Tracking changes in functionality and morphology of repopulated microglia in young and old mice.

BACKGROUND: Microglia (MG) are myeloid cells of the central nervous system that support homeostasis and instigate neuroinflammation in pathologies. Single-cell RNA sequencing (scRNA-seq) revealed the functional heterogeneity of MG in mouse brains. Microglia are self-renewing cells and inhibition of colony-stimulating factor 1 receptor (CSF1R) signaling depletes microglia which rapidly repopulate. The functions of repopulated microglia are poorly known. METHODS: We combined scRNA-seq, bulk RNA-seq, immunofluorescence, and confocal imaging to study the functionalities and morphology of repopulated microglia. RESULTS: A CSRF1R inhibitor (BLZ-945) depleted microglia within 21 days and a number of microglia was fully restored within 7 days, as confirmed by TMEM119 staining and flow cytometry. ScRNA-seq and computational analyses demonstrate that repopulated microglia originated from preexisting progenitors and reconstituted functional clusters but upregulated inflammatory genes. Percentages of proliferating, immature microglia displaying inflammatory gene expression increased in aging mice. Morphometric analysis of MG cell body and branching revealed a distinct morphology of repopulated MG, particularly in brains of old mice. We demonstrate that with aging some repopulated MG fail to reach the homeostatic phenotype. These differences may contribute to the deterioration of MG protective functions with age.

CSF1 is expressed by the intestinal epithelial cells to regulate Mφ macrophages and maintain epithelial homeostasis and is downregulated in neonates with necrotizing enterocolitis.

BACKGROUND: Colony stimulating factor 1 (CSF1) is generally expressed by immune cells in response to pro-inflammatory stimuli. The CSF1 receptor (CSFR) is activated by CSF1, and plays a key role in macrophage homeostasis. Furthermore, the CSF1R+ macrophages maintain homeostasis in the intestinal epithelium. The aim of this study was to explore the functions of CSF1-expressing and CSF1R+ macrophages in necrotizing enterocolitis (NEC), which commonly affects the ileum of neonates. METHODS: In-situ CSF1 expression in the intestines of neonates with NEC or intestinal atresia (n = 4 each) was detected by immunofluorescence staining. The CSF1 levels in the intestinal crypt-derived organoid cultures were measured by ELISA. Peripheral blood monocyte-derived Mφ macrophages were co-cultured with the organoids and stimulated with lipopolysaccharide (LPS) to mimic the inflamed state of the ileum in NEC patients. RESULTS: CSF1 was expressed in the intestinal epithelial cells of the fetal and neonatal samples, but suppressed in the NEC samples. Furthermore, CSF1 expression was downregulated in the intestinal crypt-derived organoids by LPS. CSF1R+ macrophages were detected near the intestinal crypts in the non-inflamed intestines but were absent in tissues obtained from pediatric NEC patients. Peripheral blood monocyte-derived macrophages promoted intestinal organoid proliferation in vitro following CSF1 stimulation. Finally, low concentrations of LPS slightly enhanced the proliferation of organoids co-cultured with the macrophages, whereas higher doses had a significant inhibitory effect. CONCLUSIONS: Intestinal epithelial cells express CSF1 to regulate the resident macrophages, maintain epithelial homeostasis, and resist infection. The abundant CSF1R+ macrophages in the fetal intestine may overexpress TNF-α upon activation of the TLR4/NF-κB pathway, resulting in epithelial damage and NEC induction.

Novel microglial transcriptional signatures promote social and cognitive deficits following repeated social defeat.

