The Role And Mechanism Of Heat Shock Protein 60 In Huntington’s Disease

Objective:The present study was aimed to investigate the role and underlying mechanisms of heat shock protein 60(Hsp60)in Huntington’s disease(HD).Methods: We knocked down Hsp60 in HdhQ7 cells by sh RNA to mimic the downregulation of Hsp60 in Hdh Q111 cells.Downregulation of Hsp60 was validated by western blot(WB).Control and Hsp60 knocked down Hdh Q7 cells were stained with anti-Tom20 antibodies.Mitochondrial morphology was imaged.Hdh Q111 cells were transfected with Myc or Myc-Hsp60.Control vector or Myc-Hsp60 expressed cells were stained with anti-Tom20 and anti-Myc antibodies.Mitochondrial morphology was imaged.Mitofusin 1/2(Mfn1/2),optic atrophy 1(Opa1),and dynamin-related protein 1(Drp1)were examined by WB.Drp1 association with the mitochondria was determined by WB in Hsp60 knocked down striatal cells.Control and Hsp60 knocked-down striatal cells were treated with Mdivi-1.Drp1 translocation to mitochondria was analyzed by WB.Hdh Q111 cells were transfected with Myc or Myc-Hsp60.Drp1 translocation to mitochondria was analyzed by WB.Total protein levels of Drp1 p S616 were analyzed by WB.Hdh Q111 cells were transfected with Myc or Myc-Hsp60.Total protein levels of Drp1 p S616 were determined by WB.Total protein lysates were incubated with the crosslinker DSS.Drp1 oligomers were determined by WB.Control and Hsp60 knocked-down cells were stained with Mito SOX.The mitochondrial reactive oxygen species(ROS)was determined by Flow cytometry.The cells were stained with TMRM.The mitochondrial membrane potential(MMP)was determined by Flow cytometry.Mitochondrial localization of BCL2 associated X(Bax)was analyzed by WB.Results: Western blot showed that Hsp60 protein levels were decreased by approximately 60% in Hsp60 knocked down striatal cells.Immunostaining showed that the mitochondria were elongated in Hdh Q7 cells infected with sh-Con,whereas knocked down Hsp60 significantly increased cell numbers with fragmented mitochondria.In contrast,we overexpressed Myc-tagged Hsp60 in Hdh Q111 cells and found that the mitochondria network was rescued by the presence of Hsp60.Western blot analysis showed that there was no change in total protein levels of Drp1,Mfn1,Mfn2,and Opa1 in Hsp60 knocked down striatal cells compared with control.Drp1 association with the mitochondria was dramatically increased in Hsp60 knocked down cells relative to control.Treatment of Mdivi-1 significantly blocked knockdown Hsp60 induced Drp1 translocation to the mitochondria.Expression of Myc-tagged Hsp60 in Hdh Q111 cells inhibited Drp1 association with the mitochondria.The levels of Drp1 p S616 and Drp1 oligomers were elevated in Hsp60 knocked down striatal cells.By contrast,expression of Myc-tagged Hsp60 in Hdh Q111 cells reduced the levels of Drp1 oligomers and Drp1 p S616 relative to control.We measured the MMP and ROS with fluorescence probes and observed higher ROS and lower MMP in Hsp60 knocked down striatal cells relative to control cells.Downregulation of Hsp60 had no effect on ATP production in striatal cells.Increased mitochondrial localization of Bax was seen in Hsp60 knocked down striatal cells.Conclusions: Deletion of Hsp60 causes mitochondrial fragmentation,which is closely associated with increased Drp1 translocation to mitochondrial,enhanced phosphorylation at S616 and elevated oligomers of Drp1 in wild-type Hdh Q7 striatal cells.Overexpression of Hsp60 in mutant striatal cells rescued the mitochondrial network and reduced Drp1 hyperactivation.Moreover,downregulated Hsp60 induced MMP loss,ROS production,and Bax translocation to mitochondria in striatal cells.Collectively,loss of Hsp60 evokes mitochondrial dysfunction in HD.