Neuropathology and Applied Neurobiology

Amyotrophic lateral sclerosis (ALS) is a motor neuron disease, leading to progressive muscle weakness and motor impairment. Growing evidence indicates that cerebellar Purkinje cells, which play a central role in motor coordination, are also affected in ALS. However, it is unclear whether the molecular events that initiate neurodegeneration in these ALS-relevant motor-controlling neurons are shared or distinct. Here, we used a MATR3 S85C knock-in (KI) mouse model of early-stage ALS with stage-specific motor phenotypes and selective vulnerability of motor neurons and Purkinje cells to decipher the molecular events underlying neurodegeneration in these two neuronal populations. We found that a profound reduction in detectable MATR3 S85C immunoreactivity (hereafter referred to as MATR3 loss) in both motor neurons and Purkinje cells precedes the onset of motor dysfunction and neuropathology, implicating MATR3 loss as the earliest detectable molecular event. Our bulk cerebellar RNA profiling and motor neuron-specific RNA profiling data at the onset of MATR3 loss revealed distinct molecular signatures. In the cerebellum, Ngfr expression emerged in Purkinje cells before the onset of neuronal loss and remained elevated throughout the disease course. This increase was accompanied by activation of the JNK-mediated cell death pathway. In the motor neurons, elevated Fgf21 and integrated stress response (ISR) gene expression were the first to be observed and persisted throughout disease progression, consistent with previous findings in SOD1 mouse models. Our findings provide mechanistic insights into the initiation of neurodegeneration in ALS-relevant motor-controlling neurons and implicate potential neuron type-specific targets for future therapeutics.

INTRODUCTION: Biomarkers for monitoring disease activity and treatment response in peripheral neuropathies remain limited. Big tau, a high-molecular-weight isoform of tau, is predominantly expressed in the peripheral nervous system (PNS). We investigated serum levels of big tau, brain-derived tau (BD-tau), and neurofilament light chain (NfL) in peripheral neuropathies, multiple sclerosis (MS), Alzheimer disease (AD), and healthy controls (HC). METHODS: Ultra-sensitive blood-based assays run on an HD-X Single Molecule Array analyser (Quanterix) were used to measure big tau and BD-tau in serum from patients with Guillain-Barr&eacute syndrome (GBS, n=81), Miller Fisher syndrome (MFS, n=20), Charcot-Marie-Tooth disease (CMT, n=102), chronic inflammatory demyelinating polyneuropathy (CIDP, n=43), MS (n=159), AD (n=20), and HC (n=41). NfL was measured in patients with neuropathies using an SR-X Single Molecule Array analyser (Quanterix). RESULTS: Serum big tau levels were higher in GBS than in AD (11.4 vs 2.4 pg/mL, p<0.0001) and MS (11.4 vs 9.0 pg/mL, p=0.01), and similar to CIDP and CMT. Contrarily, serum BD-tau levels in GBS were higher than in CIDP (3.0 vs 2.3 pg/mL, p=0.006) and MS (3.0 vs 1.7 pg/mL, p<0.0001), but similar to CMT, and lower than in AD (3.0 vs 9.8 pg/mL, p<0.0001). Serum NfL levels were higher in GBS than in CIDP (32.5 vs 13.0 pg/mL, p=0.0002), CMT (32.5 vs 12.3 pg/mL, p<0.0001), and HC (32.5 vs 7.6 pg/mL, p<0.0001). Compared with GBS, MFS patients showed higher BD-tau (12.7 vs 3.0 pg/mL, p=0.003), lower big tau (5.4 vs 11.4 pg/mL, p=0.002), and higher NfL levels, although the latter did not reach statistical significance (118.3 vs 32.5 pg/mL, p=0.16). The NfL/big tau ratio was significantly higher in MFS than in GBS, CIDP, and CMT. In GBS, BD-tau correlated with early clinical severity (MRC at 1 week; I-RODS at 4 weeks; maximum GBS-DS and GBS-DS at 4 weeks), whereas neither tau biomarker showed long-term clinical correlations. Higher BD-tau and big tau levels were associated with the need for mechanical ventilation (BD-tau: 8.6 vs 2.9 pg/mL, p=0.019; big tau: 19.7 vs 10.7 pg/mL, p=0.007), while higher BD-tau levels were associated with mortality (10.9 vs 2.9 pg/mL, p=0.003). CONCLUSIONS: Higher big tau levels in peripheral neuropathies than in CNS diseases support its role as a PNS-specific biomarker. In MFS, increased serum BD-tau, reduced big tau, and an elevated NfL/big tau ratio suggest CNS involvement with relative preservation of the PNS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive dysfunction and loss of upper and lower motor neurons. Although motor neuron degeneration ultimately drives paralysis, neuronal dysfunction may precede cell death by a prolonged interval, suggesting that vulnerable neurons engage stress-adaptive programs that permit survival despite impaired function. Cellular senescence represents one such persistent stress response and has increasingly been implicated in neurodegenerative disease, including disorders associated with TDP-43 pathology. Here, we investigated whether senescence-associated molecular states are present in vulnerable motor neurons in ALS and whether they differ according to anatomical region and phosphorylated TDP-43 (pTDP-43) pathology. Postmortem primary motor cortex, cervical spinal cord, and lumbar spinal cord were obtained from the Department of Veterans Affairs Biorepository Brain Bank from individuals with ALS classified as pTDP-43-positive or pTDP-43-negative, together with non-ALS controls. Targeted bulk transcriptomic profiling was combined with GeoMx Digital Spatial Profiling of individual motor neurons to characterize disease-, region-, and pathology-associated molecular phenotypes while preserving anatomical context. Across ALS cases, we identified alterations in pathways related to cell-cycle regulation, RNA processing, mitochondrial function, proteostasis, inflammation, and synaptic signaling. These signatures varied by anatomical region and pTDP-43 status, indicating substantial heterogeneity in the molecular response to ALS pathology. Despite these differences, both ALS groups exhibited convergent proteomic and transcriptomic features associated with cellular senescence. These findings identify senescence-associated molecular states within vulnerable neuronal populations in ALS and support a model in which persistent stress adaptation may permit neuronal survival while contributing to progressive cellular dysfunction. This spatially resolved analysis links neuronal phenotype to anatomical and pathological context and supports further evaluation of senescence-associated pathways as therapeutic vulnerabilities in ALS.

