Research On Protective Effect Of Hv In Cells And Transgenic Drsophila Models Of SCA3/MJD

Background:The hereditary spinocerebellar ataxias(SCAs) are a heterogeneous group of neurodegenerative disorders.To date, at least 30 gene loci responsible for SCAs have been mapped, in which 18 pathogenic genes have been cloned. Among them, spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is the most common subtype, which accounts for almost 60% SCAs in China. SCA3/MJD disease is an autosomal dominant neurodegenerative disorder caused by CAG trinucleotide repeat expansion within the coding region of MJDl gene (also called ataxin-3 gene).Normal MJD1 contains 12-40 glutamines near the C-terminus, and polyglutamine tract expands to 52-86 glutamines in disease-causing mutant MJDl.So far nine disorders have been found to be caused by CAG trinucleotide repeat expanded mutation within the open reading frame (ORF) of pathogenic gene, including SCA1,SCA2,SCA3/MJD, SCA6, SCA7,SCA17,huntington disease (HD),spinal-bulbar muscular atrophy (SBMA) and dentatorubral-pallidoluysian atrophy (DRPLA).Such diseases are referred to polyglutamine (polyQ) diseases.Up to now, the pathomechanism of polyQ diseases have not been fully explained. It is generally hypothesized that deregulation of transcription is a key contributor to the pathogenesis of DRPLA, HD and other polyQ diseases. However there are currently no known cure or effective treatment for these diseases.As a post-translational modification, the acetylation and deacetylation of histones are modulated by the interplay between histone acetyltransferase (HAT) and histone deacetylase (HDAC),which results in modification to chromatin structure and regulation of transcription. It is generally thought that HAT activity leads to an increase in gene transcription through the opening of chromatin architecture by adding acetyl groups.Conversely, HDAC removes acetyl group, which leads to gene repression through chromatin condensation. Studies in numerous have shown that aberrant activity of histone deacetylases or histone acetyltransferase might be an underlying mechanism of transcriptional dysregulation in polyQ diseases.It suggests that the balance of histone acetylation is a good target for therapeutic intervention of this kind of diseases.Consistent with this idea, numerous studies have shown a therapeutic role for HDAC inhibitors (HDACIs) such as sodium butyrate (SB),TSA, suberoylanilide hydroxamic acid(SAHA) and so on, as candidate drugs for the treatment of HD,DRPLA and SBMA.However, the effects of HDACIs on models of SCA3/MJD disease have never been tested.Hv (due to confidentiality issues,"Hv" reprensents the HDACIs we used in the study) is one of the HDACIs and belongs to short-chain fatty acids.It has been used clinically in other diseases for many years.In addition to inhibit histone acetylation and regulation of gene transcription, Hv can also play an important role in protecting neurons by a number of ways, such as inducing the express of heat shock protein (HSP).We have confirmed that expression of HSP22 protects the SCA3/MJD from neurodegeneration on Drosophila models. Based on these points, here we selected Hv to investigate the therapeutic effects of HDACIs in SCA3/MJD disease.Objective:To further investigate the mechanism of SCA3/MJD disease and examine the therapeutic potential of Hv in SCA3/MJD cells and transgenic Drosophila models.Methods:1.Fluorescence technique was utilized to study the effect of Hv on the formation of abnormal aggregate by polyglutamine expanded ataxin-3.2.Western-blot were undertaken to study the effect of Hv on the expression level of polyglutamine expanded ataxin-3 protein, acetyl-histone H3 and acetyl-histone H4 protein in SCA3/MJD cell model.3.Flow cytometry of PI/Annexin-V-FITC was used to study the effect of Hv on the apoptosis of wild-type and polyglutamine expanded ataxin-3.4.SCA3/MJD transgenic Drosophila models were constructed by GAL4/UAS system.5.Light microscope and scanning electron microscope(SEM) were used to observing the effect of Hv on the eye morphology of SCA3/MJD transgenic Drosophila models.6.Observing the effect of Hv on the climbing ability and lifespan of SCA3/MJD transgenic Drosophila models.Results:1.Using GFP fluorescence technique, we found that ataxin-3-20Q distributed scattered and ataxin-3-68Q aggregated in nucleus or cytoplasm. We analyzed the quantitation of abnormal aggregations in polyglutamine expanded ataxin-3 group without or with different doses of Hv intervention, but found no significantly difference (P>0.05).By western-blot, we also found no difference on the expression level of polyglutamine expanded ataxin-3 protein between Hv intervention group and controls.2.The result of flow cytometry showed the early apoptosis rate of pEGFP-N1-ataxin-3-68Q transfected cells was significantly higher than the empty pEGFP-N1 transfected group and pEGFP-N1-ataxin-3-20Q transfected group (P<0.05).After Hv intervention, the early apoptosis rate of pEGFP-N1-ataxin-3-68Q transfected cells was significantly decreased, and the greater the dose, the lower the apoptosis rate.3.The empty pEGFP-N1 vector transfected cells and pEGFP-N1-ataxin-3-20Q transfected cells had the same expression level of acetyl-histone H3 and acetyl-histone H4 protein, but the pEGFP-N1-ataxin-3-68Q transfected cells(SCA3/MJD cell model) had lower. After the intervention of different dosage of Hv, the expression level of acetyl-histone H3 protein of pEGFP-N1-ataxin-3-68Q transfected cells was gradually increased.4.Using GAL4/UAS transformation system, SCA3/MJD transgenic Drosophila models were constructed by gmr-GAL4 and elav-GAL4 which drive target selective gene expression in developing eyes and neurons, respectively. In no case did expression of MJDtr-Q27 protein have a phenotypic effect.But expression of MJDtr-Q78 protein caused progressive loss of pigmentation and collapse of the eye, decreasing of climbing ability and shortening of lifespan. The severity of the phenotype was relavant to the expression level and site of MJDtr-Q78 protein.5.As for SCA3/MJD transgenic Drosophila models,the loss of pigmentation and collapse of the eye was mitigated by feeding Hv.6.The climbing ability of SCA3/MJD transgenic Drosophila models was improved by feeding Hv.Especially, 1.OmM or 1.5mM Hv could obviously improve it's climbing ability. 7.We found that proper dosage of Hv (0.5mM) could extend the SCA3/MJD transgenic Drosophila lifespan. However too much Hv would shorten the lifespan of both SCA3/MJD transgenic Drosophila and wild-type Drosophila.Conclusion:1.For the first time, we find that Hv can not prevent the abnormal protein aggregation, or the expression level of polyglutamine expanded ataxin-3 protein in SCA3/MJD cell model.2.We further confirm that polyglutamine expansion mutation can induce apoptosis, and for the first time we find that Hv can inhibit the cytotoxicity of polyglutamine expanded ataxin-3 protein, thereby reduce the early apoptosis rate of SCA3/MJD cell model.3.For the first time, we find that Hv can increase the expression level of acetyl-histone H3 and acetyl-histone H4 protein which have been decresed because polyglutamine expanded ataxin-3 mutation in SCA3/MJD cell model.4.We successfully constructed SCA3/MJD transgenic Drosophila models which selective express human MJDtr-Q78 protein with different levels in developing eyes or neurons respectively. We further confirm that the expression level and site of polyglutamine expanded ataxin-3 (MJDtr-Q78)protein are concerned to the severity of SCA3/MJD symptoms. 5.For the first time, we find that proper dosage of Hv has therapeutic potential in SCA3/MJD transgenic Drosophila models by reducing pigment loss and structural damage of eyes, improving climbing ability and extending lifespan.