Chronic stress is associated with anxiety and cognitive impairment. Repeated social defeat (RSD) in mice induces anxiety-like behavior driven by microglia and the recruitment of inflammatory monocytes to the brain. Nonetheless, it is unclear how microglia communicate with other cells to modulate the physiological and behavioral responses to stress. Using single-cell (sc)RNAseq, we identify novel, to the best of our knowledge, stress-associated microglia in the hippocampus defined by RNA profiles of cytokine/chemokine signaling, cellular stress, and phagocytosis. Microglia depletion with a CSF1R antagonist (PLX5622) attenuates the stress-associated profile of leukocytes, endothelia, and astrocytes. Furthermore, RSD-induced social withdrawal and cognitive impairment are microglia-dependent, but social avoidance is microglia-independent. Furthermore, single-nuclei (sn)RNAseq shows robust responses to RSD in hippocampal neurons that are both microglia-dependent and independent. Notably, stress-induced CREB, oxytocin, and glutamatergic signaling in neurons are microglia-dependent. Collectively, these stress-associated microglia influence transcriptional profiles in the hippocampus related to social and cognitive deficits.

Microglia are not necessary for maintenance of blood-brain barrier properties in health, but PLX5622 alters brain endothelial cholesterol metabolism.

Microglia, the resident immune cells of the central nervous system, are intimately involved in the brain's most basic processes, from pruning neural synapses during development to preventing excessive neuronal activity throughout life. Studies have reported both helpful and harmful roles for microglia at the blood-brain barrier (BBB) in the context of disease. However, less is known about microglia-endothelial cell interactions in the healthy brain. To investigate the role of microglia at a healthy BBB, we used the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 to deplete microglia and analyzed the BBB ultrastructure, permeability, and transcriptome. Interestingly, we found that, despite their direct contact with endothelial cells, microglia are not necessary for the maintenance of BBB structure, function, or gene expression in the healthy brain. However, we found that PLX5622 treatment alters brain endothelial cholesterol metabolism. This effect was independent from microglial depletion, suggesting that PLX5622 has off-target effects on brain vasculature.

Plonmarlimab, a novel anti-GM-CSF blocking antibody, ameliorates disease progression in the pre-clinical model of macrophage activation syndrome.

OBJECTIVES: We aimed to characterize and investigate the safety and efficacy of Plonmarlimab, a novel anti-granulocyte-macrophage colony-stimulating factor (anti-GM-CSF) neutralizing antibody, on the treatment of macrophage activation syndrome (MAS), a life-threatening systemic inflammatory disease, in pre-clinical models. METHODS: The binding affinity was evaluated using Biacore. The neutralizing activity was measured through the blockade of ligand-receptor interaction, inhibition of STAT5 phosphorylation and suppression of TF-1 cell proliferation. The efficacy of Plonmarlimab was evaluated in a humanized MAS model, which was established by engrafting human umbilical cord blood (UCB) cells into NOG-EXL mice. Additionally, the safety profile of Plonmarlimab was investigated in cynomolgus monkeys. RESULTS: At the molecular level, Plonmarlimab showed sub-nanomolar binding affinity with human GM-CSF and effectively blocked the binding of GM-CSF to its receptor. At the cellular level, Plonmarlimab dose-dependently inhibited intracellular STAT5 phosphorylation and suppressed GM-CSF-induced TF-1 proliferation. In the UCB-engrafted NOG-EXL MAS mouse model, Plonmarlimab treatment significantly ameliorated disease progression, demonstrated by the improvements in body weight loss, anaemia and some histopathological features. Furthermore, Plonmarlimab was well tolerated up to 150 mg/kg weekly in monkeys with no reported adverse effects. CONCLUSIONS: Plonmarlimab is a highly potent GM-CSF blocking antibody and has demonstrated promising efficacy in a pre-clinical MAS model with a favourable safety profile, supporting its clinical development.

Switch to phagocytic microglia by CSFR1 inhibition drives amyloid-beta clearance from glutamatergic terminals rescuing LTP in acute hippocampal slices.

