Glioblastoma (GBM) is a devastating brain tumor with limited treatment success, partly because in vitro models poorly mimic in vivo complexity. This study introduces a high-throughput 3D culture platform utilizing modular starPEG-glycosaminoglycan (GAG) hydrogels that enable independent control of extracellular matrix (ECM) cues: stiffness, cytokine affinity, matrix metalloproteinase-responsive remodeling, and cell adhesiveness via integrin-binding RGD peptides. This platform supports encapsulation of patient-derived GBM cells, recreates physiologically relevant tumor microenvironments in 384-well plates, and enables automated drug testing on primary cells. Transcriptomic analyses show that 3D cultures recapitulate primary and recurrent GBM programs- including hypoxia-, immune-, and ECM-regulatory pathways driving growth, invasion, and resistance, without externally imposed hypoxia. The platform's versatility extends to drug screening, where single and combinatorial treatments produce reproducible cytoskeletal and transcriptomic responses. Notably, the system revealed dose-dependent reductions in invasive filaments and spheroid architecture with 5-fluorouracil/uridine and carmustine, demonstrating its potential for optimizing combinatorial therapies. This 3D model surpasses 2D cultures, capturing tumor-specific molecular programs and offering a robust tool for translational research. Despite lacking vascular or immune components, its tunability, scalability, and clinical relevance make it a strong basis for advanced co-cultures. By delivering reliable, individualized therapeutic data within a short timeframe, this model holds transformative potential for personalized GBM treatment.

Abstract Background Alzheimer’s disease (AD) involves complex molecular alterations in the cerebrospinal fluid (CSF) proteome, yet the links between these protein changes and hallmark AD pathology remain incompletely defined. We investigated the relationship between the CSF proteome with CSF biomarkers of Alzheimer’s disease (AD). Methods CSF was collected in 500 individuals of non-Hispanic white, African Americans, and Caribbean Hispanic individuals. CSF biomarkers of AD were measured including P-tau181, Aβ40, Aβ42, total-tau, neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP). CSF was depleted of abundant proteins followed by precipitation, cysteine reduction/alkylation, and proteolytic cleavage by trypsin. Peptides were measured using a Q-Exactive HF mass spectrometer (Thermo Scientific). Association of individual and co-abundant modules of proteins were tested using evelated CSF P-tau181 and reduced Aβ42/Aβ40 to confirm the diagnosis of AD. We validated results in CSF from 397 participants in the Accelerated Medicine Partnership-Alzheimer’s Disease cohort. Associated proteins were functionally validated in postmortem human brains and zebrafish. Results We detected 1030 proteins, yielding an overall data completeness value of 97%. CSF levels of 75 (7.3%) proteins were significantly associated with CSF P-tau181 levels after multiple testing correction. Notably phospholipase D3 (PLD3, p=2.41E-09), apoE (p=4.25e-08) and osteopontin (OPN p=1.4E-16) were increased and autotaxin (ATX/ENPP2, p= 8.39E-09) and ceruloplasmin (CP) (p=2.72E-07) were lower among individuals with high P-tau181 levels. These proteins were also associated with CSF Aβ42/Aβ40 ratio and total tau levels but not with NfL. OPN was also associated with CSF levels of GFAP (p=1.32e-05). Among proteins associated with P-tau181 levels, pathways related to axon development (p=2.4E-12), axonogenesis (p=1.45E-11) and regulation of axonogenesis (p=5.1E-09) were enriched. Immunostaining on postmortem human and zebrafish brain found that ENPP2 expression, the gene encoding ATX, was significantly reduced in AD brain and in the amyloidosis model in zebrafish. Reduced ENPP2 expression was consistent with reduced lysophosphatidic acid (LPA) levels in the CSF of individuals with AD. LPA administration into zebrafish CSF reduced the pathological changes in synapses and vasculature due to Aβ42. Conclusion Unbiased profiling of circulating CSF proteins among individuals with antemortem diagnosis of AD, identified key proteins PLD3, apoE, OPN, ATX, and ceruloplasmin. Validation in postmortem human brains and zebrafish models support potential roles in endosomal sorting and APP processing, inflammation, angiogenesis, lipid transport, and oxidative stress. One sentence summary Reduced autotaxin and lysophosphatidic acid were among 75 cerebrospinal fluid (CSF) proteins associated with biomarker-defined Alzheimer’s disease pathology.