Background: The Boston Criteria v2.0 represent the gold standard for diagnosing Cerebral Amyloid Angiopathy (CAA), but their application is currently precluded in mixed small vessel disease (SVD), where deep and lobar hemorrhages coexist. The aims of this study are: (i) to determine which cerebrospinal fluid (CSF) biomarker (A{beta}42, A{beta}40, A{beta}42/40 ratio) is the best candidate to support the CAA diagnosis; (ii) to define a data-driven cut-off, and (iii) to explore if a biomarker-integrated classification significantly improves the phenotypical concordance with the suspected predominant SVD (CAA vs. arteriosclerosis). Methods: We analyzed data from a retrospective multicenter cohort of patients with suspected CAA, defined as probable CAA (Boston criteria v2.0) but allowing deep hemorrhagic lesions, and with available CSF biomarkers. We visually quantified MRI-visible SVD markers (e.g., cerebral microbleeds [CMB], cortical superficial siderosis [cSS], lacunes) and their association with MRI-visible SVD features. We employed a Gaussian Mixture Model (GMM) to identify a data-driven threshold for amyloid positivity (A+). Then, we compared the prevalence of MRI-visible manifestations of SVD between subgroups applying different frameworks, namely the current MRI-based classification (probable CAA vs. mixed SVD) and a CSF biomarker-integrated classification (A+ vs. A-). Results: We enrolled 121 patients (age: 72 [66-77] years; 60% probable CAA, 40% mixed SVD with suspected CAA). The CSF A{beta}42/40 ratio showed a bimodal distribution and consistent associations with all CAA-specific radiological features. The CSF biomarker-integrated reclassification, particularly using the GMM cut-off, significantly improved the distinction between subgroups regarding CAA- and arteriosclerosis-related MRI features (e.g., cSS presence: probable CAA vs. mixed SVD: aOR=2.84 [95%CI 1.27-6.39], p=0.011; A+ vs. A-: aOR=12.68 [95%CI 4.31-37.32], p<0.001; deep lacunes presence: probable CAA vs. mixed SVD: aOR=0.20 [95%CI 0.08-0.50], p<0.001; A+ vs. A-: aOR=0.04 [95%CI 0.01-0.11], p<0.001). Notably, patients classified as A+ never demonstrated more than four deep CMBs. Discussion: A CSF biomarker-integrated classification may improve the classification of CAA compared with the current MRI-based framework. These findings are cohort-specific and would benefit from further validation, especially with a neuropathological reference. Still, these results support a future transition toward an integrated biological-radiological framework, which may refine in vivo CAA diagnosis, particularly in mixed SVD.

