Neuropathology and Applied Neurobiology

Twenty percent of familial amyotrophic lateral sclerosis (fALS) cases are linked to mutations in the Superoxide Dismutase 1 (SOD1) gene and accumulation of misfolded SOD1 aggregates. SOD1 misfolding from the broader ALS population without SOD1 mutations is less clear. Here, we report SOD1 seeding activity in antemortem cerebrospinal fluid (CSF) from ALS participants with and without SOD1 mutations during ALS progression. Antemortem CSF from controls, SOD1-ALS, and sporadic ALS (sALS) patients was subjected to SOD1 seed amplification real-time quaking induced conversion (RT-QuIC) assays. SOD1-ALS CSF exhibited shorter lag phase and increased ThioflavinT (ThT) fluorescence amplitude compared to healthy controls and those with spinal muscular atrophy. CSF from sALS participants, who had no mutations in SOD1 or nine other ALS risk genes, also displayed SOD1 seeding activity, indicating wild-type SOD1 is aggregate-prone in the broader ALS population. Longitudinal CSF data indicated that SOD1 seeding activity correlates with ALS progression via the ALS Functional Rating Scale Revised (ALSFRS-R) slope decline and CSF neurofilament light. Our sALS CSF cohort primarily comprised of participants less than 2 years from symptom onset, suggesting that SOD1 seeding activity is an early biomarker that may enable inclusion in clinical trials. With the FDA-approval of tofersen (Qalsody), a SOD1-lowering antisense oligonucleotide, new SOD1 diagnostic, prognostic and pharmacodynamic biomarkers may enable SOD1-targeting strategies that could benefit the broader ALS population.

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.

Despite important advances in understanding the etiopathology of multiple sclerosis, factors determining disease progression remain partially understood and often difficult to predict. Specific diagnostic and prognostic biomarkers are needed to optimize the risk-benefit ratio of treatment for each patient. The aim of our study was to identify a cerebrospinal fluid proteomic signature associated with diagnosis and short- to mid-term prognosis across the multiple sclerosis continuum. Our multicentric cohort study analyzed CSF samples from 120 patients using a proteomics data-independent acquisition strategy. Differentially expressed proteins were identified across diagnostic groups: 62 patients with multiple sclerosis, 15 patients with clinically isolated syndrome, and 43 healthy controls. We also compared the CSF of patients with no evidence of disease activity with those with disease activity at 2 and 5 years of follow-up. A diagnostic and prognostic classification model was built using iterative cross-validated logistic regression models on shared differentially expressed proteins across these two comparisons. A total of 1,257 proteins were quantified, and 162 differentially expressed proteins were identified across comparisons. We identified a set of ten proteins associated with the diagnosis and prognosis of multiple sclerosis, including previously identified potential biomarkers (CH3L2, IGHG1, IGKC, LAMP2, ADA2), proteins known to be involved in the pathophysiology of multiple sclerosis (A0A8J8YUT9, AT2A2, CO3A1) and two yet unreported proteins (DSC2 and MMRN2). Multivariate models based on these proteins achieved good accuracy for the diagnosis of MS compared with CIS (area under the receiver operating characteristics curve [AUROC] up to 80% using 3 proteins) and prognosis (NEDA vs. EDA; AUROC up to 96% at 2 and 5 years; using 5 proteins). These results, which will require further investigation to validate the new biomarkers, open new perspectives on multiple sclerosis pathophysiology and therapeutic targets.

