In vivo and In vitro Studies of Mitochondrial Transplantation for the Treatment of Spinocerebellar Ataxia Type 3

粒線體移植治療第三型脊髓小腦共濟失調症之體內外研究評估

張瑞芝(Jui-Chih Chang) ； 葉承翊(Cheng-Yi Yeh) ； 張子伶(Tse-Lin Chang) ； 劉科宏(Ko-Hung Liu) ； 宋秉文(Bing-Wen Soong) ； 謝明麗(Ming-Li Hsieh) ； 張瑞敏(Tiffany Chang) ； 劉青山(Chin-San Liu)

《The Changhua Journal of Medicine》 15卷4期 (2017/12) Pp. 176-193

https://doi.org/10.6501/CJM.1504.002

摘要

Spinocerebellar ataxia type 3 (SCA3) and Machado-Joseph disease (MJD) are caused by abnormal expansion of guanine-adenine- guanine (CAG) trinucleotide repeats of the ataxin 3 (ATXN3) gene located at the 14q32.1 genomic locus, which is translated into a long-chain glutamine (polyglutamine, polyQ). This results in an inability to perform autophagy or proteasomal degradation, causing accumulation of inclusion bodies and subsequent neuronal apoptosis. Although its pathological molecular mechanisms are still unclear, mitochondrial function has recently been discovered to affect cell oxidative stress and also participate in cell autophagy. To prove how mitochondria participate in autophagy and the cytotoxic effects on polyQ, a peptide-mediated mitochondrial delivery (PMD) system was employed in this study to investigate the mitochondrial role in the delay of SCA3 disease progression. SCA3 transgenic mice received a 3-point in situ cerebellum injection of mitochondria, and the effects on the survival rates of cerebellum Purkinje cells and the motor coordination of SCA3 transgenic mice were compared before and after peptide modification transplant. Eighteen weeks after the transplant, Pep-1-labeled mitochondria (P-Mito) resulted in significant improvement over unlabeled mitochondria in terms of testing mice with a rotarod to assess motor coordination and analysis of cerebellum Purkinje cell death. Based on cerebellum tissue fluorescent staining and protein tests, the mechanism may be related to the restoration of lysosome-associated membrane protein 2 (LAMP2) and may facilitate the degradation of protein accumulation of p62-mediated aggresomes. Similar results showed that human neurons possess ATXN3 genes with long glutamine chains in disease cells (MJD78). Cell experiments revealed that P-Mito can enter cells and increase in a dose-dependent manner. A low dose of 25 μg of mitochondria was unable to improve mitochondrial function, but did facilitate cell autophagy (an increased light chain 3 [LC3]-II/LC3-I ratio and decreased p62 accumulation in disease cells), decrease mutant protein accumulation of the ATXN3 gene caused by abnormal CAG trinucleotide expansion, and increase resistance to oxidative stress-induced cell apoptosis. Hence, the results of this study revealed that P-Mito transplants of appropriate doses are unable to affect mitochondrial function, but do facilitate the metabolism of mutant polyQ proteins to decrease cytotoxicity, which is beneficial in the treatment of diseases. This means that the activation of cell autophagy plays a more significant role in the treatment of SCA3 than the recovery of mitochondrial function.

關鍵字

spinocerebellar ataxia type 3 ； Pep-1 ； mitochondria ； autophagy ； lysosome

並列摘要

第三型脊髓小腦共濟失調症(spinocerebellar ataxia type 3, SCA3/Machdo-Joseph disease, MJD)主要是位於14q32.1基因座上的ataxin 3 (ATXN3)基因其guanine-adenine-guanine (CAG)三核苷酸異常擴增進而轉譯出長鏈的麩胺酸(polyglutamine, polyQ)，無法由自噬作用(autophagy)或蛋白酶體(proteasome)降解而堆積(inclusion body)導致神經細胞死亡，儘管其病理分子機轉尚未明確，近來發現粒線體功能除影響細胞氧化壓力外，也參與細胞自噬作用(autophagy)的發生，為證實粒線體如何參與細胞自噬作用的發生與其對polyQ所造成的細胞毒性之影響，本研究利用胜肽運送粒線體技術(peptide-mediated mitochondrial delivery system, PMD)，期望能透過調控粒線體功能，探討粒線體在SCA3中角色與可否減緩疾病進程。本研究先以SCA3基因轉殖鼠(SCA3-84Q)進行粒線體原位小腦三點注射，比較粒線體有無胜肽修飾對移植前後SCA3基因轉殖鼠之運動協調性與小腦浦肯野細胞(Purkinje cells)存活率之影響。18週後實驗發現，修飾粒線體(Pep-1-labelled mitochondria, P-Mito)相較未處理粒線體(mitochondrial alone, Mito)對於由轉棒試驗(rotarod test)所測得之運動協調性與小腦浦肯野細胞死亡有較明顯之改善，透過小腦組織螢光染色與蛋白檢測顯示其機轉可能與恢復溶酶體功能(增加溶酶體相關膜蛋白2, lysosome-associated membrane protein 2, LAMP2)，促進其分解p62-mediated aggresomes蛋白沉積物之清除。相同結果亦發現於人類神經細胞帶有ATXN3基因含長鏈的麩胺酸之疾病模式細胞(MJD78)。細胞實驗發現P-Mito可進入細胞並隨給與劑量增加而增加；低劑量粒線體的給與儘管無法增加粒線體功能，但卻能促進細胞自噬作用(增加的light chain 3 (LC3)-II/LC3-I比值，並且減少疾病細胞中p62的堆積)，進而減少ATXN3基因其CAG三核苷酸異常常擴增所累積之突變蛋白堆積，增加細胞低抗氧化壓力所誘發之細胞死亡。因此，本研究意外發現適當劑量胜肽移植粒線體移植儘管無法影響粒線體功能，卻能藉由促進代謝突變體PolyQ蛋白進而降低細胞毒性，有助於疾病治療，意味細胞自噬作用活化相較於粒線體功能恢復對於治療SCA3扮演更顯著性角色。

並列關鍵字

第三型脊髓小腦共濟失調症 ； 胜肽 ； 粒線體 ； 自噬作用 ； 溶酶體