Sadakata Tetsushi, Mizoguchi Akira, Sato Yumi, Katoh-Semba Ritsuko, Fukuda Mitsunori, Mikoshiba Katsuhiko, Furuichi Teiichi
Laboratory for Molecular Neurogenesis, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan.
J Neurosci. 2004 Jan 7;24(1):43-52. doi: 10.1523/JNEUROSCI.2528-03.2004.
Neurotrophins are key modulators of various neuronal functions, including differentiation, survival, and synaptic plasticity, but the molecules that regulate their secretion are poorly understood. We isolated a clone that is predominantly expressed in granule cells of postnatally developing mouse cerebellum, which turned out to be a paralog of CAPS (Ca2+-dependent activator protein for secretion), and named CAPS2. CAPS2 is enriched on vesicular structures of presynaptic parallel fiber terminals of granule cells connecting postsynaptic spines of Purkinje cell dendrites. Vesicle factions affinity-purified by the CAPS2 antibody from mouse cerebella contained significant amounts of neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), and chromogranin B but not marker proteins for synaptic vesicle synaptophysin and synaptotagmin. In cerebellar primary cultures, punctate CAPS2 immunoreactivities are primarily colocalized with those of NT-3 and BDNF and near those of a postsynaptic marker, postsynaptic density-95, around dendritic arborization of Purkinje cells. Exogenously expressed CAPS2 enhanced release of exogenous NT-3 and BDNF from PC12 cells and endogenous NT-3 from cultured granule cells in a depolarization-dependent manner. Moreover, the overexpression of CAPS2 in granule cells promotes the survival of Purkinje cells in cerebellar cultures. Thus, we suggest that CAPS2 mediates the depolarization-dependent release of NT-3 and BDNF from granule cells, leading to regulation in cell differentiation and survival during cerebellar development.
神经营养因子是多种神经元功能的关键调节因子，包括分化、存活和突触可塑性，但调节其分泌的分子却知之甚少。我们分离出一个在出生后发育的小鼠小脑颗粒细胞中主要表达的克隆，结果发现它是CAPS（钙依赖性分泌激活蛋白）的一个旁系同源物，并将其命名为CAPS2。CAPS2富集于连接浦肯野细胞树突突触后棘的颗粒细胞突触前平行纤维终末的囊泡结构上。用CAPS2抗体从小鼠小脑中亲和纯化的囊泡组分含有大量的神经营养因子-3（NT-3）、脑源性神经营养因子（BDNF）和嗜铬粒蛋白B，但不含有突触囊泡的标记蛋白突触素和突触结合蛋白。在小脑原代培养物中，点状的CAPS2免疫反应性主要与NT-3和BDNF的免疫反应性共定位，并靠近浦肯野细胞树突分支周围的突触后标记物突触后致密蛋白-95。外源表达的CAPS2以去极化依赖的方式增强了PC12细胞中外源NT-3和BDNF以及培养的颗粒细胞中内源性NT-3的释放。此外，在颗粒细胞中过表达CAPS2可促进小脑培养物中浦肯野细胞的存活。因此，我们认为CAPS2介导了颗粒细胞中NT-3和BDNF的去极化依赖性释放，从而在小脑发育过程中调节细胞分化和存活。
