臺灣博碩士論文加值系統

第三型脊髓小腦性運動失調症(Spinocerebellar ataxia type 3;SCA3)或麥克多喬瑟夫疾病(Machado-Joseph disease)是第三型運動失調質(ataxin-3)之基因突變所導致的遺傳型顯性神經病變，CAG核酸序列之增長進而增加第三型運動失調質內麩酸胺 ( glutamine)數目是引起此疾病的突變模式。正常第三型運動失調質在C-terminus含有12- 41個麩酸胺，引起第三型脊髓小腦性運動失調症的第三型運動失調質在C-terminus卻含有62- 84個麩酸胺。最近我們利用大白鼠之小腦(cerebellum)、紋狀體(striatum)及黑質(substantia nigra)神經核的培養神經元(cultured neurons)探討突變多麩酸胺第三型運動失調質引起腦部神經元死亡的分子機轉，發現突變多麩酸胺第三型運動失調質經由增加Bax表現及抑制Bcl-xL表現，進而活化由粒線體主導之凋亡蛋白酵素(caspase)路徑引起培養神經元的細胞凋亡(apoptosis)。
雖然離體細胞模式(in vitro cellular model)被廣泛地用於研究腦部疾病之病理機轉，但仍需要進一步以活體動物實驗(in vivo animal model)證實由培養神經元得到的重要發現。因此我們已成功地培養表現突變多麩酸胺 性第三型運動失調質的SCA3基因轉殖鼠，我們在本實驗中即是要利用這些基因轉殖鼠進行研究，探討突變多麩酸胺第三型運動失調質是否在活體動物也以相同的分子機轉引起腦部神經元的細胞凋亡。
依據以往對SCA3病患的研究，腦部中的橋腦核(pontine nucleus)及黑質(substantia nigra)神經核是兩個主要神經退化的區域，也因此我們的實驗主要針對此兩區域進行研究。首先利用西方碘墨法(Western blot)，結果發現在SCA3基因轉殖鼠的小腦及腦幹有突變多麩酸胺第三型運動失調質蛋白的表現，而正常非基因轉殖鼠則無法偵測到，另外針對正常老鼠以及SCA3基因轉殖鼠進行一系列的運動功能測試，包括：滾輪測試(Rotarod test)、足跡分析法(Footprint analysis)、運動活性測定(Locomotor activity)、翻正反射(Righting reflex)等等，結果顯示正常老鼠與SCA3基因轉殖鼠有顯著的差異，SCA3基因轉殖鼠不僅在體重上的增加明顯減少，運動功能上的障礙亦極為明顯。另外利用蘇木紫及伊紅染色（H &E staining）來看神經細胞死亡的狀態，跟正常老鼠比較起來，在SCA3基因轉殖鼠的橋腦核及黑質神經核有顯著的細胞死亡現像。而原位末端標記染色（TUNEL staining）顯示突變多麩酸胺第三型運動失調質會使得SCA3基因轉殖鼠的橋腦核及黑質神經核之細胞進行細胞凋亡。
既然神經細胞的死亡是藉由細胞凋亡的形式進行，其中的機轉是我們想探討的，利用西方碘墨法，結果顯示，在SCA3基因轉殖鼠中，包含有橋腦核和黑質神經核的腦幹部位，有活化caspase-3和caspase-9蛋白的表現，而在橋腦和黑質的腦組織切片中，免疫組織染色(Immunohistochemical staining)的結果亦顯示，只有在SCA3基因轉殖鼠中才有活化caspase-3和caspase-9蛋白的表現，正常非基因轉殖鼠則缺乏。這些發現顯示突變多麩酸胺第三型運動失調質很可能是經活化粒線體路徑引起細胞凋亡。
Bcl-2家族蛋白中包含Bax和Bcl-xL，此兩者是調節粒線體細胞凋亡路徑中非常重要的蛋白，利用西方碘墨法以及RT-PCR技術，結果顯示，相似於離體細胞模式，無論在蛋白或mRNA層次，於SCA3基因轉殖鼠中包含橋腦核和黑質神經核的腦幹部位，突變多麩酸胺第三型運動失調質明顯增加了Bax表現及抑制Bcl-xL的表現。
符合以往對SCA3病患的研究，我們利用SCA3基因轉殖鼠所得到的結果亦顯示，神經細胞死亡的主要區域在橋腦核和黑質神經核。綜而言之，我們的研究結果顯示，在表現突變多麩酸胺第三型運動失調質的SCA3基因轉殖鼠中，引起橋腦核及黑質神經元細胞凋亡的分子機轉很有可能是藉由提升Bax的表現以及抑制Bcl-xL的表現，進而經由粒線體路徑引起caspase的活化造成神經細胞的凋亡。藉由本實驗，我們進一步瞭解第三型脊隨小腦性運動失調症的致病機轉並可能被用於發展治療第三型脊隨小腦性運動失調症的方法。

