Acta Neuropathologica

Tau aggregation into intracellular neurofibrillary tangles (NFTs) is one of the major hallmarks of Alzheimers disease (AD). Based on neuropathological studies, NFTs have been classified into pre-tangles, mature tangles, and ghost tangles, however the ultrastructural transitions between these stages remain poorly understood. Here, we used correlative light and electron microscopy (CLEM) to structurally characterize tau tangle maturity states in post-mortem human AD brain tissue. Pre-tangles showed no consistent fibrillar ultrastructure. Mature tangles contained densely packed, highly aligned paired helical filaments (PHF) and straight filaments (SF), often organized in spatially distinct bundles within the neuronal soma. Ghost tangles lacked cellular organelles and were composed predominantly of thin fibrils compartmentalized by membranous structures, with fibril morphology differing between compartmentalized and non-compartmentalized regions. Electron tomography and fibril segmentation demonstrated that these fibrils were significantly thinner than PHFs and SFs while immunogold labeling using the 2E9 tau marker confirmed the presence of tau within both mature and ghost tangle fibrils. GFAP-positive astrocytic processes infiltrated fibril-rich compartments within ghost tangles, linking astrocytic engagement with the emergence of this distinct ultrastructural organization. Together, our findings show that ghost-tangles contain a structurally distinct population of tau fibrils, suggesting that tau aggregates undergo astrocytic-mediated structural remodeling at late stages of pathology.

Recent evidence implicates somatic mutations in disease-associated genes across multiple neurodegenerative disorders, including the identification of two somatic TARDBP variants as a putative cause of frontotemporal lobar degeneration with TDP-43 pathology type C (FTLD-TDP type C). However, these findings remain anecdotal. We performed single-nucleus amplicon sequencing of 39,800 neurons from the superior temporal gyrus of sporadic FTLD-TDP type C patients (34,738 neurons, 52 individuals) and non-demented controls (5,062 neurons, 26 individuals) using the Mission Bio Tapestri platform and investigated somatic variants in TARDBP and other FTLD-TDP-associated genes in a case-control setting. We uncovered an extensive landscape of ultra-low-frequency (<1%) somatic mutations across all analysed genes, including TARDBP. Rare somatic occurrences of known ALS/FTD-associated germline TARDBP variants, including p.N267S and p.M337V, were identified exclusively in patients. Among all targeted genes, TARDBP had the highest proportion of neurons harbouring a somatic variant, and this mutational burden was lower in individuals who died at an older age. Strikingly, the C-terminal domain of TDP-43, where most ALS/FTD germline pathogenic variants reside, exhibited a lower neuronal mutational burden than its other domains. This suggests that neurons harbouring damaging mutations may undergoprogressive loss prior to autopsy, leaving only rare survivors at frequencies too low to detect by bulk or small-scale single-cell approaches. Together, our findings provide an unprecedented resolution into the somatic mutational landscape of post-mitotic neurons in FTLD-TDP type C and reveal a gene-specific layer of neuronal mosaicism that current single-cell WGS studies fail to detect.

Amyotrophic lateral sclerosis (ALS) is a genetically and biologically heterogeneous neurodegenerative disease in which distinct pathogenic mechanisms operate across patients while overt molecular pathology is confined to only a subset of cells. Such features would act to dilute disease-associated transcriptomic signals and complicate the identification of reproducible molecular signatures across the growing number of ALS single-nucleus RNA sequencing (snRNA-seq) studies. Here, we systematically assessed cross-study reproducibility across four cortical ALS snRNA-seq datasets comprising 140 donors (87 ALS) and tested whether pathology-defined cell states improve detection of conserved molecular signatures. Cell-type annotations were harmonized prior to comparison of cell-type-specific pseudobulk differential expression using gene-level, pathway-level, gene-ranking and alternative polyadenylation analyses. We further examined nuclei exhibiting TDP-43 pathology, identified by expression of the STMN2 cryptic exon. Conventional ALS-versus-control analyses showed limited reproducibility, with minimal overlap of differentially expressed genes or enriched pathways, while fold-change patterns clustered predominantly by study rather than cell type or brain region. Nevertheless, gene-ranking analyses identified reproducible neuronal transcriptional programs, suggesting that biological signal is present but incompletely resolved by current cohort sizes. In contrast, STMN2 cryptic exon-positive nuclei showed substantially greater concordance, revealing robust TDP-43-associated signatures that partially overlapped independent models of TDP-43 dysfunction while also identifying motor cortex-specific changes, including reduced expression of the recently identified ALS risk gene UNC13C. Reproducible ALS-associated alternative polyadenylation changes were not detected, likely reflecting the higher dimensionality and sparsity of polyadenylation site analyses. Together, our findings demonstrate that pathology-defined cell states provide a more reproducible framework for studying ALS transcriptomic alterations than conventional case-control comparisons. We additionally provide an interactive browser to facilitate exploration and comparison of ALS snRNA-seq datasets.

