OPA1基因突變引發粒線體基因不穩定性以及粒線體核仁構形異常

OPA1 mutation induced mitochondrial genome instability and mitochondrial nucleoid disorganization

陳怡秀(Yi-Hsiu Chen)

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所/碩士(2015年)

摘要

第一型視神經萎縮蛋白(Optic atrophy type 1, OPA1)蛋白位於粒線體的內膜上，具有 GTPase 的功能，並且調控粒線體動態平衡(dynamic balance)。OPA1 蛋白也参與調控cytochorome c的釋放，進而影響細胞凋亡。當OPA1基因發生突變時，被證實與體染色體顯性遺傳視神經萎縮症(autosomal dominant optic atrophy, ADOA)有關。最近的研究發現在OPA1突變小鼠模式中其抗氧化基因表現被抑制，且增加活性氧自由基(reactive oxygen species)的生成。此外，當抑制(knockdown) OPA1基因 exon 4b時，則引發粒線體DNA(mitochondrial DNA，mtDNA)減量(depletion)及粒線體核仁(nucleoid)分佈異常。粒線體核仁為粒線體DNA以及一些蛋白質所組成，可幫助粒線體DNA的穩定及複製。因此，我們推測OPA1蛋白突變會增加氧化壓力生成及粒線體核仁分佈異常，進而造成粒線體基因不穩定性(mitochondrial genome instability)。本研究中，我們使用四種不同OPA1基因突變的淋巴球細胞株，並將未發病的家族成員的淋巴球細胞製成細胞株，作為internal control比較。細胞株A3為OPA1基因外顯子10 (exon 10) 突變，細胞株C3為OPA1 基因外顯子12缺失(exon 12 deletion )，細胞株D5為OPA1 基因外顯子 19單胺基酸缺失(one amino acid deletion in exon 19)，細胞株E3為OPA1 基因外顯子28點突變(exon 28 point mutation)。在本研究中，我們發現OPA1基因突變的細胞株產生高量的8-羥基去氧鳥糞嘌呤(8-hydroxydeoxyguanosine, 8-OHdG)生成，粒線體DNA發生基因斷損突變(deletions)，以及影響粒線體DNA拷貝數(mitochondrial DNA copy number)。我們利用粒線體DNA免疫沉澱法分析OPA1蛋白與粒線體DNA的結合 (interaction)，我們發現OPA1基因突變會減少OPA1蛋白與粒線體DNA的結合作用。Anti-DNA染色觀察粒線體核仁之分佈，發現粒線體核仁(nucleoid)的數量有明顯減少，OPA1基因突變的細胞其粒線體核仁呈現環核分佈(peri-nuclear distribution)，正常細胞的核仁為均勻分佈。以免疫沉澱法和西方墨點法用來測定OPA1蛋白與粒線體核仁蛋白(mitochondrial transcription factor, TFAM)的結合作用，也發現OPA1與TFAM的結合減少的情形。並且影響TFAM與prohibitin的結合能力。因此我們推測OPA1基因突變導致粒線體核仁結構異常，進而影響粒線體基因不穩定以及造成呼吸傳遞鏈功能異常。

關鍵字

顯性遺傳視神經萎縮症 ； 第一型視神經萎縮蛋白 ； 粒線體網路 ； 8-羥基去氧鳥糞嘌呤 ； 粒線體DNA斷損突變 ； 粒線體核仁

並列摘要

Optic atrophy 1 (OPA1) is the causative gene of autosomal dominant optic atrophy (ADOA) and is the central regulators of mitochondrial network dynamics and apoptosis. Recently, induced mitochondrial DNA (mtDNA) depletion and dismantled mitochondrial nucleoids was found in the OPA1 exon 4b-silenced cells. Mitochondrial nucleoids are the scaffold for mtDNA organization and replication. We hypothesized that OPA1 mutation induced disorganized distribution of mitochondrial nucleoids leads to mitochondrial genome instability. In the present study, we used four established lymphoblastoid cell lines harboring different OPA1 mutations, four from unaffected relatives (as internal control), and one unrelated normal controls (as normal control). These mutations include OPA1 whole exon 10 deletion in A3 cell lines, whole exon 12 deletion in C3 cell lines, 3 nucleotides deletion in exon 19 in D5 cell lines, and a point mutation in exon 28 in E3 cell lines. We found that these OPA1 mutations induced formation of mtDNA deletions and 8-hydroxy-deoxyguanosine, and affected mtDNA copy numbers. MtDNA immunoprecipitation was applied to address the interaction between OPA1 and mtDNA. OPA1 mutations reduced the interaction between OPA1 and mtDNA. Anti-DNA staining followed by confocal microscopy imaging was used to visualize mitochondrial nucleoid distribution. OPA1 mutations caused marked reduction in the number of mitochondrial nucleoids and induced abnormal perinuclear clustering of mitochondrial nucleoids in contrast to a homogeneous dispersed distribution in normal control cells. By immunoprecipitation and western blotting, the reduced interaction between OPA1 and mitochondrial transcription factor (TFAM, one of the mitochondrial nucleoid proteins) was also found. In addition, OPA1 mutations reduced the interaction between prohibitin and TFAM or between prohibitin and OPA1. Based on our finding, we suggested that OPA1 mutation leads to mitochondrial nucleoid disruption which may cause mitochondrial genome instability and respiratory chain dysfunction.

並列關鍵字

autosomal dominant optic atrophy ； OPA1 ； mitochondrial network ； 8-hydroxy-deoxyguanosine ； mtDNA deletion ； mitochondrial nucleoids

參考文獻

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