Acta Neuropathologica

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.

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.

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.

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.

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.

Neuroinflammation is a hallmark of several chronic neurodegenerative disorders, including Parkinson's disease (PD) and other synucleinopathies, and is closely associated with microglial activation. The inflammasome adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) undergoes oligomerization upon inflammasome activation, resulting in the formation of ASC specks. These structures are recognized by microglia as damage-associated molecular patterns (DAMPs), thereby promoting inflammatory signaling. Recent studies have demonstrated that extracellular ASC specks associate with amyloid-{beta} (A{beta}), and that uptake of ASC-A{beta} composites by microglia amplifies pro-inflammatory responses. Although ASC specks have also been shown to potentiate -synuclein (Syn) preformed fibril-induced pathology, their interaction with monomeric Syn and their effects on inflammasome activation remain poorly understood. Here, we co-stimulated murine SIM-A9 microglia with ASC-Syn assemblies and evaluated their effects on NLRP3 inflammasome activation. Electron microscopy revealed a close association between ASC fibrils and Syn monomers. Exposure of microglia to ASC-Syn enhanced NLRP3 inflammasome activation, as evidenced by increased ASC speck formation, caspase-1 activation, and secretion of IL-1{beta} and IL-18, when compared to ASC or Syn alone. In addition, immunohistochemical analyses of post mortem brain tissue from patients with dementia with Lewy bodies demonstrated ASC expression in regions exhibiting Syn pathology and showed ASC co-localization with Syn within Lewy bodies. Collectively, these findings identify ASC-Syn assemblies as potent amplifiers of NLRP3 inflammasome signaling and suggest a mechanism through which they may contribute to sustained neuroinflammation in synucleinopathies.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with ~90% of cases being sporadic. Although genome-wide association studies have identified numerous genetic risk loci, these variants account for only a portion of ALS heritability, suggesting that additional genetic and regulatory mechanisms contribute to disease risk. Furthermore, the mechanisms linking common variants to disease pathogenesis remain poorly understood. Adenosine-to-inosine (A-to-I) RNA editing is dysregulated in ALS, yet systematic identification of disease-relevant editing events and their functional consequences has been limited. We performed comprehensive RNA editing analysis across eight central nervous system regions from a Target ALS cohort, identifying 752 differentially edited sites in 304 genes. Global RNA editing was significantly reduced in ALS tissues, particularly in spinal cord and choroid plexus, with altered editing in genes involved in immune signaling, stress response and RNA processing. Through genetic colocalization analysis integrating editing quantitative trait loci (edQTLs) with ALS GWAS data, we identified common variants at the DEGS1 locus associated with both increased 3' UTR editing and elevated ALS risk (posterior probability >0.88). DEGS1 encodes dihydroceramide desaturase 1, a key enzyme converting dihydroceramide to ceramide, the precursor to all sphingolipid species. Using CRISPR-generated DEGS1 knockout iPSCs and lipidomic profiling, we demonstrated that DEGS1 is essential for ceramide synthesis and downstream sphingolipid production in astrocytes. Functional validation revealed that ALS-associated editing of the DEGS1 3' UTR significantly increased DEGS1 protein expression. Critically, astrocytes expressing the fully edited DEGS1 3' UTR increased ceramide levels and reduced cell viability compared to those expressing the unedited construct. These findings establish a novel regulatory link between RNA editing, sphingolipid metabolism and ALS pathogenesis. Our results demonstrate that common genetic variants can influence disease risk through post-transcriptional mechanisms that alter lipid homeostasis in astrocytes, a cell type increasingly recognized as central to ALS progression.

