The Role Of HDAC6 Inhibition By Tubastatin A In Parkinson’s Disease

Research BackgroundParkinson’s disease(PD)is a degenerative disorder of the central nervous system caused by extrapyramidal disorders.The clinical manifestations of PD include resting tremor,muscle rigidity,bradykinesia and ataxia,accompanied by severe memory impairment and dementia.Typical pathological features of PD are progressive degeneration of dopaminergic neurons in substantianigraparscompacta(SNpc),reduction of dopaminergic transmitters in the striatum,and accumulation of a-synuclein in the remaining dopaminergic neurons resulting the formation of the Lewy body.The pathogenesis of PD is very complex and unclear,and may involve oxidative stress,inflammatory damage,excitotoxicity,mitochondrial dysfunction,and genetic factors.Various hypotheses have been proposed,but no conclusion has been reached so far.Oxidative stress and immune inflammatory response play a key role in PD dopaminergic neuronal injury.Excessive production of reactive oxygen species(ROS),over-activation of innate immunity,and adaptive immune dysfunction can cause dopaminergic neurons to be continuously stimulated,which together constitute the pathological microenvironment ofnigrostriatal dopaminergic nerve region,is the main cause of injury of dopaminergic neurons in the mechanism.However,its upstream and downstream molecular regulation mechanisms have not yet been fully elucidated.In recent years,studies have shown that epigenetic mechanisms such as histone acetylation play an important role in the regulation of central nervous system function,affecting the multi-faceted function of cells and participating in the development of PD.The deacetylation activity of histone deacetylase 6(HDAC6)has been receiving increasing attention in oxidative stress and immune inflammatory response.HDAC6 belongs to Class 2 HDAC family,which consist of two deacetylation catalytic domain,one ubiquitination binding domain,and one dynein,binding domain.The two deacetylation catalytic domains(DD1,DD2)are the active regions of HDAC6,which have the activity of deacetylating the substrate protein.The functional characteristics of DD2 are clear,which is the target of HDAC6 specific inhibitors.The peroxidative redox protein(Prx)is a specific substrate for HDAC6.After deacetylated by HDAC6,the antioxidant capacity of deacetylated Prx has been greatly reduced,resulting in increased cell deathand oxidative damage.TubastatinA(TBA)is a HDAC6 deacetylation activity specific inhibitor.Since oxidative damage is an important causative factor of Parkinson’s disease,this experiment observes the effect of TBA to deacetylation level and the elimination of ROS.In addition to oxidative stress,innate immune-mediated inflammatory responses are also an important cause of PD.Nucleotide binding oligomerization domain and leucine rich repeat pyrin 3 domain(NLRP3),a NOD-like receptor,belongs to the intracytoplasmic pattern recognition receptor,activates the innate immune system,promotes inflammation,drives and regulates adaptive immune responses by identifying damage-related molecular patterns released by damaged cells.Studies have shown that ROS can initiate the NLRP3 inflammatory masome assembly as an endogenous pathogenic factor,and then activate caspasel,induce the mature secretion of precursor molecules such as IL-1β and IL-18 and cell death,and participate in non-infection.Its role in immune inflammatory response and central nervous system degenerative diseases has received increasing attention.Therefore,in this study,we used in vitro and in vivo PD models and aHDAC6 selective inhibitor tubastatin A(TBA)to investigate the effects ofpharmacological inhibition of HDAC6 on the activation of NLRP3 inflammatoryresponse and characterized the effects of TBA on the pathogenesis of PD.Obj ectiveThe objective of this study was to investigate the role of HDAC6 on the regulation of NLRP3inflammatory response in experimental models