Neuropathology and Applied Neurobiology

Abstract Objectives: Amyotrophic lateral sclerosis (ALS) is a clinically heterogeneous neurodegenerative disease requiring reliable biomarkers to improve patient stratification and trial design. While serum neurofilament light chain (sNfL) reflects neuroaxonal stress and disease aggressiveness, troponin T (TnT) may capture complementary aspects of neuromuscular involvement. We assessed the associations of TnT and sNfL with D50-derived measures of disease aggressiveness (D50) and disease accumulation (rD50) in ALS. Material and Methods: In this retrospective observation, TnT and sNfL levels from ALS patients in two independent German cohorts were analyzed using the D50 disease progression model; discovery cohort (Essen, n =433) and validation cohort (Bonn, n =185). Results: In both cohorts TnT demonstrated a robust correlation with rD50-defined phases across all aggressiveness subgroups (p<0.001). There was no consistent pattern regarding sNfL and the rD50 phases. sNfL concentrations demonstrated a significant and inverse correlation with D50 applied for all disease aggressiveness subgroups (p<0.001). Correlations of TnT levels with D50 disease aggressiveness groups were generally less strong and inconsistent between the two cohorts. In the discovery cohort only low aggressiveness subgroups correlated significantly (p<0.001), intermediate aggressiveness subgroups showed only a weak correlation (p<0.05) with TnT levels. High disease aggressiveness subgroups showed no significant correlation with TnT. Conclusion: In application of the D50 disease progression model, TnT was strongly associated with disease accumulation (rD50) across all disease phases, independent of disease aggressiveness (D50), whereas sNfL robustly reflected disease aggressiveness but not overall disease burden. These complementary biomarker profiles highlight the value of an integrated approach for refined disease stratification in ALS. Combining TnT and sNfL may enhance clinical decision-making, improve monitoring of disease progression and treatment response, and support optimized clinical trial design.

Early diagnosis of Parkinsons disease (PD) remains challenging because motor symptoms appear only after extensive neurodegeneration, and a definitive diagnosis still relies on post-mortem neuropathology. Increasing evidence implicates the enteric nervous system (ENS) in prodromal disease stages, but routine ENS-based diagnosis is limited by the complexity of intestinal tissue organization and the need for specific labeling strategies. Here, we developed a label-free autofluorescence (AF) imaging workflow combined with unbiased morphometric analysis to identify neurodegenerative alterations in fixed human colonic tissue. Using a correlative multiscale imaging approach, we generated a database of almost 800 high-resolution confocal images from myenteric and submucosal plexuses of controls, PD, and Alzheimers disease (AD) patients. Blind evaluation by four expert histologists showed reliable identification of control tissue but lower sensitivity for pathological cases, reflecting the heterogeneous distribution of ENS lesions. Semi-quantitative and morphometric image analyses identified a distinct population of enlarged enteric neurons, termed large neural cells (LNCs), strongly enriched in PD and AD compared with controls. LNCs contained autofluorescent cytoplasmic inclusions and frequently prominent nucleoli, both features largely absent from control tissue independent of aging. Co-localization with the amyloid-binding probe Amytracker (AmyT) demonstrated that AF granules correspond to {beta}-sheet-rich protein aggregates rather than merely age-related lipofuscin granules. Similar alterations were detected in intact three-dimensional (3-D) colonic biopsies, demonstrating the feasibility of volumetric ENS imaging without tissue clearing. Together, our results establish label-free AF imaging as a rapid and clinically compatible strategy for detecting enteric neurodegenerative pathology. This approach provides a framework for the future development of ENS-based biomarkers and supports the use of volumetric intestinal imaging for early diagnosis of neurodegenerative diseases.

Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by death of upper and lower motor neurons, usually presented with clinical heterogeneity. Fluid biomarker development remains dominated by neurofilament light chain (NEFL), a marker of neuroaxonal injury. NEFL is however unspecific to ALS and its phenotypes and there is currently a lack of biomarkers that capture ALS heterogeneity such as onset site and ALS-frontotemporal spectrum disorder (ALS-FTSD). Therefore, we investigated whether plasma proteomics could reveal pathway-level signatures that stratify and explain ALS heterogeneity. Methods: We profiled ~5,400 plasma proteins (Olink Explore HT) in 299 patients with ALS and 50 age- and sex comparable healthy controls. We used two complementary analytic frameworks: (i) differential protein abundance analysis to identify altered proteins in ALS and across clinical subgroups, and (ii) weighted gene correlation network analysis (WGCNA) to identify coordinated protein modules and relate them to ALS diagnosis and to ALS-specific clinical traits (site of onset, ALS-FTSD, ALS functional rating scale-revised (ALSFRS-R) score, and plasma NEFL). Results: Differential abundance analysis identified 56 proteins altered in ALS versus controls, of which 40 were increased. WGCNA identified 11 co-expression modules, with ALS samples having the strongest correlation to a protein module (n=51) highly enriched for muscle-related proteins. Out of the 40 proteins that had increased expression levels, 29 overlapped with the muscle-enriched protein module, indicating that muscle related proteins are the dominant circulating proteomic signature in ALS. This signal extended to clinical stratification: spinal-onset patients showed a strong positive association with the muscle-module. Further, differential abundance analysis of spinal- versus bulbar-onset ALS identified changes that mapped predominantly to the same module, supporting a molecular signature of onset phenotype. In contrast, cognitive status (ALS-FTSD) mapped to distinct modules enriched for extracellular matrix/cell-adhesion pathways, consistent with a separable biological axis of disease heterogeneity. Although multiple modules correlated with NEFL, trait-specific signatures were not fully explained by neuroaxonal injury. Notably, the muscle-enriched module increased with higher NEFL and lower ALSFRS-R, supporting its interpretation as a severity-linked, muscle-involvement proxy. Conclusions: Large-scale plasma proteomics reveals that heterogeneity in ALS reflects underlying biological structures. We identified a dominant muscle-associated protein network that distinguished ALS patients from controls and correlated with disease onset phenotype and severity, alongside distinct protein networks linked to ALS-FTSD. By integrating differential protein abundance with network-based analysis, we defined pathway-level biomarker signatures that extend beyond NEFL, enabling biologically informed patient stratification and improved therapeutic monitoring.

TDP-43 dysfunction is a defining feature of amyotrophic lateral sclerosis (ALS), yet no biofluid biomarker directly measures its functional activity. We developed a serum-based homogeneous time-resolved FRET (hTR-FRET) assay that quantifies TDP-43 RNA-binding activity using synthetic UU rich RNA probes. We analyzed 1,080 serum samples from controls, sporadic ALS, and genetic subgroups (C9orf72, SOD1) across multiple biorepositories. Cross-sectionally, TDP-43 ligation activity was elevated in ALS (mean 390 a.u.) versus controls (304 a.u.), yielding AUC = 0.79. Genotype means were 392 a.u. (sporadic), 382 a.u. (C9orf72), and 323 a.u. (SOD1); with a 366 a.u threshold achieved 95% specificity against controls. Longitudinally, Target ALS showed a modest but significant inverse correlation between TDP-43 activity and ALSFRS-R, while other cohorts exhibited similar non-significant trends. Elevated signal likely reflects increased extracellular, probe-competent TDP-43 species. This assay provides direct functional measurement of disease-relevant TDP-43 biology, supporting applications in diagnostic discrimination, genotype stratification, and progression monitoring in prospective studies.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease whose diagnosis often remains delayed. Skeletal muscle is increasingly recognized as an early contributor to ALS pathology. Using lipid imaging mass spectrometry (LIMS) in Tibialis anterior muscle from hSOD1G93A mice across disease stages, we identified fiber-type-specific and sex-dependent lipid remodeling. Lipid alterations were detected at the presymptomatic stage, preceding motor neuron loss and clinical symptoms. LIMS distinguished fast-twitch oxidative-glycolytic (type IIA) and glycolytic (type IIB/IIX) fibers and revealed their differential vulnerability to disease. Presymptomatic mutant muscles showed loss of physiological lipid signatures alongside disease-specific lipid changes. Although lipid profiles differed between sexes, ALS-associated alterations enabled accurate discrimination of mutant mice before symptom onset. Importantly, similar disease-related lipid changes were detected in serum, enabling accurate classification of presymptomatic animals. These findings establish lipid remodeling as an early ALS event and highlight novel biomarkers with potential for diagnosis and disease monitoring.

