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Mouse Astrocytes Promote Microglial Ramification by Releasing TGF-? and Forming Glial Fibers.

ABSTRACT: The morphology of microglial cells is often closely related to their functions. The mechanisms that regulate microglial ramification are not well understood. Here we reveal the biological mechanisms by which astrocytes regulate microglial ramification. Morphological variation in mouse microglial cultures was measured in terms of cell area as well as branch number and length. Effects on microglial ramification were analyzed after microinjecting the toxin L-alpha-aminoadipic acid (L-AAA) in the mouse cortex or hippocampus to ablate astrocytes, and after culturing microglia on their own in an astrocyte-conditioned medium (ACM) or together with astrocytes in coculture. TGF-? expression was determined by Western blotting, immunohistochemistry, and ELISA. The TGF-? signaling pathway was blocked by the TGF-? antibody to assess the role of TGF-? on microglial ramification. The results showed that microglia had more and longer branches and smaller cell bodies in brain areas where astrocytes were abundant. In the mouse cortex and hippocampus, ablation of astrocytes by L-AAA decreased number and length of microglial branches and increased the size of cell bodies. Similar results were obtained with isolated microglia in culture. However, isolated microglia were able to maintain their multibranched structure for a long time when cultured on astrocyte monolayers. Ameboid microglia isolated from P0 to P3 mice showed increased ramification when cultured in ACM or on astrocyte monolayers. Microglia cultured on astrocyte monolayers showed more complex branching structures than those cultured in ACM. Blocking astrocyte-derived TGF-? decreased microglial ramification. Astrocytes induced the formation of protuberances on branches of microglia by forming glial fibers that increased traction. These experiments in mice suggest that astrocytes promote microglial ramification by forming glial fibers to create traction and by secreting soluble factors into the surroundings. For example, astrocyte-secreted TGF-? promotes microglia to generate primitive branches, whose ramification is refined by glial fibers.

SUBMITTER: Zhang J

PROVIDER: S-EPMC7366495 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Mouse Astrocytes Promote Microglial Ramification by Releasing TGF-β and Forming Glial Fibers.

Zhang Jinqiang J Zhang Lijuan L Yi Saini S Jiang Xue X Qiao Yan Y Zhang Yue Y Xiao Chenghong C Zhou Tao T

Frontiers in cellular neuroscience 20200710

The morphology of microglial cells is often closely related to their functions. The mechanisms that regulate microglial ramification are not well understood. Here we reveal the biological mechanisms by which astrocytes regulate microglial ramification. Morphological variation in mouse microglial cultures was measured in terms of cell area as well as branch number and length. Effects on microglial ramification were analyzed after microinjecting the toxin L-alpha-aminoadipic acid (L-AAA) in the mo ...[more]

PMID: 32754014

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Project description:Astrocytes and microglia play an orchestrated role following spinal cord injury; however, the molecular mechanisms through which microglia regulate astrocytes after spinal cord injury are not yet fully understood. Herein, microglia were pharmacologically depleted and the effects on the astrocytic response were examined. We further explored the potential mechanisms involving the signal transducers and activators of transcription 3 (STAT3) pathway. For in vivo experiments, we constructed a contusion spinal cord injury model in C57BL/6 mice. To deplete microglia, all mice were treated with colony-stimulating factor 1 receptor inhibitor PLX3397, starting 2 weeks prior to surgery until they were sacrificed. Cell proliferation was examined by 5-ethynyl-2-deoxyuridine (EdU) and three pivotal inflammatory cytokines were detected by a specific Bio-Plex ProTM Reagent Kit. Locomotor function, neuroinflammation, astrocyte activation and phosphorylated STAT3 (pSTAT3, a maker of activation of STAT3 signaling) levels were determined. For in vitro experiments, a microglia and astrocyte coculture system was established, and the small molecule STA21, which blocks STAT3 activation, was applied to investigate whether STAT3 signaling is involved in mediating astrocyte proliferation induced by microglia. PLX3397 administration disrupted glial scar formation, increased inflammatory spillover, induced diffuse tissue damage and impaired functional recovery after spinal cord injury. Microglial depletion markedly reduced EdU+ proliferating cells, especially proliferating astrocytes at 7 days after spinal cord injury. RNA sequencing analysis showed that the JAK/STAT3 pathway was downregulated in mice treated with PLX3397. Double immunofluorescence staining confirmed that PLX3397 significantly decreased STAT3 expression in astrocytes. Importantly, in vitro coculture of astrocytes and microglia showed that microglia-induced astrocyte proliferation was abolished by STA21 administration. These findings suggest that microglial depletion impaired astrocyte proliferation and astrocytic scar formation, and induced inflammatory diffusion partly by inhibiting STAT3 phosphorylation in astrocytes following spinal cord injury.

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Mouse Astrocytes Promote Microglial Ramification by Releasing TGF-? and Forming Glial Fibers.

Publications

Mouse Astrocytes Promote Microglial Ramification by Releasing TGF-β and Forming Glial Fibers.

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