【作者】 易继平；

【导师】 唐北沙；

【作者基本信息】 中南大学 ， 神经病学， 2009， 博士

【摘要】 背景：遗传性脊髓小脑型共济失调(hereditary spinocerebellar ataxia, SCAs)是一类常见的神经系统遗传病,根据neuromuscualr website (http://neuromuscular.wustl.edu/ataxia/domatax.html)截止到2009年11月5日发布的信息,迄今至少已定位了30种基因型,其中18个疾病基因已被克隆。脊髓小脑型共济失调3型(spinocerebellar ataxia type 3/Machado-Joseph disease,SCA3/MJD)为最常见的SCAs亚型,在中国人群中几乎占常染色体显性遗传脊髓小脑型共济失调的60%,其致病基因-MJD1基因编码区的3’端含有一段CAG三核苷酸重复序列,正常人重复次数约12-40次,而SCA3/MJD患者可达52-86次,且CAG异常重复次数越多,发病年龄越早,症状越严重。迄今为止已发现九种神经退行性疾病是由于致病基因开放阅读框(open reading frame, ORF)中CAG三核苷酸重复扩展突变所致,包括SCA1、SCA2、SCA3/MJD、SCA6、SCA7、SCA17,以及亨廷顿舞蹈病(huntington disease,HD)、脊髓延髓肌萎缩症(spinal and bulbar muscular atrophy, SBMA)、齿状核红核苍白球路易体萎缩症(dentatorubral-pallidoluysian atrophy, DRPLA)。该类疾病统称为多聚谷氨酰胺(polyglutamine, polyQ)疾病。目前有关polyQ疾病的发病机制尚未完全阐明,临床上亦缺乏有效的治疗。组蛋白乙酰化是一种翻译后修饰,能导致染色质结构及转录调控的改变。目前认为组蛋白乙酰化与去乙酰化水平在组蛋白乙酰转移酶(histone acetyltransferase,HAT)和组蛋白去乙酰化酶(histone deacetylase,HDAC)的作用下保持着动态平衡,从而维持基因正常的转录调控功能。有研究表明组蛋白乙酰化水平降低可能是引起细胞毒性作用、促发polyQ疾病转录异常的关键步骤。国内外不少学者已经在体外证实组蛋白去乙酰化酶抑制剂(histone deacetylase inhibitors, HDACIs)能够抑制polyQ蛋白的毒性作用,缓解HD、SBMA、DRPLA等的病情,延缓疾病进展。然而对于同样属于polyQ病的SCA3/MJD疾病,至今国内外尚未见有关运用HDACIs进行治疗的相关研究报道。Hv(因涉及到保密性问题,本实验研究中所运用的组蛋白去乙酰化酶抑制剂统一用“Hv”表示)属于HDACIs中的短链脂肪酸类,其半衰期长,毒副作用小,已在临床上用于治疗其他疾病多年。Hv除了通过抑制组蛋白去乙酰化而调节基因转录外,还可通过多条途径,如通过诱导热休克蛋白(heat shock protein, HSP)的表达来保护神经元等。而在前期工作中,本课题组成员已证实HSP22对SCA3/MJD转基因果蝇模型具有保护作用,因此我们选用HDACIs中的Hv来进行SCA3/MJD的治疗研究。目的：探讨Hv对SCA3/MJD细胞模型及转基因果蝇模型的保护作用及其机制,为临床上运用HDACIs治疗SCA3/MJD疾病提供科学的理论依据。方法：1.应用荧光显微镜技术了解Hv对SCA3/MJD细胞模型中异常蛋白聚集体的影响。2.运用Western-blot技术检测Hv对野生型、含polyQ扩展突变型ataxin-3蛋白及乙酰化组蛋白H3和H4表达水平的影响。3.