Background: Neurofilament light chain (NfL) gained FDA recognition in amyotrophic lateral sclerosis (ALS) through SIMOA-based validation, where baseline serum NfL predicts ALSFRS-R slope and survival, and through the 2023 tofersen approval for SOD1-ALS. The commercial Roche Elecsys electrochemiluminescence immunoassay (ECLIA) reads 6- to 8-fold lower than SIMOA, and its clinical utility in ALS is uncharacterized. We assessed whether ECLIA NfL retains this correlation in routine care and whether GFAP or S-100B helps. Methods: Retrospective analysis of 58 chart-confirmed ALS patients at Georgetown University Hospital (2022-2026), biomarkers on the LabCorp Roche Elecsys ECLIA. The NfL-ALSFRS-R correlation was assessed where both measures fell within matching windows; serial NfL, in patients with repeat draws. Results: First-per-patient NfL median was 7.06 pg/mL (IQR 4.06-17.30; CV 99%). Among 31 patients with matched NfL and ALSFRS-R decline rates, Spearman r = 0.704; within 90 days (n = 17), r = 0.809 (both p < 0.0001). Fast progressors (n = 8) had mean NfL 17.10 pg/mL versus 4.64 in slow progressors (n = 21), a 3.7-fold separation. Serial NfL captured rising trajectories and stable low values. GFAP rose within patients but tracked neither progression rate, disease stage, nor motor-neuron predominance; S-100B added no value. Conclusions: Commercial ECLIA brings NfL into routine ALS care; its prognostic correlation with progression rate survives real-world fragmentation. The actionable unit is the longitudinal trajectory, not the single value, read against platform-specific reference ranges and clinical context (genotype, onset, stage). GFAP and S-100B add little. Keywords: amyotrophic lateral sclerosis, neurofilament light chain, biomarkers, implementation science, ECLIA, GFAP, monitoring, tofersen, real-world data

Amyotrophic lateral sclerosis (ALS) is a fatal, rapidly progressive neurodegenerative disease of motor neurons for which therapeutics are limited. Improved biomarkers are imperative to improve patient care and therapeutic development. Here, we employed 35-plex isobaric tandem mass tag labeling based on isobutyl-proline reporter group (TMTpro) to perform unbiased proteomic analysis of cerebrospinal fluid (CSF) and plasma from control (n= 28, n= 31) and sporadic ALS (sALS) (n= 39, n= 41), from the Target ALS Global Natural History Study (TALS GNHS). We identified 2,875 proteins in CSF and 1,118 proteins in plasma and identified known and novel differentially expressed proteins (DEPs) between controls and sALS, some of which were orthogonally validated using immunoassay. Comparison of TMTpro-MS and Olink proximity extension assay proteomics revealed common and non-overlapping differentially expressed proteins illustrating strengths unique to each platform. This initial cross-sectional proteomic study of biofluids from the TALS GNHS, with unrestricted availability of study results to the research community, highlights the potential of this resource as a potent platform for ALS biomarker discovery.

BackgroundDysregulated peptidyl deiminase 2 (PAD2) and aberrant protein citrullination (PC), a posttranslational modification (PTM), are involved in various inflammatory and neurodegenerative diseases. We previously showed in transgenic mice and postmortem human tissues that PC and PAD2 are altered in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by motor neurons loss, paralysis, and death. Herein, we investigated the role of PAD2 in ALS by PAD2 knockout in a SOD1-ALS mouse model. MethodsTo investigate the role of PAD2-induced citrullination in ALS pathogenesis, we generated PAD2 knockout (PAD2KO) in SOD1G93A ALS mouse model and investigated the consequent modulation on the neuropathology and clinical symptoms, using molecular biology techniques such as qPCR, Western blotting, confocal microscopy, and electron microscopy. Additionally, we identified C3 as being citrullinated in human ALS using ionFinder. ResultsOur results show that PAD2KO blocked the increased PC and reduced myelin basic protein (MBP) aggregates in the ALS model. PAD2KO also improved motor neuron survival and the integrity of myelin, axons, and neuromuscular junctions, and reduced microgliosis in the white matter and C3 protein levels in astrocytes. Clinically, data from monitoring the body weight changes suggests that PAD2KO modulates the course of the disease in the ALS mouse model, accelerating the onset while slowing the progression after the onset, and modestly extending the survival of male mice. ConclusionThese results show that PAD2 is responsible for the increased PC in ALS and PC contributes to neuroinflammation and degeneration of motor neurons and myelinated axons. The modest modulation of the disease phenotype suggests that the role of PC in ALS is complex, involving altered PC in numerous proteins and in multiple cell types. Future studies are needed to investigate how PC modulates individual protein functions in various cell types to understand the contribution of PC to ALS pathogenesis.

Amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) is a fatal neurodegenerative disorder that was once hyperendemic in the island of Guam (Mariana Islands, US) and a few other Pacific locales. Despite extensive investigations into its origins, the etiology of ALS/PDC remains unclear. ALS/PDC is, at the neuropathology level, characterized by tau-dominant multiple proteinopathy in brain and spinal cord. It was recently reported that Guam ALS/PDC brain extracts exhibit tau seeding activity in fluorescence resonance energy transfer (FRET)-based biosensor cells. To build upon those findings and explore the nature of tau seeds in ALS/PDC in more detail, we used an alanine mutational scanning (Ala scan) approach to determine the seeding profile of tau in nervous tissues of Guam ALS/PDC cases. First, we confirmed the detection of tau seeding activity in ALS/PDC brain samples in tau biosensor cells. Notably, we could also detect potent tau seeding activity in spinal cord. Subsequent Ala scan assays demonstrated that ALS/PDC tau displays an aggregate incorporation pattern that resembles that of chronic traumatic encephalopathy (CTE)-type tau. This result is consistent with recent electron cryo-microscopy studies of tau, which revealed that ALS/PDC tau filaments are predominantly of the CTE-type. The structural characteristics and seeding behavior of ALS/PDC tau, as well as the regional distribution of tau pathology at post-mortem, suggest that ALS/PDC is a CTE-like tauopathy. Significance StatementNeurodegenerative tauopathies are characterized by proteinaceous deposits containing microtubule-associated tau in nervous tissue. Emerging evidence suggests that disease-associated tau proteins adopt abnormal, self-propagating conformations characteristic of prions. Here, we employed alanine mutational scanning (Ala scan) to determine the nature of prion-like tau seeds in ALS/PDC, a mysterious disorder that occurred formerly in high incidence in certain regions in the western Pacific. We show that the Ala scan incorporation profile of ALS/PDC tau is similar to that of abnormal tau proteins in chronic traumatic encephalopathy (CTE). The findings lend support to the idea that ALS/PDC can be classified structurally as a CTE-like tauopathy. This work may have important implications for our understanding of ALS/PDC as well as common neurological disorders beyond the Pacific.

Blood-based proteomic profiling is now widely applied in neurodegenerative and neuroinflammatory disease, yet the choice between serum and plasma remains poorly characterised for high-multiplex platforms. Many legacy biobanks hold mainly serum, whereas most current NUcleic-acid-Linked Immuno-Sandwich Assay (NULISA) studies use plasma. We compared the 130-protein NULISAseq central nervous system (CNS) Disease Panel head-to-head in matched serum and plasma collected at the same draw from 62 participants (30 neurodegenerative, 19 demyelinating, 13 healthy controls). Agreement was measured with Spearman correlation (rho), Lin's concordance correlation coefficient (CCC), the intraclass correlation coefficient (ICC) and the mean paired serum-to-plasma difference (dNPQ). Concordance was moderate to high: 123 of 130 proteins reached significance and 18 reached rho >= 0.90, with a median rho of 0.72 (range 0.10-0.988). Proteins fell into three tiers. Cytoskeletal markers (NEFH rho=0.988; NEFL rho=0.947) and glial GFAP (rho=0.949, |dNPQ|<0.5) were interchangeable between matrices. Phosphorylated tau (pTau) species retained excellent rank concordance but carried a systematic plasma-greater-than-serum offset (pTau-181 rho=0.869, dNPQ=+0.67; pTau-217 rho=0.846, dNPQ=+0.64; pTau-231 rho=0.885, dNPQ=+0.89). Platelet-derived analytes (CD40LG rho=0.102, dNPQ=-4.74; BDNF rho=0.223, dNPQ=-2.69) and intracellular synaptic proteins (NRGN, SNAP25, ENO2) diverged markedly. For most clinically relevant neurodegeneration markers, especially cytoskeletal and glial proteins, serum is a valid substitute for plasma; absolute thresholds for phosphorylated tau and amyloid peptides require matrix-specific calibration, and platelet-sensitive analytes cannot be compared across matrices without strictly standardised pre-analytical conditions.

Nuclear depletion and cytoplasmic aggregation of TDP-43 occur in [~]97% of amyotrophic lateral sclerosis (ALS) cases and disrupt RNA processing through aberrant cryptic exon inclusion. Existing cellular models rely on partial knockdown, TARDBP mutations, or pharmacological stress, each with limitations. Here, we generated homozygous TARDBP-knockout human iPSC lines using CRISPR-Cas9 genome editing and differentiated them into spinal motor neurons (MNs). Knockout MNs demonstrated [~]16-fold lower differentiation efficiency than isogenic controls but retained neuronal marker expression. TDP-43 loss induced widespread cryptic exon inclusion and depletion of STMN2, UNC13A, and G3BP1. Integration of the CUTS splice biosensor yielded up to 4.5-fold cryptic GFP induction in knockout MNs, providing a reporter-based readout of TDP-43 dysfunction. Further, we validated the cardiac glycosides digoxin and ouabain as modulators of bortezomib-induced TDP-43 pathology. This genetically defined iPSC-derived MN model provides a platform for mechanistic and therapeutic interrogation of TDP-43-driven neurodegeneration in ALS.