Microglia, traditionally regarded as innate immune cells in the brain, drive neuroinflammation and synaptic dysfunctions in the early phases of Alzheimer disease (AD), acting upstream to Aß accumulation. Colony stimulating factor 1-receptor (CSF-1R) is predominantly expressed on microglia and its levels are significantly increased in neurodegenerative diseases, possibly contributing to the chronic inflammatory microglial response. On the other hand, CSF-1R inhibitors confer neuroprotection in preclinical models of neurodegenerative diseases. Here, we determined the effects of the CSF-1R inhibitor PLX3397 on the Aß-mediated synaptic alterations in ex vivo hippocampal slices. Electrophysiological findings show that PLX3397 rescues LTP impairment and neurotransmission changes induced by Aß. In addition, using confocal imaging experiments, we demonstrate that PLX3397 stimulates a microglial transition toward a phagocytic phenotype, which in turn promotes the clearance of Aß from glutamatergic terminals. We believe that the selective pruning of Aß-loaded synaptic terminals might contribute to the restoration of LTP and excitatory transmission alterations observed upon acute PLX3397 treatment. This result is in accordance with the mechanism proposed for CSF1R inhibitors, that is to eliminate responsive microglia and replace it with newly generated, homeostatic microglia, capable of promoting brain repair. Overall, our findings identify a connection between the rapid microglia adjustments and the early synaptic alterations observed in AD, possibly highlighting a novel disease-modifying target.

LncRNA018392 promotes the proliferation of Liaoning cashmere goat skin fibroblasts by upregulating CSF1R through binding to SPI1.

BACKGROUND: Liaoning cashmere goat is recognized as a valuable genetic resource breed, with restrictions on genetic outflow in China. Hair follicle development in the cashmere goat is influenced by melatonin and long non-coding RNAs (lncRNAs). However, the role of lncRNAs in facilitating melatonin-promoted cashmere growth remains poorly understood. Previous studies have identified a new lncRNA, lncRNA018392, which is involved in the melatonin-promoted proliferation of cashmere skin fibroblasts. METHOD: Flow cytometry and CCK-8 assays confirmed that silencing lncRNA018392 negates the effects of melatonin on cell proliferation, and that proliferation was reduced when the gene CSF1R, located near lncRNA018392, was inhibited. Further investigation using a dual-luciferase reporter assay showed that lncRNA018392 could positively regulate the promoter of CSF1R. RESULTS: Results from RNA-binding protein immunoprecipitation (RIP) and chromatin immunoprecipitation sequencing (ChIP-Seq) revealed that lncRNA018392 interacts with the transcription factor SPI1, with CSF1R being a downstream target gene regulated by SPI1. This interaction was confirmed by ChIP-PCR, which demonstrated SPI1's binding to CSF1R. CONCLUSIONS: This study found that the melatonin-responsive lncRNA018392 accelerates the cell cycle and promotes cell proliferation by recruiting SPI1 to upregulate the expression of the neighboring gene CSF1R. These findings provide a theoretical foundation for elucidating the molecular mechanisms of cashmere growth and for the molecular breeding of cashmere goats.

Discovery of an embryonically derived bipotent population of endothelial-macrophage progenitor cells in postnatal aorta.

Converging evidence indicates that extra-embryonic yolk sac is the source of both macrophages and endothelial cells in adult mouse tissues. Prevailing views are that these embryonically derived cells are maintained after birth by proliferative self-renewal in their differentiated states. Here we identify clonogenic endothelial-macrophage (EndoMac) progenitor cells in the adventitia of embryonic and postnatal mouse aorta, that are independent of Flt3-mediated bone marrow hematopoiesis and derive from an early embryonic CX3CR1+ and CSF1R+ source. These bipotent progenitors are proliferative and vasculogenic, contributing to adventitial neovascularization and formation of perfused blood vessels after transfer into ischemic tissue. We establish a regulatory role for angiotensin II, which enhances their clonogenic and differentiation properties and rapidly stimulates their proliferative expansion in vivo. Our findings demonstrate that embryonically derived EndoMac progenitors participate in local vasculogenic responses in the aortic wall by contributing to the expansion of endothelial cells and macrophages postnatally.

