The Effect Of Mutant Huntingtin On CCAAT Enhancer Binding Protein Homologous Protein

Objective: Huntington’s disease(HD)is an autosomal dominant inheritable disease caused by mutation of IT15 gene which is located in chromosome 4,the mutation of gene encodes mutant huntingtin protein(Htt)with expanded polyglutamine(Poly Qs)chain in N-terminal.Recent studies show that abnormal transcription regulation,endoplasmic reticulum stress(ER stress)and unfolded protein response(UPR)are highly related with the occurrence of HD,and the abnormal transcription regulation is central to the disease.CCAAT enhancer binding protein homologous protein(CHOP)is a main factor that induced by stress and could regulate the expression of other genes that mediate apoptosis in cells.So far,there are lots of researches about the functions of CHOP in ischemia disease,Alzheimer disease,metabolism disease and cancer,while papers published on HD are few and far between.We aim to explore whether the mutant Htt can affect the expression of CHOP and its subcellular localization,to speculate the role of CHOP in the molecular pathogenesis of HD.Methods: 1.The expression of N-Htt in the HD PC12 cells: inducible PC12 cells expressing fragment of HD exon 1 with either 23(normal)or 74(expanded)polyglutamine repeats fused to enhanced green fluorescent protein(EGFP)were cultured in standard high glucose Dulbecco’s Modified Eagle’s Medium(DMEM)supplemented with 10% heat-inactivated horse serum at 37℃ and 10%CO2.The expression of N-Htt were induced by the addition of doxycycline(Dox)and then observed the cell growth state under the fluorescence microscope.2.Total protein were extracted from different HD PC12 cells when induced by Dox at 0d,1d,3d and 5d.Western blot was performed to detect the expression level of CHOP protein,Actin was detected as an internal control.3.Total RNA was extracted from HD PC12 cells at the different induced time points above.Reverse transcription(RT)kit was used to proceed the RT response,then Real Time-PCR was conducted to explore the expression of CHOP m RNA.4.Cytoplasmic and nucleus protein were also extracted at the different induced time points.Western blot was performed to calculate the expression level of CHOP which located in cytoplasmic and nucleus.Actin was detected as internal control of cytoplasmic protein,and H3 served as internal control of nucleus protein.Results:1.Tet-on regulated EGFP-tagged N-Htt-Q23 and N-Htt-Q74 HD PC12 cell lines were observed at different induced time(0d,1d,3d,5d)under luminescence microscope,N-Htt-Q23(normal)were homogenously expressed,while N-Htt-Q74(mutant)gradually formed inclusion bodies in nucleus.2.After induced for different time,the expression level of CHOP from total protein did not change in N-Htt-Q23 HD PC12 cells,while it remarkably rose in N-Htt-Q74 HD PC12 cells.3.Normal N-Htt did not change the expression level of CHOP m RNA as induced time went on,while mutant N-Htt rose it remarkably.4.Normal N-Htt did not increase the expression level of CHOP extracted from cytoplasmic and nucleus protein of different induced time.But mutant N-Htt enhanced nucleus CHOP expression,while it was of no avail to do with the cytoplasmic protein.That was to say the mutant N-Htt could promote CHOP to translocate into nucleus.Conclusions: 1.Mutant N-Htt can remarkably raise the expression level of CHOP m RNA and protein.2.Mutant N-Htt can affect the subcellular distribution of CHOP protein and makes it prone to accumulate in nucleus.3.As an important transcription factor,the increasement of CHOP expression level and the accumulation in nucleus may have indispensable effects in the roles of transcription regulation and ER stress,as well as to the molecular pathogenesis of HD.