Table_2_Wnt/β-Catenin Signaling Promotes Differentiation of Ischemia-Activated Adult Neural Stem/Progenitor Cells to Neuronal Precursors.xlsx

Modulating endogenous regenerative processes may represent a suitable treatment for central nervous system (CNS) injuries, such as stroke or trauma. Neural stem/progenitor cells (NS/PCs), which naturally reside in the subventricular zone (SVZ) of the adult brain, proliferate and differentiate to other cell types, and therefore may compensate the negative consequences of ischemic injury. The fate of NS/PCs in the developing brain is largely influenced by Wingless/Integrated (Wnt) signaling; however, its role in the differentiation of adult NS/PCs under ischemic conditions is still enigmatic. In our previous study, we identified the Wnt/β-catenin signaling pathway as a factor promoting neurogenesis at the expense of gliogenesis in neonatal mice. In this study, we used adult transgenic mice in order to assess the impact of the canonical Wnt pathway modulation (inhibition or hyper-activation) on NS/PCs derived from the SVZ, and combined it with the middle cerebral artery occlusion (MCAO) to disclose the effect of focal cerebral ischemia (FCI). Based on the electrophysiological properties of cultured cells, we first identified three cell types that represented in vitro differentiated NS/PCs – astrocytes, neuron-like cells, and precursor cells. Following FCI, we detected fewer neuron-like cells after Wnt signaling inhibition. Furthermore, the immunohistochemical analysis revealed an overall higher expression of cell-type-specific proteins after FCI, indicating increased proliferation and differentiation rates of NS/PCs in the SVZ. Remarkably, Wnt signaling hyper-activation increased the abundance of proliferating and neuron-like cells, while Wnt pathway inhibition had the opposite effect. Finally, the expression profiling at the single cell level revealed an increased proportion of neural stem cells and neuroblasts after FCI. These observations indicate that Wnt signaling enhances NS/PCs-based regeneration in the adult mouse brain following FCI, and supports neuronal differentiation in the SVZ.

调控内源性再生过程，或可成为中枢神经系统（CNS）损伤（如脑卒中或创伤）的有效治疗策略。天然定位于成年大脑侧脑室下区（SVZ）的神经干/祖细胞（NS/PCs），可通过增殖与分化为其他细胞类型，弥补缺血性损伤带来的不良后果。发育大脑中神经干/祖细胞的命运，很大程度上受无翅/整合（Wnt）信号通路调控；但该通路在缺血条件下对成年神经干/祖细胞分化的调控作用仍不明确。在本团队此前的研究中，我们鉴定出Wnt/β-连环蛋白信号通路可在新生小鼠中促进神经发生，且该过程以牺牲胶质细胞生成为代价。本研究使用成年转基因小鼠，评估经典Wnt通路调控（抑制或过度激活）对源自侧脑室下区的神经干/祖细胞的影响，并结合大脑中动脉闭塞（MCAO）模型，以揭示局灶性脑缺血（FCI）的相关效应。基于培养细胞的电生理特性，本研究首先鉴定出体外分化的神经干/祖细胞的三类细胞亚群：星形胶质细胞、神经元样细胞及前体细胞。局灶性脑缺血造模后，抑制Wnt信号通路可检测到更少的神经元样细胞。此外，免疫组织化学分析显示，局灶性脑缺血造模后，细胞类型特异性蛋白的整体表达水平升高，提示侧脑室下区内神经干/祖细胞的增殖与分化速率提升。值得注意的是，Wnt信号通路过度激活可增加增殖细胞与神经元样细胞的丰度，而抑制Wnt通路则产生相反效应。最后，单细胞水平的表达谱分析显示，局灶性脑缺血造模后，神经干细胞与成神经细胞的比例升高。上述观察结果表明，Wnt信号通路可增强局灶性脑缺血后成年小鼠大脑中基于神经干/祖细胞的再生过程，并促进侧脑室下区内的神经元分化。