聚麩醯胺酸、聚丙胺酸、聚白胺酸對線蟲touch neurons功能影響的研究

三聯核酸重複序列擴增已知會造成人體多種遺傳性神經肌肉疾病。其中位於疾病基因密碼區之CAG及GCN (N=A，T，C，或G) 序列擴增會引起SCAs，HD，HPE，HFGS等多種病徵。其中CAG重複序列會被轉譯出聚麩醯胺酸(polyglutamine，polyQ)，而GCN重複序列會被轉譯出聚丙胺酸(polyalanine，polyA)。此外，第二型亨丁頓氏舞蹈症致病基因內CTG序列則可能被轉譯出聚白胺酸(polyleucine，polyL)。有趣的是，這些與疾病有關之序列所代表之密碼子皆為疏水性胺基酸。在此研究中，我利用線蟲作為模式動物來探討具不同疏水性質之胺基酸在各種長度下對神經細胞結構及功能的影響。為此，我先將不同長度(包括14~15，30~38，70~90)的CAG、GCA及CTG重複序列接在綠螢光蛋白質基因密碼區內，並利用mec-7基因啟動子驅動轉殖基因使其表現在線蟲的touch neurons。我發現重複40次以下之胺基酸對細胞結構及功能沒有影響，而長度70~90次的polyQ、polyA及polyL皆明顯降低touch neurons的機械刺激敏感性並使其軸突結構消失。為與相對長度的親水性胺基酸作比較我接著製造能表現76次重複由GAA及AGA序列所轉譯之聚麩胺酸(polyglutamic acid，polyE)及聚精胺酸(polyarginine，polyR)的線蟲，並發現這些親水性胺基酸不會造成細胞毒性。此外，將125次重複的CAG重複序列接在GFP基因的3’非轉譯區並表現在touch neurons亦不會造成不良效應。這些結果顯示轉譯區內擴增之CAG、GCA及CTG重複序列所引起細胞毒性是透過蛋白質機制而非由RNA效應所造成，而且胺基酸序列的毒性效應與其長度及疏水性存在密切關係。

關鍵字

線蟲；聚麩醯胺酸；聚丙胺酸；聚白胺酸；神經；聚胺基酸

並列摘要

Expansion of CAG and GCN trinucleotide repeats within the coding region of the disease genes associates with several forms of inherited neuromuscular diseases, including SCAs, HD, HPE, and HFGS etc. In addition, expanded CTG repeats found in Huntington’s disease like-2 associated gene may also be translatable. The repeat length is almost below 40 in normal population and ranges from 40 to 100 in most diseases genes. Interestingly, the amino acids encoded by these repeats, including polyglutamine (CAG), polyalanine (GCN), and polyleucine (CTG), are all hydrophobic. In this study, I established the C. elegans model system to investigate the toxicity of homopolymeric amino acid with different lengths and hydrophilicity on neuron cells. Transgenic C. elegans expressing green fluorescence protein with 14~15, 30~38, or 70~90 polyglutamine, polyalanine, and polyleucine stretches in the touch neurons were first produced and then the structure and function of neuron cells were analyzed. Our results revealed that worms expressing 70-90 residues of these hydrophobic amino acids showed touch insensitivity mechanosensory assay and axon disappearance, while those below 40 residues did not. Subsequently, we generated C. elegans expressing green fluorescence protein with 76 residues of hydrophilic amino acid stretches, including polyglutamic acid and polyarginine. We found that the structure and function of touch neurons in these transgenic animals were no affected. Furthermore, we observed that expanded CAG RNA up to 125 repeats did not cause any toxicity on neurons. These results indicate that the toxicity induced by CAG, GCN, and CTG repeat expansion in the coding region is through protein mechanism, not caused by RNA effect, which is related to the length and hydrophobicity of the encoded homopolymeric amino acids.

並列關鍵字

Caenorhabditis elegans ； HPAA ； polyQ ； polyA ； polyL ； touch neuron

參考文獻

1. Gatchel, J.R. and H.Y. Zoghbi, Diseases of unstable repeat expansion: mechanisms and common principles. Nat Rev Genet, 2005. 6(10): p. 743-55.

2. Ranum, L.P. and T.A. Cooper, RNA-Mediated Neuromuscular Disorders. Annu Rev Neurosci, 2006.

3. Orr, H.T. and H.Y. Zoghbi, Trinucleotide repeat disorders. Annu Rev Neurosci, 2007. 30: p. 575-621.

4. Greenstein, P.E., et al., Huntington's disease like-2 neuropathology. Mov Disord, 2007. 22(10): p. 1416-23.

5. Rudnicki, D.D., et al., Huntington's disease--like 2 is associated with CUG repeat-containing RNA foci. Ann Neurol, 2007. 61(3): p. 272-82.

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聚麩醯胺酸、聚丙胺酸、聚白胺酸對線蟲touch neurons功能影響的研究

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