小腦脊髓運動失調症第三型 (Spinocerebellar ataxia type 3；SCA3) ，又稱為Machado-Joseph Disease簡稱MJD，是一種晚發性體染色體顯性（Autosomal Dominant）的遺傳性疾病。而此疾病造成腦部神經細胞退化及死亡的分子機制和原因，到目前為止仍不清楚。在本篇論文中，我們利用先前已構築能夠穩定表達全長突變ataxin-3蛋白的神經細胞，SK-N-SH-MJD78細胞株，來觀察熱休克蛋白27 (Hsp27) 的動態表達。在突變ataxin-3存在的疾病過程，熱休克蛋白27在多麩醯胺酸蛋白所導致的細胞死亡中，扮演抑制的角色。實驗結果發現，相較於正常神經細胞，在SK-N-SH-MJD78早期繼代培養細胞中，熱休克蛋白27表達含量下降，但於晚期細胞中卻有明顯上升趨勢。然而，於MJD轉殖基因鼠及病人死後腦組織切片，經免疫染色法偵測熱休克蛋白27後，可發現其含量明顯比正常腦組織所偵測到的含量要高，顯示熱休克蛋白27在MJD疾病末期反而有正向調節現象。當給與細胞熱刺激處理後發現，表達突變ataxin-3蛋白的細胞和正常細胞，同樣擁有正常且相似的熱刺激反應，使熱休克蛋白27表達含量上升。然而，對於飢餓及外加氧化壓力下，表達突變ataxin-3蛋白的細胞與正常細胞相比，卻顯示較低的耐受性，使細胞有較高的死亡率。我們的結果證實，熱休克蛋白27的表達，的確受到長期表現於細胞中的全長ataxin-3蛋白所影響。我們因此提出假設，在疾病早期過程，熱休克蛋白27表達量降低，影響細胞對抗壓力所引發細胞毒性的能力；於疾病末期，多麩醯胺酸濃度增加，或其造成的核內包涵物所導致的毒性持續且長時間表達，引發細胞產生如熱刺激反應的壓力反應，提高熱休克蛋白27表達含量，來抵禦細胞毒性。但此反應引發的保護機制，最終仍無法回復細胞毒性所造成的不可逆的傷害。 Machado-Joseph disease (MJD)/Spinocerebellar Ataxia Type 3 (SCA3) is an autosomal dominant spinocerebellar degeneration characterized by a wide range of clinical manifestations. The molecular mechanisms underlying the selective neuronal death typical of MJD/SCA3 are unknown. In this study, human SK-N-SH neuroblastoma cells stably transfected with full-length MJD with 78 CAG repeats were assayed for the dynamic expression of Hsp27, known as a suppressor of poly (Q) mediated cell death, in the presence of mutant ataxin-3 during disease progression. Even though a dramatic decrease of Hsp27 expression was observed in the earlier generation of SK-N-SH-MJD78 cells, the aged generation showed a significant increase of Hsp27 to almost the same level of that of the parental cells. Furthermore, immunohistochemical analysis of MJD transgenic mice brain and post-mortem human brain tissues showed increased expression of Hsp27 compared to normal control brain, suggesting an up-regulation of Hsp27 in end stage of MJD. While heat shock was used to assess the stress response, cells expressing mutant ataxin-3 displayed normal response upon heat shock stimuli when compared to the parental cells. However, cells expressing mutant ataxin-3 showed weak tolerance upon serum depletion and oxidative stress. Our results demonstrated that the protein expression of Hsp27 is greatly influenced by the accumulation and long-term chronic exposure of expanded full-length ataxin-3. We proposed that during the early disease stage, the reduction of Hsp27 synthesis mitigated the ability of cells to cope with cytotoxicity induced by cellular stresses. In the late stage of disease, after prolonged stressful conditions of polyglutamine cytotoxicity, even though the increased concentration of polyglutamine or the appearance of nuclear inclusions of polyglutamine eventually stimulate HSP response, this increased HSP response still cannot reverse the global dysfunction of cellular proteins due to accumulation of cytotoxic effects.