BackgroundProgressive supranuclear palsy (PSP) is a primary tauopathy defined by the accumulation of 4R tau isoforms in neurons, oligodendrocytes and astrocytes. Despite evidence of genetic susceptibility operating through glial cell types, it remains poorly understood how cell type-specific epigenetic-transcriptional programs evolve with progression of tau pathology. MethodsWe conducted single-nucleus chromatin accessibility (snATACseq) and RNA sequencing (snRNAseq) on postmortem frontal cortex samples from PSP patients (n = 8) and matched controls (n = 8), yielding over 144,000 nuclei passing quality control. Tau pathology burden, including neurofibrillary tangles, coiled bodies, and tufted astrocytes, was quantified on AT8-immunostained sections from the same individuals. We integrated differential gene expression analysis, transcription factor motif enrichment, weighted gene co-expression network analysis, and pseudotime modeling anchored to cell type-specific tau pathology burden to delineate molecular pseudo-progression trajectories. ResultsIn eight cell types, 20 subclasses, and 70 subclusters, PSP brains displayed a selective depletion of certain excitatory deep-layer neurons and oligodendrocyte subclusters, with relative preservation of inhibitory neurons and vascular cells. Genetic risk enrichment was localized to astrocytes and oligodendrocytes, whereas excitatory neurons exhibited the greatest transcriptional dysregulation. Oligodendrocyte pseudo-progression indicated a transition from homeostatic myelination programs (MBP, MOBP) through glucocorticoid-responsive stress (FKBP5, ZBTB16), to compensatory myelination (PLP1, CNP) and proteostasis stress (UCHL1, CYRAB, CLU). Neuronal pseudo- progression revealed early dysregulation of synaptic (RORB2, NRG3, NPTX1), microtubule dynamics (KIF2C, RAB27B, TUBA/B), and survival (MEG3, FTX) pathways, alongside a transient increase in neuron-glia interactions (GRIP, CNTNAP4, ERBB4), converging late on ribosomal translation and vesicular trafficking modules across all neuronal subtypes. Cross-modal integration with independent cerebrospinal fluid proteomics identified a concordant subset of glial reactivity, axonal injury, and synaptic markers jointly dysregulated in inhibitory neurons, oligodendrocytes, and excitatory deep-layer neurons. ConclusionPSP pathogenesis reflects a combination of glial genetic susceptibility and staged, cell type-specific transcriptional dysfunction. Oligodendrocytes transition from myelination-competent states to FKBP5-mediated stress states, while neurons show variably timed loss of synaptic excitability and survival programs, preceded by neuron-glia interactions and followed by convergent ribosomal-proteostatic failure. These cytopathology-anchored trajectories outline a potential pathophysiological sequence and may inform candidate selection for stage-specific therapeutic interventions in PSP.

Alzheimer's disease (AD) is characterised by aberrant amyloid beta and tau aggregation, neuroinflammation, demyelination and neurodegeneration, which have been linked to changes in cell-specific gene expression signatures. Among the mechanisms driving cell-type-specific transcriptional changes, histone acetylation plays a central role in regulating gene activity. While global alterations in histone acetylation have been implicated in AD, the contribution of individual cell types to these epigenetic changes remains poorly understood. To decode cell-type-specific changes in gene regulation in AD pathogenesis, we profiled histone H3 lysine 27 acetylation (H3K27ac) in microglia, oligodendrocytes and neurons from the prefrontal cortex of individuals with late-stage AD and non-dementia controls. Oligodendrocytes had the highest number of differential H3K27ac regions in AD, followed by microglia. Genes nearest to differential H3K27ac in purified microglia were enriched for phagocytosis, lipid processing, inflammatory and disease-associated cell state signature genes. Gene network analysis revealed downregulation of homeostatic genes in AD microglia and upregulation of immune activation, including signatures of lipid-handling and monocyte-derived macrophages. Oligodendrocyte co-regulated regions were indicative of increased MHC class I antigen presentation and altered neuron-oligodendrocyte interactions in AD. We identified H3K27ac allele-specific variants (ASVs) enriched near endolysosomal and ubiquitin-proteasome-associated genes in microglia and neurons. ASVs coincided with AD genome-wide association study (GWAS) risk loci, including CLU in oligodendrocytes and HLA-DRB1 in microglia. DNA motif analysis identified putative transcription factor drivers of AD glial dysregulation, including the lysosomal-associated MITF, Cap'n'collar (CNC) family (BACH1 and NFE2) and AP-1 activation in microglia. DNA binding of the MITF protein in human microglia was localised to lysosomal-associated genes and enriched in H3K27ac regions upregulated in AD and near disease-associated microglia (DAM) genes. Collectively, these findings implicate lysosomal dysfunction and upstream transcriptional regulation via MITF as key processes in AD microglia.