Blood-brain barrier (BBB) malfunction contributes to pathophysiology of Alzheimer's disease (AD), yet the mechanisms that confer hippocampal endotheliocytes vulnerability remain unclear. Here we identify loss of endothelial major facilitator superfamily domain-containing protein 2A (MFSD2A), a suppressor of caveolae-mediated transcytosis, as an early and hippocampus-specific feature of AD-associated vascular impairment. In post-mortem human AD brains, reduction in hippocampal MFSD2A was accompanied with increased levels of caveolin-1 and perivascular albumin extravasation. Analysis of human single-nucleus transcriptomic data further demonstrated that vascular expression of MFSD2A declined during transition from mild cognitive impairment to established AD. In APP/PS1 mice, MFSD2A loss occurred early in hippocampal microvessels and coincided with disproportionately high hippocampal BBB leakage and reductions in GLUT1 and claudin-5, despite broader amyloid deposition throughout other brain regions. Restoration of endothelial MFSD2A using an AAV-BR1-based vascular vector during early stage of amyloid pathology, reduced albumin extravasation and preserved GLUT1 and claudin-5. Early intervention also reduced amyloid plaque burden and plaque-associated neuritic dystrophy, whereas this treatment during established amyloid pathology, although modifying selected endothelial markers, failed to reverse BBB leakage, amyloid pathology, or cognitive impairment. Sustained MFSD2A restoration from the early disease stage provided the greatest protection, preserving hippocampal BBB integrity, reducing amyloid and neuritic pathology, improving long-term potentiation and ameliorating spatial learning and memory deficits. These findings define endothelial MFSD2A loss as a disease stage-sensitive mechanism of hippocampal BBB vulnerability and highlight the MFSD2A-caveolae axis as a therapeutic target for early-stage AD.

INTRODUCTION: Alzheimer's disease (AD) disproportionately affects women, but the underlying mechanisms are not fully understood. We compare sex-specific molecular changes across AD with and without dementia at single-cell resolution. METHODS: We performed sex-stratified single-nucleus transcriptomic analysis of human AD to dissect sex-specific differences in dementia progression, using the SEA-AD cohort for discovery and the ROSMAP and NBB datasets, together with 5xFAD mouse models, for validation. Donors were classified as no-dementia controls, no-dementia AD, or dementia AD, followed by donor-level pathway analysis, de novo microglial clustering, communication modeling, and cytokine-response enrichment RESULTS: Females with AD dementia showed enhanced inflammatory pathway activity across glial, neuronal and vascular-associated cell types, with the strongest enrichment in microglia. This female-biased program centered on IL6-JAK-STAT3 signaling and increased STAT3 expression in microglia and astrocytes. These results were validated in the ROSMAP and NBB datasets and conserved in the 5xFAD mouse model. Microglial clustering resolved 18 states and revealed sex-divergent disease remodeling, including female-specific expansion of redox-reactive and CD163+ macrophage-primed states. Cell:cell communication analysis identified female-biased TGF{beta}/GRN signaling, male-biased CD99 signaling, and dementia-associated SPP1-integrin communication with the vasculature. Microglial cytokine-response analysis identified IL-2 response as a dementia-associated signature in both sexes, alongside a female-biased IL-17/interferon response and a male-biased IL-7 response. DISCUSSION: These findings identify a disease-emergent, female-biased STAT3-centered neuroinflammatory program in human and mouse AD, linking sex-specific microglial remodeling to cytokine responsiveness and glial-vascular communication. The results suggest that sex-specific changes in neuroinflammation pathways in microglia and other cell types are associated with progression to dementia.

Pathological tau disrupts neuronal activity and brain function in Alzheimer s disease and other tauopathies. However, the underlying mechanisms for neuronal network dysfunction remain largely unknown. Using human inducible pluripotent stem cell (iPSC) - derived neurons, we show that the familial MAPT P301L mutation leads to neuronal network hyperexcitability. Imbalanced excitation and inhibition were attributable to neuronal subtype-specific tau pathology that was associated with different alterations of tau interactomes in excitatory versus inhibitory neurons. Suppressing enhanced excitatory synaptic transmission or blocking Ca2+ influx resulting from enhanced P301L tau/AHNAK interaction in inhibitory neurons rescued the neuronal network hyperexcitability. This study expands our understanding on how disease-causing tau mutations alter the excitatory/inhibitory balance, leading to network dysfunction in human neurons.

