BMAL1 loss in oligodendroglia contributes to abnormal myelination and sleep

Myelination depends on maintenance of oligodendrocytes that arise from oligodendrocyte precursor cells (OPCs). We show that OPC-specific proliferation, morphology, and BMAL1 are time-of-day dependent. Knock out of Bmal1 in OPCs during development disrupts expression of genes associated with circadian rhythms, proliferation, density, morphology, and migration, leading to changes in OPC dynamics in a spatio-temporal manner. Furthermore, these deficits translate into thinner myelin, dysregulated cognitive and motor function, and increased sleep fragmentation. OPC-specificBmal1loss in adulthood does not alter OPC density at baseline but impairs remyelination of a demyelinated lesion driven by changes in OPC morphology and migration. Lastly, we show sleep fragmentation is associated with increased prevalence of the demyelinating disorder multiple sclerosis (MS), suggesting a link between MS and sleep that requires further investigation. These findings have broad mechanistic and therapeutic implications for brain disorders that include both myelin and sleep phenotypes. Overall design: We developed a model to specifically knock out the Bmal1 DNA-binding domain in OPCs during embryonic development by crossing clock gene knock out (Bmal1fl/fl) and cell type-specific Cre driver (NG2::Cre) mice to generate a Bmal1 transcriptional hypomorph that significantly decreased BMAL1 levels specifically in OPCs (NG2:Cre+;Bmal1fl/fl or OPC-Bmal1-KO). To test the role of BMAL1 as a transcriptional regulator in OPCs, we isolated OPCs from P6-7 OPC-Bmal1-WT and OPC-Bmal1-KO mice at 6-hr intervals for 24 hrs. OPCs were isolated at 4 different zeitgeber times: ZT0 (lights on), ZT6, ZT12 (lights off), and ZT18. For each biological replicate, 2 mice from both sexes were pooled to obtain around one million OPCs. OPCs were purified using immunopanning through 2 negative selection plates for microglia and endothelial cells with BSL1 (Vector Laboratories L1100) and one positive selection plate with an anti-PDGFRa antibody (rat anti-mouse CD140A, BD Pharmingen 558774). OPCs were immediately lysed using QIAzol Lysis Reagent. RNA extraction was performed following manufacturer's instructions and RNA purity, integrity and concentration were assessed using a Bioanalyzer. Library prep and Bulk RNA sequencing was performed using Novogene Co., Ltd. sequencing service.

髓鞘形成依赖于由少突胶质前体细胞（oligodendrocyte precursor cells, OPCs）分化而来的少突胶质细胞的维持。本研究证实，少突胶质前体细胞的特异性增殖、形态特征以及BMAL1的表达均具有昼夜节律依赖性。在发育阶段特异性敲除少突胶质前体细胞中的Bmal1基因，会扰乱与昼夜节律、增殖、细胞密度、形态及迁移相关的基因表达谱，进而以时空动态的方式改变少突胶质前体细胞的生物学特性。此外，此类缺陷最终会导致髓鞘厚度降低、认知与运动功能失调，以及睡眠碎片化程度加剧。成年阶段特异性敲除少突胶质前体细胞中的Bmal1基因，虽不会在基线状态下改变少突胶质前体细胞的密度，但会通过影响少突胶质前体细胞的形态与迁移能力，损害脱髓鞘损伤后的髓鞘再生过程。最后，本研究发现睡眠碎片化与脱髓鞘疾病多发性硬化（multiple sclerosis, MS）的患病率升高存在显著关联，提示多发性硬化与睡眠之间存在潜在关联，有待进一步深入探究。上述研究结果对兼具髓鞘与睡眠表型的脑部疾病具有广泛的机制研究与治疗转化价值。实验整体设计：本研究通过将时钟基因条件性敲除小鼠（Bmal1fl/fl）与细胞类型特异性Cre驱动小鼠（NG2::Cre）杂交，构建了可在胚胎发育阶段特异性敲除少突胶质前体细胞中Bmal1 DNA结合结构域的模型，得到了仅在少突胶质前体细胞中显著降低BMAL1表达水平的Bmal1转录低表达模型（NG2:Cre+;Bmal1fl/fl，即OPC-Bmal1-KO）。为验证BMAL1作为少突胶质前体细胞转录调控因子的功能，我们于出生后第6~7天，每隔6小时采集一次样本，连续24小时，分别从OPC-Bmal1-WT与OPC-Bmal1-KO小鼠体内分离少突胶质前体细胞。本次研究选取4个不同的授时因子时间（zeitgeber time, ZT）进行样本采集：ZT0（开灯时刻）、ZT6、ZT12（关灯时刻）与ZT18。每个生物学重复均混合2只不同性别的小鼠，以获取约100万个少突胶质前体细胞。随后通过免疫淘选法纯化少突胶质前体细胞：使用结合BSL1凝集素（Vector Laboratories L1100）的2块阴性筛选板去除小胶质细胞与内皮细胞，再通过结合抗PDGFRα抗体（大鼠抗小鼠CD140A，BD Pharmingen 558774）的1块阳性筛选板进行富集。纯化后的少突胶质前体细胞立即使用QIAzol裂解试剂进行裂解。RNA提取严格按照试剂盒说明书操作完成，并使用生物分析仪对RNA的纯度、完整性与浓度进行质控检测。文库构建与批量RNA测序由诺禾致源（Novogene Co., Ltd.）测序服务平台完成。