Fig 2: Microglial depletion results in an increased loss of neurons and oligodendrocytes leading to greater tissue damage after SCI. (a and b, d and e, g and h, j and k) Confocal immunofluorescence of astrocytes (GFAP+, blue), microglia (TdT+, red cells in a–b, d and e), blood-derived myeloid cells (LysM-eGFP+ or CD11b+, green cells in a and b, d and e, k), neurons/axons (NF-H+, green in g and h, j) and pericytes/fibroblasts (PDGFRß+, red cells in g and h, j) at the lesion epicenter at 7 (a and b), 14 (d and e) and 35 (g and h) dpi. Yellow and purple lines, respectively, delineate the contours of the primary (core) and satellite lesions, which were surrounded by astrocytic endfeet and characterized by the absence of neuronal elements and presence of cells of non-CNS origin (blood-derived myeloid cells, pericytes, and fibroblasts). Satellite lesions are shown in a microglia-depleted mouse at 35 dpi (j and k). c Quantification of the total lesion area at 7 dpi in mice fed the control diet (blue), PLX73086 (red), or PLX5622 (green) (n = 5–6 mice/group). f, i Quantification of the total area occupied by satellite lesions at 14 (f) and 35 (i) dpi (n = 5–7/group). l and m Quantification of the number of neurons (HuC/HuD+) in the uninjured spinal cord (l), as well as rostral (R) and caudal (C) to the epicenter (m), in mice treated with PLX5622, PLX73086, or control at 35 dpi (n = 5–8/group). n and o Representative confocal images taken at the lesion epicenter at 35 dpi immunostained for HuC/HuD (red). DAPI is shown in blue. p, q Quantification of the number of oligodendrocytes (Olig2+ CC1+) in the uninjured (p) and injured (q) spinal cord of mice treated with PLX5622, PLX73086 or control at 35 dpi (n = 5–8/group). Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, PLX5622 versus control; #p < 0.05, PLX73086 versus control; and &p < 0.05, &&p < 0.01, &&&p < 0.001, &&&&p < 0.0001, PLX5622 compared to PLX73086. Statistical analysis was performed using a two-way ANOVA followed by a Bonferroni’s post hoc test. Scale bars: (a and b, d and e, g and h in h) 200 µm, (j and k) 50 µm, (n and o, in o) 50 µm

Fig 3: Net outcome of early LPS effects on different types of proliferating cells. (A) Total number of BrdU cells was significantly reduced in 3xTg-AD mice but increased in the dentate gyrus of mice treated with LPS. (B) The proportion of newly-generated neurons (NeuN/BrdU cells) was diminished in 3xTg-AD mice (compared with WT mice) while the percentage of microglial/macrophage cells (CD11b/BrdU+ cells) was increased after LPS treatment. (C) Confocal microscopy images showing double stained BrdU/CD11b-positive cells in the granule cell layer/subgranular zone of the dentate gyrus (hypertrophic cells showing thick processes emerging from their cell body were considered as reactive). (D) Total number of BrdU-stained microglial/macrophage cells was increased after LPS treatment in both WT and 3xTg-AD mice. However, this increase was smaller in 3xTg-AD mice. (E) Total number of reactive BrdU/CD11b-positive cells was increased after LPS treatment. (F) Confocal microscopy images showing double stained BrdU/NeuN- positive neurons in the granule cell layer/subgranular zone of the dentate gyrus. (G) Total number of BrdU/NeuN-positive neurons was reduced in 3xTg-AD mice but increased by LPS administration. Data represent mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001 (LPS vs. saline-treated mice). +p < 0.05, ++p < 0.01, +++p < 0.001 (3xTg-AD vs. WT mice). Number of mice: WT Saline n = 4, WT LPS n = 5, 3xTg-AD Saline n = 5 and 3xTg-AD LPS n = 5.

Fig 4: Park2-/- microglia are more strongly activated than WT cells following exposure to the bacterial endotoxin LPS. (a–d) Representative images illustrating the activation of microglial cells exposed to LPS, as explored by automated microscopy assessing the cell surface area (stained for MAC-1) and Iba1 intensity (a), with the corresponding quantifications (b–d). The microglial surface area (in pixels) of LPS-treated Park2-/- microglia (n = 10) is larger than that of WT cells (n = 10; b). This modification is associated with a higher percentage of MAC-1+ microglia with an ameboid morphology (c) and stronger Iba1 staining intensity (d). (e) LPS triggers release of TNFa, IL-6, IL-1ß, IL-18, and MCP-1, quantified by ELISA with normalization according to cell numbers (AU), into the supernatant of WT microglia (n = 10). Larger amounts of IL-1ß and IL-18 are released by Park2-/- cells (n = 10). n is the number of independent experiments. Error bars indicate the SEM. *p < .05, **p < .01, ***p < .001. # versus the corresponding untreated control (UT) unless otherwise indicated. Scale bars: 10 µm

Fig 5: Maternal n-3 PUFA deficiency exacerbates microglia-mediated shaping of neuronal networks in a complement-dependent manner.a–c Representative images and quantification of C1q, CD11b, and C3aR immunostaining in coronal sections of n-3 deficient and n-3 sufficient mice hippocampus. Scale bar = 100 µm. Means ± SEM; n = 3–13 mice per group. Two-tailed unpaired Student’s t-test; t = 1.553, p = 0.135, CD11b CA1; t = 2.114, *p = 0.466, CD11b DG; t = 0.5495, *p = 0.0223 C1q CA1; t = 3.938, **p = 0.0056, C1q DG; t = 4.91, **p = 0.0027, C3aR, total hippocampus. d Protein quantification (ELISA) reveals that synaptosomes from n-3 deficient mice express more C3 than n-3 sufficient animals. Means ± SEM; n = 7–8 mice per group; Two-tailed unpaired Student’s t-test; t = 2.097, p = 0.0562. e Quantification of PSD95 protein expression in n-3 sufficient vs n-3 deficient mice treated with XVA-143 or its vehicle. Means ± SEM; n = 5–8. Two-way ANOVA followed by Bonferroni post hoc test: diet effect, F(1,21) = 11.89, p = 0.0024; treatment effect, F(1,21) = 0.1436, p = 0.708; interaction, F(1,21) = 5.977, p = 0.0234; n-3 sufficient vs n-3 deficient, **p < 0.01. f Time spent in novel vs familiar arm in the Y maze task in n-3 sufficient vs n-3 deficient mice treated with XVA-143 or its vehicle. Means ± SEM; n = 9–10 mice per group. Paired t-test: n-3 sufficient group, **p = 0.0019; n-3 deficient group, p = 0.357; n-3 sufficient + XVA-143 group, **p = 0.0013; n-3 deficient + XVA-143 group, **p = 0.0099. Source data are provided as a Source data file.