Background and Objectives To investigate the associations of blood-based tau biomarkers with clinical, electrophysiologic and prognostic measures in amyotrophic lateral sclerosis (ALS), and to determine whether they reflect distinct disease-related processes. Methods We studied 119 patients with ALS from a longitudinal observational cohort. Plasma and serum p-tau181, p-tau217, p-tau231, brain-derived tau (BD-tau), NfL and GFAP were measured using Lumipulse and Simoa assays. Associations with demographic variables, disease severity (ALSFRS-R and slow vital capacity), lower motor neuron (LMN), muscle involvement (creatine kinase [CK] and high-sensitivity cardiac troponin T [hs-cTnT]), disease progression and survival were assessed using multivariable models. Results Tau-related biomarkers, specifically p-tau217 and BD-tau, were associated with greater cross-sectional disease severity, reflected by lower ALSFRS-R scores. Plasma and serum p-tau181, p-tau217, p-tau231, and BD-tau were associated with higher CK and hs-cTnT, whereas p-tau181 and p-tau231 were also associated with greater LMN involvement. In contrast, NfL and GFAP were not associated with muscle or LMN involvement. Across analytical platforms, plasma and serum NfL were associated with faster ALSFRS-R decline and shorter survival. NfL was the only biomarker independently associated with both disease progression and survival. Discussion Blood biomarkers capture distinct dimensions of ALS. Tau-related biomarkers are associated with cross-sectional disease severity, LMN involvement and muscle injury, whereas NfL primarily reflects disease progression and survival. These findings support the complementary use of tau-related biomarkers and NfL for ALS phenotypic characterization and prognosis assessment.

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive motor neuron (MN) loss, muscle atrophy and paralysis. Although traditionally considered a MN-specific disease, accumulating evidence supports a crucial contribution of skeletal muscle pathology to disease onset and progression. Except for specific mutations, to date there is no effective treatment for ALS. FOXO transcription factors regulate programs of atrophy, metabolism and stress response in skeletal muscle, and their inhibition has shown beneficial effects in cellular and Drosophila models of ALS. MethodsIn this study, we investigated whether pharmacological FOXO inhibition (iFOXO) could modify disease progression and muscle pathology in female hSOD1G93A mice. Mice received daily oral administration of iFOXO starting at presymptomatic (P50; n=5 per group) or symptomatic (P90; n=9 mice per group) stages until end-stage. Body weight was monitored longitudinally, and motor performance was evaluated using grip strength and hanging-wire tests. Tibialis anterior and soleus muscles, representing fast- and slow-twitch muscles respectively, were analyzed by histology and immunofluorescence to assess fiber atrophy, fibrosis, lipid accumulation, satellite cell pool and fiber type composition. Quadriceps muscles (n=3 per group) were used for RNA-seq analysis. ResultsWhile histological analyses revealed severe fiber atrophy and increased fibrosis in hSOD1G93A mice, satellite cell numbers were preserved or mildly increased in a muscle and treatment onset dependent manner. iFOXO treatment did not improve motor performance, survival or attenuate muscle atrophy. Transcriptomic profiling indicated that genotype was the predominant driver of gene expression changes, while iFOXO produced only subtle, treatment onset dependent effects on pathways related to oxidative stress responses, mitochondrial function and adaptive metabolism. ConclusionOverall, FOXO inhibition alone showed limited therapeutic benefit in the hSOD1G93A ALS mouse model. These findings highlight the dominant influence of ALS driven molecular alterations over pharmacological modulation and emphasize the need for combinatorial therapeutic strategies targeting multiple disease mechanisms, including those preserving nerve health.

STRUCTURED ABSTRACT Purpose of Research: The generation of iPSC lines expressing 21, 56 and 79 glutamine repeats within the HTT protein and homozygous KO of HTT in the KOLF2.1J background as an additional disease series within the iPSC Neurodegenerative Disease Initiative (iNDI) collection. Major Findings: All iPSCs, even those expressing long repeats of 79Q or HTT KO, were capable of differentiating to striatal and cortical neurons, astrocytes and microglia using established protocols. General quality control stains and morphological analyses are described for each differentiation. A selected set of assays were carried out on differentiated cells; expanded repeat expressing astrocytes showed altered expression of astrocyte protein markers and morphological characteristics, and striatal neurons showed altered DARPP-32/CTIP2 colocalization. mRNAseq carried out for striatal neurons showed high similarities in gene expression changes between 79Q and KO lines compared to the unexpanded repeat. Conclusions: The KOLF2.1J isogenic CAG repeat series serves as a community resource to study HD mechanisms with the potential for direct comparison across other neurodegenerative diseases through the iNDI collection.

