Axonal Spheroids Research Articles

Mapping the journey of patients and care partners living with adult-onset leukoencephalopathy with axonal spheroids and pigmented glia: developing a framework for improvements in care.

Aim: To identify and raise awareness of healthcare service gaps for individuals with adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP).Materials & methods: An ALSP patient journey map from symptom onset throughout disease course was developed using existing literature, patient and clinician feedback from a structured workshop and community survey data regarding attitudes toward genetic testing.Results: ALSP diagnosis is frequently delayed due to low awareness of this rare condition and symptom overlap with more common neurological conditions. Multiple factors impact patients' decision-making regarding genetic testing for ALSP, symptom management and participation in research studies.Conclusion: These results highlight the challenges faced by individuals with ALSP and should support program development to improve patient care.

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.

Microglia’s heroic battle against neurodegeneration

In this issue of Neuron, Chadarevian etal.1 and Munro etal.2 demonstrate how the absence of homeostatic microglia leads to severe neuropathologies, including axonal spheroids, calcifications, myelination abnormalities, and gliosis, associated with leukoencephalopathy and age-related neurodegeneration. Transplantation of healthy microglia can reverse these conditions, opening new avenues for therapeutic research.

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.

NKCC1 inhibition reduces periaxonal swelling, increases white matter sparing, and improves neurological recovery after contusive SCI

Ultrastructural studies of contusive spinal cord injury (SCI) in mammals have shown that the most prominent acute changes in white matter are periaxonal swelling and separation of myelin away from their axon, axonal swelling, and axonal spheroid formation. However, the underlying cellular and molecular mechanisms that cause periaxonal swelling and the functional consequences are poorly understood. We hypothesized that periaxonal swelling and loss of connectivity between the axo-myelinic interface impedes neurological recovery by disrupting conduction velocity, and glial to axonal trophic support resulting in axonal swelling and spheroid formation. Utilizing in vivo longitudinal imaging of Thy1YFP+ axons and myelin labeled with Nile red, we reveal that periaxonal swelling significantly increases acutely following a contusive SCI (T13, 30 kdyn, IH Impactor) versus baseline recordings (laminectomy only) and often precedes axonal spheroid formation. In addition, using longitudinal imaging to determine the fate of myelinated fibers acutely after SCI, we show that ∼73% of myelinated fibers present with periaxonal swelling at 1 h post SCI and ∼ 51% of those fibers transition to axonal spheroids by 4 h post SCI. Next, we assessed whether cation-chloride cotransporters present within the internode contributed to periaxonal swelling and whether their modulation would increase white matter sparing and improve neurological recovery following a moderate contusive SCI (T9, 50 kdyn). Mechanistically, activation of the cation-chloride cotransporter KCC2 did not improve neurological recovery and acute axonal survival, but did improve chronic tissue sparing. In distinction, the NKKC1 antagonist bumetanide improved neurological recovery, tissue sparing, and axonal survival, in part through preventing periaxonal swelling and disruption of the axo-myelinic interface. Collectively, these data reveal a novel neuroprotective target to prevent periaxonal swelling and improve neurological recovery after SCI.

Frequent Alzheimer's disease neuropathological change in patients with glioblastoma.

The incidence of brain cancer and neurodegenerative diseases is increasing with a demographic shift towards aging populations. Biological parallels have been observed between glioblastoma and Alzheimer's disease (AD), which converge on accelerated brain aging. Here, we aimed to map the cooccurrence of AD neuropathological change (ADNC) in the tumor-adjacent cortex of patients with glioblastoma. Immunohistochemical screening of AD markers amyloid beta (Abeta), amyloid precursor protein (APP), and hyperphosphorylated tau (pTau) was conducted in 420 tumor samples of 205 patients. For each cortex area, we quantified ADNC, neurons, tumor cells, and microglia. Fifty-two percent of patients (N = 106/205) showed ADNC (Abeta and pTau, Abeta or pTau) in the tumor-adjacent cortex, with histological patterns widely consistent with AD. ADNC was positively correlated with patient age and varied spatially according to Thal phases and Braak stages. It decreased with increasing tumor cell infiltration (P < .0001) and was independent of frequent expression of APP in neuronal cell bodies (N = 182/205) and in tumor necrosis-related axonal spheroids (N = 195/205; P = .46). Microglia response was most present in tumor cell infiltration plus ADNC, being further modulated by patient age and sex. ADNC did not impact patient survival in the present cohort. Our findings highlight the frequent presence of ADNC in the glioblastoma vicinity, which was linked to patient age and tumor location. The cooccurrence of AD and glioblastoma seemed stochastic without clear spatial relation. ADNC did not impact patient survival in our cohort.

