Molecular basis of neurodegeneration in a mouse model of Polr3-related disease.

Pathogenic variants in subunits of RNA polymerase (Pol) III cause a spectrum of Polr3-related neurodegenerative diseases including 4H leukodystrophy. Disease onset occurs from infancy to early adulthood and is associated with a variable range and severity of neurological and non-neurological features. The molecular basis of Polr3-related disease pathogenesis is unknown. We developed a postnatal whole-body mouse model expressing pathogenic Polr3a mutations to examine the molecular mechanisms by which reduced Pol III transcription results primarily in central nervous system phenotypes. Polr3a mutant mice exhibit behavioral deficits, cerebral pathology and exocrine pancreatic atrophy. Transcriptome and immunohistochemistry analyses of cerebra during disease progression show a reduction in most Pol III transcripts, induction of innate immune and integrated stress responses and cell type-specific gene expression changes reflecting neuron and oligodendrocyte loss and microglial activation. Earlier in the disease when integrated stress and innate immune responses are minimally induced, mature tRNA sequencing revealed a global reduction in tRNA levels and an altered tRNA profile but no changes in other Pol III transcripts. Thus, changes in the size and/or composition of the tRNA pool have a causal role in disease initiation. Our findings reveal different tissue- and brain region-specific sensitivities to a defect in Pol III transcription.

Differential regulation of microglial states by colony stimulating factors.

Recent studies have emphasized the role of microglia in the progression of many neurodegenerative diseases. The colony stimulating factors, CSF-1 (M-CSF), granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF) regulate microglia through different cognate receptors. While the receptors for GM-CSF (GM-CSFR) and G-CSF (G-CSFR) are specific for their ligands, CSF-1 shares its receptor, the CSF-1 receptor-tyrosine kinase (CSF-1R), with interleukin-34 (IL-34). All four cytokines are expressed locally in the CNS. Activation of the CSF-1R in macrophages is anti-inflammatory. In contrast, the actions of GM-CSF and G-CSF elicit different activated states. We here review the roles of each of these cytokines in the CNS and how they contribute to the development of disease in a mouse model of CSF-1R-related leukodystrophy. Understanding their roles in this model may illuminate their contribution to the development or exacerbation of other neurodegenerative diseases.

Potential use of glucocorticosteroids in CSF1R mutation carriers - current evidence and future directions.

We recently found that glucocorticosteroids (GCs) have protective effects in CSF1R mutation carriers against developing symptomatic CSF1R-related leukoencephalopathy. Our findings were subsequently confirmed in a mouse model study. We have received many questions from patients, their families, patient organisations, and healthcare practitioners about the optimal type of GCs, the dose, the route of administration, and application timing. This paper attempts to answer the most urgent of these questions based on our previous studies and personal observations. Despite the promising observations, more research on larger patient groups is needed to elucidate the beneficial actions of GCs in CSF1R mutation carriers.

Trem2 Enhances Demyelination in the Csf1r+/- Mouse Model of Leukoencephalopathy.

Colony-stimulating factor-1 receptor (CSF-1R)-related leukoencephalopathy (CRL) is a neurodegenerative disease that triggers early demyelination, leading to an adult-onset dementia. Triggering receptor expressed on myeloid cells-2 (TREM2) is a microglial receptor that promotes the activation of microglia and phagocytic clearance of apoptotic neurons and myelin debris. We investigated the role of Trem2 in the demyelination observed in the Csf1r+/- mouse model of CRL. We show that elevation of Trem2 expression and callosal demyelination occur in 4-5-month-old Csf1r+/- mice, prior to the development of symptoms. Absence of Trem2 in the Csf1r+/- mouse attenuated myelin pathology and normalized microglial densities and morphology in the corpus callosum. Trem2 absence also prevented axonal degeneration and the loss of cortical layer V neurons observed in Csf1r+/- mice. Furthermore, the absence of Trem2 prevented the accumulation of myelin-derived lipids in Csf1r+/- macrophages and reduced the production of TNF-α after myelin engulfment. These data suggest that TREM2 contributes to microglial dyshomeostasis in CRL.