BACKGROUND: Increasing evidence suggests that accurate prediction of Alzheimer disease (AD) symptom onset requires more than amyloid- and tau-centric biomarkers such as cerebrospinal fluid (CSF) A{beta}42/40, total tau and p-tau181 and plasma p-tau217. Autosomal dominant AD (ADAD), caused by pathogenic PSEN1, PSEN2 and APP mutations with predictable age at symptom onset, presents a unique opportunity to characterize the chronological changes in proteins beyond amyloid and tau and clarify them as early biomarkers of disease onset or as biomarkers related to disease staging and progression monitoring. METHODS: We measured 972 CSF samples corresponding to 484 participants of the Dominantly Inherited Alzheimer Disease Network (DIAN) using the NULISASeq 120 CNS Disease Panel. We first benchmarked the technology against gold-standard measurements followed by the identification of proteins that were differentially abundant in relation to mutation status and symptomatology. Next, we determined the chronological emergence of protein changes in relation to the estimated years to onset (EYO). Finally, we assessed whether specific protein measures improved the prediction of EYO in the ADAD. FINDINGS: NULISA measurements were comparable to those previously published. We demonstrated that known early alterations in CSF amyloid and tau were followed by inflammatory and neurodegenerative responses suggesting that clinical manifestation of AD happens before the inflammatory processes is fully developed. Finally, we found a multi-protein composite approach for predicting EYO that outperformed single biomarker values. INTERPRETATION: Our results suggest that the main CSF proteomic landscape changes in ADAD are due to the presence of a pathogenic mutation and occur prior to symptom onset. Improved performance of multi-protein composite to predict EYO compared to single biomarker values highlights the added value of multiplex proteomic signatures for biomarker panel development. FUNDING: National Institute on Aging, Alzheimers Association, German Center for Neurodegenerative Diseases, Raul Carrea Institute for Neurological Research, Japan Agency for Medical Research and Development, Ministry of Health & Welfare and Ministry of Science and ICT, Republic of Korea, Spanish Institute of Health Carlos III.

Introduction: Glioblastoma multiforme (GBM) remains the most lethal primary brain tumor with median survival of 14-15 months. Current prognostic markers inadequately stratify patient outcomes. PINK1 (PTEN-induced putative kinase 1), a mitochondrial kinase regulating mitophagy and cellular stress responses, has emerged as a promising prognostic candidate. Our preliminary analysis of 20 GBM cases demonstrated significant PINK1 expression with correlation to aggressive phenotypes (Turizo Smith et al., 2025). This multicenter study aims to prospectively validate PINK1 as a prognostic biomarker for survival and functional outcomes in a Latin American cohort. Methods and analysis: PINK1-GBM Colombia is a multicenter, observational cohort study across four tertiary hospitals in Bogota, Colombia (Hospital de Kennedy, Hospital El Tunal, Hospital Santa Clara and Hospital Universitario de la Samaritana). We will enroll at least 26-50 adults (18+ years) with newly diagnosed IDH-wild type GBM undergoing surgical resection. PINK1 expression will be quantified by immunohistochemistry (IHC) on formalin-fixed paraffin embedded (FFPE) tissue using standardized protocols. Primary outcomes: overall survival (OS) and progression-free survival (PFS). Secondary outcomes: functional status trajectories (KPS/ECOG). Follow-up extends 24 months with clinical, imaging (RANO 2.0), and telephone assessments. Survival analyses will employ Kaplan-Meier methods, log-rank tests, and Cox proportional hazards models adjusted for established prognostic factors. Ethics and dissemination: Approved by Universidad Nacional de Colombia Ethics Committee (Acta 001, February 5, 2026; Ref: 2.FM.1.002-CE-002-26), Subred Sur Occidente (P-AP-19-2025, July 11, 2025), and Subred Centro Oriente (CEI 067/2025, October 24, 2025). Conducted per Declaration of Helsinki and Colombian Resolution 8430/1993. Results will be disseminated via peer-reviewed publication, international conferences, and thesis submission.

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.

