Neuropathology and Applied Neurobiology

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.

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.

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.

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.

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.

Myotonic Dystrophy type 1 (DM1) is an autosomal multisystem disorder manifested due to unstable CTG nucleotide repeat expansion within the 3'-untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Although progress towards understanding of molecular pathogenesis in muscle and heart has been made, the pathways that affect the brain in DM1 is fundamentally unknown. In addition, the congenital DM1 manifest even more complicated brain abnormalities. Despite the wealth of existing cellular and animal models, iPSCs based studies are being fostered as they replicate the human model more closely to the disease. In view of this context, we set out to characterize the differentiation potential of congenital DM1 patient derived iPSC lines towards neuronal cells. Using neurogenin2 (NGN2) induced direct reprogramming of iPSCs into neurons and chemically defined media-induced neural induction protocol, we find that congenital DM1 mutant iPSC derived neurons exhibited precocious differentiation, as evidenced by their expression of pan-neuronal markers TUJ1 and Map2, along with increased processes extension and neurite length. Moreover, unbiased RNA sequencing analyses and qPCR validation revealed precocious and enhanced expression of several neurogenic transcription factors including, Ascl1, NeuroG2, and NeuroD1. Furthermore, immunofluorescence imaging of MBNL1 and MBNL2, RNA-splicing factors, displayed enhanced nuclear aggregations, a hallmark of the DM1 disease, in the mutant lines. Moreover, investigation of RNA splicing events identified mis-splicing in many important genes/transcripts including RMST, ANK3 and MBD1 during the neural conversion of congenital DM1 lines. These studies reveal novel paradigms that may contribute to neurological pathogenesis in CDM1 patients. These studies also provide a strong foundation for future mechanistic investigation aimed at understanding CDM1 pathology and may open new avenues for the development of gene therapy approaches for individuals with DM1.

Background: Alzheimer's disease (AD) is clinicopathologically heterogeneous. A proportion of patients living with AD present clinically at a younger onset of cognitive symptoms before 65 years old and/or non-amnestic clinical syndromes. Neuropathologically, corticolimbic distribution of neurofibrillary tangle pathology occurs on a continuum with some cases having greater cortical tau pathology relative to limbic regions and others with relatively restricted accumulation in limbic structures. These patterns of corticolimbic tangle distribution are associated with clinical presentation and age at onset. This study sought to examine protein expression differences across the spectrum of clinicopathologic heterogeneity using the NULISA targeted proteomics platform. Methods: A series of thirteen neuropathologically diagnosed AD cases from Mayo Clinic prospectively followed research studies were selected to reflect heterogeneity of clinical syndromes and corticolimbic distribution of tangle pathology. Frozen postmortem brain tissue samples were isolated from inferior parietal cortex and homogenized in RIPA buffer for analysis using Alamar Biosciences NULISA CNS disease 120 panel. Applying a conservative detection threshold of 75% level of detection for the novel application of NULISA in human brain, we evaluated levels of 69 of 129 protein targets across samples. We examined associations between age at onset cognitive symptoms and corticolimbic distribution of tangles (CLix) separately with individual protein targets using linear regression analysis. Results: AD cases with a younger age at onset had higher measured levels of ubiquitin, while older age was associated with higher levels of total tau, CRH, and NPTX2. Investigations of corticolimbic heterogeneity revealed AD cases with lower CLix score (i.e., cortical predominant distribution of tau) had higher measured p-tau181, p-tau231, ubiquitin, and p62. AD cases with higher CLix (i.e., relative cortical sparing) had higher levels of total tau, CRH, NPTX2, MDH1, and HBA1. Brain-derived total tau consistently showed a stronger association in both models. Conclusion: This work demonstrates the utility of postmortem proteomics for investigating biomarkers associated with AD clinicopathologic heterogeneity. We observed proteomic differences in synapse integrity, tau post-translational modification, and ubiquitination associated with age at symptomatic onset and corticolimbic distribution of tangle pathology.

The aggregation of -synuclein (Syn) is a molecular hallmark of Parkinson's disease (PD) and other synucleinopathies. Understanding the molecular mechanisms that determine the aggregation of this protein may thus facilitate the development of disease-modifying therapies. While Syn is most commonly expressed as a 140-residue protein (Syn-140), recent evidence suggests an involvement of alternatively spliced Syn isoforms in disease onset and progression. Here, we report and characterise the interaction between Syn-140 and the aggregation-prone Syn-112 variant, one of the most abundant Syn splice isoforms. We found that amounts as low as 1% of Syn-112 accelerate the nucleation and aggregation of Syn-140. To further investigate this phenomenon, we employed MALDI-MS and NMR spectroscopy, confirming that Syn-140 and Syn-112 monomers interact strongly with one another. Furthermore, to assess the association of Syn-112 with disease pathology, we performed immunohistochemical staining combined with confocal microscopy on PD brain samples. Thereby, we found an increase in the number as well as the area of Syn-112 immunoreactive aggregates compared to healthy controls. These results illustrate how low-abundance Syn splice isoforms can modulate the aggregation landscape of Syn-140 and in turn contribute to the molecular heterogeneity of synucleinopathies.