Late-life cognitive impairment most commonly occurs in the setting of mixed neurodegenerative and cerebrovascular pathology, yet the molecular programs distinguishing vascular, neurodegenerative, and mixed pathology in the aging human brain remain incompletely defined. We performed neuropathology-stratified proteomic and transcriptomic profiling of postmortem brain tissue from participants in the Religious Orders Study and Rush Memory and Aging Project. Dorsolateral prefrontal cortex proteomics (n = 733) were analyzed alongside bulk RNA sequencing from dorsolateral prefrontal cortex (n = 938), posterior cingulate cortex (n = 569), and anterior caudate (n = 632). Participants were classified into vascular, neurodegenerative, and mixed pathology groups based on comprehensive autopsy assessment. Neurodegenerative and mixed pathology, relative to vascular pathology, showed coordinated upregulation of immune and inflammatory pathways and downregulation of mitochondrial and oxidative phosphorylation programs across molecular layers. Although few individual proteins differed between mixed and neurodegenerative groups, pathway-level analyses identified additional remodeling programs in mixed pathology, including extracellular matrix organization and vesicle-mediated transport. Proteomic co-expression network analysis identified immune-stress modules associated with amyloid burden, tau pathology, and cognitive decline, whereas a mitochondrial bioenergetic module showed relative preservation in vascular pathology. Cross-omics concordance was robust at the pathway level but limited at the level of individual genes and proteins. These findings define conserved molecular programs distinguishing vascular, neurodegenerative, and mixed pathology and demonstrate that pathway-level organization provides a stable framework for interpreting molecular heterogeneity in late-life dementia.

Evolutionary changes in cortical development were instrumental in the emergence of the mammalian neocortex. Cajal-Retzius cells, a transient neuron type discovered over a century ago, are critical players in the development of mammalian-specific cortical features such as inside-out neurogenesis. However, it is unclear whether Cajal-Retzius cells exist only in mammals or whether they are ancestral in vertebrates but acquired new functions during mammalian brain development. To trace the evolution of this cell type, we probe the presence of Cajal-Retzius cells in chicken, salamander, zebrafish, and little skate. First, through comparative transcriptomics and spatial analysis, we show the presence of cells with a Cajal-Retzius molecular identity in non-mammalian vertebrates. However, only amniote Cajal-Retzius cells gained robust expression of Reelin, a secreted glycoprotein crucial for mammalian cortical development. Second, we find that Cajal-Retzius cells are part of a larger and diverse family of neuron types and are closely related to Tp73+ external tufted cells in the olfactory bulb, which express most of the “canonical” Cajal-Retzius transcription factors. Our results indicate that Cajal-Retzius cells emerged early in vertebrate history, using a regulatory program largely shared with neurons in the olfactory system, and that transcriptomic novelties underlie their specialized roles in mammalian cortical development. We uncover an extreme example of how cell types may diverge in function over the course of evolution, even when their core transcriptomic profile is broadly preserved.

INTRODUCTION:Neuropathological studies indicate a strong association between Alzheimer's disease (AD) and stroke, yet the molecular mechanisms underlying this association remain unclear. METHODS:Local genetic correlation analysis was conducted with LAVA (Local Analysis of [co]Variant Annotation) using the results from genome-wide association studies on AD and stroke in individuals of African ancestry. Enhanced Hi-C Capture Analysis (eHiCA) examined chromatin interactions using induced pluripotent stem cell (iPSC) -derived cells from AD brain autopsy samples. RESULTS:LAVA identified a region shared between AD and stroke on chromosome 18q21.33(rg = 0.77, p = 2.41×10-6). eHiCA demonstrated that the AD and stroke loci interact with regulatory elements in PHLPP1. Variants at PHLPP1 were also associated with AD in an independent set of individuals of African ancestry (p = 4.56 × 10-5). DISCUSSION:This study identified a region on top of PHLPP1 as a locus associated with both AD and stroke. PHLPP1 inhibits protein kinase B, which contributes to both AD and stroke pathophysiology.

