinpirole treatment;PK colorimetric assay kits were used to assay PK activity;co-IP was used to detect interaction between Nrf2 and PKM1;qRT-PCR was used to analyze the mRNA level of Gclc and Gclm and the amount of anti-Nrf2 immunoprecipitated DNA with primers flanking the promoters in the Gclc and Gclm genomic regions;immunoblotting was used to detect the protein expression of Gclc and Gclm.In PTX treated astrocytes or in P-arrestinl or β-arrestin2 knockdown astrocytes followed by quinpirole administration,BN-PAGE was used to detect the dimeric and tetrameric PKM2.qRT-PCR was used to analyze the mRNA level of Gclc and Gclm and the amount of anti-Nrf2 immunoprecipitated DNA with primers flanking the promoters in the Gclc and Gclm genomic regions;immunoblotting was used to detect the protein expression of Gclc and Gclm;GSH colorimetric assay kits were used to measure GSH level.Results:1)Cabergoline promoted astrocyte GSH biosynthesis in a time and concentration-dependent manner while had no effect on neurons.2)Drdl antagonist SCH23390 had no effect on cabergoline-induced GSH biosynthesis while Drd2 antagonist sulpiride abrogated cabergoline-induced GSH biosynthesis in astrocyte.3)Drd2 agonists（quinpirole,quinelorane and bromocriptine）promoted astrocyte GSH biosynthesis in a concentration-dependent manner.Astrocyte Drd2 knockout abrogated the effect.4)Astrocytic Drd2 activation induced PKM2 dimerization followed by dimeric PKM2 binding and activating Nrf2 transactivation.5)PKM1 did not bind to Nrf2 or affect its transcriptional activity.6)Astrocytic β-arrestin2 directly bound to PKM2 and facilitated its dimerization.Conclusion:Astrocytic Drd2 activation facilitated GSH biosynthesis and the mechanism was referred to that β-arrestin2 directly bound and facilitated PKM2 dimerization followed by dimeric PKM2 bound and activated Nrf2 transactivation.Part II Drug Screening targeting PKM2 dimerization and its role in Parkinson’s diseaseObject:To screen the compounds directly dimerizing PKM2 and clarify its role in PD.Methods:Intrinsic tryptophan scanning fluorimetry was used to screen compounds directly bound with PKM2.BN-PAGE was used for further testifying the screened compounds on PKM2 isoform changes.Primary astrocytes were treated with pyridoxine or vindoline;High-content screen was used to measure GSH fluorescence intensity and PI-positive nuclei;Co-IP and PLA were used to detect the interaction between PKM2 and Nrf2.Lipofectin transfection with PKM2 siRNA was used to knockdown PKM2 in astrocyte followed by pyridoxine treatment;qRT-PCR was used to analyze the amount of anti-Nrf2 immunoprecipitated DNA with primers flanking the promoters in the Gclc and Gclm genomic regions;immunoblotting was used to detect the transfection efficiency of PKM2 siRNA and the protein expression of Gclc and Gclm;GSH colorimetric assay kits were used to measure GSH level.Primary neurons were treated with pyridoxine;qRT-PCR was used to analyze the mRNA level of Gclc and Gclm;GSH colorimetric assay kits were used to measure GSH level.Drd2flox/flox mice and Drd2hGFAPcKO mice were intraperitoneally injected with pyridoxine and quinpirole;BN-PAGE was used to detect dimeric and tetrameric PKM2 in striatum;qRT-PCR was used to analyzed the mRNA level of Gclc and Gclm in striatum;immunoblotting was used to detect the protein expression of Gclc and Gclm in striatum;GSH colorimetric assay kits were used to measure GSH level in striatum.Drd2flox/flox mice and Drd2hGFAPcKO mice were made MPTP-induced subacute PD mouse model with continuous quinpirole or pyridoxine administration.TH immunohistochemical staining and nissl staining were used to detect TH-positive neurons and nissl positive neurons in the middle brain of each group.Results:1)Pyridoxine and vindoline were detected to dimerize PKM2 in vitro.2)Pyridoxine facilitated GSH biosynthesis in astrocytes with little toxicity.3)Pyridoxine facilitated GSH biosynthesis through PKM2/Nrf2 signaling pathway.4)Astrocytic PKM2 knockdown inhibited pyridoxine-induced GSH biosynthesis.5)Pyridoxine facilitated in vivo GSH biosynthesis in a Drd2-independent manner.6)Pyridoxine reduces dopaminergic neuron loss in MPTP-induced subacute PD mouse model in a Drd2-independent manner.Conclusion:Pyridoxine facilitated GSH biosynthesis through PKM2/Nrf2 signaling pathway in astrocyte,Which exerted the neuroprotective effect on MPTP-induced PD mouse model in a Drd2-independent way.Part III The role and mechanism of neuronal PKM2 in Parkinson’s diseaseObject:To investigate the role and mechanism of neuronal PKM2 in MPTP-induced PD mouse model.Methods:Primary neurons were stimulated with MPP+,then immunoblotting was used to detect PKM,PKM1 and PKM2 protein expression.TSA staining was used to detect PKM1,PKM2 expression in the middle brain of MPTP-induced subacute PD mouse model.AV-PKM2-infected primary neurons were used for PKM2 overexpression neuron model while PKM2loxp/loxppENO2cre fetal mice were used for PKM2 deficiency primary neurons culture.CCK8 was used to analyze cell viability;LDH colorimetric kits were used to measure LDH release;Flow cytometry was used to detect cell apoptosis using Annexin V-FITC/PI kits;MAP2 immunofluorescence was used to analyze neurite length and Hoechst 33342 staining was used to detect neuron nuclei morphology.Middle brain intraperitoneal injection with corresponding AAV was used to produce neuronal specific PKM2 overexpression mice and neuronal specific PKM2 knockdown mice.Immunohistochemical was used to analyze the loss of TH-positive neurons.AV-PKM1-infected primary neurons were used for PKM1 overexpression model.The effects of PKM1 on MPP+-induced primary neurons were evaluated using CCK8 and LDH assay.BN-PAGE was used to detect the dimeric and tetrameric PKM2 under MPP+stimuli with or without DAS A administration.The effects of DASA on MPP+-induced primary neuron were evaluated using CCK8,LDH,Annexin V/PI,MAP2 immunofluorescence and Hoechst 33342 staining.qRT-PCR was used to analyze mRNA levels of Nrf2 targets in primary neurons treated with AV-PKM2 or DAS A.Results:1)MPP+/MPTP induced decreased PKM1 expression and increased PKM2 expression in vitro and in vivo.2)In vitro,PKM2 overexpression attenuated MPP+-induced primary neuron cell viability decline,LDH release,apoptosis rate increase,neuron length shortened and hoechst 33342-positive nuclei increase while PKM2 deficiency aggravated those.3)In vivo,neuronal specific PKM2 overexpression attenuated TH-positive neuron loss in MPTP-induced subacute PD model while neuronal specific PKM2 knockdown aggravated it.4)In vitro,PKM1 overexpression did not affect MPP+-damaged primary neuron.5)The overexpressed PKM2 mainly existed as dimeric form and dimeric PKM2 protected neurons against MPP+induced damage through activating Nrf2.Conclusion:Neuronal PKM2 upregulation was considered as a self-protection mechanism fighting against MPP+/MPTP-induced damage through PKM2/Nrf2 signaling pathway. |