Spinocerebellar ataxia type 3 (SCA3) or Machado-Joseph disease is an inherited autosomal dominant neurodegenerative disorder caused by abnormal expansion of CAG repeats coding for polyglutamine tract within a protein called ataxin-3. Normally, ataxin-3 contains 12- 41 glutamines near the C-terminus, and the polyglutamine tract expands to 62-84 glutamines in disease-causing mutant ataxin-3. Recently, we’ve studied the molecular mechanism underlying polyglutamine ataxin-3-induced neurodegeneration with aid of primary neuronal cultures of cerebellum, striatum and substantia nigra. The study demonstrated that mutant polyglutamine ataxin-3-Q79 activated mitochondrial apoptotic pathway and induced neuronal death by upregulating Bax expression and downregulating Bcl-xL expression.
Although in vitro cellular model has been widely used to study pathogenic mechanisms of neurological disorders, important findings observed from in vitro cellular model have to be corroborated by in vivo animal model. Thus we have generated transgenic mice expressing polyglutamine-expanded ataxin-3-Q79. In the present study, SCA3 transgenic mice were used to investigate molecular mechanism by which mutant ataxin-3-Q79-induces apoptotic neuronal death in vivo.
Previous neuropathological studies using SCA3 human brain, prominent neuronal death was found in the pontine nucleus and substantia nigra. Thus our study was mainly focus in the two major degenerative regions of SCA3 brain. Western blot analysis indicated that mutant ataxin-3-Q79HA was expressed in the cerebellum and brainstem of ataxin-3-Q79HA transgenic mouse. Ataxin-3-Q79HA was absent in the brain of control non-transgenic mouse. Transgenic mice expressing ataxin-3-Q22 or ataxin-3-Q79 and control non-transgenic mice were assessed with different behavioral tests, which include rotarod test、footprint analysis、locomotor activity and righting reflex. Behavioral tests indicated that ataxin-3-Q79 transgenic mice exhibited weight loss and several symptoms of motor dysfunction. The findings clearly indicate that ataxin-3-Q79HA transgenic mice exhibit neurological phenotypes of SCA3.
H &E staining was performed to visualize neuronal loss in the brain of ataxin-3-Q79HA transgenic mice. Compared to wild-type non-transgenic mice, a significant decrease in the number of neurons was found in the pontine nucleus and substantia nigra neurons of ataxin-3-Q79 transgenic mice. The result of TUNEL staining indicate that polyglutamine-expanded ataxin-3-Q79 causes apoptotic death of pontine and substantia nigra neurons in SCA3 transgenic mice.
Although apoptotic neuronal death is involved in SCA3, the molecular mechanism of polyglutamine-expanded ataxin-3-Q79 induce apoptotic cell death must to be further study. Immunoblotting assay was performed to visualize the formation of active caspase-3 or caspase-9 in the brainstem region containing pontine nucleus and substantia nigra of ataxin-3-Q79 transgenic mice. Cleaved active caspase-3 and active caspase-9 were present in the brainstem of ataxin-3-Q79 transgenic mice but were not found in the brainstem extract of wild-type mice. The data of immunohistochemical staining show that active caspase-3 and active caspase-9 were found in pontine and substantia nigra neurons of SCA3 transgenic mice but were not present in the neurons of wild-type mice. These findings suggest that in the brainstem of ataxin-3-Q79 transgenic mice, mutant ataxin-3-Q79 cause apoptotic neuronal death through mitochondrial apoptotic pathway.
Bcl-2 family of proteins, including Bax and Bcl-xL, are key regulators of mitochondrial apoptotic pathway in the nervous system. The results of western blot analysis and RT-PCR assay provide the evidence that similar to results observed from in vitro cellular model of SCA3, mutant ataxin-3-Q79 also induces apoptotic neuronal death by upregulating Bax expression and downregulating Bcl-xL expression in vivo.
In summary, in accordance with previous neuropathological studies using SCA3 human brain, our histological analysis indicates that in the brain of ataxin-3-Q79 transgenic mice, prominent neuronal death was found in the pontine nucleus and substantia nigra. Our results suggest that polyglutamine-expanded ataxin-3-Q79 activates mitochondrial apoptotic pathway and induces neuronal death by upregulating Bax expression and downregulating Bcl-xL expression in the pontine nucleus and substantia nigra of ataxin-3-Q79 transgenic mice. The results presented here should shed a light on the pathogenic mechanism of SCA3 and lead to the possible therapeutic strategies of SCA3.

CONTENTS
Abstract (Chinese) i
Abstract (English) iv
Abbreviations vii
Contents viii
I. Introduction 1
II. Specific aims 8
III. Materials and methods 9
3.1 Generation of transgenic mice 9
3.2 Behavioral tests 10
3.3 Western blot analysis 11
3.4 Real-time quantitative RT-PCR assay 12
3.5 Immunohistochemical staining 14
3.6 TUNEL staining 15
3.7 Statistics 15
IV. Results 16
4.1 Generation of transgenic mice expressing human
disease-causing ataxin-3 16
4.2 Transgene expression 17
4.3 Ataxin-3-Q79HA transgenic mice exhibit various ataxic
symptoms of motor dysfunction 18
4.4 Neuropathological analysis of ataxin-3-Q79HA
transgenic mice 19
4.5 Polyglutamine-expanded ataxin-3-Q79 causes apoptotic
death of pontine and substantia nigra neurons in vivo 20
4.6 Polyglutamine-expanded ataxin-3-Q79 upregulates
Bax and downregulates Bcl-xL expression in vivo 23
V. Disscussion 25
VI. References 32
VII. Figures 40

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