We investigated the relationship between the cerebrospinal fluid (CSF) proteome in Alzheimer's disease (AD) and the clinical and biomarker-assisted diagnoses, and with CSF biomarker levels of AD. CSF was collected in 500 individuals of non-Hispanic white, African Americans, and Caribbean Hispanic individuals from Dominican Republic and New York City. CSF biomarkers of AD were measured including p -tau181, Aβ40, Aβ42, total-tau, neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP). CSF was depleted of abundant proteins followed by precipitation, cysteine reduction/alkylation, and proteolytic cleavage by trypsin. Peptides were measured using a Q Exactive HF mass spectrometer (Thermo Scientific). Association of individual and co-abundant modules of proteins were tested with the clinical diagnosis of AD, as well as biologically defined AD pathological process based on CSF p -tau181 and other biomarker levels. Results from replicated in 397 participants from the Accelerated Medicine Partnership-Alzheimer's Disease CSF cohort and significantly associated proteins were functionally validated in postmortem human brains and zebrafish models. CSF levels of 41 proteins were significantly associated with p -tau181 levels after multiple testing correction. Notably phospholipase D3 (PLD3, p  = 2.41E-09), APOE ( p  = 4.25e-08) and osteopontin (OSTP, p  = 1.4E-16) were increased and autotaxin (ENPP2, p  =  8.39E-09) and ceruloplasmin (CERU, p  = 2.72E-07) were decreased among individuals with high p -tau181 levels. These proteins were also associated with CSF Aβ42/Aβ40 ratio and total Tau levels but not with NfL. OSTP was also associated with CSF levels of GFAP ( p  = 1.32e-05). We did not identify any protein association with clinical AD. Among proteins associated with p -tau181 levels, pathways related to axon development ( p  = 2.4E-12), axonogenesis ( p  = 1.45E-11) and regulation of axonogenesis ( p  = 5.1E-09) were enriched. Immunostaining on postmortem human and zebrafish brains found that ENPP2 expression reduces significantly with AD and amyloidosis, respectively. LPA administration into the zebrafish CSF mitigated Aβ42-induced vascular, neural, and glial changes. Unbiased profiling of circulating CSF proteins identified key proteins associated with β-amyloid and phosphorylated tau pathology. Biologically based diagnostic criteria may aid in the identification of unique pathogenic mechanisms.

Cerebrovascular disorders are associated with an increased risk for neurodegenerative diseases, including Alzheimer's disease (AD). However, the mechanisms by which cerebrovascular dysfunction contributes to neurodegeneration are poorly understood. We aim to dissect the complex interplay between vascular dysfunction and AD to molecular features in cerebrovascular cells (CVCs) through deep multi-omic profiling of human brains. We have previously performed single-nucleus transcriptomic profiles (snRNA-seq) and chromatin accessibility (snATAC-seq) of parietal cortex from healthy and AD donors from the Australian Brain Bank Network (ABBN; n  = 72 brains), which are richly annotated for cerebrovascular phenotypes, including cerebral amyloid angiopathy (CAA). In parallel, we generated spatially resolved transcriptomic profiles for a subset of these samples. We integrated this data with seven public snRNA-seq datasets to create a comprehensive multi-omic cerebrovascular atlas. Our cerebrovascular atlas encompassed >133K nuclei across major CVC types. This high resolution identified perturbed CVC transcriptional programs between cases and controls, including changes specific to rare AD risk variants. We identified perturbed regulatory programs associated with CAA in large-diameter endothelial cells and capillary-specific changes related to late-onset AD, including up-regulation of metallothionein genes. We validated these findings in human brains using immunohistochemistry. In addition, we identified an age-associated CVC transcriptional signature linked to APOE4 detectable before disease onset, suggesting that APOE4 associates with accelerated vascular aging. Lastly, we used snATAC-seq to prioritize multiple independent AD risk loci, including APP and APOE, where at least one fine-mapped risk variant (95% credible set) overlapped a regulatory element active in vascular cells. Finally, we observed extensive transcriptional changes in putative AD-risk genes in all CVCs subtypes, supporting a shared genetic risk between AD and cerebrovascular disease but suggesting the shared risk is mediated in a cell type-specific manner. Our work provides novel insights into the links between cerebrovascular dysfunction and AD and identifies genes and regulatory elements mediating AD genetic risk through CVCs.

