Acta Neuropathologica

Myelin oligodendrocyte basic protein (MOBP) is an abundant oligodendrocyte gene implicated in multiple neurodegenerative diseases. Genetic variation at the MOBP locus has been associated with risk for progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTD), corticobasal degeneration (CBD), Alzheimers disease (AD), Lewy body dementia (LBD), and Creutzfeldt-Jakob disease (CJD). Epigenetically, MOBP promoter hypermethylation and reduced expression have been reported in multiple system atrophy (MSA). Although MOBP is thought to play a role in oligodendrocyte morphology and myelin structure, how genetic and epigenetic variation at this locus influences gene regulation and contributes to disease risk remains poorly understood across neurodegenerative disorders. Here, we investigated whether shared or disease-specific genetic mechanisms at MOBP converge on altered DNA methylation and expression across neurodegenerative disorders. We analysed MOBP variants using summary statistics from recent GWAS for ALS, PSP, FTD, LBD, PD, MSA, AD, and CJD. Colocalisation (COLOC and SuSiE-coloc) was used to test whether disease-associated variants overlapped between diseases, and with oligodendrocyte expression quantitative trait loci (eQTLs) and bulk brain methylation quantitative trait loci (mQTLs). To further investigate mQTL effects at this locus, rs1768208, a variant previously associated with PSP, was genotyped in an overlapping brain methylation cohort, allowing direct testing of genotype-methylation associations in frontal white matter tissue. ALS and PSP GWAS demonstrated strong association at MOBP, with most strongly associated SNPs (e.g. rs631312, rs616147, rs1768208) shared between both disorders. Colocalisation analyses indicated high posterior probability that ALS and PSP share the same causal variant, with weaker overlap with FTD. mQTL colocalisation highlighted cg15069948, located near an exon junction within MOBP, as strongly colocalising with the ALS/PSP risk variants. In complementary tissue analyses, rs1768208-T carriers showed hypomethylation at cg15069948 in PSP brains. No genotype-methylation effects were detected in MSA or Parkinsons disease. Together with prior evidence of promoter hypermethylation and reduced expression in MSA, our findings identify cg15069948 as a regulatory methylation site linking ALS/PSP risk variants to altered MOBP methylation, and support MOBP dysregulation as a shared feature of neurodegeneration. However, the underlying mechanisms appear disease-specific, highlighting the complexity of involvement of this gene across neurodegenerative disorders.

Transactive response DNA binding protein 43 kDa (TDP-43) pathology, is a central molecular hallmark of amyotrophic lateral sclerosis (ALS). However, the underlying triggers are incompletely understood. Here, we show that infection with herpes simplex virus (HSV) induces molecular hallmarks of ALS in various in vitro and in vivo models and is associated with an increased risk of ALS in human population data. German healthcare provider data (n = 238,440) and herpesvirus serology of an ALS patient and control cohort (n = 1,100) showed that HSV infection elevated the ALS risk by 210% and odds by [~]65%, respectively. On a molecular level, HSV infection promoted TDP-43 pathology in neuronal cell models, human iPSC-derived motoneurons and cerebral organoids, mice, and human tissue sections. This effect was triggered by HSV-1 or 2, but not by several other related herpesviruses. Mechanistically, the infected cell protein 0 (ICP0) of HSV-1/2 drives TDP-43 pathology by disturbance of promyelocytic leukemia nuclear bodies (PML-NBs), thereby abrogating TDP-43 SUMO2/3ylation. Taken together, we reveal a previously unrecognized association between HSV infection and ALS and clarify the underlying molecular mechanism that drives TDP-43 pathology. Our data may guide future studies into therapeutic and prophylactic interventions against ALS.

