藉由RNA-Seq分析脊髓肌肉萎縮症小鼠中神經肌肉交接處失調基因

Dysregulation of Neuromuscular Junction Genes Involves in Spinal Muscular Atrophy Mice by RNA-Seq Analysis

鄭郁樺(Yu-Hua Cheng)

指導教授 ： 鐘育志

高雄醫學大學/醫學院/醫學研究所碩士班/碩士(2015年)

摘要

研究背景: 脊髓肌肉萎縮症(Spinal muscular atrophy, SMA)是一種體隱性遺傳疾病，此疾病的特徵為脊髓(Spinal cord)前角(Anterior horn)運動神經元漸進性退化而導致肌肉無力及萎縮。運動神經元存活(Survival motor neurons 1, SMN1)基因的突變使功能性SMN蛋白減少而引起SMA。最近研究顯示在神經肌肉交接處 (Neuromuscular junction, NMJ)成熟時會增加SMN蛋白的需求，且SMA的病理變化包含NMJ缺陷。然而，SMA中NMJ缺陷的分子機制還不清楚。因此本研究想要探討NMJ相關基因在SMA中的致病分子機制。
實驗方法: 為了探討基因表現程度的改變，我們收集9天大(Postnatal day,P9)3隻台灣SMA小鼠(type I; Smn-/- SMN2+/-)與3隻正常小鼠(Smn+/- SMN2+/-)的脊髓組織進行RNA-Seq分析。從分析結果找出與NMJ相關基因並檢測其mRNA表現量是否與RNA-Seq一致，接著將mRNA表現顯著差異的基因檢測其蛋白質表現量，後續找出下游相關基因是否也受影響，來探討SMA小鼠脊髓中影響NMJ基因之分子機制。
實驗結果: RNA-Seq分析結果顯示在9天大SMA小鼠中有800個基因與正常小鼠有差異，其中有642個調節下降的基因，有158個調節上升的基因，從中找出6個與NMJ相關的基因包含calcitonin-related polypeptide alpha (Calca)， collagen type IV alpha 1 (Col4a1)， coxsackie virus and adenovirus receptor (Cxadr)， integrin alpha 7 (Itga7)， integrin beta 1 (Itgb1) 和 serpin peptidase inhibitor clade E member 2 (Serpine2)，並檢測其mRNA表現量。其中， Itgb1的mRNA和蛋白質表現量在病鼠皆降低，而Itgb1的下游基因Abl-related gene (Arg, or Abl2) 活化量也進而減少，但下游基因RhoA與ROCK的下游cofilin的活化沒有因此大幅增加反而顯著減少。探討Itgb1其他下游基因protein tyrosine kinase 2 (Ptk2)和FERM, RhoGEF and pleckstrin domain protein 2 (Farp2)在RNA-Seq與mRNA表現量在病鼠皆降低，導致其下游路徑使RhoA活化降低而造成SMA小鼠中的NMJ相關致病機制。
研究結論: 藉由RNA-Seq找出在NMJ表現差異的基因而能解釋SMA的相關分子機制。Itgb1是訊息傳遞路徑中重要的上游分子，其失去調節會影響下游分子作用進而使NMJ產生缺陷，未來若能用藥物增強其調控或許能提早治療或延緩SMA的惡化。

關鍵字

脊髓肌肉萎縮症 ； 台灣脊髓肌肉萎縮症小鼠 ； RNA定序 ； Itgb1基因 ； Focal adhesion路徑

並列摘要

Background: Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease characterized by progressive degeneration in the motoneurons of anterior horn of spinal cord, and subsequently leads to generalized muscle weakness and atrophy. Mutation of the survival motor neurons 1 (SMN1) gene results in low levels of functional survival motor neuron (SMN) protein and causes SMA. Recent studies have revealed that requirement of enhanced SMN protein imposes during neuromuscular junction (NMJ) maturation and NMJ disruption is involved in SMA motoneuron pathology. However, the molecular basis of NMJ defects in SMA pathogenesis has remained unclear.
Methods: We collected the spinal cord samples from 3 Taiwanese SMA mice (type I; Smn-/- SMN2+/-) and 3 wild-type (WT, Smn+/- SMN2+/-) mice at postnatal day (P9) then performed RNA-Seq to identify RNA expression profiles. To validate the RNA-Seq analysis, transcripts from six NMJ-related genes that have significantly expression were chosen for the real-time PCR analysis. We tested the protein level which mRNA expression level had significant changes. Further, we found out related downstream effectors whether were affected by the dysregulated upstream, showing molecular events that could explain key aspects of NMJ pathology in SMA.
Results: RNA-Seq results showed that the WT mice and the type I SMA mice at P9 differentially expressed 800 affected genes, including 642 down-regulated and 158 up-regulated genes. We chose 6 NMJ-related genes included calcitonin-related polypeptide alpha (Calca), collagen type IV alpha 1 (Col4a1), coxsackie virus and adenovirus receptor (Cxadr), integrin alpha 7 (Itga7), integrin beta 1 (Itgb1) and serpin peptidase inhibitor clade E member 2 (Serpine2) and analyzed their mRNA expression level. Our results suggest that Itgb1 was down-regulated and its downstream Abl-related gene (Arg) was also downregulated compared to control. However, Itgb1 inactivated Arg and did not indirectly activates RhoA/ROCK as well as their downstream cofilin. Other results indicate that mRNA levels of protein tyrosine kinase 2 (Ptk2) and its downstream FERM, RhoGEF and pleckstrin domain protein 2 (Farp2) were decreased then inactivated RhoA/ROCK pathway.
Conclusion: In this study, we found 6 significantly differential genes related to NMJ by RNA-Seq. We suggest that the focal adhesion pathway is therapeutic relevance for SMA, and Itgb1, a crucial NMJ-related gene, may be a novel potential therapeutic target of SMA.

並列關鍵字

Spinal muscular atrophy (SMA) ； Taiwanese SMA mouse ； RNA sequencing (RNA-Seq) ； Integrin beta-1 (Itgb1) gene ； Focal adhesion pathway

參考文獻

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