Sirt3 Deacetylates TFAM And Protects Mitochondria In 6-OHDA Model Of PD

Background and objective:Parkinson’s disease(PD)is a common chronic progressive neurodegenerative disease with pathological features of selective progressive loss of dopaminergic neurons in the dense part of the substantia nigra and the appearance of Lewis bodies.Aging is the most important risk factor for PD,but the pathogenesis is not yet clear. Oxidative stress,mitochondrial dysfunction and protein misfolding accumulation may be the main factors leading to dopaminergic neuron degeneration and death.Therefore,some scholars hypothesized Parkinson disease is a mitochondrial disease.Mitochondrial dysfunction in PD mainly reflects changes in mitochondrial genetic material DNA,including deletion and mutation of mitochondrial DNA(mt DNA),mitochondrial complex I dysfunction,calcium homeostasis imbalance caused by mitochondrial endoplasmic reticulum complex dysfunction,mitochondrial division and fusion disorders,activation of mitochondrial-dependent programmed death,etc.Multiple studies have confirmed that the amount of mt DNA in dopaminergic neurons in the substantia nigra dense part of PD patients is reduced,but the specific mechanism is not completely clear.Mitochondrial DNA is located in a high ROS(Reactive oxygen species)environment within the mitochondria,and there is no DNA repair enzyme or histone encapsulation,which makes mitochondrial DNA more prone to loss and mutation.Mitochondrial transcription factor A(TFAM)belongs to a family of high mobility proteins with high affinity for mt DNA and is responsible for the transcription,replication and packaging of mt DNA.The amount of TFAM directly affects the amount and transcription of mt DNA.For example,when the mitochondrial TFAM level of TFAM heterozygous transgenic mice is reduced,the corresponding mt DNA content is also significantly reduced.However,in recent years,it was found that the amount of mt DNA in the aging mouse model was significantly reduced when there was no significant change in TFAM content,which indicates that changes in TFAM function will directly affect the amount of mt DNA.Mitochondrial acetylation is widely involved in the regulation of mitochondrial protein function.Totally,63% of mitochondrial proteins have acetylation modifications.These modifications have a negative regulatory effect on fatty acid oxidation,tricarboxylic acid cycle,electron transport chain,oxidative phosphorylation,and mitochondrial fusion and splitting.At the same time,deacetylase also exists in mitochondria to regulate excessive acetylation of proteins,which maintains the normal function of protein and protects mitochondrial function.The deacetylation function of mitochondrial proteins is mainly regulated by the mitochondrial deacetylase Sirtuin-3(Sirt3).Studies have found that the activity of Sirt3 decreases during aging,and both the number and transcription of mt DNA are reduced.However,whether Sirt3 is directly involved in the regulation of mt DNA transcription and synthesis and the role of related mechanisms in the pathogenesis of PD are currently unclear.In established PD cell and animal model of this study,we explore the following issues: whether TFAM is acetylated and the effect of acetylation on TFAM function;whether Sirt3 effectively protects TFAM transcription function and maintains mt DNA content by removing TFAM acetylation,and what are the related molecular mechanisms;verify in PD model that Sirt3 can be enhanced to maintain mt DNA content to protect neurons,and provide new strategies for PD treatment.Methods:In this study,cells were first treated with 6-OHDA,and mitochondria were separated;later lysine-acetylation levels were detected by western blot(WB).Besides,TFAM was detected by immunoprecipitation,TFAM acetylation was detected by WB.Immunofluorescence staining was used to detect the co-localization of TFAM and Ac-lysine.Sirt3,Sirt5 and Sirt1 were also detect from total cell lysis by WB.Secondly,the interaction between TFAM and Sirt3 was detected.Using Sirt3-GFP plasmid and TFAM-Flag plasmid,the binding effect of Sirt3 and TFAM in vitro was proved by immunoprecipitation;the co-localization of Sirt3-GFP and TFAM was detected by immunofluorescence staining;Sirt3 was overexpressed/knocked-out of and treated with 6-OHDA,TFAM was immunoprecipitated,and Ac-lysine was detected by immunoblotting,which proved Sirt3 regulated the acetylation of TFAM.TFAM mutant plasmids were constructed and co-transfected with Sirt3,Ac-lysine was detected by immunoprecipitation to search for the acetylated site of TFAM regulated by Sirt3.Mitochondrial light chain promoter primer probe(Biotin-LSP)primers that specifically bind to TFAM were synthesized chemically.TFAM mutants were incubated with Biotin-LSP.Then Biotin-LSP was immunoprecipitated and TFAM-Flag was detected to reflect that TFAM acetylation affects the binding of mitochondrial light chain region.Sirt3 was overexpressed/knocked-down and treated with 6-OHDA,DNA and RNA were extracted,and later mt DNA quantity and transcription were detected by q PCR.The Sirt3 agonist HKL was used to transcriptionally activate Sirt3 and observe the its intervention effect on Parkinson’s disease in vivo/in vitro.Results:Experiment 1: 6-OHDA treatment induces elevated TFAM acetylation(1)Immunoblotting found that the acetylation level of mitochondrial protein increased with the prolongation of 6-OHDA treatment time.The classic substrate Mn SOD,which is easily modified by acetylation on mitochondria,also gradually increased with the 6-OHDA treatment time.