Motor neuron (MN) loss is a hallmark of neurodegenerative disorders, yet its assessment remains variable, confounding mechanistic and therapeutic interpretation. To address this, we conducted a systematic review and meta-analysis of spinal muscular atrophy (SMA) mouse studies, revealing 60% variability in reported MN loss, largely attributable to nonspecific spinal cord sampling. Using a whole-segment approach with tissue clearing, MN tracing, and multimodal imaging, we confirmed segment-dependent differences in MN counts. Common MN markers (SMI-32, Nissl) lacked specificity, whereas choline acetyltransferase (ChAT) provided robust labeling in murine and human spinal cords. Deep learning-based whole-mount segmentation enabled unbiased MN quantification and validated manual counts. Integrating analysis with computational modeling established segment sampling as a key driver of variability and revealed degeneration patterns: widespread MN loss in amyotrophic lateral sclerosis (ALS), selective MN loss in severe SMA, and preservation in mild SMA models. These findings establish a framework for reproducible MN quantification. HighlightsO_LISpinal cord segment-specific analysis reduces variability and allows accurate MN quantification C_LIO_LIChAT is the most reliable MN marker in murine and human spinal cords C_LIO_LIDeep learning-based segmentation enables unbiased MN quantification in intact spinal cords C_LIO_LIMN degeneration is widespread in ALS but restricted to pools innervating proximal muscles in severe SMA C_LI

Tau neurofibrillary tangles are a hallmark of several neurodegenerative diseases called tauopathies, including frontotemporal dementia and Alzheimers Disease. Ongoing clinical trials for tauopathies seek to reduce Tau in the brain through immunotherapy, antisense oligonucleotides, and siRNA. MAPT codes for Tau, therefore understanding how the MAPT gene is regulated and the effect of genetic variation at its regulatory elements is likely to have high relevance for tauopathies. We screened a [~]3 Mb region including the MAPT locus using 2 different massively parallel reporter assay (MPRA) strategies in KOLF2.1J h-NGN2 neurons and HEK293FT cells, identifying previously unannotated cis-regulatory elements (CREs). Using CRISPR interference (CRISPRi) in mixed neuron cultures, we identified a new CRE for MAPT, as well as 2 CREs for another nearby gene of interest, KANSL1. Known genetic variation from the Alzheimers Disease sequencing project was tested in a separate MPRA at the top CREs near the MAPT gene, identifying variants with altered regulatory effects including those at previously identified CREs for MAPT. Using a saturation mutagenesis screen of a 2,000 bp region encompassing the MAPT promoter, we assessed regulatory effects of each possible single nucleotide variant in this region. We identified several neuron-specific regulatory variant effects at this region, including a high confidence binding site for the transcription factors EGR2, ZBTB14, and TCLF5 at a region of high MPRA activity and genetic conservation.

Although mutations in many genes cause familial amyotrophic lateral sclerosis and frontotemporal dementia, most cases are sporadic (sALS and sFTD) with unclear etiology. We tested whether somatic mutations contribute to sALS and sFTD by deep targeted sequencing of 88 neurodegeneration-related genes in postmortem brain and spinal cord samples from 399 sporadic cases and 144 controls. Predicted deleterious somatic variants in ALS/FTD genes were observed in 2.1% of sporadic cases lacking deleterious germline variants. These variants occurred at very low allele fractions (typically <2%) and were often focal and enriched in disease-affected regions. Analysis of bulk RNA-seq data from an additional cohort identified deleterious somatic variants in DYNC1H1 and LMNA, genes associated with pediatric motor neuron degeneration. Targeted long-read sequencing further identified one sFTD case with de novo somatic C9orf72 repeat expansions. Together, these findings suggest that rare, focal somatic variants can contribute to sALS and sFTD and drive widespread neurodegeneration.