Frontotemporal lobar degeneration (FTLD) is a common cause of early-onset dementias marked by progressive declines in behavior, cognition, and/or movement. FTLD neuropathologies, including TDP-43 proteinopathies and primary tauopathies, do not have reliable fluid biomarkers for in-vivo diagnosis nor biomarkers that directly correspond to FTLD clinical features. Fluid biomarkers that forecast and track FTLD clinical progression, irrespective of pathology or clinical syndrome, are urgently needed to improve clinical trial designs. We previously identified the ratio between two cerebrospinal fluid (CSF) synaptic proteins, YWHAG and NPTX2, as a prognostic biomarker of cognitive decline in Alzheimers disease (AD), independent of core AD pathologies, amyloid and tau. Here, we evaluate its utility in sporadic and familial FTLD compared to other neurodegenerative diseases. Using CSF assays from four independent cohorts (UCSF-MAC, ALLFTD, GENFI, PDBP), we find CSF YWHAG:NPTX2 is substantially elevated across all sporadic and familial FTLD syndromes, AD, and dementia with Lewy bodies. CSF YWHAG:NPTX2 robustly correlates with clinical severity across sporadic and familial FTLD (C9orf72, GRN, or MAPT mutations), independent of current gold-standard neurodegeneration biomarker neurofilament light (NfL). In presymptomatic familial FTLD, CSF YWHAG:NPTX2 is estimated to rise roughly a decade before symptom onset and improves prediction of imminent symptomatic conversion by 1.7-fold compared to plasma NfL alone, more than halving the estimated sample size required for an FTLD prevention clinical trial. These findings underscore CSF YWHAG:NPTX2 as a cross-dementia synaptic biomarker of cognitive decline and a promising biomarker for disease staging and prognosis across the clinico-pathological continuum of FTLD.

Molecular staging of Alzheimer's disease (AD) increasingly defines transition boundaries along single-cell pseudo-progression trajectories, yet whether such boundaries reproduce across brain regions, cohorts and molecular modalities is rarely tested. We present a permutation-controlled audit that combines nine boundary-detection algorithms with a fixed marker panel and four orthogonal reproducibility axes-algorithmic consensus, region, cohort and modality. On synthetic data with planted ground-truth boundaries the audit reaches 100% sensitivity and 94% specificity, rejecting four distinct artefact classes each by a different axis. Applied to the Seattle Alzheimer's Disease Brain Cell Atlas middle temporal gyrus, it localizes a transition that is robust across algorithms and recovered in most cell types but does not generalize: its leading marker is attenuated or absent in prefrontal cortex, entorhinal cortex and cerebrospinal fluid, and an apparent cross-region conservation of glial metabolic genes proves to be a global-expression offset rather than a shared program. The same audit nonetheless certifies an externally validated marker (astrocytic PTGDS) as reproducible across regions and modalities, showing that it separates generalizable anchors from dataset-specific ones rather than rejecting all signals. We provide this four-axis audit as a transferable, code-available standard to apply before a trajectory boundary is read as a biological stage, in AD and other progressive proteinopathies.

