建立第22型脊隨小腦萎縮症的果蠅模式

Abstract

脊髓小腦共濟失調症（Spinocerebellar Ataxia，SCA）為一種顯性遺傳性的神經系統疾病，若雙親其中一位患有此症，其子代不分性別均有一半的罹患機率；雖是同一家族，其發病年齡和病徵也不盡相同。台北榮總與陽明大學從一家系共四代的台灣病人，經臨床鑑定確認患有顯性遺傳的小腦運動失調症，透過進行鏈鎖分析發現不同於已知的SCA亞型致病基因位點，且基因CAG、CTG、 ATTCT並無異常重複突變，判斷此為第22型脊髓小腦共濟失調症。通過全基因組鏈鎖分析定位出SCA22突變位於1號染色體1p21-q23，基因變異是發生在編碼的鉀離子（voltage-gated potassium，Kv）通道Kv4.3的KCND3基因。其中在台灣及法國家族皆發現的基因變異為第227個胺基酸Phenylalanine缺失（p. ∆F227）；在美國猶太人及日本家族發現的則是第345個胺基酸由Glycine點突變成Valine（p.G345V）。本研究的主要目的即為建立第22型脊髓小腦共濟失調症的果蠅模式株，利用過量表現KCND3突變基因，來探討基因變異造成的Kv4.3鉀離子通道蛋白對病理症狀如運動能力及壽命的影響，並釐清SCA22的致病機制。在本研究中，我們建立的SCA22模式果蠅，確實出現年齡伴隨的病理特徵，包括細胞凋亡、運動能力下降以及縮短壽命等退化症狀。 此外，在mRNA及蛋白質表現量的測量中，發現∆F227在蛋白質的轉譯功能可能發生異常，由於前人從免疫螢光分析的實驗，發現p. ∆F227的Kv4.3鉀離子通道蛋白無法正常上到細胞膜，且大部分保留在內質網（endoplasmic reticulum，ER），前人研究指出蛋白質累積在內質網會造成內質網壓力（ER stress），我們將近一步觀察細胞是否發生為因應此壓力而產生之相關蛋白質反應。此外由於研究指出鉀離子在調控細胞質離子的恆定上扮演極重要的角色，藉由細胞膜上的鈉鉀離子幫浦（Na+/K+-ATPase）將鉀離子主動運輸至細胞內，及鉀離子通道的開閉調控鉀離子外流，兩者作用平衡可維持細胞容積和防止細胞凋亡發生，我們也將進一步研究過表現G345V基因是否造成細胞內部離子失衡導致細胞的死亡。The spinocerebellar ataxias (SCA) are a diverse group of autosomal dominant neurological disorders characterized by progressive degeneration of many nevrse systems, including cerebellum, spinocerebellar tracts, and brain stem neurons. Recent discovery of mutations in the voltage-gated potassium channel Kv4.3-encoded gene KCND3 has shown to be the cause of the autosomal dominant spinocerebellar ataxia type 22 (SCA22). Of all KCND3 mutations, the in-frame three-nucleotide deletion c.679_681delTTC p.F227del (KCND3-ΔF227) has been identified in either the French and Chinese pedigrees. The in-frame point mutation c.1304G>T p.G345V (KCND3-G345V) has been identified in either the American and Japanese pedigrees. Since the underlying pathomechanisms of SCA22 is poorly understood, we generate Drosophila models for SCA22 by overexpression of wild-type KCND3, and mutant KCND3 variants (i.e. ΔF227; G345V) using the UAS/Gal4 system to address the above question. Ectopic expression of mutant KCND3 cause various pathological features, including neurodegeneration, apoptosis, mobility defects and shortened lifespan. More detailed analysis of mRNA and protein expression level found that ΔF227 translation abnormally by decreases in protein production. Since immunocytochemistry analyses revealed that KCND3-ΔF227 retained in the endoplasmic reticulum (ER). We suspect that KCND3-ΔF227 might induces ER stress thereby inducing neurodegenerations. Additionally, KCND3 is a potassium channel, ectopic KCND3 expression may cause the imbalance intracellular potassium concentration and lead to neuronal cell death. All the above mentioned possible pathomechanisms will be investigated with the newly established models.