Highlights d ALSP is a CSF1R-deficiency dementia associated with increased CSF2 expression d In Csf1r +/À ALSP mice, CSF-2 promotes microgliosis by direct signaling in microglia d Targeting Csf2 improves cognition and myelination and normalizes microglial function d CSF-2 is a therapeutic target in ALSP

CSF-1R haploinsufficiency causes adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). Previous studies in the Csf1r +/mouse model of ALSP hypothesized a central role of elevated cerebral Csf2 expression. Here we show that monoallelic deletion of Csf2 rescues most behavioral deficits and histopathological changes in Csf1r +/mice by preventing microgliosis and eliminating most microglial transcriptomic alterations, including those indicative of oxidative stress and demyelination. We also show elevation of Csf2 transcripts and of several CSF-2 downstream targets in the brains of ALSP patients, demonstrating that the mechanisms identified in the mouse model are functional in man. Our data provide new insights into the mechanisms underlying ALSP. Since both increased CSF2 levels and decreased microglial Csf1r expression have also been reported in Alzheimer's disease and multiple sclerosis, we suggest that the unbalanced CSF-1R/CSF-2 signaling we describe in the present study may contribute to the pathogenesis of other neurodegenerative conditions. Abbreviations: CSF-1 RColony stimulating factor-1 receptor CSF-2Colony stimulating factor-2 ALSP Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia Highlights• ALSP is a CSF1R-deficiency dementia associated with increased CSF2 expression • In Csf1r+/-ALSP mice CSF-2 promotes microgliosis by direct signaling in microglia • Targeting Csf2 improves cognition, myelination and normalizes microglial function • CSF-2 is a therapeutic target in ALSP

Adult onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a dementia resulting from dominantly inherited CSF1R inactivating mutations. The Csf1r+/− mouse mimics ALSP symptoms and pathology. Csf1r is mainly expressed in microglia, but also in cortical layer V neurons that are gradually lost in Csf1r+/− mice with age. We therefore examined whether microglial or neuronal Csf1r loss caused neurodegeneration in Csf1r+/− mice. The behavioral deficits, pathologies and elevation of Csf2 expression contributing to disease, previously described in the Csf1r+/− ALSP mouse, were reproduced by microglial deletion (MCsf1rhet mice), but not by neural deletion. Furthermore, increased Csf2 expression by callosal astrocytes, oligodendrocytes, and microglia was observed in Csf1r+/− mice and, in MCsf1rhet mice, the densities of these three cell types were increased in supraventricular patches displaying activated microglia, an early site of disease pathology. These data confirm that ALSP is a primary microgliopathy and inform future therapeutic and experimental approaches.

Colony stimulating factor (CSF) receptor-1 (CSF-1R)-related leukoencephalopathy (CRL) is an adult-onset, demyelinating and neurodegenerative disease caused by autosomal dominant mutations in CSF1R, modeled by the Csf1r +/À mouse. The expression of Csf2, encoding granulocyte-macrophage CSF (GM-CSF) and of Csf3, encoding granulocyte CSF (G-CSF), are elevated in both mouse and human CRL brains. While monoallelic targeting of Csf2 has been shown to attenuate many behavioral and histological deficits of Csf1r +/À mice, including cognitive dysfunction and demyelination, the contribution of Csf3 has not been explored. In the present study, we investigate the behavioral, electrophysiological and histopathological phenotypes of Csf1r +/À mice following monoallelic targeting of Csf3. We show that Csf3 heterozygosity normalized the Csf3 levels in Csf1r +/À mouse brains and ameliorated anxiety-like behavior, motor coordination and social interaction deficits, but not the cognitive impairment of Csf1r +/À mice. Csf3 heterozygosity failed to prevent callosal demyelination. However, consistent with its effects on behavior, Csf3 heterozygosity normalized microglial morphology in the cerebellum and in the ventral, but not in the dorsal hippocampus. Csf1r +/À mice exhibited altered firing activity in the deep cerebellar nuclei (DCN) associated with increased engulfment of glutamatergic synapses by DCN microglia and increased deposition of the complement factor C1q on glutamatergic synapses. These phenotypes were significantly ameliorated by monoallelic deletion of Csf3. Our current and earlier findings indicate that G-CSF and GM-CSF play largely non-overlapping roles in CRL-like disease development in Csf1r +/À mice.

Colony stimulating factor (CSF) receptor-1 (CSF-1R)-related leukoencephalopathy (CRL) is an adult-onset, demyelinating neurodegenerative disease caused by autosomal dominant mutations in CSF1R, modeled by the Csf1r+/- mouse. The expression of Csf2, encoding granulocyte- macrophage CSF (GM-CSF) and of Csf3, encoding granulocyte CSF (G-CSF), are elevated in both mouse and human CRL brains. While monoallelic targeting of Csf2 has been shown to attenuate many behavioral and histological deficits of mouse CRL, including cognitive dysfunction and demyelination, the contribution of Csf3 has not been explored. In this manuscript, we investigate the behavioral, electrophysiological and histopathological phenotypes of CRL mice following monoallelic targeting of Csf3. We show that Csf3 heterozygosity normalized the Csf3 levels in Csf1r+/- mouse brains and ameliorated anxiety-like behavior, motor coordination and social interaction deficits, but not their cognitive impairment. Consistent with this, Csf3 heterozygosity attenuated microglial activation in the cerebellum and in the ventral but not in the dorsal hippocampus. Csf3 heterozygosity also failed to prevent demyelination. Csf1r+/- mice exhibited altered synaptic activity in the deep cerebellar nuclei (DCN) associated with increased deposition of the complement factor C1q on glutamatergic synapses and with increased engulfment of glutamatergic synapses by DCN microglia. These phenotypes were significantly ameliorated by monoallelic deletion of Csf3. Our findings indicate that G-CSF and GM-CSF play non-overlapping roles in mouse CRL development and suggest that G-CSF could be an additional therapeutic target in CRL.