Leukodystrophy with Macrocephaly, Refractory Epilepsy, and Severe Hyponatremia-The Neonatal Type of Alexander Disease.

INTRODUCTION: Alexander disease (AxD) is a rare neurodegenerative condition that represents the group of leukodystrophies. The disease is caused by GFAP mutation. Symptoms usually occur in the infantile age with macrocephaly, developmental deterioration, progressive quadriparesis, and seizures as the most characteristic features. In this case report, we provide a detailed clinical description of the neonatal type of AxD. METHOD: Next-Generation Sequencing (NGS), including a panel of 49 genes related to Early Infantile Epileptic Encephalopathy (EIEE), was carried out, and then Whole Exome Sequencing (WES) was performed on the proband's DNA extracted from blood. CASE DESCRIPTION: In the first weeks of life, the child presented with signs of increased intracranial pressure, which led to ventriculoperitoneal shunt implementation. Recurrent focal-onset motor seizures with secondary generalization occurred despite phenobarbital treatment. Therapy was modified with multiple anti-seizure medications. In MRI contrast-enhanced lesions in basal ganglia, midbrain and cortico-spinal tracts were observed. During the diagnostic process, GLUT-1 deficiency, lysosomal storage disorders, organic acidurias, and fatty acid oxidation defects were excluded. The NGS panel of EIEE revealed no abnormalities. In WES analysis, GFAP missense heterozygous variant NM_002055.5: c.1187C>T, p.(Thr396Ile) was detected, confirming the diagnosis of AxD. CONCLUSION: AxD should be considered in the differential diagnosis in all neonates with progressive, intractable seizures accompanied by macrocephaly.

Quantitative diffusion imaging and genotype-by-sex interactions in a rat model of Alexander disease.

PURPOSE: The clinical diagnosis and classification of Alexander disease (AxD) relies in part on qualitative neuroimaging biomarkers; however, these biomarkers fail to distinguish and discriminate different subtypes of AxD, especially in the presence of overlap in clinical symptoms. To address this gap in knowledge, we applied neurite orientation dispersion and density imaging (NODDI) to an innovative CRISPR-Cas9 rat genetic model of AxD to gain quantitative insights into the neural substrates and brain microstructural changes seen in AxD and to potentially identify novel quantitative NODDI biomarkers of AxD. METHODS: Multi-shell DWI of age- and sex-matched AxD and wild-type Sprague Dawley rats (n = 6 per sex per genotype) was performed and DTI and NODDI measures calculated. A 3 × 2 × 2 analysis of variance model was used to determine the effect of genotype, biological sex, and laterality on quantitative measures of DTI and NODDI across regions of interest implicated in AxD. RESULTS: There is a significant effect of genotype in the amygdala, hippocampus, neocortex, and thalamus in measures of both DTI and NODDI brain microstructure. A genotype by biological sex interaction was identified in DTI and NODDI measures in the corpus callosum, hippocampus, and neocortex. CONCLUSION: We present the first application of NODDI to the study of AxD using a rat genetic model of AxD. Our analysis identifies alterations in NODDI and DTI measures to large white matter tracts and subcortical gray nuclei. We further identified genotype by sex interactions, suggesting a possible role for biological sex in the neuropathogenesis of AxD.

Microglia sense astrocyte dysfunction and prevent disease progression in an Alexander disease model.

