Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, K1H 8L6, Canada.
Present address: Therapeutic Products Directorate, Health Canada, 1600 Scott St, Ottawa, ON, K1A 0K9, Canada.
J Neuroinflammation. 2019 Jul 4;16(1):135. doi: 10.1186/s12974-019-1530-4.
Conditional ablation of the Smarca5 gene in mice severely impairs the postnatal growth of the cerebellum and causes an ataxic phenotype. Comparative gene expression studies indicated that complement-related proteins were upregulated in the cerebellum of Smarca5 mutant mice. Complement proteins play critical roles within innate immune signaling pathways and, in the brain, are produced by glial cells under both normal and pathological conditions. The C3 complement protein-derived signaling peptide, C3a, has been implicated in contributing to both tissue damage and repair in conditions such as multiple sclerosis and stroke. Here, we investigated whether C3a receptor (C3aR) signaling promoted damage or repair in the developing cerebellum of Smarca5 mutant mice.
Brain and cerebellum lysates from single Smarca5 conditional knockout (Smarca5 cKO) mice, C3aR1 KO mice, or double mutant mice were used for qRT-PCR and immunoblotting to assess the contribution of C3aR to the Smarca5 cKO brain pathology. Immunohistochemistry was used to characterize alterations to astroglia and phagocyte cells in the developing cerebellum of each of the genotypes.
C3aR signaling was observed to limit gliosis and promote granule neuron survival during postnatal cerebellar development. In Smarca5 cKO mice, disorganized astroglia with increased GFAP expression develops concurrently with cerebellar granule neuron loss and phagocyte invasion over the first 10 days following birth. Potential ligand precursors of C3aR-VGF and C3-were found to have upregulated expression and/or altered processing during this time. Phagocytes (microglia and macrophages) in both the control and Smarca5 mutant mice were the only cells observed to express C3aR. Loss of C3aR in the Smarca5 cKO cerebellum resulted in increased numbers of apoptotic cells and early phagocyte invasion into the external granule cell layer, as well as an exacerbated disorganization of the Bergmann glia. The loss of C3aR expression also attenuated an increase in the expression of the efferocytosis-related protein, MerTK, whose transcript was upregulated ~ 2.5-fold in the Smarca5 mutant cerebellum at P10.
This data indicates that C3aR can play an important role in limiting astrogliosis and regulating phagocyte phenotypes following developmental cell loss in the brain.
条件性敲除 Smarca5 基因会严重损害小脑的出生后生长，并导致共济失调表型。比较基因表达研究表明，补体相关蛋白在 Smarca5 突变小鼠的小脑上调。补体蛋白在先天免疫信号通路中发挥关键作用，在大脑中，在正常和病理条件下由神经胶质细胞产生。C3 补体蛋白衍生的信号肽 C3a 已被牵连到多发性硬化症和中风等疾病的组织损伤和修复中。在这里，我们研究了 C3a 受体（C3aR）信号是否促进 Smarca5 突变小鼠发育中小脑的损伤或修复。
来自单个 Smarca5 条件性敲除（Smarca5 cKO）小鼠、C3aR1 KO 小鼠或双突变小鼠的大脑和小脑裂解物用于 qRT-PCR 和免疫印迹，以评估 C3aR 对 Smarca5 cKO 大脑病理学的贡献。免疫组织化学用于表征每种基因型发育中小脑的星形胶质细胞和吞噬细胞的变化。
在出生后小脑发育过程中，观察到 C3aR 信号可限制神经胶质增生并促进颗粒神经元存活。在 Smarca5 cKO 小鼠中，在出生后 10 天内，发育中的星形胶质细胞排列紊乱，GFAP 表达增加，同时发生小脑颗粒神经元丢失和吞噬细胞浸润。在此期间，C3aR-VGF 和 C3 的潜在配体前体发现表达上调和/或加工改变。在对照和 Smarca5 突变小鼠中，只有吞噬细胞（小胶质细胞和巨噬细胞）表达 C3aR。在 Smarca5 cKO 小脑中缺失 C3aR 会导致凋亡细胞数量增加，并导致早期吞噬细胞侵入外颗粒细胞层，以及 Bergmann 胶质的严重紊乱。C3aR 表达的丧失也减弱了吞噬作用相关蛋白 MerTK 的表达增加，其转录物在 Smarca5 突变小脑中的表达在 P10 时上调约 2.5 倍。
这些数据表明，在大脑发育性细胞丢失后，C3aR 可以在限制神经胶质增生和调节吞噬细胞表型方面发挥重要作用。
