F-BOX蛋白家族基因Fbxl14與精神分裂症基因Disc1在小鼠胚胎腦部交互作用之研究

Abstract

Disrupted in Schizophrenia 1 (DISC1)是一個精神分裂症的致病基因，最初在人類發現，後續研究發現本基因與躁鬱症、自閉症等精神疾病亦有關(Soares et al, 2011)。經由小鼠等模式生物研究發現，DISC1蛋白質參與了神經細胞新生(Singh et al, 2010)以及神經細胞遷徙(Ishizuka et al, 2011)等神經發育的過程。F-box and leucine-rich repeat protein 14 (FBXL14)是F-box蛋白家族的成員，可形成E3泛素連接酶複合體(E3 ubiquitin ligase complex)，辨認發育所需的特定蛋白質受質進入由蛋白酶體(proteasome)催化的蛋白質降解，是正常發育必須的過程(Cardozo et al, 2004)。本實驗室先前研究以免疫共沉澱法發現小鼠DISC1蛋白質(mDISC1)與小鼠FBXL14蛋白質(mFBXL14)之間有交互作用。由於mDISC1已知是調控神經發育的基因，因此本交互作用可能與調控神經發育有關。 本研究為了進一步了解mDISC1與mFBXL14的交互作用，首先製備帶有mDisc1與mFbxl14基因片段的質體，利用哺乳類細胞株(COS-b cell)為系統進行免疫共沉澱法，以定義兩蛋白質作用的結構域(interaction domains)。為檢驗兩蛋白質之交互作用是否為直接接觸，利用大腸桿菌為系統進行蛋白質結合試驗(GST pull-down assay)。為了解兩基因對小鼠胚胎大腦神經元發育之影響，以子宮內電穿孔法(in utero electroporation, IUEP)研究兩基因在小鼠胚胎腦部的功能，發現干擾mDisc1基因表現時會造成大腦皮質神經元遷徙速度減低，與先前報導相符(Kamiya et al, 2005)，並透過同一驗系統發現干擾mFbxl14基因表現時大腦皮質神經元聚集在中間帶(intermediate zone)。本研究進一步以IUEP操弄兩基因的體內表現量並定量分析神經元新生與神經元遷徙。本研究初步闡明mDISC1與mFBXL14交互作用的分子機制，為後續研究兩基因在小鼠胚胎大腦皮質發育功能之基礎。

Disrupted in Schizophrenia 1 (DISC1), first identified in human (Homo sapiens), is a disease-related gene that is associated with schizophrenia and other psychiatric disorders including bipolar disorder and autism spectrum disorders (Soares et al, 2011). DISC1 protein is known to be involved in neurodevelopment processes such as neuronal migration (Ishizuka et al, 2011) and neuronal progenitor proliferation (Singh et al, 2010). F-box and leucine-rich repeat protein 14 (FBXL14) is a subunit of E3 ubiquitin ligase complex involved in proteasome-mediated protein degradation (Cardozo et al, 2004). Preliminary data from our lab showed that mouse DISC1 (mDISC1) co-immunoprecipitates (co-IP) with mouse FBXL14 (mFBXL14), suggesting that these two proteins together may play a role in regulating neurodevelopment. To characterize the interaction of mDISC1 and mFBXL14, the deletion constructs of these two genes were prepared to define their respective interaction domains by co-IP assays. GST pull-down assay was also performed to address whether the interactions are via direct binding. Using in utero electroporation (IUEP), we found knock-down of mFbxl14 caused mouse embryonic cortical neurons gathering in the intermediate zone while knock-down of mDisc1 were reported to cause cortical neuron migration defects (Kamiya et al, 2005). How the interaction of mDISC1 and mFBXL14 may affect embryonic cortical neuronal migration and proliferation in vivo was also explored. Through these studies, the molecular basis of the interaction of mDISC1 and mFBXL14 was characterized, which provides insight into the developmental role of mDISC1 and mFBXL14 in the embryonic corticogenesis.