Furlan Sandra, Campione Marina, Murgia Marta, Mosole Simone, Argenton Francesco, Volpe Pompeo, Nori Alessandra
Consiglio Nazionale delle Ricerche, Institute of Neuroscience, Padova, Italy.
Department of Biomedical Sciences, University of Padova, Istituto Interuniversitario di Miologia, Padova, Italy.
Front Neuroanat. 2020 Apr 21;14:15. doi: 10.3389/fnana.2020.00015. eCollection 2020.
Calcium stores in neurons are heterogeneous in compartmentalization and molecular composition. (zebrafish) is an animal model with a simply folded cerebellum similar in cellular organization to that of mammals. The aim of the study was to identify new endoplasmic reticulum (ER) calcium store markers in zebrafish adult brain with emphasis on cerebellum and optic tectum. By quantitative polymerase chain reaction, we found three RNA transcripts coding for the intra-ER calcium binding protein calsequestrin: , , and . In brain homogenates, two isoforms were detected by mass spectrometry and western blotting. Fractionation experiments of whole brain revealed that Casq1a and Casq2 were enriched in a heavy fraction containing ER microsomes and synaptic membranes. By hybridization, we found the heterogeneous expression of and mRNA to be compatible with the cellular localization of calsequestrins investigated by immunofluorescence. Casq1 was expressed in neurogenic differentiation 1 expressing the granule cells of the cerebellum and the periventricular zone of the optic tectum. Casq2 was concentrated in parvalbumin expressing Purkinje cells. At a subcellular level, Casq1 was restricted to granular cell bodies, and Casq2 was localized in cell bodies, dendrites, and axons. Data are discussed in relation to the differential cellular and subcellular distribution of other cerebellum calcium store markers and are evaluated with respect to the putative relevance of calsequestrins in the neuron-specific functional activity.
神经元中的钙储存库在区室化和分子组成方面是异质的。斑马鱼是一种动物模型，其小脑折叠简单，细胞组织与哺乳动物相似。本研究的目的是在斑马鱼成体脑中鉴定新的内质网（ER）钙储存标记物，重点是小脑和视顶盖。通过定量聚合酶链反应，我们发现了三种编码内质网内钙结合蛋白钙网蛋白的RNA转录本：……和……。在脑匀浆中，通过质谱和蛋白质印迹检测到两种异构体。全脑分级实验表明，Casq1a和Casq2在含有内质网微粒体和突触膜的重分级中富集。通过……杂交，我们发现……和……mRNA的异质表达与通过免疫荧光研究的钙网蛋白的细胞定位一致。Casq1在表达小脑颗粒细胞和视顶盖室周区的神经源性分化1中表达。Casq2集中在表达小白蛋白的浦肯野细胞中。在亚细胞水平上，Casq1局限于颗粒细胞体，而Casq2定位于细胞体、树突和轴突。结合其他小脑钙储存标记物的不同细胞和亚细胞分布对数据进行了讨论，并就钙网蛋白在神经元特异性功能活动中的假定相关性进行了评估。