The role of the epigenome in age-related neurodegenerative disorders remains understudied. Here, we analyzed circulating cell-free DNA (cfDNA) from blood to detect methylation changes as a liquid-biopsy for Amyotrophic Lateral Sclerosis (ALS). Our study included 20 patients with sporadic ALS, 10 patients with C9orf72-associated ALS, 10 asymptomatic carriers of the C9orf72 repeat expansion mutation, and 21 non-disease controls. Following targeted enzymatic methyl-sequencing (EM-seq) of [~]4 million CpG sites, we detected numerous differentially methylated genes, including several implicated in ALS disease risk and pathogenesis. By integrating multiple epigenetic features, we delineated a distinct epigenetic signature, which achieved an average area under the curve (AUC) of 0.91 {+/-} 0.10 upon receiver operator characteristic (ROC) analysis, which enabled detection of [~]70% of ALS patients with close to 100% specificity. Furthermore, we also identified a set of genes whose methylation status significantly correlated with clinical disease progression and cerebrospinal fluid (CSF) neurofilament levels. Our results reveal the potential of cfDNA-based biomarkers to accurately diagnose ALS and potentially predict disease progression.

Hereditary ataxias are a heterogeneous group of neurodegenerative disorders characterized by impaired balance and coordination, often due to cerebellar dysfunction. Despite advances in identifying genetic causes, animal models remain essential for dissecting underlying mechanisms and testing therapeutic strategies. Here we describe a mouse model of spastic ataxia and myopathy caused by a missense mutation in Tuba4a (n.A626C, p.Gln176Pro). In an ENU mutagenesis screen, a male C57BL/6J mouse exhibiting muscle wasting and an intention tremor starting at approximately 4 weeks-of-age was identified. The male was bred by in vitro fertilization to BALB/cByJ oocyte donors. Genetic mapping determined dominant inheritance and localized the mutation to Chromosome 1. Genome sequencing revealed single nucleotide polymorphisms (SNPs) in serine threonine kinase 36 (Stk36Y1003N) and alpha-tubulin 4A (Tuba4aQ176P) in the mapping interval. These SNPs were CRISPR-engineered into C57BL/6J mice, which confirmed the Tuba4aQ176P variant as the causative mutation. Mutant mice are normal at 3 weeks, except for decrement in muscle response following repetitive nerve stimulation. However, by 30 days these mice have ataxia, Purkinje neuron degeneration, and extensive skeletal muscle defects, which contribute to a decreased lifespan. Dominant TUBA4A mutations in humans are associated with spastic ataxia type 11 (SPAX11), congenital myopathy type 26 (CMYO26), and frontotemporal dementia/amyotrophic lateral sclerosis type 9 (FTDALS9). Our mice exhibit hallmark features of SPAX11 and CMYO26, but do not show motor neuron degeneration. This specificity makes this model a valuable tool for studying cell-type selective effects of TUBA4A mutations in neurodegeneration and myopathy.

Frontotemporal lobar degeneration with tau inclusions (FTLD-tau) comprise a class of fatal heterogeneous neurodegenerative diseases. Approximately 10% arise from pathogenic MAPT mutations and often cause severe, early-onset disease with pathology that is distinct yet partially overlapping with sporadic cases. Here, we evaluated post-mortem tissue from a patient with FTLD-tau due to MAPT S305I showing neuropathology most consistent with argyrophilic grain disease (AGD), a prevalent limbic tauopathy of aging. Structures determined by cryo-electron microscopy reveal tau filament folds that differ from those found in sporadic AGD or other tauopathies and feature a 4-layer architecture stabilized by the Ile substitution within its core. Comparative structural analysis reveals conserved motifs are shared among AGD, corticobasal degeneration, and MAPT P301T. A well-defined density stacks along a cationic cleft, indicative of a bound RNA-like polyanion or small-molecule. In vitro analysis shows the S305I mutation promotes fibrilization relative to normal tau. These results demonstrate that MAPT S305I stabilizes a distinct aggregation-prone tau fold that likely contributes to disease pathology and heterogeneity beyond its known splicing defects, and underscore potential limitations of using the most pathologically similar genetic form as a model for sporadic FTLD-tau.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), where DNA methylation may play a role by connecting genetic and environmental risk factors. We performed whole-genome DNA methylation profiling of cerebrospinal fluid (CSF) cells from relapsing-remitting MS patients and matched controls, identifying 2,710 differentially methylated positions (DMPs) and 4,330 regions (DMRs). These changes were enriched in immune signaling, adhesion and migration processes, and were accompanied by corresponding RNA expression changes. MS-associated methylation changes enriched in the cohesin chromatin regulation pathway mapped to enhancers of T helper 17 (Th17) cells, whereas in other T cell types they were mapping to bivalent enhancers and repressed chromatin. Notably, this pathway comprised multiple Protocadherin (PCDH) genes, typically expressed in neuronal cells, that displayed consistent methylation and expression changes in CSF cells. Expression of shared intracellular domain of PCDH{gamma} cluster proteins was confirmed in peripheral blood T cells by flow cytometry as well as expression of PCDH{gamma} cluster genes in memory CD4+ T cell subsets. Moreover, co-expression analysis suggests a role of PCDH genes in aryl hydrocarbon receptor (AHR) signaling. In summary, DNA methylation changes in CSF resident cells reflect dysregulated T cell activation and migration in MS and suggest a novel role of protocadherin molecules in MS pathogenesis.