Background: Multiple sclerosis (MS) is a chronic neurodegenerative disorder in which inflammatory demyelinating lesions affect the brain, optic nerve and spinal cord. MS lesion formation is accompanied by profound changes to blood vessels, including the density of PDGFR{beta}+ mural cells, historically identified as pericytes. Intriguingly, in recent years, single-cell and lineage tracing studies have shown that the PDGFR{beta}+ cell population is heterogeneous, comprising both pericytes and perivascular fibroblasts. Yet, due to their overlapping expression profiles, the spatial distribution of these cell populations in MS lesions remains poorly understood. Methods: We employed multiplex immunohistochemistry for endothelial cells (CD31), basement membrane (laminin), fibroblasts (PDGFR{beta}, COL1A1, SMA), pericytes (PDGFR{beta}, SLC6A12) and immune cells (CD45, CD68) to characterize the spatial localization of fibroblasts and pericytes in MS lesions, and how this relates to perivascular space enlargement and immune cell presence. Results: We analysed 17633 individual vessels across 5 control white matter, 5 normal-appearing white matter, 4 active and 4 chronic active MS lesions. By carefully delineating endothelium and perivascular compartments, we find that perivascular space area but not number of vessels is increased in MS lesions. Through mining of publicly available sequencing datasets, we confirm COL1A1 and SLC6A12 as fibroblast and pericyte markers, respectively, in the human brain. COL1A1+ and SLCA12+ vessels were largely distinct of one another. Unsupervised clustering of the expression profile of PDGFR{beta}, COL1A1 and SLC6A12 in individual vessels distinguished three partially overlapping vessel clusters. Of these, the fibroblast-associated vessel type (COL1A1 high, SLC6A12 low) was increased in chronic active lesion rim and center. Importantly, fibroblast-associated vessels were related to increased perivascular space enlargement and more accumulation of immune cells. Conclusion: We identify distinct fibroblast- and pericyte-associated vascular phenotypes in human white matter. Notably, fibroblast-associated vessels are increased in chronic active lesions, where they are related to immune cell cuffs. These findings provide a spatial link between perivascular fibroblasts and chronic inflammation in MS.

Next-generation sequencing (NGS) for the detection of somatic variants has become the method of choice in a variety of molecular oncology fields and in the clinic. Its use ranges from sequencing entire tumor genomes and transcriptomes to targeted clinical diagnostic gene panels. The NYU Langone Genome PACT (Profiling of Actionable Cancer Targets, LG-PACT) assay is a qualitative in vitro diagnostic test that uses targeted next generation sequencing (NGS) of formalin-fixed paraffin-embedded (FFPE) tumor tissue matched with normal specimens from patients to detect gene alterations in a targeted panel covering 606 genes and the TERT promoter. Indications for testing are cancer (solid tumors and hematological malignancies) where a mutational profile from multiple genes would be informative for disease stratification, prognosis, or treatment options including targeted therapies and eligibility for clinical trials. The test is intended to provide information on somatic mutations including point mutations, small insertions/deletions (indels), and copy number aberrations for diagnostic and treatment decisions. LG-PACT is a United States Food and Drug Administration (FDA) cleared diagnostic test (510K: K202304). The clinical interpretation of sequencing data of molecular tumor markers from NGS encompasses automated variant calling tools with human interpretation. This final mostly manual review of data step is intensive, involving highly trained scientists, encompassing literature review, interpretation and clinical tier classification by pathologists, who then provide a complete molecular diagnostic report to the treating oncologists. We provide analysis of 1339 clinical genomic profiles from 31 different cancers and their subtypes, comprising of central nervous system (CNS) 792 (59%) cases (incl. meningioma, glioma and glioblastoma), with 267 (20%) cases predominantly of lung, pancreatic and colorectal and 280 of others (21%). Here, we present the technical challenges of validating an NGS oncological diagnostic targeted assay for clinical grade accuracy and sensitivity for patient care. We show how copy number alterations provide a more comprehensive description of the tumors genomic profile. We then outline the utility of targeted panel sequencing based on certified pathologist selection of reportable variants for our current patient cohort. Where analysis of variant detection has led to 49.4% (661/1339) of our clinical tumor samples containing mutations in known therapy targeted genes, 35.6% (477/1339) with mutation detected in other genes, and 15% (201/1339) cases being negative.

Immune checkpoint inhibitors targeting the PD-1/PD-L1 axis have shown great promise in treating bladder cancer and are now part of the standard treatment for advanced disease. However, many patients still fail to respond to treatment and at present many biomarkers are assessed but have yet shown only limited results. Therefore, with the advent of combination treatments and the increase of immune related adverse event, the search for reliable predictive biomarkers is paramount. Using a multiplexed enhanced FRET-FLIM based technique (QF-Pro) we quantified the interaction of PD-1/PD-L1, CTLA-4/CD80 and TIGIT/CD155 immune checkpoints in a pre-treatment TMA of 46 patients treated with atezolizumab. The association between higher PD-1/PD-L1 ICP interaction state and treatment efficacy was demonstrated in the male sample cohort, where it identified patients with better PFS. Conversely, patients exhibiting higher CTLA-4/CD80 engagement had a worse response to atezolizumab. Remarkably, the dual assessment of patients with high PD-1/PD-L1 and low CTLA-4/CD80 allowed to identify the best responders. These results indicate that the monitoring of patients immune profile in urothelial carcinoma might be critical in identifying patients who may benefit from combination therapy.