Aims/hypothesisQuantitative nanoscale analysis of insulin secretory granules (ISGs) in human pancreatic tissue has been limited by the lack of imaging methods that combine high resolution with large-scale sampling. We aimed to establish expansion microscopy (ExM) as a platform for in situ, quantitative analysis of ISG organisation in human {beta}-cells and to assess whether type 2 diabetes (T2D) is associated with alterations in granule size, abundance or spatial organisation. MethodsWe applied Magnify ExM to PFA-fixed, paraffin-embedded pancreatic tissue sections from 6 human donors, 3 non-diabetic (ND) and 3 T2D, enabling super-resolution optical imaging of insulin-labelled granules. Insulin-positive structures were segmented and analysed using a morphometric pipeline to quantitatively assess size, shape and spatial features. Granule clustering was quantified based on combined area and roundness criteria. ResultsThe diameter distribution of highly circular granules was similar between ND and T2D samples and estimates of granule number per cell indicated only a modest reduction in T2D ([~]25%). In contrast, mapping insulin-positive structures in a roundness-area space revealed a marked enrichment of large, irregular objects consistent with granule clustering in T2D. The fraction of clustered granules was significantly increased in T2D and strongly inversely correlated with insulin stimulation index (r = -0.85). Conclusions/interpretationThese results establish expansion microscopy as a powerful platform for quantitative nanoscale analysis of human pancreatic tissue and identify altered spatial organisation of insulin granules, rather than marked granule depletion, as a prominent feature associated with {beta}-cell dysfunction in T2D. Research in contextO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LI{beta}-cell dysfunction in type 2 diabetes is often attributed to reduced insulin content or {beta}-cell loss. C_LIO_LIInsulin secretory granules (ISGs) have been characterised ultrastructurally, but quantitative analysis in human tissue remains limited. C_LIO_LISuper-resolution approaches, including expansion microscopy, are emerging tools for nanoscale imaging in biological tissues. C_LI What is the key question?O_LIIs {beta}-cell dysfunction in type 2 diabetes associated with depletion of insulin granules or with altered spatial organisation? C_LI What are the new findings?O_LIInsulin granule size distribution is largely preserved in type 2 diabetes, with only a modest reduction in granule number per cell. C_LIO_LIA significant increase in insulin granule clustering is observed in diabetic {beta}-cells. C_LIO_LIGranule clustering is strongly inversely correlated with insulin secretion in the same donor tissues. C_LI How might this impact on clinical practice in the foreseeable future?O_LIIdentifying altered granule organisation as a feature of {beta}-cell dysfunction may help refine the understanding of disease mechanisms and guide future strategies targeting {beta}-cell function. C_LI

Lewy body diseases, including Parkinsons disease (PD) and dementia with Lewy bodies (DLB), are defined by neuronal accumulation of misfolded -synuclein (-Syn), yet the ultrastructural diversity of these inclusions across brain regions and disease contexts remains unclear. Here, we applied large-scale correlative light and electron microscopy (CLEM) to map -Syn pathology across cortical regions (entorhinal cortex, ENT; anterior cingulate cortex, AC; hippocampal CA2 region) and substantia nigra (SN) in clinically and pathologically confirmed PD and DLB donors. We identified pronounced regional heterogeneity in Lewy pathology, with cortical inclusions showing diverse maturation stages at the ultrastructural level, ranging from low-density fibrils interspersed with organelles to highly compact fibrillar inclusions. In the SN of DLB donors, we observed the full range of classical nigral LB morphologies previously described in PD. We additionally characterized diverse neuritic -Syn pathologies in DLB and identified a distinct population of electron-dense, degenerating, -Syn-positive cortical neurons not previously reported. Importantly, we found no significant difference in LB ultrastructure between PD and DLB in either cortical or nigral pathology. In contrast, quantitative analysis of >10,000 mitochondria revealed disease- and region-specific signatures of altered mitochondrial homeostasis. PD showed increased mitochondrial density and enlargement in the SN, whereas DLB showed increased mitochondrial density only in the ENT. Mitochondrial enlargement was exclusive to PD. These findings indicate that LB ultrastructure alone does not distinguish PD from DLB; instead, region-specific mitochondrial phenotypes may better reflect disease identity and regional susceptibility. Overall, we provide a high-resolution framework for human Lewy pathology in PD and DLB, revealing that ultrastructural responses to -Syn pathology are driven primarily by neuronal identity and regional vulnerability. Our results highlight the need for disease- and region-specific models that capture human phenotypes to advance mechanistic understanding and therapeutic targeting of synucleinopathies.