Cerebral amyloid angiopathy (CAA), characterized by cerebrovascular amyloid deposition, is strongly associated with Alzheimer's disease, ischemic injury, and lobar hemorrhages. APOE4 is the strongest known genetic risk factor for CAA, yet the cellular sources of APOE4 that drive pathogenic vascular amyloid deposition remain poorly defined. Here, we identify vascular-associated fibroblasts (VAFs) as a key source of APOE4 that mediates CAA and cerebrovascular dysfunction. In post-mortem brain tissue from APOE4/E4 patients with confirmed CAA, APOE colocalizes with COL6+ VAFs around A{beta}-laden vessels. Therefore, to study the contribution of APOE4 derived from VAFs in CAA, independently of parenchymal sources, we used 5xFAD mice in which the endogenous mouse Apoe coding exons are replaced with floxed human APOE4 sequences. This enables selective suppression of APOE4 expression in VAFs upon tamoxifen induction of Col1a2-CreERT+. APOE4 reduction in VAFs markedly reduced CAA, with the strongest effects observed in leptomeningeal vessels. Additionally, A{beta}40, which preferentially associates with vascular amyloid, was significantly reduced in the vascular fraction. VAF APOE4 depletion resulted in fewer small microbleeds and an approximately 50% reduction in parenchymal border macrophages within the leptomeningeal compartment, whereas astrocytic reactivity remained unchanged. Functional assessment using in vivo multi-parametric photoacoustic microscopy revealed improved arterial dilation and blood flow in amyloid-laden leptomeningeal arteries. Together, these findings establish VAF-derived APOE4 as a compartment-specific driver of CAA and cerebrovascular dysfunction.

Nasu-Hakola disease (NHD), a dementia with about 200 cases worldwide, is caused by loss-of-function mutation of TYROBP, encoding DAP12, or its receptor TREM2. TREM2-DAP12 is the switch that converts microglia from a surveillant to a reactive state; what DAP12-deficient microglia become and to what extent they reshape their microenvironment is unknown. We profiled prefrontal cortex and hippocampus from an NHD donor and control by single-nucleus RNA sequencing and mapped the glial changes in situ by spatial transcriptomics. NHD microglia adopted a CIITA-driven, MHC class II-high antigen-presenting state with complement, iron and glycolytic programs and Fc receptors but not the TREM2-dependent lipid program. Astrocytes became pan-reactive and lost metallothionein expression, oligodendrocytes lost mature cells and cholesterol synthesis, and excitatory neurons lost presynaptic and activity programs, consistently between regions and across a published cohort. DAP12 deficiency left microglia in an antigen-presenting state short of the TREM2-dependent arm, accompanied by a glial-to-neuronal cascade.

Variants at the CD2AP locus, which encodes a branched-actin regulator, are associated with late-onset Alzheimer's disease (AD), and CD2AP-deficient mice show increased blood-brain barrier (BBB) permeability. CD2AP is enriched in endothelial cells, but its function there is poorly understood. In endothelium, branched actin networks maintain cell-cell junctions and are also required for immune cells to cross through individual endothelial cells (transcellular transendothelial migration, TEM). We previously showed that the Arp2/3 activators N-WASP and WAVE2 contribute to these processes, but the capping protein (CP)-interacting proteins involved are unknown. Here we tested whether CD2AP, a CP-interacting protein recruited to cell-cell contacts, regulates both junction integrity and TEM. In primary human microvascular endothelial cells, shRNA knockdown of CD2AP increased TNF-induced monolayer permeability, measured by transendothelial electrical resistance, and altered VE-cadherin junction morphology. Unexpectedly, on soft hydrogels the same knockdown reduced TEM of NK-92 MI cells, lowering the proportion of transcellular events. Thus, loss of CD2AP weakens the endothelial barrier yet impairs a distinct, actin-dependent route of immune-cell passage. These findings suggest that CD2AP risk variants may affect the BBB through more than permeability, and they identify endothelial immune-cell trafficking as a testable link between CD2AP and AD.