Background: The primary neuroanatomical site of Huntington-s disease (HD) pathology resides in the striatum and its atrophy identifies important disease progression from HD-ISS Stage 0 to Stage 1. Immune-associated proteins may capture variation in HD that is incompletely represented by markers of neuroaxonal injury. Objectives: To determine whether cerebrospinal-fluid myeloperoxidase contributes information about striatal volume loss beyond genetic disease burden and neurofilament light. Methods: Cross-sectional data from 88 persons with HD were analyzed. Cerebrospinal-fluid myeloperoxidase and neurofilament light were measured with a nucleic acid-linked immunosandwich assay. Normalized putamen volume was derived from structural magnetic resonance imaging. Linear regression adjusted for genetic disease burden and sex. Results: Higher neurofilament light was associated with smaller normalized putamen volume (standardized {beta} = -0.322, (P=.0066)). Higher myeloperoxidase was associated with larger normalized putamen volume after adjustment for genetic disease burden, sex, and neurofilament light (standardized {beta} = 0.183, (P=.0386)). Adding myeloperoxidase increased explained variance in striatal loss. Conclusions: Cerebrospinal fluid myeloperoxidase contributed modest incremental information about striatal volume in this cross-sectional sample. Independent longitudinal studies are needed to determine its biological source, temporal behavior, and potential biomarker value. Findings advance efforts to characterize multicomponent biological markers of HD.

Motivation: Cell-free DNA methylation provides a minimally invasive signal for early cancer detection and tissue-of-origin prediction. Most methods represent methylation measurements as independent fixed-window features and therefore do not explicitly model relationships among genomic regions. Results: We developed PANGEM (Pan-cancer Graph-based Cancer Detection Using the Cell-free DNA Methylome), a graph-learning framework that represents genomic bins as nodes and integrates CpG context, genomic proximity, and sample-specific methylation similarity in the graph topology. Across five repeated stratified train-test splits, PANGEM achieved the highest mean performance among evaluated methods, with an AUROC/AUPR of 0.997/1.000 for binary cancer detection and macro-AUROC/AUPR of 0.977/0.870 for multiclass tissue-of-origin prediction. In the independent INSPIRE cohort, 72 of 78 cancer cases (92.3%) exceeded the binary classification threshold, and PANGEM correctly classified 9 of 17 head and neck cancer cases (52.9%), the highest accuracy among evaluated methods. Subnetwork analysis further identified recurrent, graph-connected methylation patterns, including a 111-DMR subnetwork with increased methylation in cancer samples.

Mutations in the E3 Ubiquitin (Ub) ligase RNF216 cause Gordon Holmes syndrome (GHS), a neurodegenerative disorder accompanied by neuroendocrine disruption. We developed an orthogonal ubiquitin transfer (OUT) platform to capture RNF216 substrates in neuronal cells and identified OTUD4, a deubiquitinating enzyme (DUB) mutated in GHS, and FMRP, a neuronal-enriched translational repressor. RNF216 predominantly synthesizes K6-linked Ub chains on OTUD4 to induce its degradation, forming donut-shaped structures in neurons. In return, OTUD4 removes the ubiquitination of RNF216 and FMRP. Analysis of RNF216 substrates revealed biological functions regulating protein synthesis, a shared function of the OTUD4-RNF216 substrate interaction network. Indeed, RNF216 expression increased protein synthesis rates in different cell types while Rnf216 deletion decreased dendritic development in neurons. Overall, our findings show that RNF216 and OTUD4 balance rates of protein synthesis and degradation and suggest GHS-related mutations in RNF216 or OTUD4 may offset this balance, triggering neurodegeneration.

Current approaches for imaging fibrotic remodeling have sensitivity, specificity and cost drawbacks that limit both preclinical research and clinical diagnosis. Here, we show that fast green FCF, a small molecule that binds to fibrillar collagen, enables highly sensitive and specific imaging of fibrosis in lung samples from mice and humans using fluorescence microscopy. We report strategies for using fast green FCF staining to assess fibrotic remodeling using precision-cut lung slice and whole-biopsy preparations. Our findings demonstrate that fluorescence imaging of fast green FCF-stained collagen will be useful for fibrosis research and may help to improve detection of fibrosis in clinical pathology.