Blood-brain barrier (BBB) dysfunction is a key feature of Alzheimer's disease (AD), particularly in individuals carrying the APOE-ε4 allele. This dysfunction worsens neuroinflammation and hinders the removal of toxic proteins, such as amyloid-beta (Aβ42), from the brain. In post-mortem brain tissues and in animal models, we previously reported that fibronectin accumulates at the BBB predominantly in APOE-ε4 carriers. Furthermore, we found a loss-of-function variant in the fibronectin 1 ( FN1 ) gene significantly reduces aggregated fibronectin levels and decreases AD risk among APOE-ε4 carriers. Yet, the molecular mechanisms downstream of fibronectin at the BBB remain unclear. The extracellular matrix (ECM) plays a crucial role in maintaining BBB homeostasis and orchestrating the interactions between BBB cell types, including endothelia and astrocytes. Understanding the mechanisms affecting the ECM and BBB cell types will be critical for developing effective therapies against AD, especially among APOE-ε4 carriers. Here, we demonstrate that APOE-ε4 , Aβ42, and inflammation drive the induction of FN1 expression in several models including zebrafish, mice, iPSC-derived human 3D astrocyte and 3D cerebrovascular cell cultures, and in human brains. Fibronectin accumulation disrupts astroglial-endothelial interactions and the signalling cascade between vascular endothelial growth factor (VEGF), heparin-binding epidermal growth factor (HBEGF) and Insulin-like growth factor 1 (IGF1). This accumulation of fibronectin in APOE-ε4- associated AD potentiates BBB dysfunction, which strongly implicates reducing fibronectin deposition as a potential therapeutic target for AD. Graphical abstract: Accessibility text:This image illustrates the effects of different APOE isoforms (ApoE-ε3 and ApoE-ε4) on blood-brain barrier (BBB) integrity, focusing on the molecular interactions between astrocytes and endothelial cells. This figure emphasizes the detrimental effects of ApoE-ε4 on BBB integrity via fibronectin accumulation and altered signaling pathways. The top section provides a schematic overview of the blood-brain barrier, highlighting astrocytes, endothelial cells, and their interface. The left panel represents the ApoE-ε3 condition: Normal fibronectin (FN1) levels support healthy interactions between astrocytes and endothelial cells. Growth factors, including VEGFA, HBEGF, and IGF1, maintain BBB integrity through their respective receptors (VEGFR and EGFR). Green arrows indicate activation of these signaling pathways. The right panel depicts the ApoE-ε4 condition: Elevated fibronectin (FN1) disrupts astrocyte-endothelium interactions. FN1 binds integrins and activates focal adhesion kinase (FAK), inhibiting VEGFA, which is required for endothelial HBEGF that in turn activates IGF1 signaling. Red symbols indicate inhibition of HBEGF, VEGFA, and IGF1 pathways, leading to BBB dysfunction. Highlights:APOE-ε4 drives fibronectin deposition in Alzheimer's, disrupting astrocyte-endothelia interactions. APOE-ε4 and fibronectin co-localize, forming aggregates at blood-brain barrier (BBB). Fibronectin alters the signaling between VEGF, IGF1, and HBEGF impairing BBB function. Reducing fibronectin restores BBB integrity and offsets APOE-ε4 pathology.

Genetic variations have emerged as crucial players in the etiology of Alzheimer’s disease (AD), and they serve for a better understanding of the disease mechanisms; yet the specific roles of these genetic variants remain uncertain. Animal models with reminiscent disease pathology could uncover previously uncharacterized roles of these genes. Therefore, we generated zebrafish models for AD variants to analyze the in depth molecular and biological functions of these variants. Using CRISPR/Cas9, we generated a knockout model for abca7 , orthologous to human ABCA7 . We performed single cell transcriptomics and analyzed the altered genes and molecular pathways in zebrafish. We leveraged data from multiethnic AD cohorts at Mayo Clinic and Columbia University, to perform genetic association studies, co-expression analyses, in silico interaction mapping, family based variant segregation analyses and epigenetic association studies, and the functional and histological studies in zebrafish. The abca7 ± zebrafish reduced astroglial proliferation, synaptic integrity, and microglial response after Aβ42 toxicity. We found that the abca7 loss-of-function (LOF) reduced neuropeptide Y ( npy ) expression as well as Brain-derived neurotrophic factor ( bdnf) and Nerve growth factor receptor (ngfr) . Human brain analysis showed reduced NPY in AD, regulatory interaction between NPY and BDNF , genetic variants in NPY associated with AD, and segregation of variants in ABCA7 , BDNF and NGFR in families. ABCA7 variants altered the epigenetic codes in NPY , BDNF , and NGFR promoter regions. Human results paralleled with zebrafish findings to indicate an evolutionarily conserved disease mechanism through ABCA7-NPY signalling axis. NPY administration to zebrafish rescued the phenotypes in abca7 knockout, suggesting a true biological relevance. Our results demonstrate a previously unknown link between ABCA7 and NPY in regulation of synaptic integrity and neurogenesis in AD. We propose that ABCA7-dependent NPY is a resilience factor in vertebrate brains, and this reserve mechanism is impaired in AD.