Prophylactic effect of chronic immunosuppression in a mouse model of CSF-1 receptor-related leukoencephalopathy.

Mutations leading to colony-stimulating factor-1 receptor (CSF-1R) loss-of-function or haploinsufficiency cause CSF1R-related leukoencephalopathy (CRL), an adult-onset disease characterized by loss of myelin and neurodegeneration, for which there is no effective therapy. Symptom onset usually occurs in the fourth decade of life and the penetrance of disease in carriers is high. However, familial studies have identified a few carriers of pathogenic CSF1R mutations that remain asymptomatic even in their seventh decade of life, raising the possibility that the development and severity of disease might be influenced by environmental factors. Here we report new cases in which long-term glucocorticoid treatment is associated with asymptomatic status in elder carriers of pathogenic CSF-1R mutations. The main objective of the present study was to investigate the link between chronic immunosuppression initiated pre-symptomatically and resistance to the development of symptomatic CRL, in the Csf1r+/- mouse model. We show that chronic prednisone administration prevents the development of memory, motor coordination and social interaction deficits, as well as the demyelination, neurodegeneration and microgliosis associated with these deficits. These findings are in agreement with the preliminary clinical observations and support the concept that pre-symptomatic immunosuppression is protective in patients carrying pathogenic CSF1R variants associated with CRL. Proteomic analysis of microglia and oligodendrocytes indicates that prednisone suppresses processes involved in microglial activation and alleviates senescence and improves fitness of oligodendrocytes. This analysis also identifies new potential targets for therapeutic intervention.

Elevated granulocyte colony stimulating factor (CSF) causes cerebellar deficits and anxiety in a model of CSF-1 receptor related leukodystrophy.

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+/- 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+/- 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+/- 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 the cognitive impairment of Csf1r+/- 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+/- 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+/- mice.

Inhibition of colony stimulating factor-1 receptor (CSF-1R) as a potential therapeutic strategy for neurodegenerative diseases: opportunities and challenges.

Microglia are specialized dynamic immune cells in the central nervous system (CNS) that plays a crucial role in brain homeostasis and in disease states. Persistent neuroinflammation is considered a hallmark of many neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and primary progressive multiple sclerosis (MS). Colony stimulating factor 1-receptor (CSF-1R) is predominantly expressed on microglia and its expression is significantly increased in neurodegenerative diseases. Cumulative findings have indicated that CSF-1R inhibitors can have beneficial effects in preclinical neurodegenerative disease models. Research using CSF-1R inhibitors has now been extended into non-human primates and humans. This review article summarizes the most recent advances using CSF-1R inhibitors in different neurodegenerative conditions including AD, PD, HD, ALS and MS. Potential challenges for translating these findings into clinical practice are presented.

Modeling CSF-1 receptor deficiency diseases - how close are we?

The role of colony-stimulating factor-1 receptor (CSF-1R) in macrophage and organismal development has been extensively studied in mouse. Within the last decade, mutations in the CSF1R have been shown to cause rare diseases of both pediatric (Brain Abnormalities, Neurodegeneration, and Dysosteosclerosis, OMIM #618476) and adult (CSF1R-related leukoencephalopathy, OMIM #221820) onset. Here we review the genetics, penetrance, and histopathological features of these diseases and discuss to what extent the animal models of Csf1r deficiency currently available provide systems in which to study the underlying mechanisms involved.

Colony stimulating factors in the nervous system.

Although traditionally seen as regulators of hematopoiesis, colony-stimulating factors (CSFs) have emerged as important players in the nervous system, both in health and disease. This review summarizes the cellular sources, patterns of expression and physiological roles of the macrophage (CSF-1, IL-34), granulocyte-macrophage (GM-CSF) and granulocyte (G-CSF) colony stimulating factors within the nervous system, with a particular focus on their actions on microglia. CSF-1 and IL-34, via the CSF-1R, are required for the development, proliferation and maintenance of essentially all CNS microglia in a temporal and regional specific manner. In contrast, in steady state, GM-CSF and G-CSF are mainly involved in regulation of microglial function. The alterations in expression of these growth factors and their receptors, that have been reported in several neurological diseases, are described and the outcomes of their therapeutic targeting in mouse models and humans are discussed.

