Leter, Y. M. | Search bioRxiv

bioRxiv2025

CSF1R inhibition during cranial radiotherapy reshapes glial dynamics via microglial loss, monocyte engraftment, and accelerated astrocyte reactivity

BackgroundCranial radiotherapy (cRT), a common treatment for central nervous system tumors, induces progressive cognitive problems in over half of patients. In mice, microglial depletion via CSF1R inhibition can mitigate this effect, but the underlying cellular mechanisms remain unclear. We hypothesized that CSF1R inhibition-induced microglial ablation and repopulation improves brain health by modulating microglial reactivity to radiotherapy, which attenuates glial responses to radiotherapy. MethodsNine-week-old male C57BL/6JRj mice received either a CSF1R-inhibitor supplemented diet (pexidartinib, PLX3397) or control diet, followed by fractionated CT-guided cRT (30 Gy) or sham treatment. The pexidartinib diet was discontinued 10 days post-radiotherapy. Animals were sacrificed at three intervals post-radiotherapy, allowing the assessment of temporal changes. Multiple brain regions were assessed by immunohistochemistry for markers of microglia, astrocytes, oligodendrocytes and proliferating cells. Microglial morphological changes were assessed using the semi-automated microglia morphology analysis pipeline mGlia. ResultsRadiotherapy alone reduced microglial numbers and induced a progressive reactive morphology; mild at 30 days and pronounced at 6 months post cRT. CSF1R inhibition before cRT markedly decreased microglial markers but increased general macrophage markers at 30 days and 6 months after cRT, consistent with monocyte-derived cell engraftment. Morphometric analysis revealed rapid and severe morphological change towards a reactive morphotype at 30 days that persisted until 6 months. Microglial depletion did not prevent loss of neurogenesis or oligodendrocyte progenitor cells (OPCs) and accelerated reactive astrogliosis, though partial OPC recovery in the hippocampus and thalamus was observed at 6 months. ConclusionCSF1R inhibition combined with cRT accelerates reactive gliosis and monocyte-derived macrophage engraftment without protecting vulnerable neural cell populations, though limited long-term OPC recovery occurred. Thus, with this set-up CSF1R inhibition-induced microglial ablation and repopulation does not improve overall brain health, but is beneficial for OPCs on the long term after cRT. Key pointsO_LIPexidartinib and cranial radiotherapy have synergistic effects on microglial ablation C_LIO_LIInfiltrating monocytes repopulate the irradiated brain once the pexidartinib diet is discontinued C_LIO_LIIn the absence of microglia, astrocytes show an accelerated reactivity to radiotherapy C_LIO_LILong-term after cranial radiotherapy oligodendrocyte progenitor cell repopulation is enhanced in the pexidartinib treated animals C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/681366v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@1153d64org.highwire.dtl.DTLVardef@171a100org.highwire.dtl.DTLVardef@1013e1borg.highwire.dtl.DTLVardef@949893_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

CSF1R inhibitors pexidartinib and sotuletinib induce rapid glial ablation despite their limited brain penetrability

BackgroundMicroglial reactivity, a hallmark of many neurodegenerative diseases, is thought to contribute significantly to disease pathology. In experimental models, colony stimulating factor 1 receptor (CSF1R) inhibitors transiently deplete microglia to resolve inflammation, leading to improved neuropathology. In oncology, CSF1R inhibitors modulate tumor-associated macrophages (TAMs) toward a tumor-suppressive phenotype by silencing CSF1-CSF1R signaling. As for any therapeutic, target engagement depends on effective drug delivery. In the brain a major hurdle is the limited drug delivery caused by the presence of the blood brain barrier (BBB) containing drug efflux transporters. However, the affinity to these transporters of most CSF1R inhibitors is unknown. MethodsWe assessed the brain penetrance of two CSF1R inhibitors, pexidartinib (PLX3397) and sotuletinib (BLZ945), in the absence and presence of drug transporters ABCB1 and ABCG2. We further assessed their impact on peripheral immune populations, tissue-resident macrophages, microglia and oligodendrocyte progenitor cells (OPCs). ResultsBoth compounds have a limited brain permeability (brain-to-plasma ratio: 0.1). Sotuletinib was a substrate for both ABCB1 and ABCG2, whereas pexidartinib was transported primarily by ABCB1. Despite low brain exposure, both are able to ablate microglia when given to mice at high doses, accompanied by marked depletion of OPCs and macrophage populations in the liver, intestine, and kidney, as well as non-classical monocytes in blood. Pexidartinib additionally altered splenic immune composition, increasing T cells and neutrophils, and reducing dendritic cells and non-classical monocytes. ConclusionThese findings highlight that high-dose CSF1R inhibition rapidly depletes microglia, but induces substantial off-target effects. Such systemic impacts, as well as the impact on OPCs, should be considered when interpreting experimental outcomes or translating CSF1R inhibition into clinical contexts where brain targeting is required. Key messagesO_LICSF1R inhibitors pexidartinib and sotuletinib show poor brain penetrance (brain-to-plasma ratio 0.1) C_LIO_LISotuletinib is a substrate to ABCB1 and ABCG2, pexidartinib is a substrate to ABCB1. C_LIO_LIBoth drugs rapidly deplete microglia, despite poor brain penetration C_LIO_LIMicroglia depletion is accompanied by loss of OPCs and tissue macrophages C_LI

pharmacology and toxicology↗