Neuropathological and neuroimaging studies indicate that cerebrovascular disease (CVD) is a major risk factor for Alzheimer's disease (AD), yet the molecular mechanisms underlying the association between the two traits remain unclear. To elucidate the mechanistic relationship between the two phenotypes, the current study examined the genetic correlation between stroke and AD in individuals of African ancestry. Capitalizing on the results from recent genome-wide association studies (GWAS) on AD (2,844 cases; 6,521 controls) and stroke (3,961 cases; 20,030 controls) in individuals of African ancestry, genetic correlation analysis was conducted using LAVA, which partitions the genome based on LD structure and estimates local genetic covariance within each resulting partition. Enhanced Hi-C Capture Analysis (eHiCA) was performed at identified top loci to examine chromatin interactions using 8 AD brain autopsy samples from different ancestries and iPSC-derived cells. In addition, overlapping top loci were examined in 553 African individuals with WGS data from the READD-ADSP. Genetic covariance analysis identified a locus shared between AD and stroke on chromosome 18q21.33 that includes the PHLPP1 gene (ρ = .77, p  = 2.41×10 -6 ). Examination of the LD structure and genetic association patterns at this locus identified disease-associated haplotypes exerting an effect in the same direction in both traits. eHiCA demonstrated that the two haplotypes at PHLPP1 interact with each other, and both haplotypes interact with the same chromosome regions at and around PHLPP1 . A similar pattern of chromatin interactions, which indicates a coordinated regulation of the same set of genes, was observed in brains across ancestries, and in different AD-relevant cell types (neurons, microglia, and oligodendrocytes). Variants at PHLPP1 were also nominally significant in the independent meta-analysis of African individuals that included WGS data ( p  = 4.56 × 10 -5 ). These findings nominate PHLPP1 as a shared locus for both AD and stroke in individuals of African ancestry. Notably, a genome-wide significant signal in this locus was identified in an admixture mapping study of African Americans (Rajabli et al., 2023). Identification of shared molecular mechanisms between AD and CVD in different populations is useful for understanding the relationship between these two processes in all people.

In the developing cerebral cortex, Cajal Retzius (CR) cells are early-born neurons that orchestrate the development of mammalian-specific cortical features. However, this cell type has not been conclusively identified in non-mammalian species. Here we studied neurons expressing Tp73, a transcription factor specifically expressed in most mammalian CR cells. Comparisons of chicken, salamander, zebrafish, and little skate data indicate that Tp73-expressing neurons have conserved spatial distribution and transcriptomic signatures in vertebrates. Among the conserved Tp73-expressing cell types we find CR cells andTp73+ external tufted cells (ETCs) in the olfactory bulb of jawed vertebrates. ETCs and CR cells share the expression of most "canonical" CR cell transcription factors, such as Tp73, Lhx1, Lhx5, Ebf3, and Nr2f2, indicating that they are sister cell types. Our findings suggest that CR and ETCs evolved in stem vertebrates from cells involved in olfactory processing, with CR cells progressively acquiring new specialized roles in developmental signaling.

The genetic component of early-onset Alzheimer disease (EOAD), accounting for ~10% of all Alzheimer's disease (AD) cases, is largely unexplained. Recent studies suggest that EOAD may be enriched for variants acting in the lipid pathway. The current study examines the shared genetic heritability between EOAD and the lipid pathway using genome-wide multi-trait genetic covariance analyses. Summary statistics were obtained from the GWAS meta-analyses of EOAD by the Alzheimer's Disease Genetics Consortium (n=19,668) and five blood lipid traits by the Global Lipids Genetics Consortium (n=1,320,016). The significant results were compared between the EOAD and lipids GWAS and genetic covariance analyses were performed via SUPERGNOVA. Genes in linkage disequilibrium (LD) with top EOAD hits in identified regions of covariance with lipid traits were scored and ranked for causality by combining evidence from gene-based analysis, AD-risk scores incorporating transcriptomic and proteomic evidence, eQTL data, eQTL colocalization analyses, DNA methylation data, and single-cell RNA sequencing analyses. Direct comparison of GWAS results showed 5 loci overlapping between EOAD and at least one lipid trait harboring APOE, TREM2, MS4A4E, LILRA5, and LRRC25. Local genetic covariance analyses identified 3 regions of covariance between EOAD and at least one lipid trait. Gene prioritization nominated 3 likely causative genes at these loci: ANKDD1B, CUZD1, and MS4A64.The current study identified genetic covariance between EOAD and lipids, providing further evidence of shared genetic architecture and mechanistic pathways between the two traits.

