Acta Neuropathologica Communications

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.

As we have entered the endemic phase of COVID-19, there is a need to assess the long-term effects of SARS-CoV-2 infection on the trajectory of neurodegenerative disorders. Herein, we investigate whether the SARS-CoV-2 RNA-binding nucleocapsid protein (NCP; N-protein) may influence molecular processes relevant to motor neuron degeneration in amyotrophic lateral sclerosis (ALS). We detected N-protein immunoreactivity in ALS spinal cord motor neurons, including cytoplasmic N-protein-positive puncta that colocalized with cytoplasmic TAR DNA-binding protein 43 (TDP-43). In vitro, optogenetically induced N-protein biomolecular condensates (BMCs) showed reversible, liquid-like behavior and recovered after photobleaching. Using this model, we found that these condensates colocalized with TDP-43 and the RNA-binding proteins G3BP1/2, PABPC1 and TIA1. Condensate formation was associated with TDP-43 cytoplasmic mislocalization, altered TARDBP expression, and altered exon 3 splicing of POLDIP3. The N-terminal portion of N-protein influenced BMC formation, whereas truncated C-terminal proteoforms did not form detectable BMCs or show detectable TDP-43 colocalization in this assay. Extending these observations to a human cell model, we show that optogenetically induced N-protein BMCs colocalize with TDP-43 in human iPSC-derived motor neurons. Collectively, these findings support further investigation into potential molecular links between SARS-CoV-2 exposure and the trajectory of neurodegenerative disease.

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.

CREB-regulated transcription coactivator 1 (CRTC1) regulates neuronal development, plasticity, and activity-dependent transcription, but its role in medulloblastoma (MB) remains poorly understood. We investigated expression of the CRTC1 gene during human cerebellar development and across pediatric brain tumors and examined its molecular and clinical associations in MB using public transcriptomic datasets. CRTC1 was broadly expressed in the developing cerebellum, with cell-state-specific variation within the granule-cell lineage. Among pediatric brain tumors, MB showed the highest median CRTC1 expression, which differed significantly among MB molecular groups and subtypes, with highest levels in Group 4 MB. High CRTC1 expression was associated with longer overall survival specifically in Group 3 {beta} MB and remained significant after correction across the 12 molecular subtypes. Within Group 3 {beta} tumors, CRTC1 expression was inversely associated with enrichment of mitochondrial and bioenergetic processes, including oxidative phosphorylation, mitochondrial organization, and mitochondrial translation, whereas CRTC1-high and CRTC1-low tumors displayed distinct transcriptional profiles. CRTC1 was also associated with selected cell-type-related transcriptional signatures related to the tumor microenvironment. These findings identify CRTC1 as a marker of biologically and clinically relevant transcriptional heterogeneity in Group 3 {beta} MB and support further investigation of its functional and prognostic significance.

Alexander disease (AxD) is a rare genetic leukodystrophy caused by dominant mutations in the gene coding for the astrocytic intermediate filament protein Glial Fibrillary Acidic Protein (GFAP). Understanding disease mechanisms is complicated by the fact that mutant GFAP promotes further GFAP accumulation, making it difficult to distinguish mutation-specific effects from those caused by elevated GFAP levels. Here, we performed mass spectrometry-based single cell proteomics to investigate proteomic alterations associated with increasing levels of wildtype GFAP and a common AxD-causing mutant GFAP R239C. By analyzing individual cells spanning a broad range of GFAP expression levels, we separated dosage-dependent effects from mutation-specific responses. Cells expressing high levels of wildtype GFAP increasingly resembled R239C-expressing cells at the proteomic level, supporting a role for GFAP accumulation in disease pathogenesis. In contrast, mutant GFAP specifically correlated with increased mitochondrial antioxidant enzymes and remodeling of the proteasome tailored toward ATP- and ubiquitin-independent degradation of oxidized proteins. Integration with proteomics data from patient-derived cortical organoids revealed increased abundance of nuclear-encoded mitochondrial proteins and respiratory chain assembly factors without corresponding changes in mitochondrially encoded proteins, suggesting impaired electron transport chain (ETC) assembly. Consistent with this, AxD cells displayed reduced oxygen consumption, increased glycolytic activity, enhanced utilization of the cytosolic part of the glycerol-3-phosphate (G3P) shuttle, and accumulation of triacylglycerides and lipid droplets. We propose that ETC dysfunction promotes partial uncoupling of the G3P shuttle, allowing sustained regeneration of cytosolic NAD to support glycolysis while diverting G3P toward lipid synthesis and storage. By regenerating NAD through the G3P shuttle rather than lactate fermentation, AxD astrocytes may sustain glycolytic flux while preserving lactate production, an important metabolic substrate for neighboring neurons. Together, our findings identify ETC dysfunction as a central feature of AxD and suggest that metabolic adaptation through the G3P shuttle links mitochondrial dysfunction to glycolytic reprogramming and lipid accumulation. More broadly, these results provide new insight into how astrocyte dysfunction drives molecular pathways implicated in neurodegeneration.