Pathogenic variants in leucine-rich repeat kinase 2 (LRRK2), vacuolar protein sorting 35 (VPS35), and RAB32 cause dominantly inherited parkinsonism, indistinguishable from idiopathic late-onset Parkinsons disease (PD). All three causes constitutively activate LRRK2 kinase activity to augment immune responses, enhancing immunity to fight pathogens, but similar mechanisms in the brain increase the vulnerability of dopaminergic neurons to degeneration. Although VPS35 p.D620N possess the highest constitutive increase in LRRK2 kinase activity among known variants in LRRK2 or RAB32, its effects on the immune system remain poorly understood. LRRK2 and Rab32 are highly expressed in myeloid cells including microglia; thus we examined the transcriptomic and functional consequences of Vps35 p.D620N in knock-in mice (VKI). Microglia were isolated from brains of six-month-old VKI mice and were analyzed via single-cell RNA sequencing. Differential gene expression highlighted pathways involved in antimicrobial humoral immune response, lysosomal stress sensing, and phagocytosis. Notably, genes of S100 family proteins, along with lipocalin 2 (Lcn2), were significantly upregulated, and those measures were complimented by immunohistochemistry and quantitative PCR. In contrast, pathways involved in synaptic transmission, neuronal development, and homeostatic immune signaling were downregulated. Peripheral stimulation with lipopolysaccharide amplified microglial activation and phagocytic markers in wildtype mice, and VKI mice also display enhanced morphological activation and increased synaptic engulfment. Collectively, Vps35 p.D620N drives a chronic pro-inflammatory microglial phenotype characterized by heightened innate immune signaling, lysosomal stress, and enhanced phagocytic activity. VKI microglia are sensitized to peripheral immune challenges and may promote synaptic remodeling and neurodegenerative vulnerability in PD. These results provide mechanistic insight into how retromer dysfunction and LRRK2 kinase hyperactivity intersect with microglial biology to influence PD pathogenesis.

SARM1 and NMNAT2 are two well described players in the Programmed Axon Death (PAxD) pathway. However, less is known about their transcriptional regulation, especially in humans, despite evidence that their expression levels influence axon vulnerability and thus modulation of expression presents a potential therapeutic target. Here, we used in-cell luciferase assays to functionally study the promoter regions of the human NMNAT2 and SARM1 genes. We find that human NMNAT2 expression can be driven by cAMP, acting through one cAMP response element (CRE), compared to two in mice. Naturally occurring single-nucleotide variants exist within the CRE, some of which lower NMNAT2 promoter activity by more than 50%. We also report an ultra-rare single nucleotide variant in the NMNAT2 promoter in an ALS patient in Project MinE. This variant demonstrates pathogenic potential by lowering NMNAT2 promoter activity in our assay. Project MinE also reveals a common SARM1 promoter variant that significantly increases SARM1 promoter activity in our assay. Thus, several single nucleotide changes in the NMNAT2 and SARM1 promoters modify transcription levels in the direction that would predict an increase in susceptibility to PAxD. These promoter variants refine our understanding of regulatory mechanisms affecting NMNAT2 and SARM1 expression and, together with previously reported coding variants for these genes, expand the catalogue of functionally relevant variants for future association studies in neurodegenerative diseases, including peripheral neuropathies and motor nerve disorders.

