运用CRISPR-Cas9转基因小鼠研究AQP4在青春期小鼠脑神经干细胞中的调节机制

Neurogenesis in adolescence exist a variety of developmental differences in brain, especially in hippocampus from that in adulthood, which is considered to be more homeostasis. Adolescent neurogenesis is a more highly dynamic process modulated by many intrinsic and extrinsic factors. However, the regulatory mechanism involving adolescent endogenous neural stem cell lineage progression is still unclear, due to the limitation of present animal models. Aquaporin 4 (AQP4) is the major water channel in the mammalian brain and is important for brain neurogenesis. We previously revealed that AQP4 is differentially expressed in the neural stem cells (NSCs) lineage progression from the subventricular zone, one of the main neurogenic niches. Increase in AQP4 expression levels at the cell membrane of NSCs can induce neural stem cell activation, which is essential for water transportation and brain homeostasis. Here, we apply fluorescent-tagged CRISPR-Cas9 transgenic mice model, in vivo fluorescent tracing method and lentiviral delivery system, etc, in order to examine the endogenous neurogenesis and lineage progression mapping. We aim to investigate in vivo, the changes of AQP4 during neural stem cell lineage progression in adolescence, when compared with that in adulthood, in the mouse hippocampal dentate gyrus. Furthermore, we also intend to reveal the effects of NSCs-specific AQP4 ablation on in vivo adolescent neurogenesis. The AQP4 ablation is achieved by lentiviral delivery of sgRNA for Aqp4 into the aimed brain locus-dentate gyrus of hippocampus at different developmental stages, including adolescence and adulthood. Our findings will provide a new insight into the regulation of neurogenesis in adolescence and adulthood in vivo. Furthermore, the NSCs-specific CRISPR-Cas9 system can be served as an efficient tool for gene targeting in NSCs at different development stage in vivo.

青春期中枢系统尚未发育成熟，其脑内，特别是海马区神经干细胞的发育对内外部因素刺激反应更敏感，有别于成年期稳态。但因模型限制，其神经干细胞的发育谱系尚不明确。水通道4 (AQP4)是脑内主要的水通道蛋白，参与调节脑神经干细胞发育。申请人发现，AQP4在幼年及成年期神经干细胞发育进程中表达具有特异性；激活神经干细胞中的AQP4促进神经干细胞发育，对维持脑稳态至关重要。本课题拟运用特异性诱导表达CRISPR-Cas9的转基因小鼠，通过内源荧光示踪，慢病毒表达等方法，对比成年期，在体内外研究青春期神经干细胞发育谱系中AQP4表达分布变化；针对特定发育时期，在海马区神经干细胞中特异性敲除AQP4，重点探讨AQP4对青春期神经干细胞发育谱系的影响及机制。因人鼠AQP4高同源性，此成果将为研究人青春期脑神经干细胞稳态的维持提供新的体内依据；为研究不同时期神经干细胞相关调节基因提供有效的体内动物模型。

青春期大脑正处在中枢神经发育的特殊阶段，尤其是青春期脑神经干细胞发育，对内源性和外源性刺激反应敏感，有别于成年期相对稳定的脑神经干细胞发育。然而，由于模型的局限性，我们对青春期内源神经干细胞的发育谱系尚不完全清楚。本课题运用特异性在神经干细胞中诱导表达NestinCreERT2::CRISPR-Cas9-GFP和NestinCreERT2::Rosa-tdTomato的荧光标记的转基因小鼠，运用免疫荧光、病毒感染、流式分选及PCR等技术，内源性的及体外原代培养神经干细胞水平上对特定发育阶段-青春期和成年期进行研究，A) 通过CRISPR-Cas9的基因敲除技术特异性在神经干细胞中敲除AQP4，阐明AQP4在不同发育时期体内神经干细胞发育谱系中的表达分布变化，揭示了特异性神经干细胞内敲除AQP4对青春期及成年期小鼠的神经干细胞发育谱系及脑稳态的影响；揭示特异性神经干细胞内敲除AQP4对小鼠青春期以及成年期海马神经干细胞中相关基因通路的影响机制。B) 阐明大量酒精摄入可以更显著抑制青春期小鼠神经干细胞的激活，神经干细胞分化成神经元以及抑制新生神经元的成熟过程，说明青春期小鼠对酒精更敏感。而自主跑步可以逆转这种酒精抑制作用。C）阐明人参皂苷活性单体Re和PF11对青春期和成年期神经干细胞的激活和分化有不同程度的促进作用及组蛋白甲基化调节的相关机制。此成果将为研究人青春期脑神经干细胞的稳态提供新的体内依据；为研究不同发育时期神经干细胞相关调节基因提供有效的体内动物模型。