Background: Glioblastoma (GBM) adapts to therapy through coordinated malignant-cell and microenvironmental responses, but the mechanisms linking treatment-associated inflammation to tumor-cell phenotypic plasticity remain poorly defined. Here, we investigated whether preoperative temozolomide exposure is associated with inflammatory-repair remodeling and a ROCK-linked therapeutic vulnerability in recurrent GBM using a window-of-opportunity clinical trial cohort. Methods: We integrated patient-resolved single-cell transcriptomics, spatial RNA profiling and multiplex protein imaging with experimental perturbations and orthotopic glioblastoma models. In the presurgical window-of-opportunity subgroup of NCT05236036, four patients with recurrent glioblastoma received additional preoperative temozolomide (TMZ; 150 mg/m2/day for 5 days) before resection and were compared with three recurrent comparators who did not receive additional preoperative TMZ. Results: Across seven tumors, malignant-cell inflammatory activity covaried with integrin-binding and wound-healing programs (median partial {rho} = 0.45 and 0.42, respectively). Tumors exposed to preoperative TMZ showed enrichment of inflammatory, chemokine, adhesion and wound-healing programs (GSEA q < 0.05), with the direction of enrichment preserved in all leave-one-patient-out analyses. In two tumors exposed to preoperative TMZ, CXCL12 localized to vascular territories, whereas chemokine and wound-repair programs increased near injury-reference regions. Across seven specimens analyzed by multiplex protein imaging, MYL9 abundance correlated with local CXCL12 ({rho} = 0.61), phosphorylated MYPT1 ({rho} = 0.48) and nuclear phosphorylated STAT3 ({rho} = 0.45), with positive associations in every specimen. Experimentally, TMZ increased CXCL8 and reactive-state markers and enhanced subsequent scratch closure, while CXCL8 and CXCL12 increased MLC2 phosphorylation. Fasudil attenuated TNF-NF-{kappa}B, inflammatory-response and IFN-{gamma}-response programs in TMZ-treated cells and reduced scratch closure and p-MLC2 in complementary assays. Across three orthotopic models, fasudil plus TMZ prolonged survival versus TMZ alone (model-stratified HR, 0.21; 95% CI, 0.07-0.61; P = 0.001). Conclusions: These findings link treatment-associated inflammatory-repair programs with cytoskeletal remodeling and support further translational evaluation of fasudil plus TMZ in glioblastoma.

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.

Neuronal-cancer cell interactions are a key component of brain tumor pathophysiology, yet many fundamental questions remain unanswered, with even more remaining unasked. Here, we present a comprehensive brain tumor reference map generated from bulk RNA-seq data of ~4,300 adult and pediatric brain tumors, including various glioma subtypes, medulloblastomas, ependymomas, and meningiomas, alongside ~1,400 healthy brain samples. By analyzing synaptic processes and synaptic gene expression across tumor types, we reveal the complexity of neuron-tumor crosstalk and its potential impact on patient survival. Our findings identify key synaptic signaling pathways and dysregulated patterns in brain tumors, including HNRNPH2 upregulation, a key factor in RNA processing and metabolism, in gliomas and medulloblastomas. Additionally, we highlight the role of cholinergic signaling, particularly CHRNA9, as a driver of glioma progression, which is also upregulated in WNT and Group 4 medulloblastomas, ependymomas, and meningiomas. Furthermore, P2 purinergic signaling emerges as a key player in glioma progression. These insights lay the groundwork for future studies exploring whether targeting these pathways could reprogram the tumor microenvironment toward an anti-tumor state, ultimately leading to novel therapeutic strategies and improved patient outcomes.