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disorder characterized by the cytoplasmic aggregation and nuclear depletion of the TDP-43 protein. The latter impairs TDP-43 function as an RNA-binding protein and compromises the repression of cryptic splicing events, affecting both glutamatergic upper motor neurons and cholinergic lower motor neurons. This study systematically investigated the molecular and functional consequences of TDP-43 knockdown in human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons (iGNs) and cholinergic motor neurons (iMNs) using antisense oligonucleotides. The results demonstrated that TDP-43 loss elicits widespread, cell-type-specific changes in gene expression and alternative splicing. Notably, a shared subset of ALS-associated targets, including STMN2 and UNC13A, were consistently downregulated and mis-spliced across both neuronal subtypes. Functionally, Microelectrode Array (MEA) electrophysiology recordings revealed that TDP-43 knockdown induces a hyperexcitable phenotype in both neuronal populations, though they exhibited distinct network patterns: iGNs displayed synchronized bursting and significant shifts in overall electrophysiological profiles, while iMNs showed asynchronous firing. Furthermore, the inclusion of astrocytes in co-culture models expanded the repertoire of detectable cryptic splicing, including an event in HDGFL2 previously identified in patient cerebrospinal fluid. Despite these profound molecular and functional deficits, TDP-43 depletion did not impact neuronal viability or increase susceptibility to glutamate-induced excitotoxicity. These findings validate hiPSC-derived iGNs and iMNs as relevant models for ALS and highlight the critical necessity of considering cell-type specificity when elucidating pathogenesis and developing targeted therapies.

Multiple system atrophy (MSA) is a sporadic progressive neurodegenerative disorder characterised by central nervous system alpha-synuclein inclusions. MSA pathologically most commonly shows a spectrum of two patterns, olivopontocerebellar atrophy and striatonigral degeneration, with significant overlap. Although germline variants are unlikely to play a major role, an association with the KCTD7 gene was recently reported. Somatic mutations are abundant in the brain, and may play a role in neurodegeneration. In MSA, somatic SNCA (alpha-synuclein) copy number gains occur, but single nucleotide mutations have not been investigated. In Alzheimers disease, somatic mutations in tumour suppressor genes were reported in microglia. We hypothesised that brain somatic mutations in SNCA, KCTD7, or the tumour suppressor genes mutated in Alzheimers, may contribute to MSA. To test this, we developed a targeted duplex sequencing pipeline using unique molecular identifiers, encompassing SNCA, KCTD7, and 10 tumour suppressor genes. Seven of these are involved in clonal haematopoiesis, an age-related process which predisposes to haematological malignancy, and can be subdivided into myeloid and lymphoid, based on the cell type affected, with the former much more frequent. We analysed DNA from the cerebellum, cingulate cortex, and putamen of 20 MSA cases (10 olivopontocerebellar atrophy, 10 striatonigral degeneration) and 9 controls. We observed an enrichment of clonal haematopoiesis gene mutations in MSA brains (median 1 vs 0, p=0.054). These included mutations in DNMT3A and TET2, the most frequently affected myeloid clonal haematopoiesis genes, and a recurrent mutation in three cases in KMT2D, a lymphoid clonal haematopoiesis gene. Clonal haematopoiesis mutations were often found in multiple brain regions, and multiregional mutations occurred in 12/20 MSA cases versus 1/9 controls (p=0.020), with 11 cases harbouring clonal haematopoiesis mutations in all three brain regions, compared to 0/9 controls (p=0.005). In striatonigral degeneration, clonal haematopoiesis mutations showed elevated variant allele fractions in the most pathologically affected region, the putamen, versus the cerebellum (p=0.013). MSA clonal haematopoiesis mutations included eight unique non-synonymous variants, which had higher allelic fractions than synonymous changes (p=0.076), and five of these were predicted to confer a proliferative advantage and were found in multiple brain regions. We detected no coding SNCA mutations, and the small number of KCTD7 variants, including one coding deletion, precludes any conclusions. These findings reveal enrichment of clonal haematopoiesis mutations in MSA brain, potentially due to infiltration from the periphery, suggesting a disease-associated proliferative process extending beyond peripheral haematopoiesis.