Viral infections have long been proposed as environmental contributors to neurodegenerative diseases, including Parkinson's disease (PD), yet the molecular mechanisms linking infection and neurodegeneration are not well defined. Neuroinflammation and disruption of central nervous system (CNS) homeostasis have emerged as potential mediators. In this study, we used severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, as a model pathogen to investigate convergent molecular pathways between viral infection and PD. Single-nucleus RNA sequencing (snRNA-seq) was performed on post-mortem striatal tissue from 14 individuals stratified into four groups: COVID-19 only (COVID-19), PD only (PD), comorbid PD with COVID-19 (PD/COVID-19), and controls (Control). The PD/COVID-19 group exhibited an expanded astrocytic population and a pronounced interferon-associated molecular signature characterized by increased expression of canonical interferon-stimulated genes, including IFI44L (average log2FC= 3.9; adjusted p=2.3 x 10-373), IFI44 (average log2FC=2.9; adjusted p=8.0 x 10-266), ISG15 (average log2FC=3.1; adjusted p=1.2 x 10-197), and RSAD2 (average log2FC= 3.5; adjusted p=8.6 x 10-111). Pathway analyses demonstrated activation of innate immune and antiviral signaling pathways, particularly within microglia and astrocytes, including interferon signaling, pattern-recognition receptor pathways, and complement-associated responses. In parallel, genes involved in lipid metabolism, cholesterol homeostasis, synaptic maintenance, and neuronal signaling were reduced across disease groups. Proteomic analyses independently confirmed enrichment of antiviral and interferon-associated pathways and identified convergent suppression of sterol, cholesterol, and lipid metabolic processes. Our findings identify a convergent molecular signature linking PD and COVID-19, pronounced in comorbid individuals and characterized by interferon-driven innate immune activation, glial inflammatory responses, and dysregulation of lipid metabolic homeostasis. Collectively, the data support a model in which severe viral infection amplifies biological pathways already implicated in PD pathogenesis.

Background: Brain age is a biomarker of brain tissue integrity associated with disability in multiple sclerosis. While new lesion formation is central to MS diagnosis and treatment monitoring, its direct relationship to brain aging has not been established. Methods: We analyzed 163 people with MS with clinical and MRI assessments at baseline and years 3, 6, and 8. Brain age was estimated using BrainAgeNeXt. Annualized brain age acceleration was modeled as a function of radiological activity using generalized estimating equations, adjusting for age, sex, disease duration, baseline T2 lesion volume, normalized brain volume (NBV), brain age difference (BAD), and disease-modifying therapy. Secondary analyses examined dose-response effects, post-activity recovery, paramagnetic rim lesion (PRL) associations, and disability associations. Results: 105 participants had at least one new T2 lesion over 8 years. Radiologically active intervals (138 of 333) were associated with +0.19 yr/yr greater brain age acceleration than stable intervals (95% CI: 0.03-0.37; p=0.022), scaling with lesion count (beta=+0.18; p=0.001) and volume. Older age, greater baseline BAD, and NBV were independently associated with reduced brain age acceleration. Brain age acceleration in individuals with new lesions normalized during subsequent stable intervals (0.41 vs -0.06 yr/yr; p=0.001). Both PRLs and non-PRL lesions were associated with greater brain age acceleration than stable intervals. Baseline BAD, but not annualized acceleration, predicted Expanded Disability Status Scale (EDSS) and Nine-Hole Peg Test (9HPT) worsening. Conclusions: New focal lesion formation is associated with a quantifiable, dose-response acceleration of brain aging in MS that normalizes once lesion activity is suppressed.

