Mutant Huntingtin Inhibits The Expression Of Synaptic Vesicle Protein 2C And The Transport Of Synaptic Vesicles

Huntington’s disease（HD）is an autosomal-dominant inherited neurodegenerative disease which is caused by an abnormal expansion of a CAG repeat in the exon 1 of the HD gene encoding for a protein called huntingtin（Htt）,which results in a long stretch of polygultamine.Despite the widespread distribution of Htt,the pathological characteristic feature of HD is the earliest selective deterioration of the medium spiny-projection neurons（MSNs）of the striatum.As the disease progresses,there are neuronal losses in the deep layers of cerebral cortex,hippocampus,hypothalamus,etc.MSNs,which accept the glutamatergic fibers from cortex,project their axons over relatively long distances to targets in the globus pallidus and the substantia nigra pars reticulata.The dysfunction of synapse contributes to the incipient symptoms in HD.Research data demonstrate that mutant Htt can cause the synaptic dysfunction including vesicle transport defects,synaptic vesicle structural damages,exocytosis and endocytosis defects,the expressing and posttranslational modification defects of various synaptic proteins,and so on.Synaptic vesicle proteins are of great importance to the maintaining and adjustment of synaptic vesicular functions,of which expression and dysfunction affect synaptic function.Synaptic vesicle protein 2（SV2）is a family of abundant transmembrane proteins including SV2A,SV2B and SV2C subtypes,which are mainly present on synaptic vesicles and endocrine secretory vesicles.In the three isoforms,SV2A and SV2B widely distribute in the mouse brain,however the distribution of SV2C protein is mainly in the ancient brain regions such as striatum,globus pallidus,substantia nigra,midbrain and CA3,DG area of hippocampus,which are very similar to HD pathological areas.The research data show that SV2 is an important synaptic protein involves in the nervous and endocrine vesicle exocytosis.Knock-out（KO）SV2A mice exhibited severe seizures.Electrophysiologically,the SV2A-deficient brain slices indicated a significant decrease in GABAergic synaptic transmission.The adrenal chromaffin cells from SV2A KO mice showed that both vesicle numbers and sizes were normal in reserve pool（RP）,but in the ready releasable pool（RRP）was significantly diminished.The SV2 functional research in insulin secretion demonstrated that the isoform-specific silencing of SV2A or SV2C had no influences on cytosolic[Ca²⁺]and K⁺-induced insulin releases;in contrast,the glucose-elicited secretion was significantly inhibited,specially by silencing of SV2C.The amino acid sequence of SV2 was homologous to bacterial transporters,which herald it may be a kind of transporters.Other researches suggested that SV2 maybe acted as a scaffold protein or a trafficking protein that regulated the vesicle shape and maintained the stability of transporting.So we wonder whether or not SV2 affects the vesicular transportation?The issue is unknown and unreported.Therefore,we detected the expression changes of SV2A,SV2B,and SV2C in HD models,and found that mutant Htt specially repressed the expression of SV2 isoform C.And we proved that SV2C participated in the vesicle transport.Then we revealed that mutant Htt could impair vesicle transport through inhibition of the expression of SV2C.1.Mutant Htt selectively represses SV2 isoform CMany researches prove that mutant huntingtin inhibits the expression of synaptic vesicle proteins through the transcriptional repression in HD neuropathology.To investigate the expression changes of SV2A,SV2B and SV2C isoforms in HD models,we firstly detected the expression changes of SV2 in the HD-transgenic mouse model（TG）,and found that SV2A increased slightly in the 16^th week and was no significant changes in the 14^th and 20^th week,SV2B had no changes in all periods and SV2C progressively reduced in the 14²0 weeks HD mice brains with the technique of western blotting.We also detected the morphologic changes in the TG and WT mouse brain by immunohistochemistry,the results showed that SV2A isoform widely distributed in the mouse brain and presented slight increase in the 16^th week TG mouse brain.And compared with WT,the content of SV2B isoform was unchanged in TG mouse brain.The SV2C isoform protein exhibited a striking restricted localization in the medial and lateral globus pallidus of striatum,substantia nigra pars reticulata.The expression of SV2C progressively reduced in the striatum globus pallidus and substantia nigra pars reticulata of 14²0 weeks TG mice brain.The transcriptional changes of three sv2 