Microglia promote remyelination independent of their role in clearing myelin debris.

Multiple sclerosis (MS) is an inflammatory disease characterized by myelin loss. While therapies exist to slow MS progression, no treatment currently exists for remyelination. Remyelination, linked to reduced disability in MS, relies on microglia and monocyte-derived macrophages (MDMs). This study aims to understand the role of microglia during remyelination by lineage tracing and depleting them. Microglial lineage tracing reveals that both microglia and MDMs initially accumulate, but microglia later dominate the lesion. Microglia and MDMs engulf equal amounts of inhibitory myelin debris, but after microglial depletion, MDMs compensate by engulfing more myelin debris. Microglial depletion does, however, reduce the recruitment and proliferation of oligodendrocyte progenitor cells (OPCs) and impairs their subsequent differentiation and remyelination. These findings underscore the essential role of microglia during remyelination and offer insights for enhancing this process by understanding microglial regulation of remyelination.

Single-cell microglial transcriptomics during demyelination defines a microglial state required for lytic carcass clearance.

BACKGROUND: Microglia regulate the response to injury and disease in the brain and spinal cord. In white matter diseases microglia may cause demyelination. However, how microglia respond and regulate demyelination is not fully understood. METHODS: To understand how microglia respond during demyelination, we fed mice cuprizone-a potent demyelinating agent-and assessed the dynamics of genetically fate-mapped microglia. We then used single-cell RNA sequencing to identify and track the microglial subpopulations that arise during demyelination. To understand how microglia contribute to the clearance of dead oligodendrocytes, we ablated microglia starting at the peak of cuprizone-induced cell death and used the viability dye acridine orange to monitor apoptotic and lytic cell morphologies after microglial ablation. Lastly, we treated serum-free primary microglial cultures to model distinct aspects of cuprizone-induced demyelination and assessed the response. RESULTS: The cuprizone diet generated a robust microglial response by week 4 of the diet. Single-cell RNA sequencing at this time point revealed the presence of several cuprizone-associated microglia (CAM) clusters. These clusters expressed a transcriptomic signature indicative of cytokine regulation and reactive oxygen species production with altered lysosomal and metabolic changes consistent with ongoing phagocytosis. Using acridine orange to monitor apoptotic and lytic cell death after microglial ablation, we found that microglia preferentially phagocytose lytic carcasses. In culture, microglia exposed to lytic carcasses partially recapitulated the CAM state, suggesting that phagocytosis contributes to this distinct microglial state during cuprizone demyelination. CONCLUSIONS: Microglia serve multiple roles during demyelination, yet their transcriptomic state resembles other neurodegenerative conditions. The phagocytosis of cellular debris is likely a universal cause for a common neurodegenerative microglial state.

The CD33 short isoform is a gain-of-function variant that enhances Aß1-42 phagocytosis in microglia.

BACKGROUND: CD33 is genetically linked to Alzheimer's disease (AD) susceptibility through differential expression of isoforms in microglia. The role of the human CD33 short isoform (hCD33m), preferentially encoded by an AD-protective CD33 allele (rs12459419T), is unknown. Here, we test whether hCD33m represents a loss-of-function or gain-of-function variant. METHODS: We have developed two models to test the role of hCD33m. The first is a new strain of transgenic mice expressing hCD33m in the microglial cell lineage. The second is U937 cells where the CD33 gene was disrupted by CRISPR/Cas9 and complemented with different variants of hCD33. Primary microglia and U937 cells were tested in phagocytosis assays and single cell RNA sequencing (scRNAseq) was carried out on the primary microglia. Furthermore, a new monoclonal antibody was developed to detect hCD33m more efficiently. RESULTS: In both primary microglia and U937 cells, we find that hCD33m enhances phagocytosis. This contrasts with the human CD33 long isoform (hCD33M) that represses phagocytosis, as previously demonstrated. As revealed by scRNAseq, hCD33m+ microglia are enriched in a cluster of cells defined by an upregulated expression and gene regulatory network of immediate early genes, which was further validated within microglia in situ. Using a new hCD33m-specific antibody enabled hCD33m expression to be examined, demonstrating a preference for an intracellular location. Moreover, this newly discovered gain-of-function role for hCD33m is dependent on its cytoplasmic signaling motifs, dominant over hCD33M, and not due to loss of glycan ligand binding. CONCLUSIONS: These results provide strong support that hCD33m represents a gain-of-function isoform and offers insight into what it may take to therapeutically capture the AD-protective CD33 allele.

Microglia response following acute demyelination is heterogeneous and limits infiltrating macrophage dispersion.

Microglia and infiltrating macrophages are thought to orchestrate the central nervous system (CNS) response to injury; however, the similarities between these cells make it challenging to distinguish their relative contributions. We genetically labeled microglia and CNS-associated macrophages to distinguish them from infiltrating macrophages. Using single-cell RNA sequencing, we describe multiple microglia activation states, one of which was enriched for interferon associated signaling. Although blood-derived macrophages acutely infiltrated the demyelinated lesion, microglia progressively monopolized the lesion environment where they surrounded infiltrating macrophages. In the microglia-devoid sciatic nerve, the infiltrating macrophage response was sustained. In the CNS, the preferential proliferation of microglia and sparse microglia death contributed to microglia dominating the lesion. Microglia ablation reversed the spatial restriction of macrophages with the demyelinated spinal cord, highlighting an unrealized macrophages-microglia interaction. The restriction of peripheral inflammation by microglia may be a previously unidentified mechanism by which the CNS maintains its "immune privileged" status.

Central Nervous System Remyelination: Roles of Glia and Innate Immune Cells.

In diseases such as multiple sclerosis (MS), inflammation can injure the myelin sheath that surrounds axons, a process known as demyelination. The spontaneous regeneration of myelin, called remyelination, is associated with restoration of function and prevention of axonal degeneration. Boosting remyelination with therapeutic intervention is a promising new approach that is currently being tested in several clinical trials. The endogenous regulation of remyelination is highly dependent on the immune response. In this review article, we highlight the cell biology of remyelination and its regulation by innate immune cells. For the purpose of this review, we discuss the roles of microglia, and also astrocytes and oligodendrocyte progenitor cells (OPCs) as they are being increasingly recognized to have immune cell functions.