Pathogenesis-driven treatment of primary pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is a syndrome that results from the accumulation of lipoproteinaceous material in the alveolar space. According to the underlying pathogenetic mechanisms, three different forms have been identified, namely primary, secondary and congenital. Primary PAP is caused by disruption of granulocyte-macrophage colony-stimulating factor (GM-CSF) signalling due to the presence of neutralising autoantibodies (autoimmune PAP) or GM-CSF receptor genetic defects (hereditary PAP), which results in dysfunctional alveolar macrophages with reduced phagocytic clearance of particles, cholesterol and surfactant. The serum level of GM-CSF autoantibody is the only disease-specific biomarker of autoimmune PAP, although it does not correlate with disease severity. In PAP patients with normal serum GM-CSF autoantibody levels, elevated serum GM-CSF levels is highly suspicious for hereditary PAP. Several biomarkers have been correlated with disease severity, although they are not specific for PAP. These include lactate dehydrogenase, cytokeratin 19 fragment 21.1, carcinoembryonic antigen, neuron-specific enolase, surfactant proteins, Krebs von Lungen 6, chitinase-3-like protein 1 and monocyte chemotactic proteins. Finally, increased awareness of the disease mechanisms has led to the development of pathogenesis-based treatments, such as GM-CSF augmentation and cholesterol-targeting therapies.

Deciphering glial contributions to CSF1R-related disorder via single-nuclear transcriptomic profiling: a case study.

CSF1R-related disorder (CSF1R-RD) is a neurodegenerative condition that predominantly affects white matter due to genetic alterations in the CSF1R gene, which is expressed by microglia. We studied an elderly man with a hereditary, progressive dementing disorder of unclear etiology. Standard genetic testing for leukodystrophy and other neurodegenerative conditions was negative. Brain autopsy revealed classic features of adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), including confluent white matter degeneration with axonal spheroids and pigmented glial cells in the affected white matter, consistent with CSF1R-RD. Subsequent long-read sequencing identified a novel deletion in CSF1R that was not detectable with short-read exome sequencing. To gain insight into potential mechanisms underlying white matter degeneration in CSF1R-RD, we studied multiple brain regions exhibiting varying degrees of white matter pathology. We found decreased CSF1R transcript and protein across brain regions, including intact white matter. Single nuclear RNA sequencing (snRNAseq) identified two disease-associated microglial cell states: lipid-laden microglia (expressing GPNMB, ATG7, LGALS1, LGALS3) and inflammatory microglia (expressing IL2RA, ATP2C1, FCGBP, VSIR, SESN3), along with a small population of CD44+ peripheral monocyte-derived macrophages exhibiting migratory and phagocytic signatures. GPNMB+ lipid-laden microglia with ameboid morphology represented the end-stage disease microglia state. Disease-associated oligodendrocytes exhibited cell stress signatures and dysregulated apoptosis-related genes. Disease-associated oligodendrocyte precursor cells (OPCs) displayed a failure in their differentiation into mature myelin-forming oligodendrocytes, as evidenced by upregulated LRP1, PDGFRA, SOX5, NFIA, and downregulated NKX2-2, NKX6.2, SOX4, SOX8, TCF7L2, YY1, ZNF488. Overall, our findings highlight microglia-oligodendroglia crosstalk in demyelination, with CSF1R dysfunction promoting phagocytic and inflammatory microglia states, an arrest in OPC differentiation, and oligodendrocyte depletion.

Heterozygous missense CSF1R variants hamper in vitro CD34+-derived dendritic cell generation but not in vivo dendritic cell development.

Colony stimulating factor 1 receptor (CSF1R) is an essential receptor for both colony stimulating factor 1 (CSF1) and interleukin (IL) 34 signaling expressed on monocyte precursors and myeloid cells, including monocytes, dendritic cells (DC), and microglia. In humans, dominant heterozygous pathogenic variants in CSF1R cause a neurological condition known as CSF1R-related disorder (CSF1R-RD), typically with late onset, previously referred to as adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). CSF1R-RD is characterized by microglia reduction and altered monocyte function; however, the impact of pathogenic CSF1R variants on the human DC lineage remains largely unknown. We previously reported that cord blood CD34+ stem cell-derived DCs generated in vitro originate specifically from CSF1R expressing precursors. In this study, we examined the DC lineage of four unrelated patients with late-onset CSF1R-RD who carried heterozygous missense CSF1R variants (c.2330G>A, c.2375C>A, c.2329C>T, and c.2381T>C) affecting different amino acids in the protein tyrosine kinase domain of CSF1R. CD34+ stem cells and CD14+ monocytes were isolated from peripheral blood and subjected to an in vitro culture protocol to differentiate towards conventional DCs and monocyte-derived DCs, respectively. Flow cytometric analysis revealed that monocytes from patients with late-onset CSF1R-RD were still able to differentiate into monocyte-derived DCs in vitro, whereas the ability of CD34+ stem cells to differentiate into conventional DCs was impaired. Strikingly, the peripheral blood of patients contained all naturally occurring DC subsets. We conclude that the in vitro abrogation of DC-development in patients with heterozygous pathogenic missense CSF1R variants does not translate to an impairment in DC development in vivo and speculate that CSF1R signalling in vivo is compensated, which needs further study.