Alexander disease (AxD) is an intractable neurodegenerative disorder caused by GFAP mutations. It is a primary astrocyte disease with a pathological hallmark of Rosenthal fibres within astrocytes. AxD astrocytes show several abnormal phenotypes. Our previous study showed that AxD astrocytes in model mice exhibit aberrant Ca2+ signals that induce AxD aetiology. Here, we show that microglia have unique phenotypes with morphological and functional alterations, which are related to the pathogenesis of AxD. Immunohistochemical studies of 60TM mice (AxD model) showed that AxD microglia exhibited highly ramified morphology. Functional changes in microglia were assessed by Ca2+ imaging using hippocampal brain slices from Iba1-GCaMP6-60TM mice and two-photon microscopy. We found that AxD microglia showed aberrant Ca2+ signals, with high frequency Ca2+ signals in both the processes and cell bodies. These microglial Ca2+ signals were inhibited by pharmacological blockade or genetic knockdown of P2Y12 receptors but not by tetrodotoxin, indicating that these signals are independent of neuronal activity but dependent on extracellular ATP from non-neuronal cells. Our single-cell RNA sequencing data showed that the expression level of Entpd2, an astrocyte-specific gene encoding the ATP-degrading enzyme NTPDase2, was lower in AxD astrocytes than in wild-type astrocytes. In situ ATP imaging using the adeno-associated virus vector GfaABC1D ATP1.0 showed that exogenously applied ATP was present longer in 60TM mice than in wild-type mice. Thus, the increased ATP level caused by the decrease in its metabolizing enzyme in astrocytes could be responsible for the enhancement of microglial Ca2+ signals. To determine whether these P2Y12 receptor-mediated Ca2+ signals in AxD microglia play a significant role in the pathological mechanism, a P2Y12 receptor antagonist, clopidogrel, was administered. Clopidogrel significantly exacerbated pathological markers in AxD model mice and attenuated the morphological features of microglia, suggesting that microglia play a protective role against AxD pathology via P2Y12 receptors. Taken together, we demonstrated that microglia sense AxD astrocyte dysfunction via P2Y12 receptors as an increase in extracellular ATP and alter their morphology and Ca2+ signalling, thereby protecting against AxD pathology. Although AxD is a primary astrocyte disease, our study may facilitate understanding of the role of microglia as a disease modifier, which may contribute to the clinical diversity of AxD.

Fetal-onset Alexander disease with radiological-neuropathological correlation.

Alexander disease is a leukodystrophy caused by mutations in the GFAP gene, primarily affecting the astrocytes. This report describes the prenatal and post-mortem neuroimaging findings in a case of genetically confirmed, fetal-onset Alexander disease with pathological correlation after termination of pregnancy. The additional value of fetal brain magnetic resonance imaging in the third trimester as a complementary evaluation tool to neurosonography is shown for suspected cases of fetal-onset Alexander disease. Diffuse signal abnormalities of the periventricular white matter in association with thickening of the fornix and optic chiasm can point towards the diagnosis. Furthermore, the presence of atypical imaging findings such as microcephaly and cortical folding abnormalities in this case broadens our understanding of the phenotypic variability of Alexander disease.

STAT3 Drives GFAP Accumulation and Astrocyte Pathology in a Mouse Model of Alexander Disease.

Alexander disease (AxD) is caused by mutations in the gene for glial fibrillary acidic protein (GFAP), an intermediate filament expressed by astrocytes in the central nervous system. AxD-associated mutations cause GFAP aggregation and astrogliosis, and GFAP is elevated with the astrocyte stress response, exacerbating mutant protein toxicity. Studies in mouse models suggest disease severity is tied to Gfap expression levels, and signal transducer and activator of transcription (STAT)-3 regulates Gfap during astrocyte development and in response to injury and is activated in astrocytes in rodent models of AxD. In this report, we show that STAT3 is also activated in the human disease. To determine whether STAT3 contributes to GFAP elevation, we used a combination of genetic approaches to knockout or reduce STAT3 activation in AxD mouse models. Conditional knockout of Stat3 in cells expressing Gfap reduced Gfap transactivation and prevented protein accumulation. Astrocyte-specific Stat3 knockout in adult mice with existing pathology reversed GFAP accumulation and aggregation. Preventing STAT3 activation reduced markers of reactive astrocytes, stress-related transcripts, and microglial activation, regardless of disease stage or genetic knockout approach. These results suggest that pharmacological inhibition of STAT3 could potentially reduce GFAP toxicity and provide a therapeutic benefit in patients with AxD.

Alexander disease: The story behind an eponym.

In 1949, William Stewart Alexander (1919-2013), a young pathologist from New Zealand working in London, reported the neuropathological findings in a 15-month-old boy who had developed normally until the age of seven months, but thereafter had progressive enlargement of his head and severe developmental delay. The most striking neuropathological abnormality was the presence of numerous Rosenthal fibers in the brain. The distribution of these fibers suggested to Alexander that the primary pathological change involved astrocytes. In the next 15 years, five similar patients were reported, and in 1964 Friede recognized these cases reflected a single disease process and coined the eponym "Alexander's disease" to describe the disorder. In the 1960s, electron microscopy confirmed that Rosenthal fibers were localized to astrocytes. In 2001, it was shown that Alexander disease is caused by mutations in the gene encoding glial fibrillary acidic protein, the major intermediate filament protein in astrocytes. Although the clinical, imaging, and pathological manifestations of Alexander disease are now well known, few people are familiar with Alexander's career. Although he did not make a further contribution to the literature on Alexander disease, his observations and accurate interpretation of the neuropathology have justified the continued use of the eponym "Alexander disease."