Microproteins, short functional peptides encoded by small genes, are emerging as critical regulators of cellular processes, yet their roles in mitochondrial function and neurodegeneration remain underexplored. In this study, we identify NCBP2-AS2 as an evolutionarily conserved mitochondrial microprotein with significant roles in energy metabolism and neurogenesis. Using a combination of cellular and molecular approaches, including CRISPR/Cas9 knockout models, stoichiometric co- immunoprecipitation, and advanced imaging techniques, we demonstrate that NCBP2-AS2 localizes to the inner mitochondrial space and interacts with translocase of the inner membrane (TIM) chaperones. These interactions suggest a role in ATPase subunit transport, supported by the observed reductions in ATPase subunit levels and impaired glucose metabolism in NCBP2-AS2-deficient cells. In zebrafish, NCBP2-AS2 knockout led to increased astroglial proliferation, microglial abundance, and enhanced neurogenesis, particularly under amyloid pathology. Notably, we show that NCBP2-AS2 expression is consistently downregulated in human Alzheimer's disease brains and zebrafish amyloidosis models, suggesting a conserved role in neurodegenerative pathology. These findings reveal a novel link between mitochondrial protein transport, energy metabolism, and neural regeneration, positioning NCBP2-AS2 as a potential therapeutic target for mitigating mitochondrial dysfunction and promoting neurogenesis in neurodegenerative diseases such as Alzheimer's disease.

APOEε4 significantly increases the risk of developing Alzheimer’s disease (AD). Cognitively healthy APOEε4 -carriers exist, suggesting potential protective mechanisms against APOEε4 . We hypothesized that some APOEε4- carriers may have genetic variations protecting them from developing APOEε4 -mediated AD pathology. We aim to identify these protective genetic variants. Whole genome sequencing (WGS) and cerebrospinal fluid (CSF) proteomics were performed from human cohorts to identify potential protective variants segregating exclusively among APOEε4 carriers. Bioinformatic analyses were performed to select candidate target genes. Immunohistochemistry on postmortem human brain tissues and mouse models expressing human APOE variants were performed along with in-vivo functional studies in adult zebrafish AD model. WGS analyses revealed 510 potential gene variants segregating exclusively among APOEε4 carriers, which included rare and loss-of-function (LOF) variants. Pathway analysis of these genes showed significant enrichment in extracellular matrix (ECM)-related processes, suggesting protective effects of LOF in ECM proteins. This was further validated by CSF proteome profiling and subsequent analyses in APOEε4 carriers and non-carriers. Fibronectin-1 (FN1) and Collagen-6A2 ( COL6A2 ) were prioritized as candidate target genes for postmortem validation and in-vivo functional studies. FN1 protein was increased in APOEε4 carriers resulting in thickened ECM at the basement membrane around the blood vessels, potentially impairing pathology-induced responses such as clearance and immune system activity. This observation is validated in human brains, mouse models and zebrafish model; therefore, the pathological association of FN1 to AD is evolutionarily conserved. Supporting this hypothesis, in-vivo functional study in zebrafish model with LOF mutations in fn1b revealed that fibronectin LOF enhanced gliovascular remodeling and microglial activation while reducing astrogliosis, suggesting that pathological accumulation of FN1 could impair toxic protein clearance, which is ameliorated with FN1 LOF. The vascular deposition of the ECM components FN1 and COL6A2 are increased in APOEε4 carriers. Rare variant in FN1 protect against APOEε4- mediated pathogenesis in AD. We propose a new disease mechanisms and potential therapeutic intervention targets for vascular contribution to dementia to mitigate the risk of developing AD.