Selection of regions of interest (ROIs) is often a crucial step in spatial molecular profiling and many pathology tasks, with substantial implications for research reproducibility and biological interpretability. To provide a reproducible and adaptive framework for AI-guided ROI selection, we developed a modular generalist-specialist solution across spatial profiling platforms. In a cohort comprising 55 tumor types from 160 tissue donors profiled using NanoString Digital Spatial Profiling and multiplex immunofluorescence, we first established a protein-profiling reference atlas capturing compartment-specific immune, checkpoint, stromal, and proliferation patterns. We then developed an AI Specialist Task-Oriented Model for ROI Selection (ASTROS) and tested comprehensive benchmarks considering specialist-only (ASTROS), generalist-only (PLIP/GFM), and hybrid generalist-specialist strategies, showing that the latter provides a balanced tradeoff across slide-level signal preservation, pathologist-reference concordance, within-slide placement consistency, and large-slide computational efficiency. We further demonstrated the feasibility of virtual staining for ROI preview and modular ROI placement for other spatial omics technologies, Visium and Visium HD workflows. Together, these results support our proposed framework to enable ROI selection responding to unmet needs for reducing inter-rater variability, reproducibility, and versatility in spatial profiling experiments.

Motor Neurone Disease (MND) is characterised by progressive degeneration of upper and lower motor neurons, accompanied by cytoplasmic mislocalisation and hyperphosphorylation of TDP-43, hallmarks that implicate failure of endolysosomal proteostasis. The Endosomal Sorting Complexes Required for Transport (ESCRT) pathway governs multivesicular body (MVB) formation, lysosomal cargo delivery, and autophagosome closure, yet its expression profile in human MND tissue and mechanistic contribution to disease pathology have not been established. Here, we report subunit-specific dysregulation of ESCRT proteins in postmortem motor cortex and spinal cord from MND patients: CHMP2B (ESCRT-III) is significantly upregulated in both regions, whilst TSG101 (ESCRT-I) and VPS37A (ESCRT-I) are significantly downregulated in motor cortex, indicating a region-specific remodelling of the ESCRT network. In a tunicamycin-induced ER stress model using NSC-34 motor neuron-like cells and primary cortical neurons, TSG101 overexpression reduced total and phosphorylated TDP-43, suppressed mTOR signalling, and restored autophagic flux, whereas TSG101 knockdown exacerbated TDP-43 accumulation and cytoplasmic mislocalisation. CHMP2B modulation selectively regulated TDP-43 phosphorylation without altering total TDP-43 levels, consistent with a casein kinase 1-dependent mechanism operating independently of bulk autophagy. Both TSG101 and VPS4a were required to maintain neuronal CD9 tetraspanin localisation to early endosomes; their depletion redirected CD9 to late endosomal and lysosomal compartments under ER stress. Extracellular vesicle characterisation revealed a functional divergence: TSG101 is required for general exosome biogenesis, whereas VPS4a ATPase activity specifically mediates loading of pathological TDP-43 cargo into EVs. Dynamic light scattering confirmed that ER stress and ESCRT modulation produce distinct, condition-specific alterations in EV size and polydispersity. These findings establish ESCRT dysfunction as a multifaceted contributor to MND pathogenesis and identify TSG101, CHMP2B, and VPS4a as mechanistically distinct therapeutic targets warranting preclinical validation.

Traumatic brain injury produces widespread axonal damage can be assessed histologically using amyloid precursor protein (APP) immunohistochemistry, which labels injured axonal profiles at cellular resolution [1, 2]. However, quantification of APP pathology remains a major bottleneck: annotation is manual, time-consuming, spatially localized, and variable across raters, limiting scalability and reproducibility. This limitation is particularly important in studies that use histology as a reference for neuroimaging or other tissue-level measurements, where cellular APP pathology must be quantified in a spatial form that can be aligned with imaging abnormalities. Here, we introduce PIGMENT, an annotation-efficient deep-learning framework for automated segmentation and quantification of APP-positive pathology in porcine white matter histology. PIGMENT uses a compact SegFormer-B0 architecture trained on 525 expert-annotated 512 x 512-pixel tiles from four APP-stained sections across three pigs. Because APP-positive profiles are sparse, fragmented, stain-variable, and morphologically diverse, PIGMENT combines limited expert labels with APP-specific augmentation designed to model variation in APP-positive intensity, size, continuity, fragmentation, and local tissue context. We evaluated PIGMENT using an instance-level detection rate that measures whether discrete APP-positive components are localized. Across held-out APP-stained data, PIGMENT achieved a mean instance-level detection rate of 0.86. Across the configurations tested, the highest mean detection rate was achieved by a training set that included sections from different animals, suggesting that annotation diversity may be an important factor under limited-label conditions. By extending limited high-confidence expert annotations into whole-section APP burden maps, PIGMENT provides a scalable framework for characterizing the extent and spatial distribution of traumatic axonal injury. These maps may support future studies that align histological injury burden with imaging-derived measures.