Identification of a novel de novo pathogenic variant in GFAP in an Iranian family with Alexander disease by whole-exome sequencing.

BACKGROUND: Alexander disease (AxD) is a rare leukodystrophy with an autosomal dominant inheritance mode. Variants in GFAP lead to this disorder and it is classified into three distinguishable subgroups: infantile, juvenile, and adult-onset types. OBJECTIVE: The aim of this study is to report a novel variant causing AxD and collect all the associated variants with juvenile and adult-onset as well. METHODS: We report a 2-year-old female with infantile AxD. All relevant clinical and genetic data were evaluated. Search strategy for all AxD types was performed on PubMed. The extracted data include total recruited patients, number of patients carrying a GFAP variant, nucleotide and protein change, zygosity and all the clinical symptoms. RESULTS: A novel de novo variant c.217A > G: p. Met73Val was found in our case by whole-exome sequencing. In silico analysis categorized this variant as pathogenic. Totally 377 patients clinically diagnosed with juvenile or adult-onset forms were recruited in these articles, among them 212 patients were affected with juvenile or adult-onset form carrier of an alteration in GFAP. A total of 98 variants were collected. Among these variants c.262C > T 11/212 (5.18%), c.1246C > T 9/212 (4.24%), c.827G > T 8/212 (3.77%), c.232G > A 6/212 (2.83%) account for the majority of reported variants. CONCLUSION: This study highlighted the role of genetic in AxD diagnosing. It also helps to provide more information in order to expand the genetic spectrum of Iranian patients with AxD. Our literature review is beneficial in defining a better genotype-phenotype correlation of AxD disorder.

Identification of association fibers using ex vivo diffusion tractography in Alexander disease brains.

BACKGROUND AND PURPOSE: Alexander disease (AxD) is a neurodegenerative disorder caused by heterozygous Glial Fibrillary Acidic Protein mutation. The characteristic structural findings of AxD, such as leukodystrophic features, are well known, while association fibers of AxD remain uninvestigated. The aim of this study was to explore global and subcortical fibers in four brains with AxD using ex vivo diffusion tractography METHODS: High-angular-resolution diffusion magnetic resonance imaging (HARDI) tractography and diffusion-tensor imaging (DTI) tractography were used to evaluate long and short association fibers and compared to histological findings in brain specimens obtained from four donors with AxD and two donors without neurological disorders RESULTS: AxD brains showed impairment of long association fibers, except for the arcuate fasciculus and cingulum bundle, and abnormal trajectories of the inferior longitudinal and fronto-occipital fasciculi on HARDI tractography and loss of multidirectionality in subcortical fibers on DTI tractography. In histological studies, AxD brains showed diffuse low density on Klüver-Barrera and neurofilament staining and sporadic Rosenthal fibers on hematoxylin and eosin staining CONCLUSIONS: This study describes the spatial distribution of degenerations of short and long association fibers in AxD brains using combined tractography and pathological findings.

Symptomatic care of late-onset Alexander disease presenting with area postrema-like syndrome with prednisolone; a case report.