TDP-43 dysfunction is thought to underlie frontotemporal dementia and limbic-predominant age-related TDP-43 encephalopathy, neurodegenerative dementias currently without effective therapy. Therapeutic strategies are designed to correct individual cryptic targets of TDP-43, such as UNC13A, whereby its cryptic splicing compromises synaptic function, yet the sufficiency of such an approach to prevent memory deficits is unclear. Using a forebrain neuron-specific TDP-43 knockout mouse model that recapitulates TDP-43 dysfunction occurring during early stages of human disorders, we found here that prevention of cryptic splicing to include that of Unc13a attenuated memory deficits. We show that genetic ablation of Unc13a cryptic exon solely in such TDP-43 knockout mice is sufficient to preserve cognition, supporting the clinical value of targeting UNC13A to mitigate memory deficits. Prevention of cryptic splicing of multiple targets of TDP-43 additionally attenuate neuron loss. For optimal outcomes in TDP-43 related dementias, these findings thus strongly support strategies designed to repress cryptic splicing of multiple targets of TDP-43, including UNC13A.

Structured AbstractINTRODUCTION Tau pathology typically occurs in Alzheime[r]s disease (AD), however is also frequently present in Parkinso[n]s disease dementia (PDD) and Dementia with Lewy Bodies (DLB), yet its disease-specific signature is unclear. METHODSFive tau, amyloid-{beta}, -synuclein and neuronal markers were analysed across hippocampal subfields in non-demented controls (CTLs), AD, PDD and DLB using multiplex immunohistochemistry, single-tangle classification and confocal imaging. RESULTSAT8, pTau217, and GT38 were predominatly detected in AD, while pS422 was enriched in PDD and pS396 showed a region- and disease-specific pattern. DLB resembled AD in subregional tau distribution. Tau marker correlation were different comparing AD, PDD and CTL. Single-tangle analyses revealed disease-specific immunophenotypes but conserved mature intra-tangle epitope organisation. Distinct tau signatures were associated with inhibitory interneuron vulnerability, while regional tau co-occurrence with amyloid-{beta} and -synuclein remained conserved. DISCUSSIONDisease-specific tau signatures vary across hippocampal subregions and neuronal populations, implicating the contribution of regional and cell-specific factors beyond pathology burden.

Tau aggregation, a defining feature of tauopathies, commonly appears as neuropil threads within neurites including axons. However, the affected neuronal populations and the spatial organization of axonal vulnerability remain poorly defined. Here, following identification of Tau accumulation in parvalbumin (PV) interneuron axons in primary tauopathy patients, we generated a P301S Tau model targeted to PV interneurons to track the structural integrity of their neurites and somas. We observed neuronal loss alongside axonal dystrophy, with focal swellings forming preferentially at axonal bifurcations. This spatial enrichment exceeded chance levels, and local axonal geometry predicted swelling localization. Longitudinal in vivo imaging revealed that bifurcation-associated swellings progressed to axonal severing. Analogous bifurcation-associated dystrophies were present in PV axons in human primary tauopathy tissue. These findings indicate that axonal branch geometry shapes the spatial pattern of Tau-induced neurodegeneration and identify PV axonal branch points as sites of pathology in mouse and human tauopathy.