Single-cell RNA sequencing (scRNA-seq) studies of Alzheimer disease (AD) mouse models have proliferated across independent laboratories, brain regions, and platforms, yet the cross-study reproducibility of transcriptional signatures derived from such data remains underexplored. We reanalyzed six publicly available mouse brain scRNA-seq datasets (GSE140399, GSE140510, GSE143758, GSE208683, GSE214244, and GSE296027; 360,620 cells, 102 annotated clusters) using a standardized pipeline (Figure 1) comprising quality control, Leiden clustering, confidence-tiered marker-based cell-type annotation, sample-level pseudobulk differential expression, cross-dataset consistency analysis, and pathway enrichment. Pseudobulk differential expression, performed on 32 samples (16 AD, 16 Control) from five of the six datasets, identified 301 significant genes (FDR-adjusted p < 0.05) out of 20,629 tested, of which 290 (96.3%) were upregulated and 11 (3.7%) downregulated in AD. The upregulated set was dominated by innate immune and interferon-response genes (Ccl3, Ccl4, Ccl6, Itgax, Cst7, Tlr4, Trem2) and showed extremely strong enrichment for immune system and innate immune system pathways (Reactome, p {equiv} 1.9 x 10-43 and 2.1 x 10-29 respectively), phagosome and osteoclast differentiation (KEGG), and interferon alpha/beta signaling. Of the 301 significant genes, 235 (78%) showed the same direction of effect across all datasets in which they were tested. The sixth dataset, GSE296027, was excluded from differential expression analysis due to a gene-identifier corruption artifact introduced during pseudobulk aggregation (detailed in Methods and Limitations) but contributed valid cell-type composition and microglial subclustering data, independently recovering a disease-associated microglia-like state. These findings indicate that a reproducible, strongly immune/interferon-skewed transcriptional signature, rather than a balanced up/down transcriptional shift, characterizes the mouse AD models analyzed here.

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.

Amyloid[beta]; (A[beta];) plaques are a hallmark of Alzheimer[prime]s disease (AD). A human cellular neuronal model that recaptures A[beta];-induced pathology is critical for advancing AD research. However, comprehensive proteomic profiling of A[beta];-induced cellular model remains elusive. In this study, we investigated the proteomic changes in induced pluripotent stem cell (iPSC)-derived neurons (iNs) exposed to synthetic A[beta]; (1-42) peptides (A[beta];42A) to improve our understanding of the cellular responses of A[beta]; aggregates and to establish a human-related platform for AD research. A[beta];42A formed extracellular aggregates around neuronal soma and neurites, impaired neurite outgrowth, and induced the expression of multiple AD-associated genes, such as APOE, BACE1, ADAM10. To define the molecular landscape of A[beta];-induced neuronal dysfunction, we performed proteomic analyses of whole-cell lysates as well as soma- and neurite-enriched fractions. Proteomic profiling revealed extensive gene alterations in pathways associated with synaptic function, neuronal maintenance, and AD pathogenesis, such as the upregulation of APOE. Importantly, the A[beta];42A-iNs system recapitulated molecular signatures observed in AD brain tissue and cerebrospinal fluid. Representative protein changes, including APOE upregulation and its colocalization with A[beta]; aggregates, were further validated in postmortem human AD brain tissue. Together, these findings manifest that the A[beta];42A-iNs system reproduces multiple cellular and molecular features of AD and exhibits strongly consensus with clinical observations. It provides a valuable platform for investigating A[beta];-driven neurodegeneration and for the discovery of therapeutic targets for AD.

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.

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.

Pontocerebellar hypoplasia (PCH) is a group of ultrarare, neurodegenerative disorders characterized by cerebellar and pontine hypoplasia. Genetic analysis over the last two decades has revealed an increasing number of pathogenic variants in a wide range of broadly expressed genes functioning in RNA processing, tRNA metabolism, and translation. However, the mechanisms linking these ubiquitous processes to brain region-specific vulnerability are unknown. Here, we established a multi-level variant-to-function in silico framework to predict the molecular consequences of PCH-associated variants in TSEN complex genes. These variants were predicted to have heterogeneous effects on diverse protein properties, including stability, subcellular localization, and degradation, supporting variant-specific rather than uniform disease mechanisms. Complementary transcriptomic analyses showed that PCH-associated genes were not globally enriched in the prenatal cerebellum. Instead, their expression was coordinated in a stage- and cell type-specific manner during cerebellar development. We therefore hypothesize that multiple PCH-associated genes are regulated by a common set of transcription factors, providing an explanation of the selective vulnerability of the cerebellum and to the phenotypic convergence of genetically diverse PCH subtypes. In summary, this study prioritizes candidate variants for biochemical, cellular, and in vivo validation, and identifies regulatory programs, cell lineages, and developmental windows for targeted, mechanistically informed disease modelling.