Microglial reduction of colony stimulating factor-1 receptor expression is sufficient to confer adult onset leukodystrophy.

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.

Microglial Homeostasis Requires Balanced CSF-1/CSF-2 Receptor Signaling.

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 humans. Our data provide insights into the mechanisms underlying ALSP. Because 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.

Mast cells enhance sterile inflammation in chronic nonbacterial osteomyelitis.

Chronic nonbacterial osteomyelitis (CNO) is an autoinflammatory bone disease, and patients with active or recurrent bone inflammation at multiple sites are diagnosed with chronic recurrent multifocal osteomyelitis (CRMO). The Chronic multifocal osteomyelitis (CMO) mouse model develops IL-1ß-driven sterile bone lesions reminiscent of severe CRMO. The goal of this study was to evaluate the potential involvement of mast cells in CMO/CRMO. Here, we show that mast cells accumulate in inflamed tissues from CMO mice and that mast cell protease Mcpt1 can be detected in the peripheral blood. A transgenic model of connective tissue mast cell depletion (Mcpt5-Cre:Rosa26-Stopfl/fl-DTa) was crossed with CMO mice and the resulting mice (referred to as CMO/MC-) showed a significant delay in disease onset compared with age-matched CMO mice. At 5-6 months of age, CMO/MC- mice had fewer bone lesions and immune infiltration in the popliteal lymph nodes that drain the affected tissues. In bone marrow-derived mast cell cultures from CMO mice, cytokine production in response to the alarmin IL-33 was elevated compared with wild-type cultures. To test the relevance of mast cells to human CRMO, we tested serum samples from a cohort of healthy controls and from CRMO patients at diagnosis. Interestingly, mast cell chymase was elevated in CRMO patients as well as in patients with oligoarticular juvenile arthritis. Tryptase-positive mast cells were also detected in bone lesions from CRMO patients and patients with bacterial osteomyelitis. Together, our results identify mast cells as cellular contributors to bone inflammation in CMO/CRMO and provide rationale for further study of mast cells as therapeutic targets.

CSF-1 controls cerebellar microglia and is required for motor function and social interaction.

Microglia, the brain resident macrophages, critically shape forebrain neuronal circuits. However, their precise function in the cerebellum is unknown. Here we show that human and mouse cerebellar microglia express a unique molecular program distinct from forebrain microglia. Cerebellar microglial identity was driven by the CSF-1R ligand CSF-1, independently of the alternate CSF-1R ligand, IL-34. Accordingly, CSF-1 depletion from Nestin+ cells led to severe depletion and transcriptional alterations of cerebellar microglia, while microglia in the forebrain remained intact. Strikingly, CSF-1 deficiency and alteration of cerebellar microglia were associated with reduced Purkinje cells, altered neuronal function, and defects in motor learning and social novelty interactions. These findings reveal a novel CSF-1-CSF-1R signaling-mediated mechanism that contributes to motor function and social behavior.

Regulation of Embryonic and Postnatal Development by the CSF-1 Receptor.

Macrophages are found in all tissues and regulate tissue morphogenesis during development through trophic and scavenger functions. The colony stimulating factor-1 (CSF-1) receptor (CSF-1R) is the major regulator of tissue macrophage development and maintenance. In combination with receptor activator of nuclear factor κB (RANK), the CSF-1R also regulates the differentiation of the bone-resorbing osteoclast and controls bone remodeling during embryonic and early postnatal development. CSF-1R-regulated macrophages play trophic and remodeling roles in development. Outside the mononuclear phagocytic system, the CSF-1R directly regulates neuronal survival and differentiation, the development of intestinal Paneth cells and of preimplantation embryos, as well as trophoblast innate immune function. Consistent with the pleiotropic roles of the receptor during development, CSF-1R deficiency in most mouse strains causes embryonic or perinatal death and the surviving mice exhibit multiple developmental and functional deficits. The CSF-1R is activated by two dimeric glycoprotein ligands, CSF-1, and interleukin-34 (IL-34). Homozygous Csf1-null mutations phenocopy most of the deficits of Csf1r-null mice. In contrast, Il34-null mice have no gross phenotype, except for decreased numbers of Langerhans cells and microglia, indicating that CSF-1 plays the major developmental role. Homozygous inactivating mutations of the Csf1r or its ligands have not been reported in man. However, heterozygous inactivating mutations in the Csf1r lead to a dominantly inherited adult-onset progressive dementia, highlighting the importance of CSF-1R signaling in the brain.