BackgroundNeuroinflammation is a common hallmark of primary tauopathies, and is associated with worse clinical outcomes over time. However, accurate prognosis in these disorders remains challenging, and current fluid biomarkers provide limited insight into the contribution of peripheral immune cells to PSP/CBS pathogenesis. Our study aims to characterise blood-based immune cell profiles in patients with progressive supranuclear palsy (PSP) and corticobasal syndrome (CBS), and test their associations with neurodegeneration and clinical outcomes. MethodsPeripheral blood immune cells from fresh whole blood were characterized with high-dimensional mass cytometry (29 markers) in n=60 people with PSP/CBS and n=21 age- and sex-matched controls. Cell type abundance was defined as the ratio of counts for each gated population divided by total live cells. Hierarchical clustering of cell types and principal component analysis were used to derive data-driven immune clusters. Correlation network analysis and diffusion-based network propagation integrated cell counts with plasma inflammation markers to prioritise mediators of intercellular signalling. Associations between immunological markers, plasma concentrations of neurofilament light chain (NfL), cognition, and survival were assessed using regression and Cox proportional hazards models. ResultsPatients with PSP/CBS showed a global increase in covariance among immune cell populations, indicating heightened coordination within the peripheral immune network. A monocyte-driven cluster (Cluster 1) showed higher scores in PSP/CBS, reflecting impaired phenotypic transition from classical to nonclassical monocytes, and was associated with higher NfL levels, poorer cognitive performance, and worse prognosis. In contrast, a Treg-driven cluster (Cluster 2) showed lower scores in PSP/CBS, and was associated with better cognition and longer survival. Integrated multimodal networks identified a small set of immune-regulatory molecules and cytokines mediating crosstalk between Treg/Th17-like cells and monocytic populations, supporting a dysregulated Treg-monocyte axis in PSP/CBS. ConclusionsWe identified peripheral blood-based immunophenotypic profiles of individuals with PSP/CBS that are associated with neurodegeneration, cognitive decline, and survival. Dysregulated monocyte maturation and reduced Treg-related immune configurations are enriched in patients with worse outcomes, suggesting that specific peripheral immune cell subsets may serve as fluid biomarkers and potential immunotherapy targets in primary tauopathies.

Alzheimer s disease (AD) and other tauopathies are characterized by the hyperphosphorylation of tau (pTau), leading to its aggregation in the brain, a process strongly predictive of neurodegeneration and future cognitive decline. Currently, tau positron emission tomography (PET) is the only validated method for detecting tau aggregates in vivo. However, its high cost, invasiveness, and limited accessibility restrict its use in clinical settings and preclude large-scale screening. Moreover, existing plasma biomarkers that quantify the level of pTau at specific sites (e.g., pTau217) have limited specificity for confirming AD-related tau aggregation, partly due to the heterogeneous and irregular phosphorylation patterns of pTau. Besides, the concentration of pTau is frequently elevated in the context of isolated amyloid-{beta} pathology, which is less strongly associated with cognitive decline in the absence of aggregated tau. There is therefore an urgent need for a reliable and scalable blood-based biomarker of tau pathology. A key mechanism underlying AD tau pathology is the ability of pathologically active pTau (PA pTau) to bind to and seed normal tau, facilitating prion-like propagation of insoluble tau aggregates. Here, we assessed the diagnostic performance of the VeraBIND Tau assay, the first functional assay to detect PA pTau seeding activity in plasma. Seventy-nine cognitively unimpaired (CU) and 66 cognitively impaired older adults underwent blood sampling, cognitive assessment, amyloid-PET or cerebrospinal fluid (CSF) analysis, and [18F]-MK6240 tau-PET imaging. Plasma pTau217 concentrations were quantified using the Lumipulse platform (Fujirebio). The VeraBIND Tau assay isolated PA pTau from plasma and evaluated its ability to bind recombinant normal tau using a tagged-tau chemiluminescent readout. VeraBIND Tau demonstrated 94.2% sensitivity and 96.1% specificity for predicting tau-PET positivity (AUC=0.97). It outperformed plasma pTau217 in CU individuals (PPV=85.9%), regardless of the pTau217 threshold used (maximal PPV of 57.5% using the 0.256pg/mL pTau217 threshold). This higher VeraBIND Tau diagnostic accuracy was driven by early tau-PET stages (Braak-like tau-PET stages 1-3; AUC=0.96 vs. 0.74 for pTau217, p=0.003). Moreover, both cross-sectional values and annual changes in VeraBIND Tau were significantly correlated with cognitive performance and entorhinal tau-PET signal (all absolute Spearman r[&ge;]0.23, p<0.05). These findings highlight the strong potential of VeraBIND Tau as a scalable and accurate screening tool to detect AD tau pathology in the general population. The assay may also help enrich clinical trials with tau-PET positive CU individuals, enhance clinical diagnostic workflows and support monitoring of tau-targeted therapies. Future work should evaluate its utility in optimizing triage and early-intervention strategies.

