臺灣博碩士論文加值系統

第七型脊髓小腦性運動失調症(Spinocerebellar ataxia type 7; SCA7)是一種第七型運動失調質(ataxin-7)基因突變導致多麩胺酸(polyglutamine)序列不正常增加而引起的顯性遺傳神經退化疾病。第七型運動失調質的功能目前尚不清楚，其廣泛分佈在腦與周邊組織中。正常的第七型運動失調質在靠近胺基端區域(N-terminal domain)含有4-35個麩胺酸；突變而造成疾病的第七型運動失調質其麩胺酸序列增加至37-306個。儘管第七型運動失調質廣泛表現在腦中各區域，第七型脊髓小腦性運動失調症只在小腦、橋腦、下橄欖、脊髓前角與視網膜有明顯神經退化現象發生。目前，我們藉由腺病毒載體殖入帶有75個麩胺酸的突變第七型運動失調質(ataxin-7-Q75)基因至初級小腦培養神經細胞，以體外細胞培養模式來研究第七型脊髓小腦性運動失調症致病的分子機制。
原位末端標記染色(TUNEL staining)顯示突變第七型運動失調質會引起初級培養小腦細胞死亡。突變第七型運動失調質會活化caspase-3與caspase-9，但不會使caspase-8活化。且突變第七型運動失調質在活化caspase與造成神經細胞凋亡前就引起Bax表現量增加以及Bcl-xL表現量降低。這些結果指出突變第七型運動失調質是經由粒線體路徑引起caspase的活化造成神經細胞凋亡(apoptosis)。
為了探討突變第七型運動失調質進入細胞核是否為致病機轉的必要過程，我們製造了帶有表現截除羧端(C-terminal)使其缺乏核導向信號(NLS; nuclear localization signals)的突變第七型運動失調質基因的重組腺病毒。截除羧端只表現1-601個胺基酸的突變第七型運動失調質(ataxin-7-Q75(1-601))，實驗顯示其無法進入細胞核內，但仍顯著地引起神經細胞凋亡，其神經毒性比ataxin-7-Q75略低。此結果顯示細胞核與細胞質都是突變第七型運動失調質在細胞內產生致病機轉的作用位置。
核因數κB (NF-κB；nuclear factor kappa B)藉由促進表現其下游抗凋亡因子Bcl-xL增加，被視為是維持小腦細胞存活的重要轉錄因子。電泳遷移率改變分析(Electrophoretic mobility shift assay;EMSA)顯示突變第七型運動失調質會抑制NF-κB活性。在給予NF-kB抑制劑的實驗中，有顯著的細胞死亡發生，並且caspase-3與caspase-9的活化增加，以及Bcl-xL表現量降低。這些結果顯示突變第七型運動失調質是藉由降低NF-κB轉錄活性造成下游的Bcl-xL表現量降低而導致小腦細胞凋亡。
蛋白酶體(proteasome)已知在活化NF-kB上扮演重要角色，其功能為裂解IkB蛋白質而促使NF-B二元體能進入細胞核內。實驗數據顯示ataxin-7-Q75與ataxin-7-Q75(1-601)都能降低蛋白酶體活性，而使IkB堆積在細胞質。此結果造成NF-kB次單位元p65在細胞質增加其表現量，在細胞核則有降低的現象。此結果代表突變第七型運動失調質藉由抑制蛋白酶體作用與IkB過量堆積在細胞質，造成NF-kB活性降低導致細胞凋亡。
目前的研究顯示突變第七型運動失調質是透過促進表現Bax與抑制表現Bcl-xL來活化粒線體細胞凋亡路徑來引起小腦神經細胞死亡。突變第七型運動失調質在細胞內作用位置包含細胞核與細胞質。在細胞質內，突變第七型運動失調質藉由影響蛋白酶體功能使NF-kB活性降低來引起Bcl-xL表現降低。造成小腦細胞內Bax表現增加的分子機轉仍待進一步闡明。藉由本實驗，我們進一步瞭解第七型脊髓小腦性運動失調症的致病機轉並可能被用於發展治療第七型脊髓小腦性運動失調症的方法。