BACKGROUND: Alexander disease (AxD) is classified into AxD type I (infantile) and AxD type II (juvenile and adult form). We aimed to determine the potential genetic cause(s) contributing to the AxD type II manifestations in a 9-year-old male who presented area postrema-like syndrome and his vomiting and weight loss improved after taking prednisolone. CASE PRESENTATION: A normal cognitive 9-year-old boy with persistent nausea, vomiting, and a significant weight loss at the age of 6 years was noticed. He also experienced an episode of status epilepticus with generalized atonic seizures. He showed non-febrile infrequent multifocal motor seizures at the age of 40 days which were treated with phenobarbital. He exhibited normal physical growth and neurologic developmental milestones by the age of six. Occasionally vomiting unrelated to feeding was reported. Upon examination at 9 years, a weak gag reflex, prominent drooling, exaggerated knee-deep tendon reflexes (3+), and nasal tone speech was detected. All gastroenterological, biochemical, and metabolic assessments were normal. Brain magnetic resonance imaging (MRI) revealed bifrontal confluent deep and periventricular white matter signal changes, fine symmetric frontal white matter and bilateral caudate nucleus involvements with garland changes, and a hyperintense tumefactive-like lesion in the brain stem around the floor of the fourth ventricle and area postrema with contrast uptake in post-contrast T1-W images. Latter MRI at the age of 8 years showed enlarged area postrema lesion and bilateral middle cerebellar peduncles and dentate nuclei involvements. Due to clinical and genetic heterogeneities, whole-exome sequencing was performed and the candidate variant was confirmed by Sanger sequencing. A de novo heterozygous mutation, NM_001242376.1:c.262 C > T;R88C in exon 1 of the GFAP (OMIM: 137,780) was verified. Because of persistent vomiting and weight loss of 6.0 kg, prednisolone was prescribed which brought about ceasing vomiting and led to weight gaining of 3.0 kg over the next 3 months after treatment. Occasional attempts to discontinue prednisolone had been resulting in the reappearance of vomiting. CONCLUSIONS: This study broadens the spectrum of symptomatic treatment in leukodystrophies and also shows that R88C mutation may lead to a broad range of phenotypes in AxD type II patients.

A novel in-frame GFAP p.E138_L148del mutation in Type II Alexander disease with atypical phenotypes.

Alexander disease (AxD) is a neurodegenerative astrogliopathy caused by mutation in the glial fibrillary acidic protein (GFAP) gene. A 42-year-old Korean man presented with temporary gait disturbance and psychiatric regression after a minor head trauma in the absence of bulbar symptoms and signs. Magnetic resonance images of the brain and spinal cord showed significant atrophy of the medulla oblongata and the entire spinal cord as well as contrast-enhanced T2 hypointensity in the basal ganglia. DNA sequencing revealed a novel 33-bp in-frame deletion mutation (p.Glu138_Leu148del) within the 1B rod domain of GFAP, which was predicted to be deleterious by PROVEAN analysis. To test whether the deletion mutant is disease-causing, we performed in vitro GFAP assembly and sedimentation assays, and GFAP aggregation assays in human adrenal carcinoma SW13 (Vim-) cells and rat primary astrocytes. All the assays revealed that GFAP p.Glu138_Leu148del is aggregation prone. Based on these findings, we diagnosed the patient with Type II AxD. This is a report that demonstrates the pathogenicity of InDel mutation of GFAP through functional studies. This patient's atypical presentation as well as the discrepancy between clinical symptoms and radiologic findings may extend the scope of AxD.

Alexander disease: models, mechanisms, and medicine.

Alexander disease is a primary disorder of astrocytes caused by gain-of-function mutations in the gene for glial fibrillary acidic protein (GFAP), which lead to protein aggregation and a reactive astrocyte response, with devastating effects on the central nervous system. Over the past two decades since the discovery of GFAP as the culprit, several cellular and animal models have been generated, and much has been learned about underlying mechanisms contributing to the disease. Despite these efforts, many aspects of Alexander disease have remained enigmatic, particularly the initiating events in GFAP accumulation and astrocyte pathology, the relation between astrocyte dysfunction and myelin deficits, and the variability in age of onset and disease severity. More recent work in both old and new models has begun to address these complex questions and identify new therapeutics that finally offer the promise of effective treatment.

Metabolic Enzyme Alterations and Astrocyte Dysfunction in a Murine Model of Alexander Disease With Severe Reactive Gliosis.

