Dynamics of reactive astrocytes fosters tissue regeneration after cuprizone-induced demyelination [RNA-seq I]

Demyelination and dysregulated myelination in the CNS are hallmarks of many neurodegenerative diseases such as multiple sclerosis (MS) and leukodystrophies. Here, we studied GFAP+ astrocytes during de- and remyelination in the cuprizone mouse by exploiting the ribosomal tagging (RiboTag) technology. Analyses were performed 5 weeks after cuprizone feeding, at the peak of demyelination in the corpus callosum, and 0.5 and 2 weeks after cuprizone withdrawal, when remyelination and tissue repair is initiated. After 5 weeks of cuprizone feeding, reactive astrocytes showed inflammatory signatures with enhanced expression of genes that modulate leukocyte migration (Tlr2, Cd86, Parp14,Cxcl10). Furthermore, demyelination-induced reactive astrocytes expressed numerous ligands including Cx3cl1, Csf1, Il34, and Gas6 that act on homeostatic as well as activated microglia and thus potentially mediate activation and recruitment of microglia as well as enhancement of their phagocytosis. During early remyelination, region-specific astrocytes displayed reduced inflammatory response signatures as indicated by shut down of CXCL10 production. During late remyelination, the signatures of GFAP+ astrocytes shifted towards resolving inflammation by active suppression of lymphocyte activation and differentiation and support of glia cell differentiation. Astrocytes showed enhanced expression of osteopontin (SPP1) as well as of factors that are relevant for tissue remodelling (Timp1), regeneration and axonal repair. In conclusion, we detected highly dynamic astroglial transcriptomic signatures in the cuprizone model, which reflects excessive communication amongst glia cells and highlights different astrocyte functions during neurodegeneration and regeneration. Overall design: Male GFAP-Cre+/-RPL22HA/wt aged 8-9 weeks fed with 0.2% (w/w) of cuprizone blended into standard rodent chow for a total of 5 weeks to induce demyelination . Animals were fed ad libitum until defined experimental endpoints. After 5 weeks post-cuprizone feeding, mice were put on regular chow for additional 2 weeks (total 7 weeks) to examine remyelination. Natural remyelination occurs starting 0.5 weeks after Cuprizone withdrawal (total 5.5wks). For this study, brains were collected at 5, 5.5 and 7 weeks, which corresponds, to acute demyelination, earlier remyelination and late remyelination respectively. Along with this, aged-matched control mice were kept of a normal diet.At the experimental endpoint, animals were euthanized, brain harvested and corpus callosum was micro-dissected. Thereafter, corpus callosum was homogenized and lysate used to pull down HA-tagged ribosomes and to isolate RNA from the immunoprecipitate (IP) for bulk RNA-sequencing.

中枢神经系统（central nervous system, CNS）的脱髓鞘与髓鞘调控异常，是多发性硬化（multiple sclerosis, MS）、脑白质营养不良（leukodystrophies）等多种神经退行性疾病的标志性病理特征。本研究利用核糖体标签（ribosomal tagging, RiboTag）技术，对双环己酮草酰二腙（cuprizone）小鼠模型中脱髓鞘与髓鞘再生过程中的胶质纤维酸性蛋白阳性（glial fibrillary acidic protein, GFAP+）星形胶质细胞展开研究。

我们在双环己酮草酰二腙喂食5周（即胼胝体（corpus callosum）脱髓鞘峰值期）、停药0.5周及2周（髓鞘再生与组织修复启动阶段）分别开展分析。双环己酮草酰二腙喂食5周后，反应性星形胶质细胞呈现炎症特征，其调控白细胞迁移的基因（Tlr2、Cd86、Parp14、Cxcl10）表达显著上调。此外，脱髓鞘诱导的反应性星形胶质细胞还表达多种配体，包括Cx3cl1、Csf1、Il34及Gas6，这些配体可作用于稳态及活化状态的小胶质细胞（microglia），从而潜在介导小胶质细胞的活化、募集，并增强其吞噬功能。

在髓鞘再生早期，区域特异性星形胶质细胞的炎症应答特征减弱，表现为CXCL10的产生被抑制。髓鞘再生晚期，GFAP+星形胶质细胞的转录特征向炎症消退方向转变：一方面主动抑制淋巴细胞的活化与分化，另一方面支持胶质细胞的分化。星形胶质细胞的骨桥蛋白（osteopontin, SPP1）、组织重塑相关因子（Timp1）以及再生与轴突修复相关因子的表达均显著上调。

综上，本研究在双环己酮草酰二腙模型中检测到高度动态的星形胶质细胞转录组特征，该特征反映了胶质细胞间的广泛通讯，并揭示了星形胶质细胞在神经退行性变与再生过程中的不同功能。

实验设计：选取8~9周龄的GFAP-Cre+/-RPL22HA/wt雄性小鼠，喂食添加0.2%（质量分数，w/w）双环己酮草酰二腙的标准啮齿类动物饲料，持续5周以诱导脱髓鞘。小鼠自由进食直至预设实验终点。双环己酮草酰二腙喂食5周后，更换为普通饲料继续饲养2周（总饲养时长7周），以观察髓鞘再生过程。自然髓鞘再生于双环己酮草酰二腙停药后0.5周（即总饲养5.5周时）启动。本研究分别在5周、5.5周及7周三个时间点采集脑组织，分别对应急性脱髓鞘期、早期髓鞘再生期与晚期髓鞘再生期。同期设置年龄匹配的正常饮食对照组小鼠。

实验终点处，对小鼠实施安乐死，采集脑组织并显微分离胼胝体。随后将胼胝体匀浆，利用裂解液沉淀带有HA标签的核糖体，从免疫沉淀产物（immunoprecipitate, IP）中提取RNA，用于批量RNA测序（bulk RNA-sequencing）。