林炎壽Yenshou Lin莊仁華Jen-Hua Chuang2019-09-052018-8-212019-09-052013http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22GN0895430072%22.&%22.id.&http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/104452神經細胞的生長與分化需要許多因子傳遞訊息及相關蛋白快速且大量新合成，以因應如發育過程或環境的刺激。目前有一些訊息傳遞路徑已被報導參與在神經的生長，如CAMKII路徑，又如另一與蛋白轉譯息息相關的一重要因子- mTOR。以細胞腦組織初級培養的實驗證實mTOR的活化有助於長期記憶的形成及神經纖維的突觸可塑性等。然而對於mTOR是否影響由胚胎幹細胞所分化出的神經細胞之生長等則所知甚少。另外，對於近年來所發現mTOR藉由和不同蛋白而組合成不同之蛋白複合體，其中mTOR蛋白複合體II對於神經之生長或型態的作用也是鮮少被探討，因此我們旨在探討mTOR蛋白複合體I與mTOR蛋白複合體II對於由胚胎幹細胞所分化出的神經細胞生長之影響。首先，我們建立小鼠胚胎幹細胞分化成神經的模式，發現新鮮的胰蛋白酶在特定的時間內作用，可以有效地將形成的胚胎體分解成較小的球體，並且得到平均約87%均一性的神經細胞，且這些神經細胞屬於可分泌谷氨酸的興奮性神經元。利用這些神經細胞進而來探討mTOR蛋白複合體I對其生長之影響。先以藥物處理方式給予分化後的神經細胞mTOR蛋白複合體I的抑制劑，0.2μM或1μM雷帕拉霉素（rapamycin），均在處理後第三天明顯導致神經纖維的斷裂與神經細胞的死亡；在分子層級方面，以病毒包裹RNAi感染胚胎幹細胞、以抑制其mTORC1複合體I中raptor基因表達的實驗中發現雖然這些幹細胞仍可形成胚胎體，但體積明顯地比對照組的胚胎體小很多，甚至導致分化失敗。至於mTORC2對於神經細胞所扮演角色之探討，我們以胚胎幹細胞分化成之神經細胞以及小鼠大腦皮質神經元之初級培養細胞二者為模式加以研究。除了研究mTOR蛋白複合體II中專一蛋白rictor之外，之前在rictor基因剃除的胚胎纖維細胞，本實驗室又發現一與rictor有關的蛋白，暫時稱之為RICAP。以免疫沉澱方式已驗證HA-rictor與FLAG-RICAP得以結合，目前亦發現rictor/RICAP對神經的生長極具影響。此等研究了解了mTOR蛋白複合體I及II在小鼠胚胎幹細胞所分化出的神經細胞之重要性，並且於訊息傳遞領域開創了可能之新穎範疇。Neuronal growth and differentiation need many signal cues and de novo protein synthesis to convey information in order to respond to various environmental stimulations. Some signal pathways have been demonstrated to participate in the neuronal growth, such as Ca2+/calmodulin-dependent protein kinase II (CaMKII) and cell division cyclin 42 (Cdc 42) pathway. Previous studies suggested that mTOR, mammalian target of rapamycin, is important in the formation of long-term potential (LTP)/long-term depression (LTD) by using animal model or primary neuronal cells. However, much less is known regarding the role of mTOR and its complexes in the neurons differentiated from mouse embryonic stem cells (mESCs). In addition, the upstream regulators, downstream molecules, and roles of mTORC2, a newly identified mTOR complex, are also largely unknown. Hence, we aim to investigate the roles of mTORC1 and mTORC2 in the progression of neuronal growth/morphological change by using neurons differentiated from mESCs. First of all, we established a cellular model in which glutamatergic neurons can be uniformly differentiated from mESCs. We found that applying fresh trypsin/EDTA solution to dissociate embryonic bodies (EBs) in critical timing determines that >87% of cells differentiated into glutamatergic neurons. By employing these neurons, we found that neurites loss as well as soma shrinkage after 0.2μM or 1μM rapamycin treatment for 48 to 72 hr. Likewise, the EBs formation from mESCs infected with raptor shRNAs showed a smaller size, even fail to differentiate into neurons. Interestingly, phosphorylation of ribosomal protein S6 kinase (S6K), but not 4E-binding protein 1(4E-BP1), was decreased in rapamycin- or shRNA- treated neurons. On the other hand, a novel rictor associated protein, named RICAP, is recently revealed in our laboratory through immunoprecipitation (IP) and mass spectrometry analysis. FLAG-RICAP and HA-rictor were demonstrated to be able to associate with each other by using IP. The regulation/ function/ morphology of this complex remains further investigation. Taken together, this study provides a new insight to reveal Mtorc1 dependent mechanism which is involved in neuronal growth. The observation of a difference between S6K and 4E-BP1 in neurons suggests that additional regulation might be involved. Equally important, a groundbreaking research regarding Mtorc2 and its novel partner in neuroscience might shed a light on signal transduction as well.mTOR蛋白複合體ImTOR蛋白複合體II小鼠胚胎幹細 胞胚胎體雷帕拉霉素mTORC1mTORC2mouse embryonic stem cellsembryonic bodiesrapamycin