Huntington&'s disease (HD) is characterized by progressive accumulation of mutant huntingtin (mHTT), neurodegeneration and motor dysfunction. Increasing evidence implicates impaired glymphatic function and loss of perivascular aquaporin 4 (AQP4) polarization in HD, suggesting that defective brain waste clearance may contribute to disease pathogenesis. Whether restoration of AQP4&-mediated glymphatic transport can ameliorate HD pathology remains unknown. We tested whether genetic restoration of AQP4 improves glymphatic function and HD&-associated phenotypes in heterozygous zQ175 knock-in mice. At 2 months of age, premanifest zQ175 mice received a single retro-orbital injection of BBB-penetrant AAV9&-PHP.eB encoding the AQP4 (M1 isoform). Glymphatic transport was assessed by intracisternal injection of fluorescent BSA 647 and quantification of parenchymal influx and drainage to the deep cervical lymph nodes. Perivascular AQP4 localization, mHTT aggregation, gliosis, DARPP32 immunoreactivity, CSF neurofilament light chain (NfL) and total Tau, structural brain volumes by 11.7T MRI, and motor behavior were assessed longitudinally through 12 months of age. AAV9&-PHP.eB&-AQP4 produced widespread brain expression and restored perivascular AQP4 localization in the striatum and cortex of zQ175 mice. AQP4 overexpression significantly increased glymphatic tracer influx into brain parenchyma and BSA-647 drainage to the deep cervical lymph nodes. At 12 months, AQP4 restoration reduced CSF NfL and total Tau concentrations, mHTT aggregate number and mean aggregate size, and striatal Iba1 positive microglia and GFAP-positive astrocytes (P < 0.05). AQP4 overexpression also preserved striatal DARPP32 immunoreactivity and significantly attenuated striatal atrophy measured by MRI. Female zQ175 mice showed improved balance-beam performance and locomotor activity, while effects on motor performance in males did not reach statistical significance. AQP4 overexpression attenuated body-weight loss in both sexes. No detectable structural, behavioral or body-weight abnormalities were observed following sustained AQP4 overexpression in wild-type mice. In summary, restoration of AQP4 enhances glymphatic transport and is associated with reduced proteopathic, neuroinflammatory and neurodegenerative phenotypes in zQ175 HD mice. These findings provide proof-of-concept that impaired glymphatic function is a modifiable component of HD pathology and support AQP4&-mediated restoration of brain waste clearance pathways as a potential disease-modifying therapeutic strategy.

Extracellular cold-inducible RNA-binding protein (eCIRP) is a crucial neuroinflammatory mediator in ischemic stroke and alcohol-induced memory impairment. We have recently discovered that amyloid{beta} causes microglia to release eCIRP, prompting us to investigate its role in Alzheimer's disease (AD). We found that eCIRP levels were significantly elevated in the cerebrospinal fluid (CSF) and plasma of AD patients compared with age-matched non-AD subjects, as well as in hTau.P301S mice (a model of AD tauopathy) compared with wildtype control mice. Plasma eCIRP strongly correlated with astrocyte activation marker glial fibrillary acidic protein (GFAP) in AD patients. eCIRP induced neurotoxic astrocyte-specific genes and astrocyte release of proinflammatory and neurotoxic factors in C8-D1a cells, primary murine astrocytes and intracerebroventricular eCIRP-injected C57BL/6 mice brains. In particular, eCIRP increased Complement 3, an astrocytic marker involved in neuroinflammation-associated neurodegeneration. Primary astrocytes from TREM-1 knockout mice were resistant to eCIRP induction of neurotoxic astrocytes. eCIRP increased TREM-1 expression and activation in astrocytes. Blocking CIRP/TREM-1 interaction using peptide M3, effectively attenuated eCIRP's induction of neurotoxic astrocytes. Thus, eCIRP strongly correlates with astrocyte reactivity in AD patients, and eCIRP induces neurotoxic astrocytes via TREM-1, which is attenuated by M3, suggesting a novel therapeutic opportunity to target neurotoxic astrocytes in AD.