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.

Decreasing ganglioside synthesis delays motor and cognitive symptom onset in Spg11 knockout mice

Biallelic variants in the SPG11 gene account for the most common form of autosomal recessive hereditary spastic paraplegia characterized by motor and cognitive impairment, with currently no therapeutic option. We previously observed in a Spg11 knockout mouse that neurodegeneration is associated with accumulation of gangliosides in lysosomes. To test whether a substrate reduction therapy could be a therapeutic option, we downregulated the key enzyme involved in ganglioside biosynthesis using an AAV-PHP.eB viral vector expressing a miRNA targeting St3gal5. Downregulation of St3gal5 in Spg11 knockout mice prevented the accumulation of gangliosides, delayed the onset of motor and cognitive symptoms, and prevented the upregulation of serum levels of neurofilament light chain, a biomarker widely used in neurodegenerative diseases. Importantly, similar results were observed when Spg11 knockout mice were administrated venglustat, a pharmacological inhibitor of glucosylceramide synthase expected to decrease ganglioside synthesis. Downregulation of St3gal5 or venglustat administration in Spg11 knockout mice strongly decreased the formation of axonal spheroids, previously associated with impaired trafficking. Venglustat had similar effect on cultured human SPG11 neurons. In conclusion, this work identifies the first disease-modifying therapeutic strategy in SPG11, and provides data supporting its relevance for therapeutic testing in SPG11 patients.

A novel CSF1R missense mutation in a Chinese family with adult-onset leukoencephalopathy with axonal spheroids and pigmented glia: A case report

Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a rare autosomal dominant disorder caused by mutations in the colony-stimulating factor 1 receptor (CSF1R) gene. We report a case of a 41-year-old man with rapid decline in cognition within 7 months of onset. Magnetic resonance imaging showed periventricular confluent white matter changes and atrophy of the corpus callosum. Clinical exome sequencing showed a mutation (c.2390T&gt;G) in exon 18 of the CSF1R gene. In conclusion, the differential diagnosis of adult-onset leukodystrophy is extensive. Neuroimaging and genetic analysis greatly aid in the differential diagnosis of leukoencephalopathy.

Bilateral stringhalt associated with Hypochaeris radicata in Uruguayan horses

We reported thirteen cases of bilateral stringhalt associated with Hypochaeris radicata that occurred in horses in Uruguay during a severe drought in the summer of 2023. All horses were affected chronically and progressively by bilateral hyperflexion of hindlimbs. In two severely affected horses, the main histological lesions included neuronal chromatolysis and axonal spheroids in the ventral gray horn in the lumbar and sacral spinal cord and axonal degeneration and digestion chambers in ventral roots fibers and long peripheral nerves. We suggest that in addition to injuries to peripheral nerves, lesions in the spinal cord play an important role in the clinical signs of stringhalt in horses.

Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP): Estimation of pathological lesion stage from brain images

BackgroundAdult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a disease responsible for cognitive impairment in adult humans. It is caused by mutations in the colony stimulating factor 1 receptor gene (CSF1R) or alanyl-transfer (t) RNA synthetase 2 (AARS2) gene and affects brain white matter. Settlement of stages of the pathological brain lesions (Oyanagi et al. 2017) from the findings of brain imaging will be inevitably essential for prognostication. MethodsMRI images of eight patients with ALSP were analyzed semiquantitatively. White matter degeneration was assessed on a scale of 0 to 4 (none, patchy, large patchy, confluent, and diffuse) at six anatomical points, and brain atrophy on a scale 0 to 4 (none, slight, mild, moderate, and severe) in four anatomical areas. The scores of the two assessments were then summed to give total MRI scores of 0–40 points. Based on the scores, the MRI features were classified as Grades (0–4). Regression analysis was applied to mutual association between mRS, white matter degeneration score, brain atrophy score, the total MRI score and disease duration. ResultsWhite matter degeneration score, brain atrophy score, and the total MRI score were significantly correlated with the disease duration. MRI Grades (2–4) based on the total MRI scores and the features of the images were well correlated with the pathological lesion stages (II - IV); i.e., ‘large patchy’ white matter degeneration in the frontal and parietal lobes (MRI Grade 2) corresponded to pathological Stage II, ‘confluent’ degeneration (Grade 3) to Stage III, and ‘diffuse’ degeneration (Grade 4) to Stage IV. ConclusionMRI Grades (2–4) resulted from the total MRI scores were well correlated with the pathological lesion Stages (II - IV).