Alexander disease (AxD) is a rare and fatal neurodegenerative disorder caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). In this report, a mouse model of AxD (GFAPTg;Gfap+/R236H) was analyzed that contains a heterozygous R236H point mutation in murine Gfap as well as a transgene with a GFAP promoter to overexpress human GFAP. Using label-free quantitative proteomic comparisons of brain tissue from GFAPTg;Gfap+/R236H versus wild-type mice confirmed upregulation of the glutathione metabolism pathway and indicated proteins were elevated in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which had not been reported previously in AxD. Relative protein-level differences were confirmed by a targeted proteomics assay, including proteins related to astrocytes and oligodendrocytes. Of particular interest was the decreased level of the oligodendrocyte protein, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (Ugt8), since Ugt8-deficient mice exhibit a phenotype similar to GFAPTg;Gfap+/R236H mice (e.g., tremors, ataxia, hind-limb paralysis). In addition, decreased levels of myelin-associated proteins were found in the GFAPTg;Gfap+/R236H mice, consistent with the role of Ugt8 in myelin synthesis. Fabp7 upregulation in GFAPTg;Gfap+/R236H mice was also selected for further investigation due to its uncharacterized association to AxD, critical function in astrocyte proliferation, and functional ability to inhibit the anti-inflammatory PPAR signaling pathway in models of amyotrophic lateral sclerosis (ALS). Within Gfap+ astrocytes, Fabp7 was markedly increased in the hippocampus, a brain region subjected to extensive pathology and chronic reactive gliosis in GFAPTg;Gfap+/R236H mice. Last, to determine whether the findings in GFAPTg;Gfap+/R236H mice are present in the human condition, AxD patient and control samples were analyzed by Western blot, which indicated that Type I AxD patients have a significant fourfold upregulation of FABP7. However, immunohistochemistry analysis showed that UGT8 accumulates in AxD patient subpial brain regions where abundant amounts of Rosenthal fibers are located, which was not observed in the GFAPTg;Gfap+/R236H mice.

Principles of Astrogliopathology.

The role of astrocytes in the nervous system pathology was early on embraced by neuroscientists at end of the nineteenth and the beginning of the twentieth century, only to be pushed aside by neurone-centric dogmas during most of the twentieth century. However, the last decade of the twentieth century and the twenty-first century have brought the astroglial "renaissance", which has put astroglial cells as key players in pathophysiology of most if not all disorders of the nervous system and has regarded astroglia as a fertile ground for therapeutic intervention.Astrocytic contribution to neuropathology can be primary, whereby cell-autonomous changes, such as mutations in gene encoding for glial fibrillary acidic protein, can drive the pathologic progression, in this example, Alexander disease. They can also be secondary, when astrocytes respond to a variety of insults to the nervous tissue. Regardless of their origin, being cell-autonomous or not, changes in astroglia that occur in pathology, that is, astrogliopathology, can be contemporary and arbitrary classified into four forms: (i) reactive astrogliosis, (ii) astrocytic atrophy with loss of function, (iii) pathological remodelling of astrocytes and (iv) astrodegeneration morphologically manifested as clasmatodendrosis. Inevitably, as with any other classification, this classification of astrogliopathology awaits its revision that shall be rooted in new discoveries and concepts.

Antisense therapy in a rat model of Alexander disease reverses GFAP pathology, white matter deficits, and motor impairment.

Alexander disease (AxD) is a devastating leukodystrophy caused by gain-of-function mutations in GFAP, and the only available treatments are supportive. Recent advances in antisense oligonucleotide (ASO) therapy have demonstrated that transcript targeting can be a successful strategy for human neurodegenerative diseases amenable to this approach. We have previously used mouse models of AxD to show that Gfap-targeted ASO suppresses protein accumulation and reverses pathology; however, the mice have a mild phenotype with no apparent leukodystrophy or overt clinical features and are therefore limited for assessing functional outcomes. In this report, we introduce a rat model of AxD that exhibits hallmark pathology with GFAP aggregation in the form of Rosenthal fibers, widespread astrogliosis, and white matter deficits. These animals develop normally during the first postnatal weeks but fail to thrive after weaning and develop severe motor deficits as they mature, with about 14% dying of unknown cause between 6 and 12 weeks of age. In this model, a single treatment with Gfap-targeted ASO provides long-lasting suppression, reverses GFAP pathology, and, depending on age of treatment, prevents or mitigates white matter deficits and motor impairment. In this report, we characterize an improved animal model of AxD with myelin pathology and motor impairment, recapitulating prominent features of the human disease, and use this model to show that ASO therapy has the potential to not only prevent but also reverse many aspects of disease.

Pathology of the neurovascular unit in leukodystrophies.