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.

Motor performance and coordination deficits are hallmarks of spinocerebellar ataxias, yet effective disease-modifying therapies remain limited. Here, we investigate the expression of the interleukin 17 receptor subunit A (IL-17RA) in cerebellum and assess the therapeutic potential of its ligand in a mouse model for Spinocerebellar Ataxia Type 2 (SCA2). We found that IL-17RA is highly enriched in cerebellar molecular layer interneurons (MLIs), which provide inhibitory input to Purkinje neurons. In-vitro electrophysiological recordings revealed that symptomatic SCA2 mice exhibited increased spontaneous inhibitory synaptic input onto Purkinje neurons, which was normalized by IL-17A application to control levels. Concomitantly, IL-17A application restored Purkinje neuron firing, a parameter characteristically reduced in SCA2 mice. Behaviorally, intranasal administration of IL-17A restored motor performance of symptomatic SCA2 mice to control levels in both rotarod and beam-crossing assays. Collectively, our results indicate that IL-17A rescues Purkinje neuron dysfunction and motor deficits in SCA2 mice, highlighting IL-17A signaling as a promising therapeutic target for spinocerebellar ataxia.

Riluzole is the most commonly prescribed among the limited approved therapies for amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder characterized by progressive motoneuron loss and paralysis. It is thought to act by suppressing motoneuron excitability and glutamate release, but its clinical benefits are modest and often diminish over time. We previously showed that homeostatic mechanisms in the SOD1G93A (mSOD1) mouse model of ALS are hyperactive and prone to overcompensation. Here, we tested whether such dysregulated homeostasis antagonizes the effects of riluzole. Wild-type (WT) and presymptomatic mSOD1 mice received therapeutic doses of riluzole in drinking water for 10 days, with untreated littermates of both genotypes serving as controls. Motoneuron excitability and synaptic inputs were then examined using intracellular recordings from the isolated sacral spinal cord. The data showed that chronic riluzole treatment increased motoneuron excitability and polysynaptic inputs in mSOD1 mice but produced no detectable changes in WT motoneurons. These results suggest that hyperactive homeostatic mechanisms in ALS counteract the suppressive effects of riluzole. Notably, mSOD1 motoneurons exhibited larger membrane capacitance than WT, consistent with their increased cell size at this disease stage. Riluzole treatment reduced motoneuron membrane capacitance in mSOD1 mice to the range observed in WT animals, indicating normalization of cell size and potentially reduction in metabolic demand. Together, these findings help explain the limited clinical efficacy of riluzole while revealing a previously unrecognized neuroprotective mechanism of the drug in ALS.

Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis involves dynamic changes in glutamatergic signalling. Magnetic resonance spectroscopy can monitor these changes but lacks temporal resolution and cell-type specificity. We investigated whether urinary astrocyte-derived extracellular vesicles (ADEVs) could serve as a non-invasive proxy for brain receptor dynamics. We prospectively collected longitudinal urine and cerebrospinal fluid (CSF) samples from a 30- 35-year-old female patient during 34 days of treatment. We isolated ADEVs using a specific protocol and measured GluN1 protein levels. A 30-35-year-old healthy female provided control samples. Wavelet transform analysis of the patient's GluN1 time series revealed two distinct patterns. First, a low-frequency trend showed declining GluN1 levels over the treatment period, which mirrored the reduction in CSF GluN1 concentrations. Second, a high-frequency oscillation appeared to be coupled with methotrexate infusions, with GluN1 peaks occurring approximately 48 hours after each dose. This secondary increase may reflect drug-induced p53 activation, which promotes the exosomal release of internalised receptors. These findings suggest that urinary ADEVs provide a feasible and informative method to monitor real-time molecular fluxes in the brain.