Spinal muscular atrophy (SMA) is usually considered a lower motor neuron disease; however, growing evidence from both experimental models and clinical studies indicates that SMN deficiency affects the whole CNS, beyond the spinal and brainstem motor system. Here, we investigated the temporal progression and selective vulnerability of cortical projection neurons in the SMN{Delta}7 mouse model at early (P5) and late (P11) symptomatic stages. Combining subtype-specific immunohistochemistry, DiI tracing associated with morphometric reconstruction, and dendritic spine analysis, we identified stage- and subtype-dependent cortical alterations. At P11, corticospinal and infragranular callosal projection neurons were selectively reduced in number and showed marked dendritic remodeling and altered excitatory synaptic organization, whereas corticothalamic neurons and supragranular SATB2-positive populations were comparatively preserved. Notably, cortical abnormalities were already detectable at P5, before overt neuronal loss: indeed, corticospinal neurons exhibited reduced soma size and dendritic complexity, while dendritic spine density remained preserved, but spine morphology was shifted toward immature phenotypes. These early maturation defects persisted during disease progression and preceded the reduction in spine density observed at P11. Together, our findings identify cortical projection neurons as selectively vulnerable to constitutive SMN deficiency and suggest that impaired neuronal and synaptic maturation represents an early feature of cortical pathology preceding subtype-specific neurodegeneration. These results expand the pathophysiological spectrum of SMA beyond lower motor neurons and highlight early brain alterations as a potential component of disease progression.

Background: Dysregulated innate immune activation is a key feature of Parkinson disease (PD), but the upstream triggers and cell-type-specific mechanisms remain unclear. Double-stranded RNA (dsRNA) has emerged as a potential driver of innate immune responses. Adenosine deaminase acting on RNA 1 (ADAR1), particularly its interferon-inducible p150 isoform, edits dsRNA to limit aberrant immune activation. Whether this dsRNA-ADAR1 axis contributes to neuroinflammation in PD in a cell-type-specific manner is unknown. Methods: We performed immunofluorescence on post-mortem cingulate cortex tissue from PD cases and controls to localise dsRNA, ADAR1 p150 and pathological alpha-synuclein protein. We integrated these findings with single-nucleus RNA sequencing data from three independent human brain datasets. An eight-module gene scoring framework was applied to assess dsRNA processing, sensing, and downstream innate immune pathways in neurons and astrocytes. Results: In PD cortex, the proportion of dsRNA-positive astrocytes was higher than in controls, whereas neuronal dsRNA positivity did not differ between groups. ADAR1 p150 positivity increased in both astrocytes and neurons, and the proportion of ADAR1 p150 positive astrocytes correlated with astrocytic -synuclein burden. Transcriptomic analyses showed that neurons were enriched for dsRNA editing and processing machinery, whereas astrocytes exhibited higher expression of dsRNA sensing pathways, innate immune responses, and transposable element control. While overall pathway activity did not differ between PD and controls after covariate adjustment, network analyses revealed cell-type-specific coupling patterns. Notably, complement gene expression showed a strong and selective association with dsRNA-related pathways in astrocytes, whereas interferon and NF-{kappa}B signalling were shared across cell types. In both cell types, ADAR1-associated gene programmes were enriched for oxidative phosphorylation and PD-related pathways. Conclusions: These findings identify astrocytes as a central site of relative dsRNA accumulation and ADAR1 p150 induction in the PD cortex. The selective coupling of dsRNA sensing to complement activation in astrocytes highlights a cell-type-specific innate immune pathway that is constitutively present rather than induced by disease. Together, our protein-level and network-level findings converge on a coherent and testable model in which a constitutive astrocytic circuit is engaged by disease-associated dsRNA accumulation, with potential relevance for disease progression and therapeutic targeting.

Extracellular vesicles (EVs) play a critical role in the propagation of Tau pathology in Alzheimer's disease (AD). The selective identification and characterization of Tau seed-carrying EVs would help developing new diagnostic tools and therapeutic strategies. While pathogenic forms of Tau can be detected biochemically in EV-enriched fractions, only super-resolution fluorescence imaging provides both morphological and compositional information at the level of single EVs on account of their small size and the low number of target molecules. This was the object of our study. Post-mortem human brain-derived EVs from AD patients and non-demented control individuals were characterized in depth using biochemistry, transmission electron microscopy (TEM) and single molecule localization microscopy (SMLM). Dual-colour SMLM of immobilized EVs, displaying size distributions that were consistent with TEM, enabled statistically meaningful measurements of thousands of EVs in parallel. Owing to the high spatial resolution and capability of SMLM to detect sparse epitopes, we identified pathological Tau proteins in individual EVs from AD samples. Reconstructed super-resolution images of permeabilized EVs demonstrate that pathological Tau forms are frequent cargo proteins of EVs from AD brains. Our data illustrate the power of SMLM to determine the ultrastructure, molecular composition and topology of single EVs and EV populations.