Local and CNS-wide astrocyte intracellular calcium signalling attenuation in vivo with CalExflox mice: experimental and computational exploration

In this study, we generated knock-in CalExflox mice (Calcium Extrusion) for Cre-dependent expression of mCherry-hPMCA2w/b to attenuate astrocyte calcium signaling in genetically defined cells in vivo. We crossed CalExflox mice to astrocyte specific Aldh1l1-Cre/ERT2 mice in order to achieve inducible global CNS-wide calicum signaling attenuation. Within six days of induction in the bigenic mice, we observed profoundly altered ambulation in the open field, disrupted motor coordination and gait, and premature lethality, which based on imaging, behavioural, and RNA-seq analyses was likely to be partly due to significant cerebellar defects. Overall design: CalExflox mice were crossed to astrocyte specific Aldh1l1-Cre/ERT2 mice to generate bigenic mice carrying both alleles (CalExflox; Aldh1l1-Cre/ERT2). CalExflox mice were used as controls. Both groups of mice received a single dose tamoxifen (75 mg/kg) and the cerebellar and the striata were dissected out 5 days later. The tissues were homogenized in ice-cold lysis buffer and the cerebellar RNA was extracted using a mirVana miRNA isolation kit. RNA samples with RNA integrity number (RIN) greater than 7 were used for multiplexed library preparation with Nugen Ovation RNA-Seq System V2. All samples were multiplexed into a single pool in order to avoid batch effects, and sequencing was performed on Illumina NextSeq 4000 for 2 × 75 yielding at least 45 million reads per sample.

本研究构建了敲入型CalExflox小鼠（Calcium Extrusion，钙排出），其可介导Cre依赖性的mCherry-hPMCA2w/b表达，以在体内遗传定义的细胞中减弱星形胶质细胞的钙信号通路。我们将CalExflox小鼠与星形胶质细胞特异性Aldh1l1-Cre/ERT2小鼠进行杂交，以实现可诱导的全中枢神经系统（CNS）钙信号通路减弱。在双转基因小鼠诱导后的六天内，我们观察到其旷场实验中的自主活动发生显著改变，运动协调能力与步态受损，并出现早发性致死表型；结合成像、行为学与RNA测序（RNA-seq）分析结果，该表型部分可能源于显著的小脑结构缺陷。

实验整体设计：将CalExflox小鼠与星形胶质细胞特异性Aldh1l1-Cre/ERT2小鼠杂交，获得同时携带两种等位基因的双转基因小鼠（CalExflox; Aldh1l1-Cre/ERT2），以CalExflox小鼠作为对照组。两组小鼠均单次给予他莫昔芬（75 mg/kg），并于给药后5天分离小脑与纹状体组织。将组织置于冰浴的裂解缓冲液中匀浆，随后使用mirVana miRNA分离试剂盒提取小脑RNA。选取RNA完整性指数（RNA Integrity Number, RIN）大于7的RNA样本，采用Nugen Ovation RNA-Seq System V2进行多重文库制备。为避免批次效应，所有样本均合并为单一混合文库；随后在Illumina NextSeq 4000测序平台上开展2×75 bp双端测序，每个样本至少产出4500万条测序读段（reads）。