Distinct transcriptomic and epigenomic responses of mature oligodendrocytes during disease progression in a mouse model of multiple sclerosis [ATAC-seq]

Multiple sclerosis (MS) is a chronic demyelinating autoimmune disease which targets mature oligodendrocytes (MOLs) and their myelin. MOLs are transcriptionally heterogeneous, and can transition to immune-like states in the context of MS. However, the intricacies of their dynamics throughout disease progression remain poorly understood. Here, we employed simultaneous single-cell multiome ATAC and RNA sequencing targeting oligodendroglia (OLGs) from the experimental autoimmune encephalomyelitis (EAE) MS mouse model, at different stages of the disease course. We found that the transition to immune OLGs states occurs already at early stages of EAE and these persist at late stages of the disease. Interestingly, transcription factor activity suggested immunosuppression activity in MOLs at early stages of EAE and we also observed a transitory activation of a regenerative programme in MOLs at this stage. Importantly, different MOLs present a differential responsiveness to EAE, with MOL2 exhibiting stronger transcriptional immune response than MOL5/6. Moreover, we observed divergence responses at the epigenetic level of MOL2 and MOL5/6 during disease evolution. Thus, our single-cell multiomic resource highlights dynamic and distinct responses of OLG subpopulations to the evolving environment in EAE, which might modulate their response to regenerative therapeutic interventions in MS. Overall design: Mice brains from P3-6 pups were dissociated using the Neural Tissue Dissociation Kit (Miltenyi Biotec, 130-092-628) according to the manufacturer's protocol. OPCs were obtained with MACS with CD140a microbeads following the manufacturer's protocol (CD140a Microbead kit, Miltenyi Biotec, 130-101-547). Cells were seeded in Poly-L-lysine (Sigma, P4707)-coated dishes and grown on OPC proliferation media comprising DMEM/F-12/GlutaMAX (ThermoFisher, 10565018), N2 media (ThermoFisher, 17502048), penicillin–streptomycin (ThermoFisher, 15140122), NeuroBrew (Miltenyi, 130-097-263), bFGF 40 ng/ml (R&D, 233-FB) and PDGF-AA 20 ng/ml (Peprotech, 315-17).

多发性硬化（Multiple Sclerosis, MS）是一种以成熟少突胶质细胞（Mature Oligodendrocytes, MOLs）及其髓鞘为靶标的慢性脱髓鞘性自身免疫疾病。成熟少突胶质细胞在转录层面具有异质性，且在多发性硬化的病理环境中可转变为免疫样状态。然而，学界对其在疾病全程中的动态变化机制仍知之甚少。
本研究针对多发性硬化小鼠模型——实验性自身免疫性脑脊髓炎（Experimental Autoimmune Encephalomyelitis, EAE）——在疾病进程不同阶段的少突胶质细胞（Oligodendroglia, OLGs），开展了同步单细胞多组学ATAC与RNA测序。研究发现，向免疫样少突胶质细胞状态的转变早在实验性自身免疫性脑脊髓炎早期便已发生，且该状态可持续至疾病晚期。
值得注意的是，转录因子活性分析显示，实验性自身免疫性脑脊髓炎早期的成熟少突胶质细胞存在免疫抑制活性；同时本研究还观察到该阶段的成熟少突胶质细胞中，再生程序存在一过性激活现象。尤为关键的是，不同成熟少突胶质细胞亚群对实验性自身免疫性脑脊髓炎的响应存在差异，其中MOL2相较于MOL5/6表现出更强的转录层面免疫应答。此外，本研究还观察到在疾病演进过程中，MOL2与MOL5/6的表观遗传层面响应模式存在分化。
综上，本研究的单细胞多组学数据集揭示了少突胶质细胞亚群对实验性自身免疫性脑脊髓炎中动态变化的微环境所产生的动态且特异的响应模式，该模式或可调控成熟少突胶质细胞对多发性硬化再生治疗干预手段的应答效果。

整体实验设计：
采用神经组织解离试剂盒（Neural Tissue Dissociation Kit, Miltenyi Biotec, 130-092-628），按照厂商说明书流程解离P3-6天幼鼠的脑组织。使用CD140a微珠试剂盒（CD140a Microbead kit, Miltenyi Biotec, 130-101-547）通过磁激活细胞分选（Magnetic Activated Cell Sorting, MACS）获取少突胶质前体细胞（Oligodendrocyte Progenitor Cells, OPCs）。将细胞接种于多聚赖氨酸（Poly-L-lysine, Sigma, P4707）包被的培养皿中，使用少突胶质前体细胞增殖培养基进行培养，该培养基配方包含：DMEM/F-12/GlutaMAX（ThermoFisher, 10565018）、N2培养基（ThermoFisher, 17502048）、青霉素-链霉素（ThermoFisher, 15140122）、NeuroBrew（Miltenyi, 130-097-263）、40 ng/ml 碱性成纤维细胞生长因子（bFGF, R&D, 233-FB）以及20 ng/ml 血小板衍生生长因子-AA（PDGF-AA, Peprotech, 315-17）。