新穎型Charcot-Marie-Tooth氏症之致病基因 Inscuteable突變之作用機轉及潛在治療方向

A Novel Pathogenic Mutation in Inscuteable Causes Charcot-Marie-Tooth Neuropathy: Investigating the Molecular Mechanism and Potential Therapy

葉芮妤(Jui-Yu Yeh)

指導教授 ： 詹智強

國立臺灣大學/醫學院/生理學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202100859

摘要

Charcot-Marie-Tooth(CMT)氏病為遺傳性周邊神經病變最常見的疾病之一，造成患者在青壯年時期遠端肢體出現感覺、運動覺或本體感覺的喪失。目前約有90種以上的CMT致病基因已被發掘，但仍有近三成的患者其基因仍屬未知，導致在疾病治療上出現困難。臨床研究的團隊利用全外顯子定序技術對於台灣CMT的家族進行檢測，發現一個新穎型的致病點突變-Inscuteable(Insc)c.209T>G(p.INSC M70R)。Insc是在演化上高度保守性的基因，而先前文獻對於其功能認知大多在發育學上藉由調控微管，使神經母細胞進行不對等分裂;而對於此基因在成體是否參與維持神經穩定性之能力仍屬未知。為了能探討該突變對於成體神經系統之影響，我同時利用果蠅與人類細胞作為疾病模式，發現人類的 INSCWT能夠救援果蠅之神經元喪失，代表果蠅成體中的肢體感覺神經與人類呈現功能上的保守。而若於成體時期，對於果蠅之Insc進行抑制，發現其運動及感覺神經都出現明顯退化，代表Insc具有穩定神經系統之功能。人類之INSCM70R無法救援果蠅的神經元喪失，顯示INSCM70R為一種loss-of-function突變。另一方面，用ppk-Gal4在野生型(wild type)的個體單獨過表達INSCM70R也會造成神經出現明顯退化，顯示INSCM70R突變同時 也具有gain-of-toxicity的特性。藉由觀察果蠅Insc 基因的表現，我發現Insc會專一性的表現在本體感覺受體-弦音感受器(一種微管含量極為豐富的組織)之中，顯示Insc可能具有調控感覺輸入以及運動輸出的能力。在分子層次，藉由改變Insc的表現量，我同時在果蠅與細胞模式之下都觀察到自噬體堆積與微管形態異常，而堆積的情況卻能夠因為加入抑制 mTORC1的藥物而瓦解。總結而言，我認為INSCM70R會造成弦音感受器之中微管之穩定性降低，進而使堆積物無法於微管上進行正常的傳輸，導致物質無法獲得有效清除而大量滯留於細胞之中，進而出現神 經退化的狀況，而此現象卻能夠因為施予mTORC1的抑制性藥物而獲得改善，故或許本研究能夠帶給未來CMT氏病進行藥物治療的一個新方向。

關鍵字

Charcot-Marie-Tooth 氏病 ； Inscuteable ； 細胞自噬 ； 神經退化 ； 聚集體 ； 本體感覺

並列摘要

Charcot-Marie-Tooth (CMT) disease is the most common form of inherited peripheral neuropathy characterized by progressive sensory loss and motor weakness especially in the proximal portion of the limb. Although over 90 genes have been associated with CMT, the genetic linkage of approximately 30% of the CMT patients remained uncharacterized. We have discovered a novel pathogenic variant, c.209T>G (p.INSC M70R) of Inscuteable (Insc) gene in a Taiwanese family of CMT neuropathy using whole-exome sequencing. Insc is an evolutionarily-conserved gene known for regulating the mitotic spindle during neuroblast development, but its post-mitotic function remained largely unexplored. Here we present a Drosophila model of CMT with M70R mutation in INSC. Flies with neuronal Insc knockdown exhibit a reduction in the number of sensory neuron cell body, which is rescued by human INSC overexpression, indicating the functional conservation between human INSC and its fly orthologue. Knockdown of Insc specifically in adulthood caused a progressive loss of the sensory neurons and a decline in locomotor activity, demonstrating that Insc exerted the post- mitotic function for neuronal maintenance. Human INSCM70R failed to rescue the neuronal loss of the Insc knockdown flies in adulthood, suggesting that the fly model can serve as a platform to discern the mechanistic difference between Wild-Type and M70R. Also, overexpression of INSCM70R with ppk-Gal4 in fly sensory neuron caused the progressive loss of cell bodies. Taken together, INSCM70R mutation causes CMT likely by a combination of gain-of-toxicity and loss-of-function. Furthermore, Insc specifically expressed in the chordotonal organ, the Drosophila proprioceptors in the leg, implying its role in coordinating the sensory and motor functions. At the cellular level, we found that both overexpression and knockdown of Insc caused the accumulation of autophagosome in the neuron, supporting that mutation of Insc may impaired autophagy. Activation of autophagy by treatment with mTORC1 inhibitor, Rapamycin, could reverse the CMT-associated defects in both the fly model and human neuroblastoma cell. Overall, this study demonstrates a novel role of Insc in post-mitotic neuronal maintenance and provides a potentially treatable cause of CMT neuropathy.

並列關鍵字

Charcot-Marie-Tooth neuropathy ； Inscuteable ； autophagy ； neurodegeneration ； aggrephagy ； proprioception

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