Astrocytes are crucial mediators of diverse aspects of brain function, including energy metabolism and synapse formation and maturation. Calcium is the primary information carrier in astrocytes, reporting cellular health and activity, and can be measured using fluorescent indicators. However, this readout is not yet widely used to screen and evaluate disease models and drug candidates. Here, we adapted a simple automated calcium imaging pipeline with key output parameters that characterize changes in astrocytic calcium signaling. We compared calcium responses in mouse astrocyte monocultures and astrocyte-neuron cocultures using GFAP-driven membrane-targeted GCaMP6f, with human astrocytes differentiated from two different induced pluripotent stem-cell lines using the calcium dye Cal520-AM. Event-based analysis reported similarities and differences in mean fluorescence, amplitude, frequency, duration, and area of calcium responses. We benchmarked the pipeline using the purinergic receptor agonist ATP to increase astrocyte activity, and the ER calcium pump blocker CPA to decrease activity across all culture models. Glutamatergic and serotonergic receptor function was tested with glutamate and lysergic acid diethylamide (LSD). LSD decreased activity in mouse cocultured astrocytes, but increased activity in human astrocytes. Furthermore, the addition of human recombinant Tau oligomers, an in vitro model of Alzheimers disease pathology, decreased activity in both mouse and human astrocytes. This pipeline can be used to quickly and easily characterize effects of astrocyte-targeting compounds, effects of non-astrocyte-targeting compounds on astrocyte activity, and rescue of disease models that affect astrocyte function, in mouse and human astrocytes and astrocyte-neuron cocultures.

BackgroundPeri-synaptic astrocyte processes (PAPs) play a fundamental role in synapse formation and function. Central afferent synapse loss and astrocyte dysfunction greatly impede sensory-motor circuitry in spinal muscular atrophy (SMA) disease progression, however mechanisms underpinning tripartite synapse dysfunction remains to be fully elucidated. The aims of this study were to further define PAP and motor neuron synaptic defects in human SMA disease pathology and implement a therapeutic intervention strategy to improve motor neuron function. MethodsWe derived astrocyte monocultures and motor neuron astrocyte co-cultures from healthy and SMA patient induced pluripotent stem cell (iPSC) lines to assess intrinsic astrocyte filopodia defects and phenotypes occurring at the synapse-PAP interface, respectively, using cell surface capture mass spectrometry proteomics, confocal and super resolution microscopy, synaptogliosome isolation, and electrophysiology. ResultsSMA astrocytes demonstrated intrinsic filopodia actin defects featuring low abundance of actin-associated cell surface N-glycoproteins, and decreased filopodia density and CDC42-GTP levels after actin remodeling stimulation. This phenotype is likely driven by the significant reduction of CD44 and phosphorylated ezrin, radixin and moesin ERM proteins (pERM) within SMA astrocyte filopodia. The dual combination of SMN1 gene therapy and forskolin treatment, an adenylyl cyclase activator leading to increased cyclic adenosine monophosphate (cAMP) levels and actin signaling pathway stimulation, led to extensive branching and increased filopodia density of SMA astrocytes during actin remodeling. SMA patient-derived motor neuron and astrocyte co-cultures, particularly samples derived from male patient iPSC lines, demonstrated a significant decrease in synapse number, actin-associated pre-synaptic neurotransmitter release protein, synapsin I (SYN1), and PAP-associated expression of pERM and glutamate transporter, EAAT1. Our astrocyte-targeted SMN1 augmentation and forskolin treatment paradigm restored SYN1 protein levels within the SMA synaptogliosome, resulting in significant increases in motor neuron synapse formation and function, but did not fully restore PAP-associated proteins levels at the synapse. ConclusionsSMA astrocytes demonstrate intrinsic actin-associated defects within filopodia, which correlates with decreased pERM levels at tripartite motor neuron synapses. We also define a SMN- and cAMP-targeted treatment paradigm that significantly increases pre-synaptic neurotransmitter release protein levels to improved SMA motor neuron synapse formation and function. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/714618v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1257ab8org.highwire.dtl.DTLVardef@19c0010org.highwire.dtl.DTLVardef@c84552org.highwire.dtl.DTLVardef@3f1e62_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

