Elucidation of physiological significance of a novel regulator of neuronal functions and its application to functional regeneration of brain

V-1是一种新的锚蛋白重复序列蛋白，在出生后大脑发育过程中表现出短暂的表达水平增加。该蛋白还具有控制PC12D细胞中酪氨酸羟化酶（TH）基因表达和儿茶酚胺释放的活性，并与肌动蛋白封顶蛋白CapZ结合，在体外调节肌动蛋白聚合。然而，V-1在控制肌动蛋白动力学中的生理意义尚不清楚。在本研究中，我们首次发现在v -1过表达的PC12D转染物中，与病媒控制的转染物相比，细胞骨架部分的CapZ数量减少，而细胞质部分的CapZ数量增加。与capz相关的肌动蛋白丝的数量也减少，导致V-1转染物中F-/ g -肌动蛋白的比例增加。此外，在用forskolin处理PC12D细胞后5min， CapZ的capping活性受到刺激，并伴随其从细胞质部分向细胞骨架部分的移位。因此，V-1可能是一种新的胞质锚点，通过与V-1的相互作用来控制CapZ的活性可能是一种新的pka依赖的肌动蛋白动力学调节机制。此外，作为寻找体内具有正或负控制V-1与CapZ相互作用活性的天然产物的第一步，我们筛选了20种天然草药提取物，以研究这些草药是否影响V-1与CapZ的相互作用。因此，几种草药显示出这种药理肌动蛋白。构建了V-1逆转录病毒载体，成功地在神经干细胞中过表达了V-1蛋白。在生长培养基中，神经干细胞中V-1的过表达明显产生星形胶质细胞和神经元。此外，V-1逆转录病毒抑制前脂肪细胞系向脂肪细胞的分化。

V-1 has been identified as a novel ankyrin repeat protein which exhibits a transient increase in the protein expression level during the postnatal brain development. This protein also has the activity to control both tyrosine hydroxylase (TH) gene expression and catecholamine release in PC12D cells, and associates with CapZ, an actin capping protein to regulate actin polymerization in vitro. However the physiological significance of V-1 in controlling actin dynamics remains unknown. In this study, we for the first time showed that in V-1-overexpressing PC12D transfectant, the amount of CapZ in the cytoskeletal fraction was decreased, whereas that in the cytoplasmic fraction was increased, as compared with of vector control transfectant. The amount of CapZ-associated actin filaments was also decreased to produce an increment of the ratio of F-/G-actin in V-1 transfectant. Moreover the capping activity of CapZ was stimulated at 5 min after treatment with forskolin, accompanied by its translocation from the cytoplasmic to cytoskeletal fractions in PC12D cells. It is thus suggested that V-1 serves as a novel cytoplasmic anchor of CapZ and that control of CapZ activity by its interaction with V-1 probably acts as a novel PKA-dependent regulatory mechanism of actin dynamics in vivo. Furthermore, as the first step to searching for natural products with the activity to positively or negatively control the interaction between V-1 and CapZ in vivo, we screened twenty natural herbal extracts to examine whether these herbs affected the interaction between V-1 and CapZ. Consequently several herbs showed such a pharmacological actin.Also we constructed a retrovirus vector for expression of V-1 to successfully overexpress its protein in neural stem cells. Notably overexpression of V-1 in neural stem cells appreciably produced astrocytes and neurons in the growth medium. In addition the V-1 retrovirus inhibited differentiation of a preadipocyte cell line to adipocytes.