Cspg4high microglia contribute to microgliosis during neurodegeneration

Abstract

Microglia play a critical role in the pathogenic process of neurodegenerative diseases, such as Parkinson's disease (PD) and Alzheimer's disease (AD). Upon pathological stimulation, microglia are converted from a surveillant to an overactivated phenotype. However, the molecular characters of proliferating microglia and their contributions to the pathogenesis of neurodegeneration remain unclear. Here, we identify chondroitin sulfate proteoglycan 4 (Cspg4, also known as neural/glial antigen 2)-expressing microglia as a specific subset of microglia with proliferative capability during neurodegeneration. We found that the percentage of Cspg4⁺ microglia was increased in mouse models of PD. The transcriptomic analysis of Cspg4⁺ microglia revealed that the subcluster Cspg4^high microglia displayed a unique transcriptomic signature, which was characterized by the enrichment of orthologous cell cycle genes and a lower expression of genes responsible for neuroinflammation and phagocytosis. Their gene signatures were also distinct from that of known disease-associated microglia. The proliferation of quiescent Cspg4^high microglia was evoked by pathological α-synuclein. Following the transplantation in the adult brain with the depletion of endogenous microglia, Cspg4^high microglia grafts showed higher survival rates than their Cspg4^- counterparts. Consistently, Cspg4^high microglia were detected in the brain of AD patients and displayed the expansion in animal models of AD. These findings suggest that Cspg4^high microglia are one of the origins of microgliosis during neurodegeneration and may open up a avenue for the treatment of neurodegenerative diseases.

Keywords: Alzheimer’s disease; Cspg4; Parkinson’s disease; microglia; neurodegenerative diseases.

Fig. 1.

CSPG4⁺ microglia exist in the human brain and their numbers in mice are decreased during aging. (A) UMAP showing the cell populations in the human brain based on the data extracted from the Human Protein Atlas. (B) CSPG4 expression in neuronal and glial cells of the human brain. The data are extracted from the Human Protein Atlas. (C) Representative immunofluorescence microphotographs showing colocalization of NG2 and IBA1 in WT mice at various ages, from postnatal day 15 (P15, n = 3), 2 mo (n = 4) and 9 mo (n = 4) of age. White arrow heads indicate NG2⁺ microglia. (Scale bar, 20 μm.) (D) Quantitative data shown in C. (E) Cytometric analysis of NG2⁺/IBA1⁺ cells in the mouse brain at P15 (n = 5), 2 mo (n = 5) and 9 mo (n = 5) of age. (F) Quantitative data shown in E. (G) Representative graph showing relative mRNA levels of Cspg4 in microglia of WT mice at P15 (n = 10), 2 mo (n = 10), and 9 mo (n = 10) of age. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2.

The percentage of NG2⁺ microglia is increased in the pd models. (A) Representative immunofluorescent micrographs showing colocalization of NG2 and IBA1 in the SN of mice injected with vehicle (PBS) or α-syn PFFs. Arrows indicate NG2⁻ microglia. Arrowheads indicate NG2⁺ microglia. (Scale bar, 20 μm.) (B) Quantitative data showing the percentage of NG2⁺ microglia in the SN of mice with vehicle (n = 4) or α-syn PFF injection (n = 4). (C) Representative immunofluorescent micrographs showing colocalization of NG2 and IBA1 in the SN of A30P Tg mice or WT mice. Arrows indicate NG2⁻ microglia. Arrowheads indicate NG2⁺ microglia. (Scale bar, 20 μm.) (D) Quantitative data showing the percentage of NG2⁺ microglia in the SN of A30P Tg mice (n = 4) or WT mice (n = 4). (E) Representative immunofluorescent microphotographs showing colocalization of NG2 and IBA1 in primary cultured cells of rat brain exposed to α-syn PFFs. (Scale bar, 20 μm.) (F) Quantitative data shown in E, vehicle (n = 8) or α-syn PFF (n = 8). (G) Representative immunoblots showing expression of NG2 in primary cultured microglia exposed to α-syn PFFs. (H) Quantitative data shown in G, vehicle (n = 4) or α-syn PFF injection (n = 4). Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

The single-cell transcriptomic analysis for egfp-labeled Cspg4⁺ cells isolated from A30P;NG2-EGFP double Tg mice. (A) The clusters of Cspg4⁺ cells. # denotes cluster 9, Cspg4⁺ microglia. (B) A dotplot for glial marker genes in cluster 0 to 14. (C) Expression of microglial marker genes in clusters. (D) Cluster 9 (circled) in A30P mice and control. The data represent SN tissues pooled from both NG2-EGFP Tg mice (n = 6) and A30P α-syn;NG2-EGFP double Tg mice (n = 6).

