| Abstract: | Machdo-Joseph Disease (MJD)是屬於體染色體顯性遺傳神經退化性疾病其中一種，又名為小腦脊髓運動失調症第三型(SCA3)，發病原因與其基因座內CAG三核酸重覆序列倍增突變有關。而對於此段核酸重覆序列所轉譯出的多醯胺麩胺酸(polyglutamine)的蛋白產物之正常功能與致病機轉尚未明瞭。只知道此段核酸重覆序列愈長則病症愈嚴重；而且經由病人腦部切片以細胞免疫染色實驗得知MJD/ataxin-3 蛋白會聚積於腦部神經核內形成包涵物(inclusion body)，並猜測此核內包涵物會影響神經退化的病程發展。而核內包涵物究竟是促使神經細胞走向細胞程序性凋亡(apoptosis)亦或是保護神經細胞其作用並不清楚。本實驗中是將會表達全長MJD致病蛋白的質體藉由脂蛋白包裹的方式送入人類神經腫瘤母細胞中，由於此質體本身具有G418抗藥性基因，所以於培養液中添加G418藥物，若此質體DNA能嵌入人類神經腫瘤母細胞的染色體DNA中，長期培養後可以篩選出可穩定表達MJD致病蛋白的細胞株。實驗結果篩選三株可存活於含G418藥物培養液的人類神經腫瘤母細胞株。進一步以西方點墨法實驗證明其中一株人類神經腫瘤母細胞可偵測到全長MJD致病蛋白產生。而所篩選出可存活含G418藥物培養液的人類神經腫瘤母細胞經11代的繼代培養，以細胞免疫染色實驗目前尚無發現核內包涵物的形成。
Machado-Joseph 疾病是晚發性的體染色體顯性遺傳疾病，一種小腦脊髓漸進性神經退化性疾病，此疾病的徵狀在臨床上變化很多，顯現範圍很廣，主要病徵有：運動失調、漸進性眼外肌麻痺、錐體及外錐體路徑的病徵、肌張力的異常及僵硬末稍肌萎縮、臉部及舌的顫搐。MJD的基因位於在第14對染色體長臂上，在靠近3端轉譯區(translated region)內，有一段不穩定的CAG核酸重覆序列倍增突變。有關MJD疾病的治療至目前為止仍根據臨床徵狀來治療。Lamotrigine是一種新的抗癲癇藥物，被廣泛用來治療單純性或複合性的痙攣。根據臨床的觀察發現Lamotrigine能夠減輕 MJD臨床的症狀，先前研究顯示含致病MJD蛋白的淋巴母細胞處理lamotrigine 2天後發現lamotrigine的濃度增加到300 M時，會降低致病MJD蛋白至多30%的表現，顯示lamotrigine對淋巴母細胞中所表現的致病MJD蛋白的具有抑制性。
本論文延續先前的研究利用已建立的人類神經母細胞株(SK-N-SH-MJD78)，其中可表達正常MJD蛋白及致病MJD蛋白，此細胞培養後以不同濃度的lamotrigine處理2天之後，再以西方轉漬法分析致病MJD蛋白表現 。 發現lamotrigine的濃度增加到200 M時，會降低致病MJD蛋白的表現，顯示lamotrigine在人類神經母細胞株中對致病MJD蛋白的表現也具有抑制性。這個結果與先前淋巴母細胞實驗及臨床觀察的現象相似。
另一方面，病人於服用lamotrigine藥物前後，以西方轉漬法分析突變MJD蛋白表現，發現有的病人服藥後，其突變MJD蛋白的表現增加，而有的病人服藥後，其突變MJD蛋白的表現有減少的現象，這種血液中MJD蛋白表達的不穩定性可能與病人個體於抽血時的生理情況有相互關係。因此，需進一步的研究以釐清此藥物作用於人體MJD蛋白的機制。
Machado-Joseph Disease (MJD)是屬於體染色體顯性遺傳神經退化性疾病其中一種，又名為小腦脊髓運動失調症第三型(SCA3)，發病原因與其基因座內CAG三核酸重覆序列倍增突變有關。而對於此段含有三核酸重覆序列所轉譯出的多醯胺麩胺酸(poly- glutamine)蛋白產物之正常功能與致病機轉尚未明瞭。只知道此段核酸重覆序列愈長則病症愈嚴重且經由病人腦部病理切片以細胞免疫染色實驗得知MJD/ataxin-3 蛋白會聚積於腦部神經核內形成包涵物並猜測此核內包涵物會影響神經退化的病程發展而核內包涵物究竟是促使神經細胞走向細胞程序性凋亡(apotosis)，亦或是保護神經細胞，其作用並不清楚。目前只知道此核內包涵物組成含有全長MJD致病蛋白、致病MJD蛋白片段、正常MJD蛋白、泛激素(Ubiqutine)以及一些熱休克蛋白等等、是否還包括其它蛋白並不清楚。我們利用之前實驗室所製備的MJD多株抗體與正常的人類神經腫母細胞株(SK-N-SH)和含突變MJD蛋白淋巴母細胞株進行免疫沉澱反應，希望藉由蛋白和蛋白間的交互作用來尋找會與MJD蛋白有相互作用的蛋白。結果發現有許多非特異性反應的蛋白出現，與實驗對照組比較後，雖然尚未如預期得到明顯特異性的蛋白出現，然而在大約70kDa蛋白位置有一個比較弱的反應，值得後續研究。許多非特異性反應的蛋白出現可能與所用的抗體純度不夠有關，因此，我們將進一步純化此MJD多株抗體，再者，亦有可能是神經細胞所表達的MJD蛋白量太少，所以反應太弱無法被測定出來，因此，將MJD蛋白表達量增加也是未來改進的方法之一。
Machado-Joseph disease(MJD) is an autosomal dominant spinocerebellar degeneration characterized by cerebellar ataxia and pyramidal signs associated in varying degrees with a dystonic-rigid extrapyramidal syndrome or peripheral amyotrophy as major neurologic signs. Unstable CAG trinucleotide repeat expansion in MJD gene has been identified as the pathologic mutation of MJD. It is known that expanded polyglutamine tracts cause ataxin-3 and other proteins to accumulate and aggregate in neuronal nuclei. Whether the intranuclear aggregation of a polyglutamine protein initiates cellular pathology remains controversial. In this study, we established stably transfected human neuroblastoma SK-N-SH cells expressing the full length of ataxin-3 with expanded polyglutamine (78Q), named SK-N-SH-MJD78. Western blot analysis and immunostaining experiments demonstrated that the expanded ataxin-3 is stably expressed and predominantly present in the cytoplasm. Compared to parental SN-K-SH cells expressing 26Q, cells expressing 78Q display normal morphology. No intranuclear aggregation was observed in SK-N-SH-MJD78 cells after 11 passages. Further analysis will apply to this cellular model to better understand the role(s)of other environmental stress may play in the pathogenesis of MJD.
Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar degeneration characterized by a wide range of clinical manifestations, including ataxia, progressive external ophthalmoplegia, pyramidal and extrapyramidal signs, dystonia with rigidity, and distal muscular atrophies. Unstable CAG trinucleotide repeat expansion in MJD gene on long arm of chromosome 14 has been identified as the pathologic mutation of MJD. The treatment of MJD has so far been purely symptomatic. Lamotrigine, an antiepileptic drug, was found to specifically relieve some major symptoms of MJD patients. Encouraged by the clinical observation and Lymphoblastiod cell lines (LCLs) from patients, we carried out the expression analysis ataxin-3 in SK-N-SHMJD78 cells to understand the molecular basis for this observation.
We have investigated in the present study the effect of lamotrigine (LTG) on the Machado-Joseph disease protein (ataxin-3) in cultured lymphoblastiod cells and demonstrated that the LTG-treated MJD cells express less mutant ataxin-3 proteins. Extracellular application of LTG 200 M decreased the expression of mutant ataxin-3 protein in SK-N-SH-MJD78。This observation is consistent with the clinical phenomena and the preliminary results 。Further analysis is underway for better understanding of the role(s) which lamotrigine may play in the pathogenesis of MJD. The molecular analysis of the drug effect on MJD expression will be certainly benefits the MJD patients.
Machado-Joseph disease(MJD) is caused by unstable CAG trinucleotide repeat expansion in the coding region of the MJD gene. It is known that expanded polyglutamine tracts cause ataxin-3 and other proteins to accumulate and aggregate in neuronal nuclei of affected brain regions. Whether the intranuclear aggregation of a polyglutamine protein initiates cellular pathology remains controversial. It was shown previously that intranuclear inclusions contain the full-length ataxin-3, truncated ataxin-3, ubiquitin, heat shock proteins and other unknown proteins. In order to understand the functional role(S) of mutant ataxin-3 through it’s interacted protein(s), we performed immunoprecipitation assay to search for any putative protein which may interact with the mutant ataxin-3. Polyclonal antisera against ataxin-3 were used to precipitate ataxin-3 together with associated proteins, if any, from the mixture of total protein extracts from SK-N-SH cells and patient’s lymphoblastoid cells. Compared to control experiment, however, no clear specific signals were observed in the assay except one weak signal around 70 kDa. Further improvement in antibody preparation and enrichment of protein concentration will be untaken in the near future. |