SummaryFrontotemporal dementia (FTD), a leading cause of young-onset dementia, is characterized by progressive behavioral and cognitive decline associated with frontotemporal cortical atrophy. Nearly 40% of cases exhibit tauopathy, yet the molecular drivers of tau aggregation leading to synaptic dysfunction remain poorly understood. Here, we investigated whether Phospholipase D1 (PLD1, a lipid signaling enzyme), implicated in Alzheimers disease (AD), and amyotrophic lateral sclerosis (ALS), contributes to tau pathology dependent synaptic deficits in FTD. Postmortem temporal (BA38) and frontal (BA9) cortices from clinically diagnosed FTD and age-matched control subjects were analyzed using fluorescence-assisted single synaptosome long-term potentiation (FASS-LTP), immunofluorescence, proximity ligation assays (PLA), and PLD1-interactome proteomics. FASS-LTP revealed markedly reduced glutamatergic potentiation in BA38 and BA9 crude synaptoneurosomes from FTD brains compared to controls. Western blotting demonstrated elevated PLD1 expression in both crude synaptoneurosomal and cytosolic fractions from FTD subjects in BA38, but not BA9. Bielschowsky staining confirmed increased Pick body burden in FTD temporal cortex. Immunofluorescence and PLA showed robust PLD1 co-localization with total tau (HT7), hyperphosphorylated tau (AT8), and acetylated tau oligomers (TOMA2), indicating a strong spatial association between PLD1 and pathological tau species. PLD1 also exhibited enhanced co-localization with astrocytic GFAP and synaptic markers (PSD95, Nrx1{beta}), suggesting compartmentalized involvement in glial and synaptic remodeling. Proteomic profiling of PLD1-associated complexes revealed compartment-specific alterations with cytosolic fractions enriched for metabolic enzymes, stress-response proteins, and GFAP, while crude synaptoneurosomal fractions showed depletion of presynaptic scaffolds, vesicle-trafficking regulators, and proteostasis components. Cross-compartment integration indicated that over one-third of proteins were redistributed from synapses to cytosol, consistent with trafficking and degradative impairments. Gene Ontology analysis highlighted lipid metabolism, astrocyte activation, and proteasome dysfunction as dominant pathways. Collectively, these findings identify PLD1 as a critical mediator of synaptic dysfunction and tau pathology in FTD, acting through astroglial activation and disrupting synaptic proteostasis. This study provides the human clinical relevance towards PLD1 attenuation as a therapeutic target for FTD and related tauopathies to mitigate tau-driven neurodegeneration and restore synaptic integrity.

Charcot-Marie-Tooth (CMT) neuropathy is a clinically and genetically heterogeneous group of diseases characterised by the length dependent axonal degeneration of peripheral nerves. We previously mapped a rare form of X-linked CMT, CMTX3, to a 5.7-Mb interval on chromosome Xq26.3-q27.1 and excluded the coding region of all known genes in the linkage interval for mutations. Whole genome sequencing subsequently identified a 78-kb region of chromosome 8q24.3, that had been duplicated and inserted into the CMTX3 locus between the genes HAPSTR2 and SOX3. The 78-kb insertion, which contains a partial transcript of ARHGAP39, fully segregated in families with CMTX3 and was absent in neurologically normal controls. To retain the CMTX3 insertion and investigate its consequences in appropriate neuronal tissue, we generated induced pluripotent stem cells (iPSC) from CMTX3 fibroblasts. Using bulk RNA sequencing of patient-derived spinal motor neurons, ARHGAP39 was deemed non-pathogenic by excluding both the formation of novel fusion transcripts and dosage effects from the partial duplication. Subsequent NanoString expression analyses of candidate genes within the CMTX3 locus, across different stages of neuronal differentiation, identified spatiotemporal dysregulation of SOX3. NanoString showed reduced SOX3 expression in patient iPSC. RNA sequencing detected SOX3 downregulation in CMTX3 neuroepithelial progenitor cells, which was further confirmed by quantitative proteomics. Given the early onset and relatively rapid progression of CMTX3, these data prioritise SOX3 as a leading candidate gene, consistent with its role as one of the earliest transcription factors expressed in the developing nervous system and a key regulator of neuronal fate.

