Unkempt Is Negatively Regulated by mTOR and Uncouples Neuronal Differentiation from Growth Control

Neuronal differentiation is exquisitely controlled both spatially and temporally during nervous system development. Defects in the spatiotemporal control of neurogenesis cause incorrect formation of neural networks and lead to neurological disorders such as epilepsy and autism. The mTOR kinase integrates signals from mitogens, nutrients and energy levels to regulate growth, autophagy and metabolism. We previously identified the insulin receptor (InR)/mTOR pathway as a critical regulator of the timing of neuronal differentiation in the Drosophila melanogaster eye. Subsequently, this pathway has been shown to play a conserved role in regulating neurogenesis in vertebrates. However, the factors that mediate the neurogenic role of this pathway are completely unknown. To identify downstream effectors of the InR/mTOR pathway we screened transcriptional targets of mTOR for neuronal differentiation phenotypes in photoreceptor neurons. We identified the conserved gene unkempt (unk), which encodes a zinc finger/RING domain containing protein, as a negative regulator of the timing of photoreceptor differentiation. Loss of unk phenocopies InR/mTOR pathway activation and unk acts downstream of this pathway to regulate neurogenesis. In contrast to InR/mTOR signalling, unk does not regulate growth. unk therefore uncouples the role of the InR/mTOR pathway in neurogenesis from its role in growth control. We also identified the gene headcase (hdc) as a second downstream regulator of the InR/mTOR pathway controlling the timing of neurogenesis. Unk forms a complex with Hdc, and Hdc expression is regulated by unk and InR/mTOR signalling. Co-overexpression of unk and hdc completely suppresses the precocious neuronal differentiation phenotype caused by loss of Tsc1. Thus, Unk and Hdc are the first neurogenic components of the InR/mTOR pathway to be identified. Finally, we show that Unkempt-like is expressed in the developing mouse retina and in neural stem/progenitor cells, suggesting that the role of Unk in neurogenesis may be conserved in mammals.

神经系统发育过程中，神经元分化在空间与时间维度上均受到精密调控。神经发生的时空调控异常会导致神经网络构建错误，并引发癫痫、孤独症等神经系统疾病。哺乳动物雷帕霉素靶蛋白激酶（mTOR kinase）可整合丝裂原、营养物质与能量水平的信号，从而调控细胞生长、自噬与代谢过程。此前我们已在黑腹果蝇（Drosophila melanogaster）的视网膜中证实，胰岛素受体（InR）/mTOR通路是神经元分化时序的关键调控因子。后续研究证实，该通路在脊椎动物的神经发生过程中同样发挥保守的调控作用。但目前完全未知的是，介导该通路神经调控功能的下游效应因子究竟为何物。为鉴定InR/mTOR通路的下游效应因子，我们以感光神经元的神经元分化表型为筛选指标，对mTOR的转录靶标进行了筛查。我们筛选得到了保守基因unkempt（unk），其编码一种含锌指/RING结构域的蛋白质，可作为感光神经元分化时序的负调控因子。unk基因敲除可模拟InR/mTOR通路激活的表型，且unk通过该通路的下游参与调控神经发生。与InR/mTOR信号通路不同的是，unk并不参与细胞生长的调控。因此，unk可将InR/mTOR通路在神经发生中的功能与其生长调控功能解耦联。我们同时还鉴定出了headcase（hdc）基因，其作为第二个下游调控因子参与InR/mTOR通路对神经发生时序的调控。Unk可与Hdc形成蛋白复合物，且hdc的表达受unk与InR/mTOR信号通路的调控。共过表达unk与hdc可完全抑制Tsc1基因敲除所引发的神经元过早分化表型。综上，Unk与Hdc是首个被鉴定出的InR/mTOR通路神经源性调控组分。我们还证实，Unkempt-like蛋白在发育中的小鼠视网膜以及神经干细胞/前体细胞中均有表达，这提示Unk在神经发生中的调控功能在哺乳动物中同样具有保守性。