The role of mTOR complexes signaling pathway in neurons differentiated from mouse embryonic stem cells

• Main Authors: Jen-Hua Chuang, 莊仁華
• Other Authors: Yenshou Lin
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/24508814680614177299

博士 === 國立臺灣師範大學 === 生命科學研究所 === 101 === 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.