OPA1基因突變引發粒線體功能缺損

OPA1 Mutation Cause Mitochondrial Dysfunction

葉依綾(Yi-Ling Yeh)

指導教授 ： 高淑慧

臺北醫學大學/醫學科技學院/醫學檢驗暨生物技術學系/碩士(2011年)

摘要

顯性遺傳視神經萎縮症 (autosomal dominant atrophy , ADOA)，是由於視網膜神經節細胞 (retinal ganglion cells , RGC) 病變而造成的視力退化，為最常見的遺傳性視神經疾病，ADOA 與位於粒線體內膜上的optic atrophytype1 , OPA1 蛋白有關，該蛋白被證實具有 GTPase 的功能並且可以調控粒線體動態平衡(mitochondrial dynamic)。此外OPA1 蛋白也直接調控cytochorome c 的釋放，影響細胞凋亡的情形，研究證實當OPA1 基因發生
突變時，會引發ADOA。為了瞭解罹患視神經萎縮症的病人其OPA1 基因的突變，是否影響細胞及粒線體的功能，因此我們蒐集了四個不同家族中罹患有ADOA 的病人，將病人的淋巴球細胞製備成細胞株培養，另外也將家族成員中沒有發病的淋巴球細胞製成細胞株，作為internal control 比較。
本實驗主要討論第三個和第四個家族的病人，其OPA1 基因突變分別為第三個家族是OPA1 基因外顯子 19 缺失 (exon 19 deletion)，第四個家族是OPA1 基因外顯子28 點突變 (exon 28 point mutation) 。實驗結果發現在OPA1 基因突變的細胞株上，其OPA1 mRNA 和蛋白的表達比正常人或是同家族中未發病的人減少約20 ~ 30 % 且ATP 生合成量也降低約30 ~ 40 %，此外利用免疫螢光染色觀察細胞中粒線體形態，結果發現帶有OPA1 基因突變的細胞其粒線體形態主要呈現分裂型(fission form)約佔70 ~ 80%，相較於正常人或是同家族中沒有突變基因的人，其粒線體型態則以融合型(fusion form)為主，而分裂型約為20~30%，以流式細胞儀檢測細胞膜電位，粒線體總量和細胞氧化性傷害時發現，帶有OPA1 基因突變的細胞使細胞膜電位降低約20 ~ 30%，粒線體總量減少約30%，有較高的氧化性傷害堆積。
因此綜合本實驗室之前和這次的研究，我們確認OPA1 基因對於細胞能量的生合成或是粒線體的動態平衡都扮演著重要的角色，因此推測由於OPA1基因突變影響了細胞產生能量的效率和粒線體網絡，進而造成對能量需求較大的神經細胞產生病變，導致視神經萎縮的病症。

關鍵字

顯性遺傳視神經萎縮症,視神經萎縮蛋白,粒線體

並列摘要

Autosomal dominant atrophy (ADOA) is the most common hereditary optic neurotrophy due to the degeneration of the retinal ganglion cell accompanied with the lost of visual. ADOA is in concerned with a protein named OPA1, which located in the mitochondrial inner membrane. OPA1 gene is located on chromosome 3 q arm 28 locus. OPA1 is a dynamin – related GTPase, which plays a role in mitochondrial cristae remodeling. In order to understand the effect of different
OPA1 gene mutation in mitochondrial function, we analyzed four ADOA families and use the lymphocyte from the patients and relatives to establish cell lines. In this study, we focus on ADOA family III with the deletion in exon 19 and ADOA family IV with point mutation in exon 28. Study results show that the ADOA affected lymphocyte have lower OPA1 mRNA and protein expression. In addition, cell harboring OPA1 mutations show the depleted ATP synthesis, dissipated mitochondrial membrane potential and decreased mitochondrial mass, but enhanced oxidative damages. Confocal microscopy image revealed that the cells with OPA1 gene mutation have more fragmented mitochondria. Moreover, by using SeaHorse
XF 24 analyzer, we analyze the mitochondrial respiratory function of the lymphocytes from patients. All the affected lymphocyte in both families show lower energy capacity in max respiration and reserve capacity. Therefore we demonstrate that OPA1 gene defects cause mitochondrial dysfunction, energy dissipation, and fragmented mitochondrial network. It may be the reason of disease and pathology of ADOA and the degree of disease may differ from the mutation site in OPA1 gene.

並列關鍵字

Autosomal dominant atrophy (ADOA), OPA1, mitochondria

參考文獻

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