Involvement of aquaporin-4 in laminin-enhanced process formation of mouse astrocytes in 2D culture: Roles of dystroglycan and α-syntrophin in aquaporin-4 expression
Involvement of aquaporin-4 in laminin-enhanced process formation of mouse astrocytes in 2D culture: Roles of dystroglycan and α-syntrophin in aquaporin-4 expression
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DOI：
10.1111/jnc.14548
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发表时间：
2018-11-01
影响因子：
4.7
通讯作者：
Rikitake, Yoshiyuki
中科院分区：
文献类型：
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作者：
Sato, Junya;Horibe, Sayo;Rikitake, Yoshiyuki
在中枢神经系统中，星形胶质细胞延伸终足突起以包鞘突触和微血管。然而，这种星形胶质细胞突起延伸的机制仍不清楚。使用2D培养的星形胶质细胞进行此类研究的局限性在于，它们显示出平坦的上皮样形态，没有或很少有突起，这与体内观察到的星状形态明显不同。在这项研究中，我们获得了2D培养的星形胶质细胞与丰富的复杂性的过程中使用分化的神经球在体外。使用这些过程轴承星形胶质细胞，我们表明，层粘连蛋白，细胞外基质分子丰富的血管周围网站，有效地诱导过程的形成和分支。具体而言，第一和第二阶分支过程和最大突起长度的星形胶质细胞的数量增加时，培养层粘连蛋白，与当他们培养的聚-L-鸟氨酸或IV型胶原蛋白。使用小干扰RNA敲低肌营养不良蛋白聚糖或α-突触营养蛋白（肌营养不良蛋白-糖蛋白复合物的组成蛋白，其提供层粘连蛋白和细胞骨架之间的联系）抑制星形胶质细胞过程形成和分支，并下调水通道水通道蛋白-4（AQP 4）的表达。直接敲低和特异性抑制剂的AQP 4也抑制，而过表达的AQP 4增强星形胶质细胞过程的形成和分支。AQP 4的敲低降低了与肌动蛋白重塑密切相关的粘着斑激酶（FAK）的磷酸化。总的来说，这些结果表明层粘连蛋白-肌营养不良蛋白聚糖-α-突触营养蛋白-AQP 4轴对于2D培养的星形胶质细胞的突起形成和分支是重要的。
In the central nervous system, astrocytes extend endfoot processes to ensheath synapses and microvessels. However, the mechanisms underlying this astrocytic process extension remain unclear. A limitation of the use of 2D cultured astrocytes for such studies is that they display a flat, epithelioid morphology, with no or very few processes, which is markedly different from the stellate morphology observed in vivo. In this study, we obtained 2D cultured astrocytes with a rich complexity of processes using differentiation of neurospheres in vitro. Using these process-bearing astrocytes, we showed that laminin, an extracellular matrix molecule abundant in perivascular sites, efficiently induced process formation and branching. Specifically, the numbers of the first- and second-order branch processes and the maximal process length of astrocytes were increased when cultured on laminin, compared with when they were cultured on poly-L-ornithine or type IV collagen. Knockdown of dystroglycan or alpha-syntrophin, constituent proteins of the dystrophin-glycoprotein complex that provides a link between laminin and the cytoskeleton, using small interference RNAs inhibited astrocyte process formation and branching, and down-regulated expression of the water channel aquaporin-4 (AQP4). Direct knockdown and a specific inhibitor of AQP4 also inhibited, whereas over-expression of AQP4 enhanced astrocyte process formation and branching. Knockdown of AQP4 decreased phosphorylation of focal adhesion kinase (FAK) that is critically implicated in actin remodeling. Collectively, these results indicate that the laminin-dystroglycan-alpha-syntrophin-AQP4 axis is important for process formation and branching of 2D cultured astrocytes.