Neurofibrillary tangles (NFTs) are a hallmark of Alzheimer's disease. However, the molecular basis underlying their spatial heterogeneity and biochemical complexity remain unclear. To address this, we performed mass spectrometry-based proteomic profiling of two neocortical regions with different susceptibilities to NFT pathology -- the inferior temporal (IT) and middle frontal (MF) cortices -- from 27 autopsy cases stratified by Braak stage (0-II, n=8; III-IV, n=11; V-VI, n=8). Brain homogenates were sequentially fractionated using buffers of increasing solubility (TBS, Na2CO, and Urea), followed by trypsin digestion and analysis by nanoLC-MS/MS with data-independent acquisition. Proteins were identified using PEAKS Studio 12 and linear mixed-effects models were applied to assess Braak stage-associated changes in protein abundance and solubility. A total of 5,573 protein groups were identified, including 452 differentially expressed proteins (DEPs), 183 bidirectional expression proteins (BEPs), and 693 differentially soluble proteins (DSPs) associated with NFT pathology. These proteins were enriched in pathways related to cytoskeletal organization, metabolism, vesicular trafficking, and proteostasis. Marked regional differences were observed, with limited overlap between IT and MF across DEPs, BEPs, and DSPs. Protein change in MF was dynamic with a higher number of BEPs and suggested active cytoskeletal remodeling, whereas IT showed fewer BEPs and evidence of structural disruption, including alterations in intermediate filament components, consistent with vulnerability of IT and possible compensatory/plasticity changes in MF cortex. MF cortex had the greatest downregulation of DEPs between Low and Mid Braak stages, while the transition from Mid to High Braak stage showed upregulation of most of DEPs in both regions. In addition, widespread changes in protein solubility were observed, particularly decreased solubility at later Braak stages, implicating disruption of proteostasis. Together, these findings highlight region- and stage-dependent proteomic alterations associated with tau pathology and underscore their relevance for understanding disease mechanisms and informing biomarker development.

Two PSEN1 E280A carriers have presented extreme protection against autosomal dominant Alzheimers disease (ADAD), with over two decades of delay for dementia onset. One of them, a male heterozygous for the RELN-COLBOS protective variant showed increased neuronal density in the entorhinal cortex 1. We conducted a deep phenotyping and genotyping study of the entorhinal cortex in protected and unprotected PSEN1 E280A cases, sporadic AD, and non-demented controls. We used single nuclei and spatial transcriptomics, whole genome sequencing, and candidate genotype-associated expression changes (GAEC) analysis. Our results showed unique neuronal and oligodendrocytic populations in the male RELN-COLBOS patient. Unique RELN positive inhibitory interneurons were enriched in cortical Layer I, while unique abundant ADAMTSL1 positive excitatory neurons were distributed in Layers II/III and Layer Va. These neurons and mature myelinated oligodendrocytes benefitted from increased expression of LRP6 receptor, functioning as a non-canonical receptor for the mutated Reelin protein encoded by RELN-COLBOS. Finally, GAEC and pathway enrichment analyses suggested that other mutations enhanced RELN-COLBOS effects in oligodendrocytes in the male RELN-COLBOS patient, explaining the phenotypic differences with his sister, a RELN-COLBOS carrier with no evident protection from ADAD. Our findings suggest that extreme deviations of the PSEN1 E280A phenotype are more likely attributed to oligogenic effects, including simultaneous mutations occurring in genes including ITGA2, involved in single molecular pathways, such as the Integrins / Focal Adhesion pathway, as potential disease modifiers for Alzheimers disease (AD).

INTRODUCTIONMicroglia regulate amyloid plaque-associated microenvironments that contribute to downstream tau pathology in Alzheimers disease (AD). Variants within the MS4A locus are strongly associated with AD risk and resilience and are linked to microglial biology; however, the functional role of MS4A4A in plaque-associated tau pathology remains poorly understood. METHODSSingle-nucleus RNA sequencing (snRNA-seq) was performed on hippocampi from non-transgenic, Ms4a4a knockout (4A-KO), 5xFAD, and 5xFAD 4A-KO mice at 6 months of age. To assess plaque-associated tau pathology, AD-derived tau aggregates were injected into the hippocampus of 5xFAD and 5xFAD 4A-KO mice at 6 months, and histological analyses were performed 3 months later. RESULTSAmyloid pathology was the dominant driver of microglial state transitions, while Ms4a4a loss selectively remodeled activated microglial transcriptional programs enriched for interferon, lysosomal, autophagic, and proteostatic pathways. Activated microglia from 5xFAD 4A-KO mice exhibited altered expression of genes linked to immune signaling and protein handling. Following AD-tau inoculation, Ms4a4a loss did not significantly alter overall phospho-tau burden but selectively reduced dense-core plaque-associated neuritic plaque tau (NP-tau), particularly in the contralateral hemisphere. This phenotype was strongest surrounding X-34-positive fibrillar plaques and occurred without major changes in plaque-associated microgliosis. DISCUSSIONThese findings identify Ms4a4a as a regulator of plaque-associated microglial programs linked to NP-tau accumulation in the amyloid-bearing brain. More broadly, this work supports a model in which AD resilience-associated microglial pathways selectively shape plaque-associated microenvironments that promote downstream tau pathology.