Parkinsons disease (PD) arises through disruption of multiple interconnected cellular processes, but the genetic contributions to these processes may differ across ancestries. We investigated functional convergence among genes harboring pathogenic or likely pathogenic (P/LP) variants and variants of uncertain significance (VUS) in a multicenter Indian cohort recruited through the Genetics of Parkinsons Disease in India Young Onset Parkinsons Disease project (GOPI YOPD). The cohort included 668 participants (463 males 69.3%) with a mean age at motor onset of 39.4+/-8.8 years. P/LP variants and VUS identified through previously reported whole-exome or whole genome sequencing were retained as separate evidential categories. The P/LP-associated gene set comprised 11 unique genes and the VUS associated set comprised 40 unique genes. Separate STRING functional-enrichment analyses evaluated Gene Ontology Biological Process, Molecular Function and Cellular Component terms, KEGG pathways, WikiPathways and STRING local network clusters. Terms meeting a Benjamini Hochberg false discovery rate threshold of <0.05 were organized into eight non-mutually-exclusive ontology/pathway categories. Gene to pathway mappings were subsequently projected to individual participants to estimate pathway representation and examine clinical associations. At least one reportable P/LP variant or VUS was identified in 336/668 participants (50.3%): 35 had a P/LP variant alone, 282 had VUS alone and 19 had a P/LP variant together with VUS in one or more additional genes. The most frequently represented categories were mitochondrial organization (247/336, 73.5%), autophagy related processes (228/336, 67.9%) and regulation of synaptic vesicle transport (201/336, 59.8%). PRKN was the most frequent P/LP-associated gene, occurring in 29/54 P/LP carriers, followed by PLA2G6 and PINK1. Lysosomal transport was represented exclusively by VUS-associated genes, particularly GBA1, VPS13C and LRRK2. Among P/LP carriers, additional VUS in distinct genes were not associated with age at onset (P = 0.81) or family history (52.6% versus 31.4%; P = 0.15). No pathway phenotype association remained significant after correction for multiple testing. Genetic findings in this Indian cohort converged across an interconnected mitochondrial autophagic lysosomal vesicular network, with different contributions from P/LP-associated and VUS associated gene sets. This study provides the first pathway resolved South Asian genetic profile and a framework for comparative studies across populations.

Service members with traumatic brain injury are at approximately two- to four-fold higher risk of Alzheimer's disease or related dementias than those without such an injury, with risk increasing with injury severity. The amyloid/tau/neurodegeneration biomarker framework treats amyloid, tau, and neurodegeneration as independent axes but omits astroglial injury, despite evidence that reactive astrogliosis (indexed by glial fibrillary acidic protein, GFAP) must be elevated for cognitive decline to occur in amyloid-positive individuals. Total GFAP immunoassays aggregate intact protein with multiple calpain- and caspase-cleaved proteoforms, blurring the biological signal. We compared a calpain-cleaved GFAP neoepitope, the glial fibrillary acidic protein neoepitope (neoGFAP), against total GFAP across the full traumatic brain injury--mild cognitive impairment--Alzheimer's disease continuum in Veterans using a two-stage plasma-to-cerebrospinal-fluid biomarker approach. A plasma triage gate combining phosphorylated tau 217 and amyloid beta 42 was applied to 367 unique subjects; a cerebrospinal-fluid benchmarking cohort of 57 subjects (controls, chronic blast traumatic brain injury, mild cognitive impairment, and Alzheimer's disease) received head-to-head neoGFAP and total GFAP measurement. In the whole benchmarking cohort, neoGFAP discriminated mild cognitive impairment plus Alzheimer's disease from non-Alzheimer subjects with an area under the receiver-operating-characteristic curve of 0.81 versus 0.73 for total GFAP, a trend-level advantage that did not reach nominal significance. Within the gate-positive, amyloid-committed subset of 23 subjects, neoGFAP dominance became significant by McNemar's exact test (six discordant subjects favored neoGFAP, none the reverse). Across diagnostic contrasts, neoGFAP outperformed total GFAP for Alzheimer's disease versus control and, importantly for Veterans, for mild cognitive impairment versus chronic blast-exposed Veterans without cognitive impairment. In chronic blast injury, neoGFAP was paradoxically depleted relative to controls, consistent with tissue sequestration of aggregated proteoform fragments. Unbiased proteomic profiling confirmed coordinated elevation across astrocytic, neuronal, mitochondrial, and microglial compartments. An exploratory subject-level reclassification improved accuracy from 71.1 percent using plasma alone to 79.5 percent with added cerebrospinal-fluid markers and age. In a same-cohort ProQuantum replication (n=57), CSF neoGFAP preserved its discrimination advantage over total GFAP for MCI+AD versus non-AD (AUROC 0.76 vs 0.72; cross-platform Spearman {rho}=0.84), while plasma neoGFAP achieved AUROC 0.90, comparable to pTau217 (0.92) and exceeding A{beta}42/40 (0.84). In this small sample, neoGFAP is a superior proteoform-resolved diagnostic and prognostic biomarker across the continuum and supports adding an astroglial-proteoform axis to amyloid/tau/neurodegeneration biomarker frameworks in high-risk populations.