BackgroundAmyotrophic lateral sclerosis (ALS) is characterized by profound metabolic reprogramming, yet the lack of biomarkers for specific druggable targets remains a major hurdle for precision medicine. We hypothesized that peripheral lipid biosynthetic signatures could serve as both prognostic indicators and a roadmap for identifying novel disease-modifying targets. MethodsWe assessed serum fatty acid (FA) metabolic pathways in two independent longitudinal cohorts (n = 37 and n = 38) using high-dimensional CoxBoost modeling. Primary outcomes were survival and functional staging milestones, including non-invasive ventilation and gastrostomy. The biological relevance of the identified candidate was further assessed through correlation with plasma neurofilament light-chain (NfL) levels. Causality and therapeutic potential were validated in Drosophila melanogaster models of TDP-43 proteinopathy via genetic ablation and pharmacological inhibition. ResultsOur multi-parametric model, comprising two clinical variables and the estimated ELOVL6 (elongation of very long-chain fatty acids protein 6) activity, demonstrated robust prognostic accuracy (Unos C 0.69) across both cohorts; ELOVL6 activity served as a strong independent predictor of mortality and functional decline. Notably, high ELOVL6 activity significantly correlated with elevated plasma NfL levels (p < 0.01), linking peripheral elongation imbalances to central axonal damage. In Drosophila, ELOVL6 overactivation was identified as a conserved pathological consequence of TDP-43 dysfunction, characterized by an increased C18:0/C16:0 ratio in both loss-of-function and gain-of-function models. Inhibition of ELOVL6, either genetically or pharmacologically, rescued neuromuscular junction integrity, prolonged survival, and significantly reduced pathological TDP-43 phosphorylation in glial models. ConclusionThese findings position ELOVL6 as a promising modifiable metabolic node with potential for disease-modifying intervention in ALS. Beyond its potential utility for identifying high-risk metabolic profiles and assisting in prognostic counseling, ELOVL6 bridges systemic lipid dysregulation with TDP-43 proteinopathy. Targeting this pathway offers a dual opportunity: as a biological marker to supplement clinical staging and as a druggable enzymatic target to ameliorate motor neuron degeneration. HIGHLIGHTSO_LISystemic ELOVL6 activity is a robust independent predictor of ALS survival. C_LIO_LIHigh ELOVL6 levels correlate with plasma NfL and functional decline. C_LIO_LIInhibition of ELOVL6 rescues NMJ integrity and survival in Drosophila models. C_LIO_LIPharmacological targeting of ELOVL6 reduces glial TDP-43 phosphorylation. C_LIO_LIELOVL6 represents a druggable metabolic node linking lipids to proteinopathy. C_LI

The clinical and molecular heterogeneity observed in amyotrophic lateral sclerosis (ALS) presents a challenge for diagnosis, prognosis, and treatment. RNA sequencing of post-mortem brain samples from ALS patients has identified several subtypes with distinct molecular signatures. We sought to evaluate these subtypes across diverse tissues and datasets and assess the feasibility of supervised machine learning models for sample classification. Unsupervised clustering and pathway analysis were performed to confirm the presence of ALS subtypes in motor cortex samples. Three machine learning strategies were then used to create models based on post-mortem motor cortex expression data of 112 people with ALS from the London Neurodegenerative Diseases Brain Bank. These models were subsequently improved through feature selection and evaluated in independent cohorts from motor cortex (n = 257, NYGC ALS Consortium) and blood (n = 96, Macquarie University Neurodegenerative Disease Biobank) samples. Multi-class linear discriminant analysis (LDA) models were then used for subtype classification. Clustering of ALS post-mortem motor cortex samples confirmed the presence of three subtypes: neuroinflammation (ALS-Neu), extracellular matrix organisation and muscle contraction (ALS-OxA), and synaptic and neuropeptide signalling (ALS-SNs). Among all machine learning strategies, random forests produced the most accurate and stable models for binary classification ([~]93% accuracy across the three subtypes). After feature selection, random forest models were able to classify samples from an independent post-mortem motor cortex cohort in their respective subtypes (AUC of [~]0.98 across the three subtypes). When these models were evaluated in blood using LDA, we found consistent clustering patterns, with samples aligning in the same subtype regions of the post-mortem motor cortex samples, with ALS-SNs being the subtype in which samples were classified with the highest confidence (LDA class probability [~]86%). Moreover, classification for this subtype improved when blood samples were collected closer to death. Our findings support the presence of three gene expression-based ALS subtypes in motor cortex samples and the utility of machine learning strategies for subtype classification. We also observed that the subtypes identified in the brain partially match those in the blood, with samples from the late stages of the disease more likely to be correctly predicted into the ALS-SNs cluster. This suggests a longitudinal effect in subtype identification that requires further investigation.

