Biochemical and physiological assessment of neural visinin-like calcium-binding protein 3 in rodent cerebellum.

神经粘蛋白样钙结合蛋白3是神经细胞钙传感器蛋白(NCS)家族的成员，在小脑中高表达。免疫印迹和免疫组织化学方法检测NVP3在大鼠脑内的表达。3个月时，NVP3主要定位于浦肯野细胞，胞体、树突和轴突中有较强的染色。小脑颗粒细胞和基底核神经元染色较淡。在小脑发育过程中，NVP3阳性浦肯野细胞最早出现在出生后第14天(P14)。染色强度随后增加，并在P28时趋于平稳。标记有在树突和神经末梢内以细小颗粒状聚集的趋势。在衰老过程中，NVP3水平在12个月时下降了43%，24个月时下降了68%，而NVP1、突触素和Drebrin的水平则优先得到保留。这些结果表明NVP3是inv…。小脑浦肯野细胞的树突分枝和突触后功能得到了更多的解决，突触前神经末梢是该蛋白的另一个功能部位。为了揭示其在体内的生理作用，我们产生了NVP3缺失突变(-/-)小鼠。首先，我们克隆了小鼠NVP3基因和cDNA，小鼠NVP3基因由4个外显子和3个内含子组成，全长约7kb。对小鼠基因组DNA的Southern杂交分析表明，阳性条带与克隆的基因序列完全一致，表明小鼠NVP3基因是以单拷贝基因的形式存在的。然后，利用新霉素抗性基因和白喉毒素A基因构建了NVP3基因敲除载体。将载体DNA导入CCE胚胎干细胞。筛选阳性克隆，显微注射C57BL/6囊胚产生嵌合体。嵌合体的F1杂合子相互杂交，产生零突变(-/-)小鼠。零突变小鼠在光镜下未见明显的小脑结构异常。NVP3的缺失不能通过同源蛋白NVP1、2和海马素的上调来弥补。运动活动和昼夜周期也未观察到明显异常。小脑Purkije细胞的电生理反应和运动协调学习能力将在进一步的研究中进行研究。较少

Neural visinin-like calcium-binding protein 3, a member of neuronal calcium sensor protein (NCS) family, is highly expressed in the cerebellum. Expression of NVP3 was assessed by immunoblot and immunohistochemical analyses in rat brain. NVP3 was markedly expressed in the cerebellum, at a concentration of 9.5μM.At 3 months, NVP3 was primarily localized in the Purkinje cells, with intense staining in the cell bodies, dendrites and axons. The cerebellar granule cells and basal nuclear neurons were faintly stained. During development of the cerebellum, NVP3-positive Purkinje cells first appeared on post-natal day 14 (P14). The staining intensity then increased and plateaued on P28. Labeling showed a tendency to accumulate in the dendrites and nerve terminals in a fine granular pattern. During aging process, NVP3 levels decreased by 43% at 12 months and 68% at 24 months, while the levels of NVP1, synaptophysin and drebrin were preferentially preserved. These results suggest that NVP3 is inv … More olved in dendritic arborization and postsynaptic function in cerebellar Purkinje cells and that presynaptic nerve terminals are another functional sites of the protein.To reveal its physiological roles in vivo, we generated NVP3 null mutant (-/-) mice. Firstly, we isolated the mouse NVP3 gene and cDNA.The mouse NVP3 gene contains 4 exons and 3 introns and spans approximately 7 kb. Southern blot analysis of the mouse genomic DNA demonstrated that positive bands exactly coincide with those expected from sequences of the cloned genes, indicating that the mouse NVP3 gene is present as a single copy gene. Then, we constructed NVP3 knockout vector using neomycin-resistance gene and Diphtheria toxin A gene. The vector DNA was introduced into CCE embryonic stem (ES) cells. Positive clones were selected and microinjected into C57BL/6 blastocysts to generate the chimeras. F1 heterozygotes form the chimeras were intercrossed with each other to yield null mutant (-/-) mice. Null mutant mice exhibited no apparent structural abnormality of the cerebellum in light microscopic levels. The lack of NVP3 was not compensated by possible up-regulation of homologous proteins NVP1, 2 and hippocalcin. No apparent abnormalities were also observed in motor activity and day-night cycles. Electrophysiological responses of the cerebellar Purkije cells and motor coordination learning abilities will be investigated in further studies. Less