Spinal muscular atrophy (SMA) is characterized by motor neuron degeneration caused by deficiency of the survival motor neuron (SMN) protein. However, evidence increasingly supports broader systemic involvement. This study aimed to examine cardiac pathology in SMA patients and to investigate how reduced SMN levels impact cardiomyocyte homeostasis. We analyzed postmortem data from 14 SMA type I patients from the pre-treatment era, integrating gross anatomical, histopathological, and clinical findings. To investigate cardiomyocyte-intrinsic effects of SMN deficiency, healthy human cardiomyocytes were subjected to SMN knockdown and assessed using metabolic assays and transcriptomic profiling. Key findings were further investigated in vivo using the Smn2B/- mouse model of SMA. We found heterogeneous cardiac involvement in SMA patients, including cardiomegaly, variable fat deposition and interstitial fibrosis. SMN knockdown in human cardiomyocytes induced a metabolic shift and widespread transcriptional dysregulation, with pathway analyses identifying selective upregulation of PTEN signaling. Elevated PTEN protein levels were observed in a subset of human SMA hearts and in early postnatal hearts of Smn2B/- mice. Our results demonstrate that the heart remains a biologically relevant target of SMN deficiency and highlights cardiomyocyte-specific metabolic and PTEN signaling alterations as potential contributors to cardiac involvement in SMA.

Background: Neurite orientation dispersion and density imaging (NODDI) shows promise in providing specific insights into the neurite morphology underlying white matter (WM) damage in neurodegenerative diseases. This study aimed to advance the currently limited knowledge by characterizing NODDI-derived microstructural WM alterations in Wilson disease (WD) and examining their relationships with clinical symptoms. Methods: 30 WD patients, including 19 with predominant neurological involvement (neuro-WD) and 11 with hepatic manifestation (hep-WD), and 30 matched healthy controls underwent multi-shell diffusion-weighted magnetic resonance imaging. NODDI metrics, including neurite density index (NDI), orientation dispersion index (ODI), and isotropic volume fraction (ISOVF), and diffusion tensor imaging-based fractional anisotropy (FA) were estimated. Group differences in diffusion parameters across the WM skeleton were determined using tract-based spatial statistics. Additionally, voxel-wise correlations with neurological and cognitive scores were investigated. Results: We observed widespread NDI and ODI reductions in neuro-WD patients and ISOVF increases in hep-WD patients compared with healthy controls, particularly involving the corpus callosum, corona radiata, superior longitudinal fasciculus, external and internal capsule, and superior fronto-occipital fasciculus. A comparable yet more subtle pattern was found when comparing phenotypes. Distinct NDI and ODI constellations were identified as the microstructural determinants of FA alterations. Decreased NDI in the aforementioned fibers were correlated with neurological impairment, processing speed, and visual attention. Conclusions: Phenotype-specific microstructural WM alterations were identified, characterized by globally reduced axonal density and fiber organization in neuro-WD and excess free water in hep-WD. NODDI could be useful as an imaging biomarker for forecasting conversion to neurological WD manifestations and monitoring of disease progression.

Alzheimers disease (AD) is a neurodegenerative disorder characterised by progressive dementia, brain atrophy, and ultimately death. Using cerebral organoids derived from human induced pluripotent stem cells (hiPSCs) carrying the familial PSEN1 A246E variant, we investigated the temporal relationship between amyloid-{beta} (A{beta}) dysregulation and spontaneous neuronal activity. Multielectrode array recordings from the differentiation day 60 (DD60) to at least DD130 revealed that AD organoids exhibited transient hyperexcitability and hypersynchrony compared with wild-type (WT) controls, followed by a gradual decline in activity. During the enhanced excitability stage, both elevated A{beta}42/40 and A{beta} aggregate size showed positive correlations with the percentage of active electrodes and the global synchrony index (GSI) in AD organoids. These findings indicate that A{beta} dysregulation might contribute to transient network hyperexcitability in early AD. The results also suggest that patient-derived cerebral organoids may serve as a translational model to examine early network dysfunction and inform future investigations of potential A{beta}-induced changes in excitability during the preclinical stages of AD.