Amyotrophic lateral sclerosis (ALS) shows well-established sex differences in incidence, age at onset and progression, but their biological basis is unclear. We hypothesised that the DNA methylation changes associated with ALS differ between the sexes, which would point to sex-specific disease processes. To test this, we performed a blood-based epigenome-wide association meta-analysis of ALS cases and controls from Project MinE, an international ALS genomics consortium, and an independent Australian cohort, comprising 8,946 individuals after quality control (5,129 males and 3,817 females; 8,942 with complete covariates in the primary models). We analysed 781,648 autosomal CpGs using complementary sex-stratified case-control models and an ALS-by-sex interaction model that directly tested whether ALS-associated methylation effects differed by sex. Sex-stratified analyses identified no genome-wide significant differences between male and female ALS effects. In contrast, interaction testing identified seven robust sex-dependent ALS-associated differentially methylated positions (P < 9 x 10-8), mapping to KCNQ1, ADPRH, HLA-F, GHRLOS, IRF5, RAD51B and one intergenic region. The strongest signal was at KCNQ1 (P = 3.1 x 10-9). Six of the seven remained genome-wide significant in a four-stratum sensitivity analysis, and none was detected in previous conventional ALS or population sex-associated methylation analyses. Together, these findings suggest a distinct sex-conditional component of the ALS blood methylome, implicating immune, metabolic and ion-channel loci and providing new candidates for investigating biological heterogeneity between males and females with ALS.

Synapse loss is the strongest pathological correlate of cognitive decline in Alzheimers disease, yet the molecular basis of synaptic vulnerability across disease progression remains poorly understood. We performed proteomic profiling of isolated synaptoneurosomes from post-mortem middle temporal gyrus tissue from 67 individuals, comprising 40 individuals spanning all Alzheimers disease Braak stages and 27 non-diseased controls. Differential abundance and weighted gene co-expression network analyses were combined with pathway, cell-type and gene set variation analyses to characterise stage-associated changes in the synaptic proteome. Brain-derived candidate proteins were subsequently evaluated using receiver operating characteristic and regression analyses against cognitive examination scores in two independent plasma proteomics datasets. We identified six distinct stage-associated synaptic proteomic signatures. Mitochondrial function and synaptic signalling declined by mid-stage Alzheimers disease, while clathrin-mediated endocytosis increased at mid-stage without further change at later stages. By contrast, proteostasis and perisynaptic astrocytic processes increased progressively throughout the disease course. Global excitatory and inhibitory protein signals also showed a progressive shift in their relative balance (P = 7.80 x 10-3; adjusted R{superscript 2} = 0.072). These findings guided targeted investigation of candidate plasma markers linked to distinct synaptic processes in two independent proteomic datasets. We nominated 42 plasma proteins mapping to synaptic signatures of progression, which showed associations with disease stage and cognitive measures including Mini-Mental State Examination, Montreal Cognitive Assessment and Clinical Dementia Rating. PHGDH was prioritised across analyses through associations with cognitive impairment from early disease stages (Clinical Dementia Rating transition 0 to 0.5, false discovery rate = 8.87 x 10-18), neuropathological progression (P = 6.85 x 10-7) and excitatory-inhibitory protein imbalance (P = 5.56 x 10-3). Together, our findings define the stage-associated molecular changes at the AD synapse and connect distinct brain pathways to accessible plasma candidates, establishing a mechanistically anchored framework for future patient stratification and evaluation of synapse-directed therapies.

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.