Emerging Roles for CSF-1 Receptor and its Ligands in the Nervous System.

The colony-stimulating factor-1 receptor (CSF-1R) kinase regulates tissue macrophage homeostasis, osteoclastogenesis, and Paneth cell development. However, recent studies in mice have revealed that CSF-1R signaling directly controls the development and maintenance of microglia, and cell autonomously regulates neuronal differentiation and survival. While the CSF-1R-cognate ligands, CSF-1 and interleukin-34 (IL-34) compete for binding to the CSF-1R, they are expressed in a largely non-overlapping manner by mature neurons. The recent identification of a dominantly inherited, adult-onset, progressive dementia associated with inactivating mutations in the CSF-1R highlights the importance of CSF-1R signaling in the brain. We review the roles of the CSF-1R and its ligands in microglial and neural development and function, and their relevance to our understanding of neurodegenerative disease.

Therapeutic targeting of macrophages in lupus nephritis.

Systemic lupus erythematosus (SLE) is an autoimmune disease which results in multiple different end organ pathologies, including the kidney. Lupus nephritis (LN) is one of the most serious complications of SLE, and a leading cause of morbidity and mortality. Current treatment options are suboptimal, involving non-specific immunosuppression which exposes patients to potentially serious side effects with no guarantee of remission. More targeted therapeutic approaches may improve treatment results. Many studies have implicated macrophages as actively contributing to LN pathogenesis in both human and murine disease. Indeed, various studies have shown that depletion of macrophage populations, inhibition of macrophage recruitment, and disruption of inflammatory macrophage activation and polarization have significantly ameliorated nephritis in several different murine LN models. The current literature explores targeting macrophages by several different means, including the CSF-1/CSF-1R signaling axis, the CX3CL1/CX3CR1 signaling axis, the CCL2/CCR2 signaling axis, and Bruton's Tyrosine Kinase (BTK), all of which hold promise as targets for future LN treatments. These studies highlight the potential benefit of targeting macrophages in LN, and emphasize the need for future investigations to discern the ideal mean(s) for targeting macrophages in LN.

Macrophage depletion ameliorates nephritis induced by pathogenic antibodies.

Kidney involvement affects 40-60% of patients with lupus, and is responsible for significant morbidity and mortality. Using depletion approaches, several studies have suggested that macrophages may play a key role in the pathogenesis of lupus nephritis. However, "off target" effects of macrophage depletion, such as altered hematopoiesis or enhanced autoantibody production, impeded the determination of a conclusive relationship. In this study, we investigated the role of macrophages in mice receiving rabbit anti-glomerular antibodies, or nephrotoxic serum (NTS), an experimental model which closely mimics the immune complex mediated disease seen in murine and human lupus nephritis. GW2580, a selective inhibitor of the colony stimulating factor-1 (CSF-1) receptor kinase, was used for macrophage depletion. We found that GW2580-treated, NTS challenged mice did not develop the increased levels of proteinuria, serum creatinine, and BUN seen in control-treated, NTS challenged mice. NTS challenged mice exhibited significantly increased kidney expression of inflammatory cytokines including RANTES, IP-10, VCAM-1 and iNOS, whereas GW2580-treated mice were protected from the robust expression of these inflammatory cytokines that are associated with lupus nephritis. Quantification of macrophage related gene expression, flow cytometry analysis of kidney single cell suspensions, and immunofluorescence staining confirmed the depletion of macrophages in GW2580-treated mice, specifically within renal glomeruli. Our results strongly implicate a specific and necessary role for macrophages in the development of immune glomerulonephritis mediated by pathogenic antibodies, and support the development of macrophage targeting approaches for the treatment of lupus nephritis.