Cardio and cerebrovascular risk factors (CVRFs) increase the risk of cerebrovascular disease and clinical Alzheimer’s Disease (AD), and over 70% of the patients with AD coincident cerebrovascular pathology. We previously found that FMNL2 interacts with a burden score of hypertension, diabetes, heart disease, and body mass index (BMI) by altering the normal astroglial-vascular mechanisms that underly amyloid clearance. Stroke, defined by history of a clinical stroke or brain imaging, is a moderately robust risk factor for AD and dementia. The goal here was to identify genes that interact with CVRFs, incorporating stroke as an additional factor, on AD in multi-ethnic cohorts. We conducted a genome-wide gene-CVRF score interaction analysis for AD, in 7,939 AD patients and 9,631 controls from eight multi-ethnic cohorts of non-Hispanic Whites, African Americans, and Hispanics including ADNI, NACC, NOMAS, WHICAP, EFIGA, and ROSMAP. A CVRF score was created from the first principal component of history of clinical stroke, hypertension, diabetes, and heart disease, and measured BMI. Gene-based interaction test was performed with the adaptive gene-environment interaction test. Results were summarized using a meta-analysis. We investigated the association of pathological AD, amyloid-β, or brain infarcts with gene expression and protein expression from the frontal cortex in ROSMAP using a generalized linear model. Age, sex, and the first three principal components were adjusted in the models. The interaction of CVRF score with FMNL2 on AD ( p = 1.02E-05) was identified and additional genes were identified to interact with CVRF score, including SLC22A14 ( p = 1.44E-06), AMMECR1L ( p = 2.74E-06), PRG3 ( p = 2.76E-06), CFAP99 ( p = 5.22E-06) , ADPGK-AS1 ( p = 8.58E-06) and BRINP1 ( p = 6.29E-06). ADPGK-AS1 and FMNL2 gene expressions were associated with pathological AD (p = 0.004 and p = 0.0002). FMNL2 and BRINP1 gene expressions were higher in the brains of patients with brain infarcts (p = 0.025 and p = 0.006). BRINP1 protein expression was associated with pathological AD (p = 0.0002) and was higher in the brains of patients with brain infarcts (p = 0.022). We identified novel candidate genes that interact with CVRFs on AD in multi-ethnic cohorts. Understanding the interplay between genes, CVRFs, and AD has the potential to reveal novel molecular targets for prevention and treatment for AD.

The apolipoprotein E ε4 allele ( APOE-ε4 ) is the strongest genetic risk factor for late-onset Alzheimer's disease (AD), yet its molecular impact on cerebrovascular biology remains inconclusive, particularly in underrepresented populations with elevated vascular burden. Individuals from Hispanic ancestry experience disproportionately high rates of cerebrovascular pathology, offering a unique opportunity to investigate the mechanisms of cerebrovascular pathology in AD. Here, we performed single-nucleus RNA sequencing (snSeq) on 413,175 nuclei from 52 postmortem Hispanic brains to determine APOE-ε4 -associated cell type specific transcriptomic changes in a population with elevated cerebrovascular risk. We identified a conserved molecular signature marked by dysregulated extracellular matrix deposition and focal adhesion signaling in astrocytes. These findings were replicated in the non-Hispanic ROSMAP cohort (n = 424) snSeq. Findings were validated in isogenic human iPSC-derived astrocytes, humanized APOE targeted replacement mouse brains, and post-mortem human brains at protein and chromatin accessibility level. Our data suggest that APOE-ε4 astrocytes adopt a hyper-adhesive, mechanically rigid phenotype that may exacerbate cerebrovascular pathology.

[This corrects the article DOI: 10.1371/journal.pbio.3002468.].

The proper functioning of the heart relies on the intricate interplay between the central nervous system and the local neuronal networks within the heart itself. While the central innervation of the heart has been extensively studied, the organization and functionality of the intracardiac nervous system (IcNS) remain largely unexplored. Here, we present a comprehensive taxonomy of the IcNS, utilizing single-cell RNA sequencing, anatomical studies, and electrophysiological techniques. Our findings reveal a diverse array of neuronal types within the IcNS, exceeding previous expectations. We identify a subset of neurons exhibiting characteristics akin to pacemaker/rhythmogenic neurons similar to those found in Central Pattern Generator networks of the central nervous system. Our results underscore the heterogeneity within the IcNS and its key role in regulating the heart's rhythmic functionality. The classification and characterization of the IcNS presented here serve as a valuable resource for further exploration into the mechanisms underlying heart functionality and the pathophysiology of associated cardiac disorders.