Fig. 4.

Cspg4^high microglia exhibit a high expression of genes associated with cell proliferation but not disease-associated response in A30P mice. (A) Two-dimensional UMAP embedding of the scRNA-seq of cluster 9 data. Dots are colored based on the scRNA-seq clustering. Subcluster 3 represents Cspg4^high microglia indicated with asterisk (*). (B) Expression of P2ry12 and Cspg4 in cluster 9. (C) A dotplot for glial marker genes in subclusters. (D) A dotplot for marker genes of Cspg4^high microglia. (E) A dotplot showing the relative expression of genes responsible for cell cycle (x axis) across all subclusters (y axis). (F) A dotplot showing the relative expression of genes associated with neuroinflammation (x axis) across all subclusters (y axis). (G) A dotplot for genes related to homeostasis and DAM (x axis) across all subclusters (y axis).

Fig. 5.

NG2⁺ microglia are in a state of active proliferation and contribute to microgliosis during microglial repopulation. (A) A diagram showing timeline for EdU administration in mice with injection of α-syn PFFs. (B) Immunofluorescent histochemical staining for NG2, IBA1, and EdU in α-syn PFF-induced PD model. (Scale bar, 50 μm.) (C) Quantitative data showing the percentage of EDU⁺ cells in NG2⁻ microglia (n = 5) or NG2⁺ microglia (n = 5) in α-syn PFF-treated animals. (D) Cytometric analysis of EdU⁺/NG2⁺/IBA1⁺ cells in α-syn PFF-induced PD model. (E) Quantitative data shown in D, NG2⁻ microglia (n = 5) and NG2⁺ microglia (n = 5). (F) Representative immunofluorescent staining for NG2 and IBA1 in WT mice during microglial repopulation post PLX5622 treatment. (Scale bar, 30 μm.) (G) Quantitative data shown in F, Day 0 post PLX5622 (n = 2), Day 3 post-PLX5622 (n = 3), Day 5 post-PLX5622 (n = 3). (H) A diagram showing timeline for microglia transplantation. (I) Immunofluorescent histochemical staining for IBA1 in the SN of mice received transplantation of NG2⁺ or NG2⁻ CX3CR1-EGFP microglia (P7). G indicates the graft. (Scale bar, 100 μm.) (J) Quantitative data showing the percentage of transplanted microglia derived from NG2⁺ microglia (n = 8) or NG2⁻ microglia (n = 8) in the region surrounding the graft (circled with dashed white lines). Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6.

Presence of CSPG4^high microglia in the brain of AD patients and the expansion of Cspg4⁺ microglia in animal models of AD. (A) Single-cell analysis for microglial CSPG4 level in the entorhinal cortex of patients with AD. (B) Microglial clusters (m1 to m5) in the entorhinal cortex of subjects with AD. Cluster m3 represents Cspg4^high microglia indicated with asterisk (*). (C) Representative immunofluorescent staining for NG2 and IBA1 in WT and 5×FAD mice. Arrows indicate NG2⁻ microglia. Arrowheads indicate NG2⁺ microglia. (Scale bar, 15 μm.) (D) Quantitative data shown in C, WT (n = 4), 5×FAD mice (n = 5). (E) Representative immunofluorescent staining for NG2 and IBA1 in mice received injection of Aβ PFF or vehicle. Arrows indicate NG2⁻ microglia. Arrowheads indicate NG2⁺ microglia. (Scale bar, 20 μm.) Hip-DG, hippocampal dentate gyrus. (F) Quantitative data shown in E, vehicle (n = 3), Aβ PFF (n = 3). (G) Representative graph showing relative mRNA levels of Cspg4 in microglia of 5×FAD mice at 3 mo (n = 4), 6 mo (n = 6), and 9 mo (n = 6) of age. (H) A dotplot showing the relative expression of genes responsible for cell cycle (x axis) across all subclusters (y axis). (I) A dotplot showing the relative expression of genes associated with neuroinflammation (x axis) across all subclusters (y axis). LPS, lipopolysaccharide. (J) A dotplot for genes related to homeostasis and DAM (x axis) across all subclusters (y axis). Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.