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

神经视素样钙结合蛋白3（Neural visin-like calcium-binding protein 3，NCL 3）是神经元钙传感蛋白（neuronal calcium sensor protein，NCS）家族的一员，在小脑中高表达。通过免疫印迹和免疫组织化学分析评估大鼠脑中NVP 3的表达。NVP 3在小脑中显著表达，浓度为9.5μM。在3个月时，NVP 3主要定位于浦肯野细胞中，在细胞体、树突和轴突中具有强染色。小脑颗粒细胞和基底核神经元呈淡染。在小脑发育过程中，NVP 3阳性的浦肯野细胞在出生后第14天（P14）首次出现。然后染色强度增加，并在P28达到平台。标记显示倾向于聚集在树突和神经末梢中的细颗粒图案。在衰老过程中，NVP 3水平在12个月和24个月时分别下降了43%和68%，而NVP 1、突触素和突触素的水平优先保留。这些结果表明，NVP 3是inv ...更多信息 NVP 3蛋白在小脑浦肯野细胞中的树突状分支和突触后功能中发挥重要作用，突触前神经末梢是NVP 3蛋白的另一个功能位点。首先，我们克隆了小鼠NVP 3基因及其cDNA，该基因由4个外显子和3个内含子组成，全长约7 kb。小鼠基因组DNA的Southern印迹分析表明，阳性条带与克隆基因序列中预期的条带完全一致，表明小鼠NVP 3基因以单拷贝基因存在。然后，我们利用新霉素抗性基因和白喉毒素A基因构建了NVP 3基因敲除载体。将载体DNA导入CCE胚胎干（ES）细胞中。选择阳性克隆并显微注射到C57 BL/6胚泡中以产生嵌合体。将来自嵌合体的F1杂合子彼此杂交以产生无效突变（-/-）小鼠。在光镜下，突变小鼠的小脑结构无明显异常。NVP 3的缺乏并没有通过同源蛋白NVP 1、2和钙调蛋白的可能上调来补偿。在运动活动和昼夜周期中也未观察到明显异常。小脑浦肯野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。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