应用PI/Annexin-V-FITC双染流式细胞仪技术分析Hv对SCA3/MJD细胞模型凋亡率的影响。4.利用GAL4/UAS系统构建在眼睛及神经系统表达致病蛋白的SCA3/MJD转基因果蝇模型。5.利用光学显微镜和扫描电镜观察Hv对SCA3/MJD转基因果蝇模型复眼的影响。6.通过表型观察,了解Hv对SCA3/MJD转基因果蝇模型爬行能力及寿命的影响。结果：1.野生型ataxin-3蛋白在细胞中呈弥散分布,而polyQ扩展突变型ataxin-3蛋白能在细胞中形成异常蛋白聚集体；不同剂量Hv干预后,polyQ扩展突变型SCA3/MJD细胞模型中异常蛋白聚集体阳性细胞率及polyQ扩展突变型ataxin-3蛋白的表达水平较其未给Hv干预对照组相比无明显变化。2.PI/Annexin V-FITC双染流式细胞仪结果显示：未予以Hv干预的空载体pEGFP-N1转染组及野生型pEGFP-N1-ataxin-3-20Q转染组细胞早期凋亡率无明显差异,而pEGFP-N1-ataxin-3-68Q转染组细胞的早期凋亡率明显高于空载体组及野生型组细胞；不同剂量的Hv干预polyQ扩展突变型SCA3/MJD细胞模型后,其早期细胞凋亡率明显下降,且剂量越大凋亡率下降越明显。3.Western-blot检测发现：未予以Hv干预的空载体pEGFP-N1转染组及野生型pEGFP-N1-ataxin-3-20Q转染组细胞中乙酰化组蛋白H3、H4表达水平无明显差别,而polyQ扩展突变型SCA3/MJD细胞模型中乙酰化组蛋白H3、H4表达水平低于空载体组和野生型组。不同剂量Hv干预后,随着Hv剂量的增加,polyQ扩展突变型SCA3/MJD细胞模型的乙酰化组蛋白H3、H4表达水平逐渐升高。4.利用GAL4/UAS系统分别构建了在果蝇眼睛和神经系统中以不同水平表达MJDtr-Q78蛋白的SCA3/MJD转基因果蝇模型。该模型表现为进行性的复眼结果破坏、色素脱失,爬行能力下降及寿命缩短,且其严重程度与MJDtr-Q78蛋白的表达量及表达部位有关。5.Hv干预SCA3/MJD转基因果蝇模型后,其复眼结构破坏及色素脱失好转,且Hv剂量越大,复眼改善越明显。6.Hv干预SCA3/MJD转基因果蝇模型后,其爬行能力得到一定程度的改善,且Hv剂量为1.0mM到1.5mM时爬行能力改善最明显。7.一定剂量的Hv (0.5mM)能延长SCA3/MJD转基因果蝇模型的寿命,而当Hv剂量过大时反而会缩短SCA3/MJD转基因果蝇模型及野生型果蝇的寿命。结论：1.进一步证实polyQ扩展突变能导致细胞内异常蛋白聚集体的形成,发现Hv不能减少SCA3/MJD细胞模型中异常蛋白聚集体的形成和ataxin-3蛋白的表达。2.进一步证实polyQ扩展突变能诱导细胞凋亡,发现Hv可抑制polyQ扩展突变型ataxin-3蛋白的细胞毒性作用,从而降低SCA3/MJD细胞的早期凋亡率。3.发现SCA3/MJD细胞模型中存在组蛋白H3、H4乙酰化水平的降低,而Hv可提高SCA3/MJD细胞模型的组蛋白H3、H4乙酰化水平。4.成功地构建了在果蝇眼睛和神经系统中以不同水平表达MJDtr-Q78蛋白的SCA3/MJD转基因果蝇模型,进一步证实扩展突变型polyQ蛋白的表达量及表达部位与SCA3/MJD转基因果蝇模型的表型及严重程度存在相关性。5.发现一定剂量的Hv能减轻SCA3/MJD转基因果蝇模型的复眼结构破坏与色素脱失,并能改善SCA3/MJD转基因果蝇模型的爬行能力和延长其寿命。即一定剂量的Hv在一定程度上具有保护SCA3/MJD转基因果蝇模型的作用。更多还原

【Abstract】 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.更多还原