Accurate, reproducible interpretation of kidney allograft biopsies is critical for diagnosis of graft injury to guide prognosis and management. The international Banff classification is a consensus diagnostic system based on semiquantitative histological lesion scoring on either extent or severity of kidney transplant biopsies. However, pathologist scoring is limited by substantial interobserver variability, constrained scalability, and the inherent nature of the scoring system itself. Here we present BanffNET, a weakly supervised, probabilistic deep learning framework that combines self-supervised feature extraction with a novel Bayesian multiple-instance learning framework to predict (continuously) the full spectrum of Banff lesion scores directly from whole-slide images (WSIs). Using lesion-specific aggregation functions tailored to localized (modeling lesion severity) and diffuse pathologies (modeling lesion extent), BanffNET generates interpretable, patch-level probability maps and calibrated slide-level scores. BanffNET's performance was assessed relative to consensus, biological correlates of rejection and clinical outcome, demonstrating superior consistency, transportability and generalization. Trained on 7,249 WSIs from three cohorts, BanffNET demonstrates consistent performance on 11,028 WSIs across five external test sets, performing on par or exceeding expert consensus across lesions. BanffNET scores align more closely than pathologist Banff scores with molecular profiles of rejection, offering a transparent, biologically grounded framework for computational pathology with relevance beyond transplantation.

Spatial-omics workflows enable molecular analysis within tissue spatial context. Despite the prognostic value of tissue stiffness, these approaches have not incorporated direct, mechanical measurements. This omission reflects several challenges, including sample requirements, low throughput, specialized equipment, and complex data registration. Here, we introduce mechanoMaST (mechanics mapped to spatial transcriptomics), the first workflow to combine absolute mechanical measurements with spatial-omics. It pairs atomic force microscopy-based nanoindentation stiffness maps with spatial transcriptomics maps from adjacent tissue cryosections. The two modalities are then computationally co-registered to enable direct spatial correlation at 100 um resolution, with mapping accuracy quantified through error propagation, providing ground-truth mechanical data directly linked to spatial gene expression. We demonstrate mechanoMaST in human colorectal cancer liver metastasis, generating a spatial resource from 10 patients and revealing a four-gene stiffness signature. mechanoMaST is readily adaptable to other tissues across development and disease, and extendable to additional spatial-omics modalities in adjacent sections.

Cryo-electron tomography (cryo-ET) enables near-native visualization of subcellular architectures, yet applying it to moderately thick, multilayered tissues such as the retina is hampered by inadequate vitrification and inaccurate depth-targeting. Here, we developed PLCT, an integrated approach combining modified high-pressure freezing, cryo-ultramicrotome trimming, and plasma-based cryo-FIB milling to overcome these barriers. PLCT reliably vitrified <100 m retinal strips with minimal ice artifacts, navigates precisely to the outer plexiform layer using morphological landmarks, and produces high-quality lamellae suitable for high-resolution cryo-ET. Subtomogram averaging (STA) analysis identified microtubules at 16.33 [A] within retinal horizontal cell processes. Importantly, STA also resolved a 10-nm-diameter filamentous structure at 24.81 [A] in the same processes, featuring six peripheral strands surrounding an elongated central density with continuous intervening cavities, an architecture consistent with intermediate filaments. Together with its native localization and immunoreactivity, these features collectively identify the filaments as neurofilaments. Separately, 3D reconstruction of synaptic ribbons uncovered a previously unrecognized "mahjong tile"-like fine ultrastructure. These results demonstrate that PLCT-produced lamellae are of sufficient quality to support structural analysis in native tissue. Although demonstrated on retinal photoreceptor synapses as a proof-of-principle, PLCT is inherently generalizable, with its depth-navigation and vitrification strategies directly applicable to any multilayered tissues. This work establishes PLCT as a robust, reproducible platform for depth-resolved in situ cryo-ET of multilayered tissues.