Background Despite WHO grade and IDH status, significant survival differences remain in diffuse gliomas. We hypothesized that a brain-aging transcriptomic signature, reflecting neuroinflammation, myeloid infiltration, and synaptic loss, would independently predict survival and allow for molecular reclassification. Methods A neurodegeneration score was derived via PCA of brain MRI volumes from 1,057 OASIS-3 subjects and projected onto 888 TCGA-LGG/GBM (discovery) and 693 CGGA gliomas (validation). A 14-gene signature of glial/myeloid (GFAP, AQP4, TYROBP, TREM2, C1QA, CD68, ITGAM) and neuronal (SYP, DLG4, GRIN1, GRIA1, SNAP25, SYN1, RBFOX3) genes were computed. Elastic-net Cox regression identified a 3-gene panel (C1QA, CD68, GRIA1). Kaplan-Meier, multivariate Cox, decision curve, and single-cell RNA-seq analyses were performed. Results High brain-aging scores predicted poorer overall survival (p < 0.0001) and remained an independent prognostic factor after adjusting for WHO grade and IDH status (z = 4.72, p < 0.001); chronological age was non-significant (p = 0.231). In IDH-mutant gliomas, significance was confirmed in both cohorts (TCGA p = 0.027; CGGA p < 0.0001). Bidirectional reclassification showed high-risk Grade 2 tumors with Grade 3-like survival (p = 0.00089), and indolent Grade 3 tumors resembling Grade 2 by Ki-67. Single-cell RNA-seq confirmed macrophage localization of signature genes; DCA demonstrated net benefit over grade alone at 5-30% probability thresholds. Conclusions A brain-aging transcriptomic signature independently predicts glioma survival beyond WHO grade and IDH status, validated in an independent Chinese cohort, with clinical utility for identifying high-risk Grade 2 and sparing over-treatment of indolent Grade 3 tumors.

Guillain-Barre syndrome is an acute immune-mediated polyradiculoneuropathy with heterogeneous outcomes and limited molecular biomarkers for diagnosis, disease monitoring, and prognosis. To elucidate the circulating proteomic profile of this disorder and identify candidate biomarkers associated with disease activity and recovery, we measured over 6,500 proteins using an aptamer-based proteomic platform. We analysed paired, longitudinal sera from 20 patients at disease onset and one-year follow-up, alongside 15 healthy controls. Unbiased differential protein abundance and gene-set enrichment analyses were performed. Candidate proteins were validated using conventional immunoassays in a cohort including healthy and disease controls. We identified 39 differentially abundant proteins between the acute and recovery phases and 248 proteins altered in acute Guillain-Barre syndrome compared to controls. The acute phase was characterised by a marked enrichment in systemic immune cascades and muscle sarcomere proteins, alongside a significant depletion of axonal adhesion molecules. Serum amyloid A1 (SAA1) emerged as the most strongly increased protein in the acute phase. Validation through independent immunoassays confirmed robust serum amyloid A elevations at disease onset relative to the one-year recovery phase, healthy controls, and relevant post-infectious and neuromuscular disease controls (acute disseminated encephalomyelitis and myasthenia gravis), underscoring a peripheral nerve-specific inflammatory response. Furthermore, unexpected elevations of cardiac troponin T (cTnT) were observed at disease onset. Clinical validation using high-sensitivity assays demonstrated that cTnT exceeded the diagnostic 99th percentile upper reference limit in 25.5% of acute Guillain-Barre syndrome patients. A similarly high frequency of elevation in the myasthenia gravis disease control group (42.1%) suggests these increases predominantly reflect neuromuscular damage rather than myocardial injury. Finally, Mendelian randomisation provided causal genetic evidence linking specific systemic proteins to disease susceptibility, identifying robust roles for SERPING1 (plasma protease C1 inhibitor), CNDP1 (an antioxidant protein), and CRISPLD2 (a lipopolysaccharide-binding protein that regulates endotoxin function). Together, this comprehensive proteomic characterisation reveals distinct, stage-specific molecular signatures in Guillain-Barre syndrome. Importantly, it suggests SAA1 as a robust marker of acute peripheral nerve inflammation and challenges the conventional interpretation of elevated cTnT in severe neuropathies and neuromuscular disorders. Furthermore, this work provides a novel dataset to explore future targeted therapeutic development in Guillain-Barre syndrome.