The blood-brain barrier is a dynamic endothelial cell barrier in the brain microvasculature that separates the blood from the brain parenchyma. Specialized brain endothelial cells, astrocytes, neurons, microglia and pericytes together compose the neurovascular unit and interact to maintain blood-brain barrier function. A disturbed brain barrier function is reported in most common neurological disorders and may play a role in disease pathogenesis. However, a comprehensive overview of how the neurovascular unit is affected in a wide range of rare disorders is lacking. Our aim was to provide further insights into the neuropathology of the neurovascular unit in leukodystrophies to unravel its potential pathogenic role in these diseases. Leukodystrophies are monogenic disorders of the white matter due to defects in any of its structural components. Single leukodystrophies are exceedingly rare, and availability of human tissue is unique. Expression of selective neurovascular unit markers such as claudin-5, zona occludens 1, laminin, PDGFRß, aquaporin-4 and α-dystroglycan was investigated in eight different leukodystrophies using immunohistochemistry. We observed tight junction rearrangements, indicative of endothelial dysfunction, in five out of eight assessed leukodystrophies of different origin and an altered aquaporin-4 distribution in all. Aquaporin-4 redistribution indicates a general astrocytic dysfunction in leukodystrophies, even in those not directly related to astrocytic pathology or without prominent reactive astrogliosis. These findings provide further evidence for dysfunction in the orchestration of the neurovascular unit in leukodystrophies and contribute to a better understanding of the underlying disease mechanism.

Elevated GFAP isoform expression promotes protein aggregation and compromises astrocyte function.

Alexander disease (AxD) caused by mutations in the coding region of GFAP is a neurodegenerative disease characterized by astrocyte dysfunction, GFAP aggregation, and Rosenthal fiber accumulation. Although how GFAP mutations cause disease is not fully understood, Rosenthal fibers could be induced by forced overexpression of human GFAP and this could be lethal in mice implicate that an increase in GFAP levels is central to AxD pathogenesis. Our recent studies demonstrated that intronic GFAP mutations cause disease by altering GFAP splicing, suggesting that an increase in GFAP isoform expression could lead to protein aggregation and astrocyte dysfunction that typify AxD. Here we test this hypothesis by establishing primary astrocyte cultures from transgenic mice overexpressing human GFAP. We found that GFAP-δ and GFAP-κ were disproportionately increased in transgenic astrocytes and both were enriched in Rosenthal fibers of human AxD brains. In vitro assembly studies showed that while the major isoform GFAP-α self-assembled into typical 10-nm filaments, minor isoforms including GFAP-δ, -κ, and -λ were assembly-compromised and aggregation prone. Lentiviral transduction showed that expression of these minor GFAP isoforms decreased filament solubility and increased GFAP stability, leading to the formation of Rosenthal fibers-like aggregates that also disrupted the endogenous intermediate filament networks. The aggregate-bearing astrocytes lost their normal morphology and glutamate buffering capacity, which had a toxic effect on neighboring neurons. In conclusion, our findings provide evidence that links elevated GFAP isoform expression with GFAP aggregation and impaired glutamate transport, and suggest a potential non-cell-autonomous mechanism underlying neurodegeneration through astrocyte dysfunction.

Pexidartinib treatment in Alexander disease model mice reduces macrophage numbers and increases glial fibrillary acidic protein levels, yet has minimal impact on other disease phenotypes.

BACKGROUND: Alexander disease (AxD) is a rare neurodegenerative disorder that is caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP), an intermediate filament that is primarily expressed by astrocytes. In AxD, mutant GFAP in combination with increased GFAP expression result in astrocyte dysfunction and the accumulation of Rosenthal fibers. A neuroinflammatory environment consisting primarily of macrophage lineage cells has been observed in AxD patients and mouse models. METHODS: To examine if macrophage lineage cells could serve as a therapeutic target in AxD, GFAP knock-in mutant AxD model mice were treated with a colony-stimulating factor 1 receptor (CSF1R) inhibitor, pexidartinib. The effects of pexidartinib treatment on disease phenotypes were assessed. RESULTS: In AxD model mice, pexidartinib administration depleted macrophages in the CNS and caused elevation of GFAP transcript and protein levels with minimal impacts on other phenotypes including body weight, stress response activation, chemokine/cytokine expression, and T cell infiltration. CONCLUSIONS: Together, these results highlight the complicated role that macrophages can play in neurological diseases and do not support the use of pexidartinib as a therapy for AxD.