of PD.MethodsIn vitro1.Human neuroblastoma SH-SY5Y cells were were cultured in DMEM-F12 supplementedwith 10%FBS at 37℃ in a humididfied atmosphere of 5%CO 2.Cells wereexposed to 50 μM 6-OHDA and were harvested at different time points.Western blot was used to observe the protein expression of NLRP3.2.SH-SY5Y cells were pretreated with TBA for 2 h and exposed to 6-OHDA for another 24 h.Western blot was used to observe the expression pattern of NLRP3,pro-caspase-1,caspase-1,pro-IL-1βand IL-1β.3.SH-SY5Y cells were treated with different concentrations of TBA.CCK-8 was used to observe whether TBA had an effect on cell proliferation.and whether TBA protected cells against 6-OHDA-induced cell death.4.SH-SY5Y cells were pretreated with TBA for 2 h and exposed to 6-OHDA for another 24 h.Flow cytometry and TUNEL staining were used to observe whether TBA could protect cells from 6-OHDA-induced cell death.5.SH-SY5Y cells were transfected with HDAC6 si-RNA or pc-DNA-HDAC6 plasmid,to knockdown or overexpress HDAC6,cells were then exposed to 6-OHDA for 24 h.Flow cytometry was used to observe apoptotic rate.6.SH-SY5Y cells were pretreated with TBA for 2 h and exposed to 6-OHDA for another 24 h.IP was used to observe the effect of TBA on the level of Prx-II acetylation,and the impact of ROS levels by TBA was observed by flow cytometry.In vivo:1.The animal models of PD were established by sterologically injection of 6-OHDA into the mice striatum.TBA were intraperitoneally injected for 7 days after 6-OHDA lesion.The tissues of SNpc were taken for frozen section and immunofluorescent staining were performedto observe the activation of astroglia and microglia.TH immunostaining was applied to observe the protective effect of TBA on dopaminergic neurons.2.The animal model of PD was established by stereological injection of 6-OHDA into the mice striatum.21 days after the injection of 6-OHDA,mice were sacrificed and the striatum were collected for the measurement of dopamine(DA),dihydroxyphnylacetic acid(DOPAC)and homovanillic acid(HVA)by HPLC-MS.3.The animal model of PD was established by stereological injection of 6-OHDA into the mice striatum.TBA was injected intraperitoneally for 7 daysafter 6-OHDA lesion.Tissue of SNpc and striatum were taken for frozen section.Immunofluorescent staining and western blot were applied to detect the effect of TBA on the expression of 4-HNE.4.The animal model of PD was established by stereological inj ection of 6-OHDA into the mice striatum.TBA was injected intraperitoneally for 7 days after 6-OHDA lesion.SNpc were taken for frozen section.Immunofluorescence staining was used to observe the expression of NLRP3 and HDAC6.5.The animal model of PD was established by stereological injection of 6-OHDA into the mice striatum.TBA was injected intraperitoneally for 7 days after 6-OHDA lesion..The protein and mRNA expression of NLRP3 were observed by western blot and real time RT-PCR.6.The animal model of PD was established by stereological inj ection of 6-OHDA into the mice striatum.TBA was inj ected intraperitoneally for 7 days after 6-OHDA lesion.The tissues of the striatum were taken.Western blot and real time RT-PCR were used to observe the protein and mRNA expression of NLRP3,caspase-1,TH,IL-18,IL-1βand IL-6.Results1.TBA inhibits 6-OHDA-induced NLRP3 inflammasome activation in SH-SY5Y cells.2.The pharmacological inhibition of HDAC6 by TBA attenuates 6-OHDA-induced SH-SY5Y cell death.Knockdown or overexpression of HDAC6 has no effect on cell apoptosis.3.In 6-OHDA-induced PD mouse model,TBA inhibits the inflammatory response and inhibit the expression of NLRP3.4.TBA attenuates 6-OHDA-induced dopaminergic neurotoxicity in PD mouse model.5.TBA enhances the level of acetylation of PRX-II and reduces ROS levels in cell and animal models of PD.ConclusionTBA specifically inhibits the level of HDAC6 deacetylation and ameliorates 6-OHDA-induced neurotoxicity by reducing oxidative stress and inflammatory response.