Aberrant neuronal cell cycle re-entry (NCCR) is an alternative pathogenic mechanism in Alzheimer disease (AD) that has gained substantial support in the literature. The pathogenic role of ectopic NCCR is supported by our past work demonstrating that SV40T-mediated NCCR in adult mice can induce numerous pathologies associated with AD. Since NCCR is chronically induced for an extended period in the mouse model which gives rise to numerous pathologies including neuroinflammation, many of these neuropathological changes could simultaneously participate in driving disease progression. We hypothesized that the NCCR is a primary pathogenic driver and that halting this disease process at a later age could be sufficient for preventing the progression of AD-related pathologies. Here we show that modulation of NCCR at a later age prevents the progression of AD pathologies, including Abeta; and tau pathologies. Furthermore, functional genomics analysis demonstrates the late-onset AD (LOAD)-relevance of NCCR. Our findings suggest that our NCCR mouse model could help identify novel therapeutic targets that could aid in preventing AD progression.

Frontotemporal lobar degeneration (FTLD), predominantly comprised of tau and TDP-43 proteinopathies, is a common cause of early-onset dementia. Biomarkers that reliably classify FTLD subtypes are limited, thereby hindering in-vivo diagnosis and clinical trial stratification. We conducted large-scale, multi-platform cerebrospinal fluid proteomics in 566 individuals, including 274 FTLD-spectrum (48 autopsy-confirmed FTLD-TDP, 90 autopsy-confirmed FTLD-tau), 171 Alzheimers disease, and 95 controls. In neuropathological subtype contrasts, elevated matrix metalloproteinase MMP10 classified Picks disease (PiD) versus other subtypes with near-perfect accuracy across proteomic platforms. We further validated CSF MMP10 as a potential PiD diagnostic biomarker via a targeted immunoassay in an independent, autopsy-confirmed FTLD cohort. Post-synaptic proteins HOMER1 and DLG4 exhibited elevations across FTLD-TDP subtypes (A, B, and C) versus FTLD-tau. Network analysis identified RNA metabolism and pro-inflammatory signatures in FTLD-TDP, and cytoskeletal and heat shock signatures in progressive supranuclear palsy. These findings reveal candidate diagnostic biomarkers and proteomic pathways underlying FTLD clinico-pathological heterogeneity.

Charcot Marie Tooth type 1A (CMT1A) is the most common inherited peripheral neuropathy, caused by duplication of the peripheral myelin protein 22 (PMP22) gene and characterized by dysmyelination and defects in myelin maturation. Hypercellularity is a prominent feature of the disease, although its origin, persistence, and underlying mechanisms remain controversial. Here, we performed a longitudinal analysis of sciatic nerves from wild-type (WT) and CMT1A rats between 2 and 12 months of age. We demonstrated that CMT1A nerves exhibit persistent cellular proliferation extending into adulthood. This phenotype is confirmed by bulk RNA-seq data showing sustained enrichment of proliferation-associated gene signatures. Cell type signature analysis identifies overrepresentation of immature, neural crest related, and epithelial mesenchymal transitional (EMT) transcriptional programs in CMT1A nerves. Among the most upregulated genes, RUNX2 showed strong and prolonged expression at both transcript and protein levels. Consistently with the key role of RUNX2 in the activation of the injury response, CMT1A nerves also showed a marked enrichment in SOX10+JUN+ cells, that only partially overlap with SOX10+RUNX2+ cells, indicating a maladaptive activation of the injury-like Schwann cell (SC) program. Interestingly, we detected robust upregulation of SMOC2, an extracellular matrix protein selectively expressed by endoneurial fibroblasts, pointing to activation of non-canonical cell types and altered extracellular matrix remodeling. Together, these data demonstrate that CMT1A peripheral nerves are characterized by chronic hypercellularity driven by persistent proliferation, EMT, and dysregulated injury transcriptional programs, collectively pointing at progressive and multicellular nerve remodeling as a relevant pathomechanism in the disease.

MAPT (Tau) dysregulation is implicated in several neurodegenerative diseases, but its contribution to amyotrophic lateral sclerosis (ALS) is poorly understood. Here we show that mRNA isoforms encoding 4-repeat (4R) Tau are upregulated and cytoplasmically enriched in iPSC-derived motor neurons (MNs) from VCP-mutant and sporadic ALS, without a corresponding change in Tau protein. Using splice-switching antisense oligonucleotides and isoform-specific siRNAs, we find that enhanced 4R expression reduces MN viability, whereas its selective knockdown improves survival, with kinetics more consistent with an RNA-intrinsic effect than altered protein synthesis. Exon 10-containing MAPT RNA shows increased predicted secondary structure, self-association and altered Tau biocondensation in vitro. In post-mortem ALS cervical spinal cord, increased relative exon 10 usage is associated with a higher-risk clinical phenotype and shorter disease duration These findings identify an isoform-specific contribution of MAPT to MN vulnerability in ALS and nominate 4R MAPT RNA as a therapeutic target.

