br Acknowledgements We extend our sincere

Acknowledgements We extend our sincere thanks to Cindy Cheung for her help with the immunostaining, Yuhuan Wang for her help with daily cell culture, and Atena Zahedi for growing the GFP-RAC activated plasmid. The pcDNA3-EGFP-Rac1-Q61L plasmid (Plasmid # 12981) was created by Gary Bokoch and purchased from Addgene (Sunauste et al., 2000).
Introduction Obtaining and manipulating neurons are critical for neural biology mechanism studies, drug discovery and cell therapy applications. To date, several methods have been established to derive neurons including direct isolation from neural tissues (Barker et al., 2013; Goldberg et al., 2002; MacLaren et al., 2006), differentiation from neural stem retinoic acid receptor (NSCs) (Chen et al., 2016; Gage and Temple, 2013), or gradual generation from pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) (Kriks et al., 2011; Qian et al., 2016; Tao and Zhang, 2016). Each method has not only its advantages but also its caveats. For example, although primary neurons are naturally occurring cells that do not need further in vitro differentiation manipulations, their sources are very limited making them impractical for most neural biology translational applications. Although NSCs can be expanded in vitro, both their proliferation and differentiation capacities are far from ideal. Similarly, despite the rising interest in applying ESCs/iPSCs due to their tremendous amplification and differentiation capacities, their tedious preparation procedures and tumor formation concerns remain an issue in ES/iPS-based cell therapy applications (Fox et al., 2014; Steinbeck and Studer, 2015). Recently, it has been shown that terminally differentiated somatic cells can be directly reprogrammed to become functional neuronal cells, which not only demonstrated a much greater cell fate plasticity of terminally differentiated cells than previously thought, but also expanded the cell sources accessible for basic and translational neural biology studies (Pang et al., 2011; Vierbuchen et al., 2010). In 2010, Wernig lab demonstrated that by overexpressing Ascl1, Brn2 and Myt1l (BAM), mouse fibroblasts can be directly converted into functional neuronal cells, termed iNs for ‘induced neuronal cells’, that express multiple neuron specific genes, exhibit neural membrane activities, and establish synaptic communications with other neurons (Vierbuchen et al., 2010). By adding an additional neuron determining transcription factor-NeuroD1(BAMN), they later demonstrated that human iNs can also be generated from embryonic and postnatal fibroblasts, though at a lower efficiency than in mice (Pang et al., 2011). Based on this, iN direct reprogramming has been extensively explored by the field, and a number of direct reprogramming protocols for specific types of neuron have been established (Ambasudhan et al., 2011; Blanchard et al., 2015; Caiazzo et al., 2011; Hu et al., 2015; Kim et al., 2011; Ladewig et al., 2012; Li et al., 2015; Liu et al., 2012; Pfisterer et al., 2011; Son et al., 2011; Victor et al., 2014; Wainger et al., 2015; Xue et al., 2013; Yoo et al., 2011). Among a variety of iN induction transcription factors, Ascl1 seems to be the most critical one: it is included in most iN induction protocols; it alone is able to induce iNs from fibroblasts, though at a lower efficiency, and requires the help from glial cells (Caiazzo et al., 2011; Chanda et al., 2014; Karow et al., 2012; Kim et al., 2011; Liu et al., 2012; Pang et al., 2011; Pfisterer et al., 2011; Son et al., 2011; Vierbuchen et al., 2010; Wainger et al., 2015). More importantly, Ascl1 has been reported to functions as an ‘on target’ pioneer factor that immediately occupies its cognate genomic targets to initiate the reprogramming process during iN induction, while other factors participate later to ensure the neural reprogramming route (Treutlein et al., 2016; Wapinski et al., 2013).