Mutant Huntingtin Inhibits Expression Of Synaptic Vesicle Protein 2C Through Repressor Element Silencing Transcription Factor

Huntington’s disease (HD) is a autosomal dominant inherited neurodegenerative disorder which characteized by abnormal motor symptoms, cognitive decline and psychiatric disturbances. The pathological characteristic feature of HD were projective neurons selective death in deep layers of the cerebral cortex and striatum in the early stage. However, in advanced stages there are obvious degeneration in hypothalamus, hippocampus and other brain regions. HD is caused by the the mutantion of HD gene which encode huntingtin(Htt). We have no effective treatment exists, due in part insufficient understanding of the underlying pathogenic mechanisms of HD.The is emerging evidence that abnormal synaptic transmission underlies the early symptoms of HD, mutant huntingtin elicits toxicity to the functon of the synapse, mutant Htt aggregates in axonal terminals are found in association with a lower density of synaptic vesicles, mutant Htt can affect glutamate uptake and release of synaptic vesicles, and mutant Htt increase its sensitivity of the NMDA agonists.Synaptic vesicles is an important organelles in the presynaptic component, which modulate the synaptic transportation through the release of neurotransmitters. The function of Synaptic vesicles is regulated by a variety of synaptic proteins. The expression and/or post-translational modifications of various synaptic proteins is modulated by mutant Htt which have an abnormal relation with synaptic proteins.Synaptic vesicle protein 2 (SV2) is transmembrane glycoprotein which is present only in secretory vesicles of neural and endocrine cells. Three isoforms, called SV2A, SV2 B and SV2C.SV2C is concentrated in old brain regions, such as the striatum, pallidum, the substantian nigra, the midbrain, olfactory bulb, CA3 and dentate gyrus of the hippocampus which plays a important role in neurotransmitter release and synaptic transmission.The selective expression of S2C in the striatum, pallidum, the substantian nigra hippocampus which were the HD affected area suggests that the effect of mutant Htt on the expression and/or post-translational modifications of SV2C or abnormal interaction is possibly relative to HD.The relation of mutant Htt and SV2C is unclear and there is no report about whether SV2C expression is affected by mutant Htt.Using bioinformatics analysis, we found that SV2C promoter contained a REST (repressor element silencing transcription factor) regulator site which is NRSE(neuron restrictive silencer element). REST mediates transciptional repression of many genes in both neural and non-neural cells by recruitment to its cognate the NRSE regulator sites.Many studies indicate that mutatnt Htt can interact with transcription factors and affect gene transcription. Normal Htt can bind to REST and retain in the cytoplasm, which play a positive regulatory role, while mutant Htt leads to REST nuclear translocation which suppresses the expression of NRSE-controlled genes. whether REST can modulate SV2C expression is not reported.On the basis of whether muatnt Htt has effect on the expression of SV2C, we compared the effect of different REST level on the expressoion of SV2C, analysised wherther REST regulates the transcription of SV2C by binding to the promoter of SV2C, examed the influence of mutant Htt on the actving of SV2C promoter, and furtherly observed wherther mutant Htt promotes the nuclear translocation of REST. Mutant Htt down-regulates expression of synaptic vesicle protein 2CIn order to observe whether mutant Htt affect the SV2C expression. Firstly we used Western blotting and RT-PCR methods to survey the expression of SV2C protein and mRNA in transgenic mouse and HD cell models.We observed that the levels of SV2C decreased in 14w, and progressively reduced in 18w,20w in hippocampus, striatum and cortex of the lines of transgenic mouse models of HD expressing exon 1 of the human HD gene with 82Q CAG repeats(TG mouse). We used immunochistochemistry method to detect that the progressive depletion of SV2C immunoreactivity in the brain of the lines of transgenic mouse models of HD expressing exon 1 of the human HD gene with 150Q CAG repeats(R6/2 mouse). We also found that the progressive down-regulation of SV2C protein expression and mRNA were in 48,72, 96h in transfected plasmids with150Q CAG repeats.The results suggest that mutant Htt down-regulates expression of synaptic vesicle protein 2CREST repress the transcription of SV2C through its binding to the promoter of SV2CIn order to observe whether REST can regulate the expression of SV2C, we researched the different expression of REST effect on the SV2C. Using RT-PCR and Western blotting we demonstrated that overexpression of REST reduced SV2C mRNA and protein levels, and suppressing the expression of REST using REST-SiRNA could up-regulate SV2C mRNA and protein levels.To determine whether REST can bind to the SV2C promoter.We performed a ChIP (Chromatin Imunoprecipitation) assay. The binding of REST site on the SV2C promoter Which included the RNSE (nucleotids-83 to-105bp)was confirmed by the ChIP assay.We next isolated the 5’-flanking region (-322 to +3bp), SV2C(-105 to +3bp), SV2C(-83 to +3bp) of the SV2C gene by PCR of mouse genomic DNA, and the fragments were subcloned into the luciferase reporter plasmid pGL3-basic.We next investigated whether REST could effect the luciferase activity. We found the luciferase activity expressed from the SV2C(-322 to +3bp), SV2C(-105 to +3bp) promote reporter construct significantly reduced in the presence of REST compared with the luciferase activity in the presence of the empty vector. Whereas there was no reduced with SV2C(-83 to +3bp) construct.These results strongly suggest that the SV2C promoter region between nucleotides (-83 to -105)contains the NRSE motif is necessary for the REST-mediated transcriptional regulation of SV2C.The result indicates that REST binds to the cis-acting elements in the SV2C promoter and repress the transcription of SV2C.Mutant Htt inhibits the transcription of SV2C gene via promoting the nuclear translocation of RESTIn order to test whether mutant Htt could repress promoter region of SV2C. SV2C proximal promote fragment of SV2C(-322 to +3bp) were cloned into the pGL3 basic luciferase reporter vector, The luciferase activity significantly reduced when we transfected mutant Htt, compared with normal Htt. But also positively correlated to the concentration. however when we co-transfected siRNA-REST and mutant Htt, there were significant increasing effect on luciferase activity compared with normal Htt. we showed that siRNA-REST can reverse mutant Htt inhibit the promote of SV2C. these suggest that mutant Htt inhibits the transcription of SV2C gene via promoting the nuclear translocation of REST.We next examed whether mutant Htt can increase the binding of REST with the promoter reigon of SV2C. we transfected N2a cells with mutant Htt and normal Htt.then using chromatin immunoprecipitation assay to exame the the extent binding of promote reigon of SV2C. the result showed that REST increased the binding of promote reigon of SV2C transfected mutant Htt compared with normal Htt. This suggest that mutnat Htt can led to increasing of the binding REST with the promote reigon of SV2C and repress the transcription of SV2C. we examed the mRNA and protein of REST using RT-PCR and Western blotting methods and found that no significant change in total protein, but increased expression of nuclear protein in TG and R6/2 mouse and HD cell models. immunohistochemistry analysed that aberrant acumulating of REST in the neuclear in TG and R6/2 mouse and when N2a cells were transfected mutant Htt, compared with normal Htt. these result suggests that mutant Htt do not change the level of REST, but it can cause REST translocate from the cytoplasm to the nucleus.