Cerebrovascular dysfunction is increasingly recognized as a central contributor to the initiation and progression of Alzheimer's disease (AD). The mammalian/mechanistic target of rapamycin (mTOR) pathway has emerged as a key driver of AD-related vascular dysfunction. Consistent with this role, attenuation of mTOR signaling restores multiple aspects of cerebrovascular function in AD models, including vascular density, cerebral blood flow, vascular reactivity, and neurovascular coupling. However, the molecular mechanisms linking mTOR dysregulation to cerebrovascular dysfunction in AD remain poorly understood we assessed cerebrovascular levels of RNA-regulatory proteins in hAPP(J20) mice treated with rapamycin from 6 to 12 months of age. Rapamycin partially restored disease-suppressed heterogeneous nuclear ribonucleoprotein (hnRNP) family members, including hnRNP D1 (AUF1) and hnRNP E1 (PCBP1). Rapamycin-mediated restoration of hnRNP extended to the cerebrovasculature of PS19 (P301S) tauopathy mice, indicating that mTOR-dependent hnRNP suppression is shared across amyloid- and tau-driven models of AD. Consistent with these findings, hnRNP E1 and hnRNP L each showed a nominally significant inverse relationship with Braak stage in AD patient brains, with a similar but non-significant trend for hnRNP R and hnRNP M; The coordinated pattern of decline suggests network-level disruption of RNA-binding protein homeostasis in AD. Together, our studies uncover a previously unrecognized link between mTOR signaling and the regulation of RNA-regulatory proteins and identify a previously unrecognized, reversible mTOR-dependent RNA regulatory program in the cerebrovasculature that may contribute to neurovascular dysfunction in AD and reveal new targets for therapeutic intervention.

Frontotemporal lobar degeneration (FTLD) encompasses clinically and biologically heterogeneous syndromes that are unlikely to be captured by a single blood biomarker. Although plasma neurofilament light (NfL) chain reflects neuroaxonal injury, multiplex profiling may identify complementary inflammatory, vascular and proteostatic processes relevant to clinical phenotype and progression. We characterised plasma CNS-related and inflammatory proteomic signatures across FTLD-related clinical syndromes and tested their associations with neuroaxonal injury, clinical severity, regional brain atrophy and survival. Plasma samples from 177 participants with behavioural variant frontotemporal dementia (bvFTD), primary progressive aphasia (PPA), progressive supranuclear palsy (PSP) or corticobasal syndrome (CBS) and 67 healthy controls were analysed using the high-sensitivity nucleic acid-linked immuno-sandwich assay (NULISA) platform. We quantified 130 CNS-related and 250 inflammation-related proteins. Differential expression and multivariate classification analyses were complemented by hierarchical clustering, penalised regression and survival analyses. Canonical correlation analysis tested multivariate associations between disease-associated proteins and regional MRI brain volumes. Sixty-one CNS-related and 61 inflammation-related proteins differed between participants with FTLD-related syndromes and controls after false-discovery-rate correction. NfL was the strongest individual discriminator between all patients and controls, with an area under the receiver operating characteristic curve of 0.91. In exploratory full-cohort models, the multivariate CNS signature achieved an area under the curve of 0.93, compared with 0.82 for the multivariate inflammation signature. However, inflammatory proteins provided stronger discrimination for selected syndrome comparisons, including PPA versus PSP. Among inflammation-related proteins, SPP1 and CD276 showed the strongest positive associations with NfL. Higher CD276 and SPP1 were associated with increased mortality, whereas higher amyloid-{beta}42 was associated with lower mortality. A multivariate protein signature weighted towards tumour necrosis factor and immune signalling was associated with lower regional brain volumes across predominantly frontotemporal and limbic regions (canonical correlation 0.43; permutation p=0.018). Plasma multiplex proteomics identified complementary dimensions of neuroaxonal injury and immune dysregulation across frontotemporal lobar degeneration-related syndromes. CNS-related proteins, particularly NfL, predominantly captured case-control neurodegeneration, whereas inflammatory signatures provided additional information about syndrome heterogeneity, structural brain changes and survival. SPP1, CD276 and broader immune-related signatures are candidates for independent biomarker validation and mechanistic investigation.