Figure 3. Extensive CSF1R inhibition unveils the presence of CSF1Ri-resistant myeloid cells in the subventricular zone and white matter tracts.

Two-month-old WT mice treated with vehicle or PLX3397 (600 ppm in chow) for 14 days to evaluate extent of microglial depletion. (A) Brain section schematic of SVZ and WM areas where CSF1Ri-resistant myeloid cells are present following 14 day PLX3397 (600ppm in chow) treatment. (B) Representative whole brain slice image of CSF1Ri-resistant IBA1⁺ cells (white arrows) near the SVZ/WM areas (white box) surrounding the lateral ventricle. (C–D) Representative tile scan (C) and high-resolution confocal (D) images of control and 14 day PLX3397 mice showing the deposition of surviving SVZ/WM IBA1⁺ (green) cells in WM areas (white dotted lines). (E–G) Representative tile scan (E–F) and confocal immunofluorescence image of IBA1⁺ cells co-stained with microglial-specific markers TMEM119 (E) and P2RY12 (F–G) in control and 14 day PLX3397 mice, showing that CSF1Ri-resistant cells are TMEM119^- and P2RY12^-. Higher resolution images illustrate atypical morphological profile of CSF1Ri-resistant cells. (H) Representative 20x images of myeloid cells (Cd11b, red) and P2RY12 (green). (I) Quantification of % colocalization of CD11b⁺ and P2RY12⁺ cells as seen in (H). (J) Control, 14 day PLX3397, 7 day recovery, and 28 day recovery mouse hemispheres were collected and analyzed for bulk-tissue gene expression changes using Nanostring Immune Profile. (K–L) Volcano plots displaying the fold change of genes (log2 scale) and their significance (y axis, -log10 scale) between 14 day PLX3397 depleted vs. 7 day recovery mice (K) and control vs 28 day recovery (L). Data are represented as mean ± SEM (n=3–5). *p < 0.05, ** p < 0.01, *** p < 0.001; significance symbols represent comparisons between groups: control *, 0d #, 3d ⋄, 7d Δ, 14d Φ. CC, corpus callosum; CP, caudoputamen; CTX, cortex; HC, hippocampus; LV, lateral ventricle; SVZ, subventricular zone; WM, white matter.

Figure 3—figure supplement 1. Extensive CSF1R inhibition unveils the presence of CSF1Ri-resistant myeloid cell in the subventricular zone/white matter areas.

Two-month-old WT or Cx3cr1^CreERT2 mice were treated with vehicle or PLX3397 (600 ppm in chow) for 14 days to evaluate extent of microglial depletion. (A–C) Representative whole brain slice images of IBA1⁺ cells co-stained with microglial-specific markers P2RY12 (A) and TMEM119 (B), or YFP, to detect microglia or cells of Cx3cr1 lineage (C), and ultimately microglial depletion efficiency. For Cx3cr1 lineage tracing studies, Cx3cr1^CreERT2 mice were crossed with YFP reporter mice, treated with tamoxifen and then allowed to recover for 21 days prior to PLX3397 treatment. Inserts show 20x high-resolution images of brain regions highlighted by white boxes. (D) Total IBA1⁺ cells were quantified throughout the entire brain taking every 6^th section (i.e. stereology-style) along the rostral-caudal axis of 2-month-old mice treated for 14 days with PLX3397 (600 ppm in chow). (E) Representative 20x images of myeloid cells (Cd11b, red) and a microglial-specific marker (TMEM119, green). (F–H) Two-month-old WT mice were treated with vehicle or PLX3397 (600 ppm in chow) for 3.5 months. (F–G) Representative tile scan confocal images of IBA1⁺ (green) cells in control and 3.5 month PLX3397 mice. G1 is a higher resolution image showing the absence of SVZ/WM myeloid cells following prolonged CSF1Ri treatment. (H) Quantification of the number of IBA1⁺ cells in the whole brains of control and 3.5 month PLX3397-treated mice. (I) Experimental schematic depicting dose and duration of CSF1Ri resistance study. Two-month-old WT mice were placed on PLX3397 (600 ppm) for 14 days, allowed to recover for 7 or 14 days on control diet (allowing for WM repopulation), and then placed back on CSF1Ri diet (PLX3397 600 ppm) for 7 days. Controls, 14 day PLX3397, and 7 day Recovery were also included for comparison. (J) Representative immunofluorescent images of IBA1⁺ cell (green) and DAPI staining (blue, used to distinguish structures), showing the susceptibility of WM repopulating cells once migrated from SVZ/WM areas. White dotted lines illustrate WM areas. Data are represented as mean ± SEM (n=4–5). *** p < 0.001. CP, caudoputamen; CTX, cortex; LV, lateral ventricle; WM, white matter.