Abstract
AbstractMicrogliosis is a prominent pathological feature in many neurological diseases including multiple sclerosis (MS), a progressive auto‐immune demyelinating disorder. The precise role of microglia, parenchymal central nervous system (CNS) macrophages, during demyelination, and the relative contributions of peripheral macrophages are incompletely understood. Classical markers used to identify microglia do not reliably discriminate between microglia and peripheral macrophages, confounding analyses. Here, we use a genetic fate mapping strategy to identify microglia as predominant responders and key effectors of demyelination in the cuprizone (CUP) model. Colony‐stimulating factor 1 (CSF1), also known as macrophage colony‐stimulating factor (M‐CSF) ‐ a secreted cytokine that regulates microglia development and survival—is upregulated in demyelinated white matter lesions. Depletion of microglia with the CSF1R inhibitor PLX3397 greatly abrogates the demyelination, loss of oligodendrocytes, and reactive astrocytosis that results from CUP treatment. Electron microscopy (EM) and serial block face imaging show myelin sheaths remain intact in CUP treated mice depleted of microglia. However, these CUP‐damaged myelin sheaths are lost and robustly phagocytosed upon‐repopulation of microglia. Direct injection of CSF1 into CNS white matter induces focal microgliosis and demyelination indicating active CSF1 signaling can promote demyelination. Finally, mice defective in adopting a toxic astrocyte phenotype that is driven by microglia nevertheless demyelinate normally upon CUP treatment implicating microglia rather than astrocytes as the primary drivers of CUP‐mediated demyelination. Together, these studies indicate activated microglia are required for and can drive demyelination directly and implicate CSF1 signaling in these events.
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📋 Methods
Animals and administration of tamoxifen –
All rodent experiments were carried out under an approved animal protocol in accordance with the guidelines of the Institutional Animal Care and Use Committee of New York University School of Medicine. Mice were maintained under standard husbandry conditions at an NYU vivarium. Details on all mouse strains used are described in Table 1 ; all strains were maintained on a C57BL/6 background. Genotyping Methods: Small (~0.5 cm) tail trimmings from mouse pups (
📊 Figures
Figure 1.
Genetic labeling identifies microglia in the cuprizone model of demyelination.
A. High power micrographs of the corpus callosum of mice on control and cuprizone (5 week) diets stained for CD11b and TMEM119; the latter marker is markedly downregulated despite a striking increase ...
Figure 2:
CSF1 is upregulated during cuprizone-mediated demyelination
A. Micrographs of the CC from mice on a control or a CUP diet were stained for Iba1 and probed for expression of IL34 (red) and CSF1 (green) transcripts by RNAscope. Scale bar, 20 u00b5m. Boxed region...
Figure 3.
Depletion of microglia via CSF1R inhibition delays demyelination.
A. Schematic of experimental design. CX3CR1 CreER-iresGFP/+ ;Rosa26 stop-DsRed mice were treated with tamoxifen and after an additional month were placed on CUP for 3 or 5 weeks, PLX for 5 weeks, or C...
Figure 4.
Prophylactic depletion of microglia prevents CUP-mediated demyelination and oligodendrocyte loss
A. Schematic of experimental design. CX3CR1 CreER-iresGFP/+ ;Rosa26 stop-DsRed mice were treated with tamoxifen. After an additional month, one group of mice received PLX for 7 weeks, PLX for 2 weeks ...
Figure 5.
Electron microscopic analysis of CUP-treated myelin in the presence or absence of microglia
A. Electron micrographs of the CC of mice fed a control diet or diets of CUP (5 weeks), CUP/PLX (5 weeks), and PLX for 2 weeks then PLX/CUP (5 weeks). There is substantial demyelination of the CC of m...
Figure 6.
Focal injection of CSF1 is sufficient to induce demyelination only in the presence of microglia.
A. Schematic of the experimental design for the stereotactic injection of vehicle or rCSF1 into the CC of CX3CR1 CreER-iresGFP/+ ;Rosa26 stop-DsRed mice on a control diet or on a PLX diet (pretreated ...
Figure 7:
Demyelination is accompanied by astrogliosis
A. Schematic of experimental design to assess the impact of ablating microglia on astrogliosis. B. Representative coronal sections from mice on control, PLX, cuprizone or PLX + CUP/PLX diets were stai...
Figure 8.
Demyelination remains robust in a mouse model of defective astrocyte activation
A. Micrographs of coronal sections of the CC of WT and TNFu03b1/IL1/C1q (TIC) triple knockout mice on control or cuprizone diets were stained for microglia (CD11b; green), reactive astrocytes (GFAP; r...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
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