were the same by reverse transcription semiquantitative PCR（RT-PCR）detection.Then we detected the same changes in transfected Htt-120Q N2a cell by western blotting（WB）and RT-PCR techniques.These data showed that mutant Htt selectively repressed SV2 isoform C.2.SV2C affects the vesicle transportThe selectively high expression of SV2C in HD relevant brain regions and the selective repression of SV2C by mHtt indicated that the lesions of HD neuropathology was likely related to SV2C dysfunction resulted from mutant Htt.It is known that vesicle transport is very important to normal synaptic function,but it is defective in HD.To investigated whether SV2C involved in synaptic vesicular transport,the distributions of SV2C in different vesicles were firstly determined in the differentiated N2a cell,then used fluorescence recovery after phptobleaching（FRAP）technique to detect the synaptic vesicle trafficking after silencing endogenous SV2C in N2a.The pDsRed-NPY plasmid（marking large dense core vesicles,LDCV）,the pEGFP-VAchT plasmid（marking small vesicles,SV）or pEGFP-VAMP2（marking both vesicles）was respectively transfected into N2a cells.The cell immunofluorescence results showed that SV2C located not only in vast majority of SVs but also in many LDCVs by laser scanning confocal miscroscope.After that,pEGFP-VAMP2 and pmCherry-SV2C-siRNA plasmids were cotransfected into N2a,FRAP showed that the intensity ratios and speed of fluorescence recovery were significantly declined,namely the vesicle transport slowed in si-SV2C cells compared with control,which indicated that SV2C played a role in the vesicle axonal transport.3.Mutant Htt impairs the vesicle transport through inhibition of the expression of SV2CIn order to prove that mutant Htt impairs the vesicle transport through inhibition of the expression of SV2C,we adopted reversing experiments by upregulating expression of SV2C on the basis of observating vesicle transport dysfunction in transfected mutant Htt N2a cells.3.1 Overexpressing SV2C can significantly rescue the dysfuntion of vesicle transport resulting from mutant HttThe plasmids pmCherry-Htt-20Q+pEGFP-VAMP2 or pmCherry-Htt-160Q+pEGFP-VAMP2 were cotransfected in N2a,FRAP data showed that the vesicles marked by EGFP-VAMP2 significantly slowed in N2a expressing mutant Htt,compared with control which expressedmCherry-Htt-20Q.Then the plasmids pmCherry-Htt-160Q+pEGFP-VAMP2+pCFP-SV2CorpmCherry-Htt-160Q+pEGFP-VAMP2+pCFP-C1 were cotransfected into N2a,FRAP data showed that the vesicles marked by EGFP-VAMP2 signifi-cantly speeded up in the axonal transport by overexpressing SV2C,which pro-ved that overexpressing SV2C can significantly correct the inhibition of vesicu-lar transport by mutant Htt.3.2 Sodium butyrate can significantly reverse transcriptional inhibition of SV2C,and then reduce the damage of vesicle transport resulted from mutant Htt Our previous study found that repressor element silencing transcription factor（REST）can inhibit expression of SV2C.REST,as a transcript repressor,can recruit histone deacetylase1/2（HDAC1/2）and suspress transcription of many genes.Mutant Htt promotes the HDAC activity.To investigate whether mutant Htt repressed SV2C through promoting the HDAC activity,the effects on SV2C expression of the drugs antagonizing HDAC1/2,for example sodium butyrate,were detected.Applying the different concentrations of sodium butyrate（10¹00μmol/L）to the N2a cells expressing mutant Htt,the results demonstrated that the concentration of 20 to30μmol/L sodium butyrate could significantly reverse SV2C expressing inhibition resulting from mutant Htt by WB and RT-PCR techniques.And the lower concentration（<10μmol/L）or the higher concentration（50¹00μmol/L）played no obvious effects on it.So,to a certain extent,the appropriate concentration of sodium butyrate can correct the the transcriptional inhibition of SV2C causing by mutant Htt.In order to further clarify that mutant Htt impaired vesicle transport through inhibition of SV2C expression and then to analyze the effects of sodium butyrate on the dysfunction of vesicle transport resulting from mutant Htt by FRAP technique.The FRAP data showed that 30μmol/L sodium butyrate effectively rescued the vesicle trafficking dysfunction through reversing SV2C transcriptional dysregulation in N2a cells cotransfected the plasmids pmCherry-Htt-160Q+pEGFP-VAMP2.Conclusion:The mutant Htt selectively repressed the transcriotion of SV2 isoform C,and SV2C participated in the vesicle transport.The mutant Htt can inhibit the expression of SV2C and transport of synaptic vesicle through promoting the HDAC activity which can be activated by REST.