Neurodegenerative diseases (NDDs) are driven by multiple related yet distinct pathological processes that evolve over decades, resulting in substantial clinical and neuropathological heterogeneity. Postmortem examinations of older individuals frequently reveal multiple coexisting neuropathologies, however, the underlying pathological processes cannot be easily assessed directly in living individuals. To quantify pathology-specific genetic signatures, we analysed a spectrum of NDDs using polygenic risk scores (PRSs) in the UK Biobank, restricting analyses to genes reported to be expressed in major brain cell-types, including microglia, astrocytes, oligodendrocytes, and neurons. We found substantial overlap among purportedly cell-type specific gene sets (21-29%), with APOE or neighbouring genes from the APOE locus represented in all gene sets, indicating that current cell-type specific annotations capture considerable shared genetic variation across cell-types. Jointly, cell-type specific PRSs explained a large proportion (60-93%) of the genetic liability for NDDs in which dementia is a major clinical manifestation. Alzheimer's disease (AD) showed associations across all cell-types, with oligodendrocyte- and astrocyte-based PRSs demonstrating strong effects independent of APOE. AD PRS and APOE status independently differentiated Parkinson's disease (PD) with dementia from PD without dementia. PD without dementia and amyotrophic lateral sclerosis (ALS) exhibited distinct genetic profiles, with limited overlap with other NDDs. Collectively, these findings reveal both shared and disease-specific genetic contributions across neurodegenerative disorders and demonstrate that currently available cell-type specific gene sets capture substantial overlapping genetic risk. The imperfect alignment between clinical phenotypes and underlying pathology, coupled with the extensive overlap of gene expression across these cell types, underscores the persistent challenges in resolving the genetic architecture of neurodegenerative diseases and elucidating their underlying biological mechanisms.

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.

C9orf72 encodes a protein that is important for both typical neurodevelopment and homeostatic processes across the lifespan. Pathogenic C9orf72 repeat expansions constitute the most common genetic cause of frontotemporal lobar degeneration (FTLD). Collectively, preclinical models and clinical neuroimaging studies have observed smaller thalamic volumes in C9orf72-associated FTLD, including in asymptomatic young adult C9orf72 repeat expansion carriers across 18 to 30 years old, supportive of potential neurodevelopmental origins. However, it is unknown whether asymptomatic young adult C9orf72 expansion carriers demonstrate volume differences in specific thalamic nuclei, as reported in older asymptomatic carriers and symptomatic patients; if so, normative transcriptomic patterns could reveal relevant neurodevelopmental and/or FTLD-related neurodegenerative processes. This study investigated volumetric MRI differences of the thalamic nuclei in asymptomatic young adult C9orf72 expansion carriers compared to non-carriers, and examined spatial associations with normative thalamic transcriptomics from 8 weeks post-conception through middle age. Young adult C9orf72 expansion carriers (n=72) and non-carriers (n=48) were enrolled in the Frontotemporal Dementia Prevention Initiative across Europe, Canada, and the USA (ages: 18-30 years; sex: 57% female). Spatial associations between young adult thalamic nuclei volumes and normative transcriptomics from the Allen Human Brain Atlas were examined using complementary data-driven and hypothesis-driven approaches. Spatial expression patterns of transcripts of interest were characterized across prenatal and postnatal neurodevelopment using BrainSpan data. Smaller volumes of the pulvinar, mediodorsal, and lateral nuclei were observed in young adult C9orf72 expansion carriers compared to non-carriers. These nuclei showed higher normative expression of transcripts involved concurrently in neurodevelopmental and C9orf72-associated FTLD neurodegenerative processes (including lysosomal, autophagic, and small GTPase functions), and lower normative expression of CALB1, a marker of prenatal human thalamic development. Normative expression of CALB2 (another human thalamic development marker) yielded positive correlations with C9orf72 across prenatal development to adulthood. These findings identify candidate transcripts that may provide insight into the molecular architecture leading to smaller thalamic nuclei volumes in C9orf72-associated FTLD.

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.