Multiple sclerosis (MS) is a chronic autoimmune demyelinating disease of the central nervous system with a complex etiology. Recent genomic studies highlight the contribution of expression quantitative trait loci (eQTLs) in modulating gene expression and disease susceptibility. Given the emerging role of circular RNAs (circRNAs) in MS, we hypothesized that genetic variants may regulate circRNA expression through circRNA-specific eQTLs (circ-eQTLs). We performed a cis-circ-eQTL analysis integrating circRNA expression and whole-genome genotyping data from 30 MS patients and 18 healthy controls using a linear regression model adjusted for disease status and sex. Candidate circ-eQTLs were prioritized based on MS-associated regions and known splicing QTLs (sQTLs) from GTEx and validated in an independent cohort (67 MS, 64 controls). Association analysis in a larger cohort (2831 MS, 3191 controls) evaluated two candidate variants for MS risk. We identified 42,077 significant cis-circ-eQTLs and validated three. Two SNPs, rs7214410 and rs11079784, modulated hsa_circ_0106983 expression, and rs7214410 also acted as an sQTL affecting EFCAB13 splicing. rs7214410 showed stronger association with MS than rs11079784. Our findings reveal extensive genetic regulation of circRNA expression and highlight rs7214410 as a dual-function variant refining the MS susceptibility locus on chromosome 17.

Microglia, resident immune cells of the brain, are important players in neurodegeneration. While microglial activation is a hallmark of many neurodegenerative diseases, the specific role of microglia intrinsic factors in microglial activation and disease pathogenesis remains unknown. Spinocerebellar ataxia type-1 (SCA1) is an inherited autosomal dominant neurodegenerative disease characterized by severe neuronal loss and early microglial activation in the cerebellum. SCA1 is caused by CAG repeat expansion in the ubiquitously expressed ATAXIN1 (ATXN1) gene. Using human microglia differentiated from SCA1 patient derived iPSCs, we found that mutant ATXN1 is sufficient to alter morphology, gene and protein expression in human microglia in a cell-autonomous manner. Moreover, compared to controls, human SCA1 microglia exhibited increased phagocytosis and pro-inflammatory cytokine production, indicating an immune priming. To determine the extent to which mutant ATXN1 in microglia contributes to SCA1 pathogenesis and behavioral symptoms, we removed mutant ATXN1 from microglia and macrophages in a novel conditional SCA1 mouse model, f-ATXN1146Q/2Q mice. Microglial mutant ATXN1 reduction led to a marked correction in microglia phenotype, in particular in the transcriptomic signature of interferon type 1 mediated immune response, reduced microglial density and resulted in smaller microglia with reduced branching in the cerebellum. Pathology of Purkinje neurons and cerebellar astrogliosis were also ameliorated. Utilizing a battery of behavioral tests, we found that microglia and macrophage mutant ATXN1 reduction ameliorated cognitive, mood, and motor deficits in SCA1 mice. Together, these results indicate that mutant ATXN1 directly impacts microglial phenotype in SCA1, contributing to SCA1 pathology and behavioral deficits.