Assessing Chitinases and Neurofilament Light Chain as Biomarkers for Adult-Onset Leukodystrophies.

Leukodystrophies represent a large and complex group of inherited disorders affecting the white matter of the central nervous system. Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a rare leukodystrophy which still needs the proper identification of diagnostic, prognostic, and monitoring biomarkers. The aim of this study was to determine the diagnostic and prognostic value of chitinases and neurofilament light chain as biomarkers for ALSP. A cross-sectional study was performed to analyze cerebrospinal fluid levels of chitinases (chitotriosidase and chitinase 3-like 2) and neurofilament light chain in five different groups: (i) normal health individuals; (ii) patients with definitive diagnosis of ALSP and genetic confirmation; (iii) asymptomatic patients with CSF1R variants; (iv) patients with other adult-onset leukodystrophies; and (v) patients with amyotrophic lateral sclerosis (external control group). Chitinase levels showed a statistical correlation with clinical assessment parameters in ALSP patients. Chitinase levels were also distinct between ALSP and the other leukodystrophies. Significant differences were noted in the levels of chitinases and neurofilament light chain comparing symptomatic (ALSP) and asymptomatic individuals with CSF1R variants. This study is the first to establish chitinases as a potential biomarker for ALSP and confirms neurofilament light chain as a good biomarker for primary microgliopathies.

Iluzanebart Phase 2, IGNITE Study: First Interventional Study in Patients with Adult-Onset Leukoencephalopathy with Axonal Spheroids and Pigmented Glia (ALSP)

Iluzanebart Phase 2, IGNITE Study: First Interventional Study in Patients with Adult-Onset Leukoencephalopathy with Axonal Spheroids and Pigmented Glia (ALSP)

VGL101: An Immunotherapy that Enhances Microglial Survival for Adult-onset Leukoencephalopathy with Axonal Spheroids and Pigmented Glia (ALSP) (P9-9.013)

VGL101: An Immunotherapy that Enhances Microglial Survival for Adult-onset Leukoencephalopathy with Axonal Spheroids and Pigmented Glia (ALSP) (P9-9.013)

Findings from the ILLUMINATE Prospective Natural History Study (NHS) in Individuals with Adult-onset Leukoencephalopathy with Axonal Spheroids and Pigmented Glia (ALSP) (P3-9.001)

Findings from the ILLUMINATE Prospective Natural History Study (NHS) in Individuals with Adult-onset Leukoencephalopathy with Axonal Spheroids and Pigmented Glia (ALSP) (P3-9.001)

Interim Results on VGL101 from IGNITE: First Interventional Phase 2 Study in Patients with Adultonset Leukoencephalopathy with Axonal Spheroids and Pigmented Glia (ALSP) (S28.005)

Interim Results on VGL101 from IGNITE: First Interventional Phase 2 Study in Patients with Adultonset Leukoencephalopathy with Axonal Spheroids and Pigmented Glia (ALSP) (S28.005)

An adapted protocol to derive microglia from stem cells and its application in the study of CSF1R-related disorders

BackgroundInduced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial function in health and disease. Yet, since differentiation and survival of iMGL are highly reliant on colony-stimulating factor 1 receptor (CSF1R) signaling, it is difficult to use iMGL to study microglial dysfunction associated with pathogenic defects in CSF1R.MethodsSerial modifications to an existing iMGL protocol were made, including but not limited to changes in growth factor combination to drive microglial differentiation, until successful derivation of microglia-like cells from an adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) patient carrying a c.2350G > A (p.V784M) CSF1R variant. Using healthy control lines, the quality of the new iMGL protocol was validated through cell yield assessment, measurement of microglia marker expression, transcriptomic comparison to primary microglia, and evaluation of inflammatory and phagocytic activities. Similarly, molecular and functional characterization of the ALSP patient-derived iMGL was carried out in comparison to healthy control iMGL.ResultsThe newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to cultured primary human microglia and with higher scavenging and inflammatory competence at ~ threefold greater yield compared to the original protocol. Using this protocol, decreased CSF1R autophosphorylation and cell surface expression was observed in iMGL derived from the ALSP patient compared to those derived from healthy controls. Additionally, ALSP patient-derived iMGL presented a migratory defect accompanying a temporal reduction in purinergic receptor P2Y12 (P2RY12) expression, a heightened capacity to internalize myelin, as well as heightened inflammatory response to Pam3CSK4. Poor P2RY12 expression was confirmed to be a consequence of CSF1R haploinsufficiency, as this feature was also observed following CSF1R knockdown or inhibition in mature control iMGL, and in CSF1RWT/KO and CSF1RWT/E633K iMGL compared to their respective isogenic controls.ConclusionsWe optimized a pre-existing iMGL protocol, generating a powerful tool to study microglial involvement in human neurological diseases. Using the optimized protocol, we have generated for the first time iMGL from an ALSP patient carrying a pathogenic CSF1R variant, with preliminary characterization pointing toward functional alterations in migratory, phagocytic and inflammatory activities.Graphical