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disease caused by mutant polyglutamine-expanded ataxin-7. Ataxin-7 is a novel protein of unknown function and widely distributed in the brain and peripheral tissues. Wild-type ataxin-7 contains 4-35 glutamines at the N-terminal domain, polyglutamine tract of disease- causing ataxin-7 expands to 37-306 glutamines. Despite the wide distribution of ataxin-7 in the brain, SCA7 neurodegeneration is only prominent in the cerebellum, pons, inferior olive, anterior horn of spinal cord and retina. In the present study, we used in vitro cellular model of SCA7 to investigate the molecular mechanisms by introducing adenovector encoding ataxin-7-Q75 into primary cerebellar cultured neurons.
TUNEL staining studies showed that mutant ataxin-7-Q75 induced apoptotic death of cultured cerebellar neurons. Mutant polyglutamine ataxin-7-Q75 activated caspase-3 and caspase-9 formations without inducing the formation of active caspase-8. Upregulation of Bax and downregulation of Bcl-xL expression caused by ataxin-7-Q75 preceded caspase activation and neuronal death. These findings strongly suggest that mutant ataxin-7-Q75 activates caspases and induces apoptotic neuronal death via mitochondria-mediated apoptotic pathway.
In order to investigate whether nuclear localization is an essential process for pathological mechanism, recombinant adenovirus containing the cDNA of C-terminal truncated ataxin-7-Q75(1-601), which lacks nuclear localization signals(NLS), was prepared. Ataxin-7-Q75(1-601) failed to enter into the nucleus and still induced significant neuronal death. Compared with full-length ataxin-7-Q75, the neurotoxic effect of ataxin-7-Q75(1-601) was less pronounced. The results imply that both nucleus and cytoplasm are the subcellular action sites of ataxin-7- Q75.
NF-kB has been regarded as an important transcription factor in supporting viability of cerebellar neurons by inducing downstream anti-apoptotic factor Bcl-xL expression. Electrophoretic mobility shift assay (EMSA) showed that mutant ataxin-7-Q75 inhibited NF-kB activity. Treatment of NF-kB inhibitor resulted in significant neuronal loss, caspase-3 and caspase-9 activation, and Bcl-xL downregulation. These results suggest that ataxin-7-Q75 reduce Bcl-xL expression by impairing NF-kB transcription level, leading to induces apoptotic death of cultured cerebellar neurons.
Proteasome-mediated degradation of IkB proteins has been known to play an important role in NF-kB activation by permitting nuclear translocation of NF-kB dimer. Our data revealed that ataxin-7-Q75 or ataxin-7-Q75(1-601) inhibited proteasome activity and accumulated IkBa in the cytoplasm. As a result, both ataxin-7-Q75 and ataxin-7-Q75 (1-601) upregulated NF-kB subunit p65 in the cytosolic fraction and downregulated NF-kB p65 level in the nuclear fraction. Our data strongly suggest that mutant ataxin-7-Q75 downregulates NF-kB activity and causes cerebellar neuronal death by impairing proteasome activity and increasing the cytosolic level of IkBa protein.
In summary, the present study provides the evidence that polyglutamine-expanded ataxin-7-Q75 activates mitochondria-mediated apoptotic cascade and induces neuronal death by upregulating Bax expression and downregulating Bcl-xL expression of cerebellar neurons. Our results indicate that subcellular action sites of mutant ataxin-7-Q75 include the nucleus and cytoplasm. Within the cytoplasm, ataxin-7-Q75 downregulates Bcl-xL level by impairing the proteasome function and NF-kB activity. Further studies are required to elucidate the molecular mechanism by which mutant ataxin-7-Q75 upregulates Bax expression of cerebellar neurons. The results presented here should shed a light on the pathogenic mechanism of SCA7 and lead to the possible therapeutic strategies of SCA7.

Acknowledgements iv
Abstract (Chinese) v
Abstract (English) viii
Abbreviations xi
Contents xii
I. Introduction 1
1.1 Spinocerebellar ataxia type 7 and apoptotic neuronal death 1
1.2 Subcellular action site of mutant polyglutamine ataxin-7 3
1.3 The involvement of downregulated NF-kB function in mutant
ataxin-7-induced neuronal death 4
II. Specific aims 7
III. Materials and methods 8
3.1 Preparation of cultured cerebellar neurons 8
3.2 Preparation of recombinant adenoviruses 8
3.3 Immunoblotting analysis 9
3.4 Analysis of neuronal death 10
3.5 Immunofluorescence staining and confocal microscopy 11
3.6 Proteasome activity assay 12
3.7 Preparation of nuclear and cytosolic extract 12
3.8 Electrophoretic mobility gel shift assay (EMSA) 13
3.9 Statistics 13
IV. Results 14
4.1 Mutant polyglutamine-expanded ataxin-7-Q75 induces apoptotic death of cerebellar neurons by upregulating Bax expression and downregulating Bcl-xL expression 14
4.2 Subcellular action site of mutant ataxin-7-Q75-induced
neurotoxicity 17
4.3 Mutant ataxin-7-Q75 downregulates Bcl-xL expression by
impairing proteasome function and NF-kB activity 19
V. Disscussion 23
VI. References 30
VII. Figures 39

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