Background: CD276 has been proposed as a candidate gene associated with the biological characteristics of meningioma, but its predictive position and interpretive significance within a transcriptomic classifier have not yet been clearly established. Accordingly, this study aimed to evaluate CD276 stepwise across internal model development, external validation, calibration, decision-analytic assessment, feature stability, and robustness analyses using public transcriptomic cohorts. Methods: The analyses in this study were organized into two interconnected notebooks. In Notebook A, we reconstructed the internal training cohort (GSE183653), evaluated the CD276 single-gene signal, and then developed a transcriptome-wide multigene classifier. We also performed permutation importance, bootstrap confidence interval, label permutation test, repeated cross-validation, CD276 ablation, and internal calibration analyses. In Notebook B, we reproduced the external validation cohort (GSE136661) in a fixed common-gene space, applied train-only recalibration and train-only threshold transfer, and extended the interpretation through decision curve analysis, stability analysis, enrichment analysis, and one-factor-at-a-time robustness analysis. Results: The internal training cohort consisted of 185 samples and 58,830 genes, of which 25 were WHO grade III cases. CD276 expression showed a significant association with WHO grade, but the internal discrimination of the CD276-only baseline was limited (ROC-AUC 0.628, average precision 0.323, balanced accuracy 0.540). In contrast, the initial transcriptome-wide model showed ROC-AUC 0.834 and PR-AUC 0.509, and under 5-fold cross-validation, the canonical fulltranscriptome model and the CD276-forced 5,001-feature branch showed mean ROC-AUC/PR-AUC of 0.854/0.564 and 0.855/0.606, respectively, outperforming the CD276-only baseline at 0.644/0.391. CD276 was not included in the initial 5,000-feature filtered set and ranked 900th among 5,001 features even in the forcibly included 5,001-feature branch. In paired ablation analysis, the performance difference attributable to inclusion of CD276 was effectively close to zero (delta ROCAUC 0.000062, delta PR-AUC 0.000056). Internal calibration analysis showed an overconfident probability pattern (Brier score 0.10501, intercept -1.421392, slope 0.413241). In external validation, the fixed multigene pipeline achieved ROC-AUC 0.928 and PR-AUC 0.335. Train-only recalibration improved calibration metrics while preserving discrimination, and decision curve analysis showed threshold-dependent but limited external utility. Stability analysis showed overlap between core-stable genes and high-impact genes, but CD276 was not supported as a dominant stable core feature and remained in the target-of-interest tier. In robustness analysis, some perturbations preserved the primary interpretation, whereas others revealed transform sensitivity or an alternative high-performing feature-space solution. Conclusions: CD276 is a gene of interest associated with meningioma grade, but it was difficult to interpret it as a strong standalone predictor or a dominant stable classifier feature. In this study, the main basis of predictive performance lay not in CD276 alone but in a broader multigene transcriptomic structure, and probability output needed to be interpreted conservatively with calibration taken into account. These findings position CD276 not as a direct single-gene classifier but as a biologymotivated target-of-interest that should be interpreted within a broader transcriptomic program.

Optimized histological techniques are crucial for visualizing cellular morphology across zebrafish tissues. Here, we report a rapid and reliable hematoxylin and Oil Red O (H-ORO) staining protocol for frozen sections that can be completed in less than three minutes. Mayers hematoxylin is used for nuclear staining, followed by Oil Red O (ORO) to visualize lipid-rich structures such as the endomysium surrounding myofibers, white matter of the brain, and myelin layers of major axonal tracts. Importantly, our optimized H-ORO protocol preserves tissue integrity and minimizes artifacts such as myofiber shrinkage commonly observed with ethanol-based hematoxylin and eosin (H&E) staining in both frozen and paraffin sections.