CSF1R Inhibition in Patients with Advanced Solid Tumors or Tenosynovial Giant Cell Tumor: A Phase I Study of Vimseltinib.

PURPOSE: Tenosynovial giant cell tumor (TGCT) is a locally aggressive neoplasm caused by dysregulation of the colony-stimulating factor 1 (CSF1) gene and overexpression of the CSF1 ligand. Surgery is the standard of care for most patients, but there are limited treatment options for patients with TGCT not amenable to surgery. This study evaluates vimseltinib, an investigational, oral, switch-control tyrosine kinase inhibitor designed to selectively and potently inhibit the CSF1 receptor. PATIENTS AND METHODS: This first-in-human, multicenter, open-label phase I/II study of vimseltinib in patients with malignant solid tumors (N = 37) or TGCT not amenable to surgery (N = 32) followed a pharmacologically guided 3 + 3 study design (NCT03069469). The primary objectives were to assess safety and tolerability, determine the recommended phase II dose, and characterize the pharmacokinetics; exploratory objectives included pharmacodynamics and efficacy. RESULTS: Vimseltinib was well tolerated; the majority of non-laboratory treatment-emergent adverse events were of grade 1/2 severity. There was no evidence of cholestatic hepatotoxicity or drug-induced liver injury. The recommended phase II dose was determined to be 30 mg twice weekly (no loading dose), and vimseltinib plasma exposure increased with the dose. In patients with TGCT, the median treatment duration was 25.1 months (range, 0.7-46.9), and the objective response rate as assessed by independent radiological review using RECIST version 1.1 was 72%. CONCLUSIONS: Vimseltinib demonstrated long-term tolerability, manageable safety, dose-dependent exposure, and robust antitumor activity in patients with TGCT not amenable to surgery.

Redefining the ontogeny of hyalocytes as yolk sac-derived tissue-resident macrophages of the vitreous body.

BACKGROUND: The eye is a highly specialized sensory organ which encompasses the retina as a part of the central nervous system, but also non-neural compartments such as the transparent vitreous body ensuring stability of the eye globe and a clear optical axis. Hyalocytes are the tissue-resident macrophages of the vitreous body and are considered to play pivotal roles in health and diseases of the vitreoretinal interface, such as proliferative vitreoretinopathy or diabetic retinopathy. However, in contrast to other ocular macrophages, their embryonic origin as well as the extent to which these myeloid cells might be replenished by circulating monocytes remains elusive. RESULTS: In this study, we combine transgenic reporter mice, embryonic and adult fate mapping approaches as well as parabiosis experiments with multicolor immunofluorescence labeling and confocal laser-scanning microscopy to comprehensively characterize the murine hyalocyte population throughout development and in adulthood. We found that murine hyalocytes express numerous well-known myeloid cell markers, but concomitantly display a distinct immunophenotype that sets them apart from retinal microglia. Embryonic pulse labeling revealed a yolk sac-derived origin of murine hyalocytes, whose precursors seed the developing eye prenatally. Finally, postnatal labeling and parabiosis established the longevity of hyalocytes which rely on Colony Stimulating Factor 1 Receptor (CSF1R) signaling for their maintenance, independent of blood-derived monocytes. CONCLUSION: Our study identifies hyalocytes as long-living progeny of the yolk sac hematopoiesis and highlights their role as integral members of the innate immune system of the eye. As a consequence of their longevity, immunosenescence processes may culminate in hyalocyte dysfunction, thereby contributing to the development of vitreoretinal diseases. Therefore, myeloid cell-targeted therapies that convey their effects through the modification of hyalocyte properties may represent an interesting approach to alleviate the burden imposed by diseases of the vitreoretinal interface.