(2)Using immunoprecipitation technology,it was found that after 6-OHDA treatment,the TFAM acetylation level increased with the extension of the treatment time.Using immunofluorescence dual-label technology,it was found that TFAM and Ac-lysine co-localization increased after 6-OHDA treatment.It also showed that TFAM has increased acetylation.(3)Immunoblotting revealed that the level of mitochondrial deacetylase Sirt3 decreased with the increase of 6-OHDA treatment time.Experiment 2: Sirt3 regulates TFAM acetylation and affects the content and transcription of mt DNA(1)The interaction between Sirt3 and TFAM was explored.In vitro immunoprecipitation found that Sirt3-GFP and TFAM-Flag can combine with each other;endogenous immunoprecipitation found that Sirt3 and TFAM can also combine with each other;immunofluorescence staining found that Sirt3-GFP and TFAM can colocalize.(2)Sirt3 was overexpressed in cells and then treated with 6-OHDA.The acetylation level of TFAM was detected after immunoprecipitation.The results showed that overexpression of Sirt3 can significantly reduce 6-OHDA-and thus induce acetylation of TFAM,and while Sirt3 was interfered by si RNA,the acetylation level of TFAM induced by 6-OHDA was significantly increased.(3)In order to clarify the acetylation site of TFAM,TFAM mutant plasmids were constructed and co-transfected with the Sirt3-GFP plasmid.TFAM-Flag was purified by immunoprecipitation,and the acetylation of flag was detected by Ac-lysine.It was found that Sirt3 regulates TFAM deacetylation at the 62 lysine.(4)The effect of TFAM-K62 deacetylation on the binding of mitochondrial light chain promoter(Biotin-LSP)was explored.The results of co-immunoprecipitation found that TFAM-K62 R has a stronger binding capacity with Biotin-LSP.(5)The influence of Sirt3 on the quantity and transcription of mt DNA,was explored.q PCR results showed that Sirt3 overexpression can alleviate the decrease in mt DNA quantity and transcription caused by 6-OHDA treatment,while Sirt3 interference can aggravate the decrease in mt DNA quantity and transcription caused by 6-OHDA treatment.Experiment 3: Sirt3 agonist HKL regulates mt DNA content and transcription(1)Sirt3 agonist HKL can increase Sirt3 protein level and m RNA level in cells was confirmed.(2)The immunoprecipitation was used to detect the acetylation of TFAM,and it was found that under normal conditions,HKL treatment can reduce the acetylation level of TFAM in cells;HKL can significantly reduce the acetylation of TFAM induced by 6-OHDA treatment;q PCR results showed that HKL treatment can reduce6-OHDA induced decrease in the number of mt DNA and transcription level,while Sirt3 interference would block the protective effect of HKL on TFAM function.Experiment 4: Sirt3 agonist HKL protects neurotoxin-induced cell death(1)Western blot,and CCK8 and TUNEL staining results showed that HKL protected cells from 6-OHDA-induced death.After interfering with Sirt3 expression,HKL lost its protective effect.(2)The 6-OHDA mouse model was constructed,and the protective effect of HKL on DA neurons was tested by in vivo animal model.The immunofluorescence staining results showed that HKL can significantly protect DA neurons,the TH staining positive neurons in the substantia nigra dense part;WB results showed that HKL increased Sirt3 levels and reduced TFAM acetylation levels.The q PCR results also showed that HKL could alleviate mt DNA decrease caused by 6-OHDA.Conclusion:This study found that acetylation damages the function of mitochondrial DNA transcription factor TFAM,and the deacetylase Sirt3 can reduce the TFAM acetylation level and protect its function.The deacetylation site was identified.It is also found that increasing Sirt3 expression can significantly reduce neurotoxin6-OHDA-induced TFAM acetylation,which in turn maintains mt DNA content and transcription.The establishment of cell and animal models verified the maintenance effect of Sirt3-specific agonist HKL on mitochondrial TFAM function,and the amount and transcription of mt DNA.HKL can effectively protect DA neurons in midbrain SNc region from damage caused by 6-OHDA.The study identified the important role of Sirt3-TFAM-mt DNA regulation in maintaining mitochondrial function.Impaired Sirt3-TFAM regulation may be involved in mitochondrial impairment in the pathogenesis of PD.