Background: Ewing sarcoma (EwS) is a highly aggressive bone and soft tissue cancer mainly affecting children, adolescents, and young adults. The rarity of the disease, relatively small cohort sizes of prior studies, and overall low mutational burden of EwS have limited the ability to establish robust correlations of genomic findings and clinicopathological factors. Methods: To overcome these limitations, we integrated genomic and clinical data from the seven major sequencing studies encompassing 538 EwS patients. Mutational profiles (SNV, indels and CNVs), and their correlation with clinicopathological features in the aggregated cohort were systematically analyzed to provide an integrated view of the EwS genomic landscape. Results: This study compiles the largest EwS genomic dataset reported to date. In the aggregated cohort (n=538) bone tumors were more common (65.4%) than soft-tissue tumors (34.6%), the latter being more frequent in older male patients and associated with poorer outcomes. EWSR1::FLI1 was the most prevalent fusion (87.2%). No major clinicopathological differences were identified between fusion types. The mutational landscape was dominated by STAG2 (15.6%) and TP53 (7.1%) alterations, associated with younger or with older age at diagnosis and poor survival, respectively. Strikingly, the coexistence of STAG2 and TP53 mutations, although rare (n=12), was associated with lethal outcome in all cases. CDKN2A loss (9.1%) was associated with older age, poor survival, and linked to a higher frequency of TP53-mutations in soft tissue EwS. Among frequent CNVs, gain of chr1q (25.2%) and loss of chr16q (21.9%) were per se frequently associated with fatal outcome and their co-occurrence further increased the risk of lethality. Conclusions: We delineate recurrent genomic alterations with important clinicopathological associations, including a uniformly lethal STAG2/TP53 co-mutation and CNV signatures marking aggressive disease. This comprehensive pooled analysis of EwS genomic studies provides a foundation for refined biological risk-stratification.

Apolipoprotein E (ApoE) is the strongest genetic predictor of Alzheimers disease (AD) risk, with ApoE4 increasing and ApoE2 decreasing risk relative to ApoE3. Using a global LC-MS proteomic approach, we integrated protein abundance and kinetics in Human-APOE knock-in mice for young (3-month) and aged (18-month) cohorts to quantify the changes in steady-state proteostasis. By mapping 6,052 identified proteins and 3,986 associated turnover rates into ontological groups, we observed that vesicle trafficking and mitochondrial dysregulation occur as early as 3 months in ApoE4 mice accompanied by hyperactive metabolism that eventually reduces with age. In contrast, young and old ApoE2 mice retain similar signatures to ApoE3 mice in metabolic, mitochondrial, cellular regulation, and membrane trafficking ontologies. We found that females had more isoform-induced ontological changes relative to ApoE3, providing insight into sex-dependent vulnerabilities. Our global proteomic approach for ApoE proteostasis crucially unifies independent literature observations while providing turnover kinetics to uncover the underlying mechanism behind abundance changes. Data are available via ProteomeXchange with identifier PXD079261. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/735293v1_ufig2.gif" ALT="Figure 1000"> View larger version (45K): org.highwire.dtl.DTLVardef@54c14borg.highwire.dtl.DTLVardef@5e490dorg.highwire.dtl.DTLVardef@df5b1org.highwire.dtl.DTLVardef@7d7717_HPS_FORMAT_FIGEXP M_FIG C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/735293v1_ufig1.gif" ALT="Figure 1001"> View larger version (48K): org.highwire.dtl.DTLVardef@115c4cdorg.highwire.dtl.DTLVardef@2baac8org.highwire.dtl.DTLVardef@d965e6org.highwire.dtl.DTLVardef@b0e49a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Quantifying blood-brain barrier (BBB) integrity from fluorescence microscopy remains limited by subjective scoring and categorical classification methods that lack reproducibility. For objective and consistent BBB phenotyping, we present two semi-automated image-analysis pipelines that replace manual scoring with quantitative, continuous-variable measurements. Our in vitro pipeline, implemented in Python, quantifies the connectivity of tight junction structures by measuring discrete ZO-1 fragment objects within manually traced junction regions. It outputs continuous metrics including average fragment area, total junctional area, and a junctional fragmentation ratio that captures degree of ZO-1 continuity versus discontinuity. In human brain microvascular endothelial cells subjected to glycocalyx component knockdown, the pipeline detected significantly reduced fragment area (37% decrease for both CD44 and syndecan-1 (SDC1) knockdown, p = 0.0148 and 0.0084) and junctional fragmentation ratio (p = 0.0061 and 0.0137). Our in vivo pipeline integrates ilastik-based pixel classification with FIJI macro automation to quantify vascular marker colocalization and to separate vessel signal from microglial contamination within a single fluorescence channel, eliminating the need for dedicated counterstains. Applied across four mouse cohorts [young, aged, Alzheimer's, traumatic brain injury (TBI)] and three brain regions [prefrontal cortex (PFC), hippocampus, midbrain], the pipeline revealed concurrent ZO-1 loss and ICAM-1 elevation in the PFC and hippocampus of aged and Alzheimer's mice, with Alzheimer's-specific doubling of eNOS occurring in the PFC (p = 0.0013). TBI mice showed persistent ZO-1 loss with transient ICAM-1 and eNOS changes. Both deterministic pipelines are available on GitHub and designed for adoption beyond the specific markers and systems analyzed here.