Targeting CSF1R in myeloid-derived suppressor cells: insights into its immunomodulatory functions in colorectal cancer and therapeutic implications.

OBJECTIVE: This study aimed to investigate the critical role of MDSCs in CRC immune suppression, focusing on the CSF1R and JAK/STAT3 signaling axis. Additionally, it assessed the therapeutic efficacy of LNCs@CSF1R siRNA and anti-PD-1 in combination. METHODS: Single-cell transcriptome sequencing data from CRC and adjacent normal tissues identified MDSC-related differentially expressed genes. RNA-seq analysis comprehensively profiled MDSC gene expression in murine CRC tumors. LNCs@CSF1R siRNA nanocarriers effectively targeted and inhibited CSF1R. Flow cytometry quantified changes in MDSC surface markers post-CSF1R inhibition. RNA-seq and pathway enrichment analyses revealed the impact of CSF1R on MDSC metabolism and signaling. The effect of CSF1R inhibition on the JAK/STAT3 signaling axis was validated using Colivelin and metabolic assessments. Glucose and fatty acid uptake were measured via fluorescence-based flow cytometry. The efficacy of LNCs@CSF1R siRNA and anti-PD-1, alone and in combination, was evaluated in a murine CRC model with extensive tumor section analyses. RESULTS: CSF1R played a significant role in MDSC-mediated immune suppression. LNCs@CSF1R siRNA nanocarriers effectively targeted MDSCs and inhibited CSF1R. CSF1R regulated MDSC fatty acid metabolism and immune suppression through the JAK/STAT3 signaling axis. Inhibition of CSF1R reduced STAT3 activation and target gene expression, which was rescued by Colivelin. Combined treatment with LNCs@CSF1R siRNA and anti-PD-1 significantly slowed tumor growth and reduced MDSC abundance within CRC tumors. CONCLUSION: CSF1R via the JAK/STAT3 axis critically regulates MDSCs, particularly in fatty acid metabolism and immune suppression. Combined therapy with LNCs@CSF1R siRNA and anti-PD-1 enhances therapeutic efficacy in a murine CRC model, providing a strong foundation for future clinical applications.

CNS-wide repopulation by hematopoietic-derived microglia-like cells corrects progranulin deficiency in mice.

Hematopoietic stem cell transplantation can deliver therapeutic proteins to the central nervous system (CNS) through transplant-derived microglia-like cells. However, current conditioning approaches result in low and slow engraftment of transplanted cells in the CNS. Here we optimized a brain conditioning regimen that leads to rapid, robust, and persistent microglia replacement without adverse effects on neurobehavior or hematopoiesis. This regimen combines busulfan myeloablation and six days of Colony-stimulating factor 1 receptor inhibitor PLX3397. Single-cell analyses revealed unappreciated heterogeneity of microglia-like cells with most cells expressing genes characteristic of homeostatic microglia, brain-border-associated macrophages, and unique markers. Cytokine analysis in the CNS showed transient inductions of myeloproliferative and chemoattractant cytokines that help repopulate the microglia niche. Bone marrow transplant of progranulin-deficient mice conditioned with busulfan and PLX3397 restored progranulin in the brain and eyes and normalized brain lipofuscin storage, proteostasis, and lipid metabolism. This study advances our understanding of CNS repopulation by hematopoietic-derived cells and demonstrates its therapeutic potential for treating progranulin-dependent neurodegeneration.

CSF1R inhibition depletes brain macrophages and reduces brain virus burden in SIV-infected macaques.