Clinical presentation and diagnosis of adult-onset leukoencephalopathy with axonal spheroids and pigmented glia: a literature analysis of case studies.

Because adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a rare, rapidly progressive, debilitating, and ultimately fatal neurodegenerative disease, a rapid and accurate diagnosis is critical. This analysis examined the frequency of initial misdiagnosis of ALSP via comprehensive review of peer-reviewed published cases. Data were extracted from a MEDLINE search via PubMed (January 1, 1980, through March 22, 2022) from eligible published case reports/series for patients with an ALSP diagnosis that had been confirmed by testing for the colony-stimulating factor-1 receptor gene (CSF1R) mutation. Patient demographics, clinical symptoms, brain imaging, and initial diagnosis data were summarized descriptively. Categorical data for patient demographics, symptoms, and brain imaging were stratified by initial diagnosis category to test for differences in initial diagnosis based on each variable. Data were extracted from a cohort of 291 patients with ALSP from 93 published case reports and case series. Mean (standard deviation) age of symptom onset was 43.2 (11.6) years. A family history of ALSP was observed in 59.1% of patients. Cognitive impairment (47.1%) and behavioral and psychiatric abnormalities (26.8%) were the most frequently reported initial symptoms. Of 291 total cases, an accurate initial diagnosis of ALSP was made in 72 cases (24.7%) and the most frequent initial misdiagnosis categories were frontotemporal dementia (28 [9.6%]) and multiple sclerosis (21 [7.2%]). Of the 219 cases (75.3%) that were initially mis- or undiagnosed, 206 cases (94.1%) were later confirmed as ALSP by immunohistology, imaging, and/or genetic testing; for the remaining 13 cases, no final diagnosis was reported. Initial diagnosis category varied based on age, family history, geographic region, mode of inheritance, and presenting symptoms of pyramidal or extrapyramidal motor dysfunction, behavioral and psychiatric abnormalities, cognitive impairment, and speech difficulty. Brain imaging abnormalities were common, and initial diagnosis category was significantly associated with white matter hyperintensities, white matter calcifications, and ventricular enlargement. In this literature analysis, ALSP was frequently misdiagnosed. Improving awareness of this condition and distinguishing it from other conditions with overlapping presenting symptoms is important for timely management of a rapidly progressive disease such as ALSP.

A missense mutation in the Hspa8 gene encoding heat shock cognate protein 70 causes neuroaxonal dystrophy in rats.

Neuroaxonal dystrophy (NAD) is a neurodegenerative disease characterized by spheroid (swollen axon) formation in the nervous system. In the present study, we focused on a newly established autosomal recessive mutant strain of F344-kk/kk rats with hind limb gait abnormalities and ataxia from a young age. Histopathologically, a number of axonal spheroids were observed throughout the central nervous system, including the spinal cord (mainly in the dorsal cord), brain stem, and cerebellum in F344-kk/kk rats. Transmission electron microscopic observation of the spinal cord revealed accumulation of electron-dense bodies, degenerated abnormal mitochondria, as well as membranous or tubular structures in the axonal spheroids. Based on these neuropathological findings, F344-kk/kk rats were diagnosed with NAD. By a positional cloning approach, we identified a missense mutation (V95E) in the Hspa8 (heat shock protein family A (Hsp70) member 8) gene located on chromosome 8 of the F344-kk/kk rat genome. Furthermore, we developed the Hspa8 knock-in (KI) rats with the V95E mutation using the CRISPR-Cas system. Homozygous Hspa8-KI rats exhibited ataxia and axonal spheroids similar to those of F344-kk/kk rats. The V95E mutant HSC70 protein exhibited the significant but modest decrease in the maximum hydrolysis rate of ATPase when stimulated by co-chaperons DnaJB4 and BAG1 in vitro, which suggests the functional deficit in the V95E HSC70. Together, our findings provide the first evidence that the genetic alteration of the Hspa8 gene caused NAD in mammals.