Background: Single cell RNA sequencing provides a wealth of information to explore the complexities of the tumour microenvironment, but crucially the spatial topology of the tumour is lost and studying cellular interactions is limited. Spatial transcriptomics aims to address this however the technique remains cost prohibitive for the generation of data from meaningfully-sized clinical cohorts. In contrast, spatial proteomic profiling with multiplex immunofluorescence, preserves spatial interactions, is relatively cost accessible, and is scalable for large clinical cohorts to address powerful translational questions. Whilst multiplex approaches have advanced in recent years, we note that cancer-associated fibroblasts (CAFs) have been explored in less detail, potentially due to difficulties associated with CAF heterogeneity and the diversity of markers used to define them. Methods: We designed, optimised, and validated a multiplex immunofluorescence panel that combines four frequently used CAF markers; alpha smooth muscle actin (aSMA), fibroblast activation protein (FAP), podoplanin (PDPN) and platelet-derived growth factor receptor alpha (PDGFRa) with CD8 and pan-cytokeratin. Here we share our methodology and the practical considerations taken to inform the final panel design. We also highlight the benefits of robust optimisation experiments.

Laminin-2 (gene: LAMA2) is a key protein in the basement membrane of muscle and Schwann cells. A complete lack of this protein results in LAMA2-related congenital muscular dystrophy (LAMA2-RD), a severe muscle disease characterized by progressive muscle weakness, respiratory insufficiency, failure to thrive and shortened life span. One key signature of this disease is early onset of fibrosis coupled with poor muscle growth. We previously showed that TGF-{beta} and its activator, integrin-V, are elevated in dystrophic fibers of DyW mice, a mouse model of LAMA2- RD. Other than activating TGF-{beta}, integrin-V is also known to facilitate the transdifferentiation of various cell types to myofibroblasts. In this study we present evidence for transcriptional dysregulation of genes driving myofibroblast transdifferentiation and extracellular matrix (ECM) remodelling during the early development of DyW mice that is also reflected in muscle biopsies from young LAMA2-RD patients. We hypothesize that the early ECM remodelling, seen in both DyW mice and LAMA2-RD children, may explain the congenital onset of fibrosis with poor muscle growth seen in the disease.

Background and Objective: Due to the heterogeneity of bladder cancer, minimally invasive molecular profiling may improve tumor characterization at the time of diagnosis. We evaluated whether integrated genomic and fragmentomic profiling of plasma and urinary circulating tumor DNA (ctDNA) detects BC-derived signals for diagnosis and disease stratification across all tumor stages. Methods: In this real-world cohort, 202 plasma and urine samples were obtained from 33 patients with non-muscle-invasive BC (NMIBC), mostly Ta tumors, and 15 patients with muscle-invasive BC (MIBC), as well as from 58 cancer-free controls. Low-coverage whole-genome sequencing was performed to assess ctDNA fragmentation, chromosomal instability and copy number variations. Matched tumor tissue was analyzed to evaluate concordance between liquid biopsy and tissue-derived molecular alterations. Key Findings and Limitations: Complementary genomic and fragmentomic profiling of cfDNA achieved detection rates of 75.8% in NMIBC patients and 91.7% in MIBC patients with paired plasma and urine. Distinct differences were observed between MIBC, NMIBC and cancer-free controls, consistent with increasing ctDNA signals during disease progression. Tumor tissue analysis confirmed BC-associated molecular alterations. Limitations include the single-center design and limited sample size. Conclusions and Clinical Implications: Multimodal profiling of plasma and urinary cfDNA enabled the detection of tumor-derived molecular signals for all bladder cancer stages, including early-stage disease. By integrating genomic and fragmentomic features, this minimally invasive approach provides molecular tumor characterization at the time of diagnosis and may support future risk-adapted diagnostic, therapeutic and surveillance strategies.