Perivascular macrophages (PVMs) and, to a lesser degree, microglia are targets and reservoirs of HIV and simian immunodeficiency virus (SIV) in the brain. Previously, we demonstrated that colony-stimulating factor 1 receptor (CSF1R) in PVMs was upregulated and activated in chronically SIV-infected rhesus macaques with encephalitis, correlating with SIV infection of PVMs. Herein, we investigated the role of CSF1R in the brain during acute SIV infection using BLZ945, a brain-penetrant CSF1R kinase inhibitor. Apart from three uninfected historic controls, nine Indian rhesus macaques were infected acutely with SIVmac251 and divided into three groups (n = 3 each): an untreated control and two groups treated for 20-30 days with low- (10 mg/kg/day) or high- (30 mg/kg/day) dose BLZ945. With the high-dose BLZ945 treatment, there was a significant reduction in cells expressing CD163 and CD206 across all four brain areas examined, compared with the low-dose treatment and control groups. In 9 of 11 tested regions, tissue viral DNA (vDNA) loads were reduced by 95%-99% following at least one of the two doses, and even to undetectable levels in some instances. Decreased numbers of CD163+ and CD206+ cells correlated significantly with lower levels of vDNA in all four corresponding brain areas. In contrast, BLZ945 treatment did not significantly affect the number of microglia. Our results indicate that doses as low as 10 mg/kg/day of BLZ945 are sufficient to reduce the tissue vDNA loads in the brain with no apparent adverse effect. This study provides evidence that infected PVMs are highly sensitive to CSF1R inhibition, opening new possibilities to achieve viral clearance.

Novel variants in CSF1R associated with adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP).

The CSF1R gene, located on chromosome 5, encodes a 108 kDa protein and plays a critical role in regulating myeloid cell function. Mutations in CSF1R have been identified as a cause of a rare white matter disease called adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP, also known as CSF1R-related leukoencephalopathy), characterized by progressive neurological dysfunction. This study aimed to broaden the genetic basis of ALSP by identifying novel CSF1R variants in patients with characteristic clinical and imaging features of ALSP. Genetic analysis was performed through whole-exome sequencing or panel analysis for leukodystrophy genes. Variant annotation and classification were conducted using computational tools, and the identified variants were categorized following the recommendations of the American College of Medical Genetics and Genomics (ACMG). To assess the evolutionary conservation of the novel variants within the CSF1R protein, amino acid sequences were compared across different species. The study identified six previously unreported CSF1R variants (c.2384G>T, c.2133_2919del, c.1837G>A, c.2304C>A, c.2517G>T, c.2642C>T) in seven patients with ALSP, contributing to the expanding knowledge of the genetic diversity underlying this rare disease. The analysis revealed considerable genetic and clinical heterogeneity among these patients. The findings emphasize the need for a comprehensive understanding of the genetic basis of rare diseases like ALSP and underscored the importance of genetic testing, even in cases with no family history of the disease. The study's contribution to the growing spectrum of ALSP genetics and phenotypes enhances our knowledge of this condition, which can be crucial for both diagnosis and potential future treatments.

Therapeutic potential of human microglia transplantation in a chimeric model of CSF1R-related leukoencephalopathy.

Microglia replacement strategies are increasingly being considered for the treatment of primary microgliopathies like adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). However, available mouse models fail to recapitulate the diverse neuropathologies and reduced microglia numbers observed in patients. In this study, we generated a xenotolerant mouse model lacking the fms-intronic regulatory element (FIRE) enhancer within Csf1r, which develops nearly all the hallmark pathologies associated with ALSP. Remarkably, transplantation of human induced pluripotent stem cell (iPSC)-derived microglial (iMG) progenitors restores a homeostatic microglial signature and prevents the development of axonal spheroids, white matter abnormalities, reactive astrocytosis, and brain calcifications. Furthermore, transplantation of CRISPR-corrected ALSP-patient-derived iMG reverses pre-existing spheroids, astrogliosis, and calcification pathologies. Together with the accompanying study by Munro and colleagues, our results demonstrate the utility of FIRE mice to model ALSP and provide compelling evidence that iMG transplantation could offer a promising new therapeutic strategy for ALSP and perhaps other microglia-associated neurological disorders.