Recent genetic studies have underscored the central role of microglia in orchestrating neurodegenerative pathology, particularly in Alzheimers disease (AD). However, contributions of other cell types are complex and poorly understood. Studying specific genetic mutations related to the disease is limited by the lack of robust in vitro models. To address this, we investigated the impact of the high-risk AD-associated TREM2-R47H variant using iPSC-derived forebrain organoids co-cultured with microglia, comparing mutant and isogenic control lines. Organoids were cultured up to 173 days, co-cultured with microglia and profiled using single-cell transcriptomics, bulk RNA sequencing, and confocal microscopy. Our findings demonstrate that the mutant co-culture model exhibits AD-specific signatures in vitro. Notably, confocal imaging revealed that control microglia internalized phosphorylated-Tau throughout the tissue, while R47H microglia showed no such uptake. Single-cell and bulk RNA profiling uncovered alterations in gene expression associated with oxidative phosphorylation, lysosomal activity and pathways of neurodegeneration. Interestingly, signs of neurodegeneration appeared as early as day 139 in variant organoids cultured without microglia, whereas wild-type (WT) organoids did not exhibit comparable changes at the whole-organoid level. By day 163, robust neurodegenerative profiles spanning neuronal and glial populations were evident exclusively in TREM2-R47H samples. These data suggest that the TREM2-R47H variant impairs microglial clearance of pathological proteins and may affect broader cellular networks beyond microglia, challenging current assumptions about its role and opening avenues for redefining AD pathogenesis.

Cerebral amyloid angiopathy (CAA), a major vascular contributor to cognitive decline, is present in 85-95% of Alzheimers disease (AD) patients. Despite its high prevalence, the mechanisms by which CAA contributes to neurodegeneration remain poorly understood. Triggering receptor expressed on myeloid cells 2 (TREM2), an innate immune receptor expressed exclusively by microglia, regulates activation, phagocytosis, and amyloid clearance, thereby shaping neuroinflammation. Loss-of-function mutations in TREM2 markedly increase AD risk, but its role in CAA pathology remains unknown. To investigate this, we crossed the Familial Danish Dementia (Tg-FDD) mouse model, which accumulates robust vascular amyloid, with TREM2 knockout (TREM2KO) mice to generate Tg-FDD/TREM2KO animals. Histological and transcriptomic analyses revealed region-specific effects of TREM2 deficiency. In the cortex, TREM2 loss markedly reduced vascular amyloid deposition, accompanied by decreased tau pathology. In contrast, in the cerebellum, TREM2 deletion exacerbated vascular amyloid accumulation, promoted astrogliosis, and enhanced tau pathology. Transcriptomic profiling further identified distinct neuroinflammatory signatures between cortex and cerebellum, particularly in cytokine signaling, matrix remodeling, and lipid metabolism. Together, these findings demonstrate that TREM2 deficiency leads to region-specific effects on CAA, revealing extensive regional variability in vascular amyloid pathology and underscoring the importance of considering these differences when developing TREM2-based therapies.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease with a heterogeneous clinical presentation, complicating early diagnosis and therapeutic monitoring. To identify disease-specific biomarkers, we performed an unbiased cerebrospinal fluid (CSF) proteomic analysis in 87 ALS patients, 89 healthy controls, and 61 neurological controls using mass spectrometry. Across all quantified proteins, 399 were significantly dysregulated in ALS, including established neurodegeneration (NEFL, NEFM, UCHL1) and neuroinflammatory (CHIT1, CHI3L1, CHI3L2) markers. Correlation and pathway analyses uncovered dysregulation of immune, synaptic, and metabolic processes, with aberrant complement activation emerging as a hallmark. Complement proteins increased progressively with declining ALS Functional Rating Scale-Revised and longer disease duration, whereas early-stage markers (CLSTN3, CHAD, RELN) indicated pre symptomatic neuronal and synaptic disruptions. Machine learning identified a minimal five protein CSF panel (MB, ITLN1, YWHAG, FCGR3A, PGAM1) that accurately distinguished ALS patients from healthy controls, capturing disease-specific pathophysiology beyond general neurodegeneration. Our findings define a robust ALS-specific CSF proteomic signature, reveal prognostic protein candidates across disease stages, and provide a framework for diagnostic biomarker development, enabling earlier intervention and monitoring.