Lee, Annie

Abstract Histopathological assessment has served as the gold standard for diagnosing Alzheimer’s disease (AD). Emerging technological advancements, including the development of amyloid positron emission tomography (PET), have enabled early detection of amyloid pathology, one of the neuropathological hallmarks of AD. Genome-wide association study (GWAS) across cohorts of aging and AD, leveraging different measurements of amyloid burden, may facilitate the identification of novel genetic variants that drive the earliest neuropathological changes in AD. This study presents the largest GWAS of brain amyloidosis to date, leveraging amyloid β (Aβ) measured by in vivo amyloid PET and postmortem histopathology from 13,555 individuals of European ancestry. Amyloid positivity was defined as moderate or frequent neuritic plaques according to the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) staging scores for each postmortem cohort. A Gaussian mixture model (GMM) was applied to each amyloid PET cohort to identify the cohort and tracer-specific cut-offs that differentiate amyloid positive and negative populations. In silico and ex vivo analyses further characterized implicated loci, including interrogating the association between bulk and single-nucleus gene expression profiles and AD-related traits. Genetic covariance analysis assessed the extent amyloid PET and postmortem measures reflect the shared genetic architecture of brain amyloidosis. Our combined amyloidosis GWAS identified three established AD risk loci: BIN1 (rs6733839, OR = 1.20, 95% CI 1.14-1.26, P = 1.32 × 10−11), CR1 (rs4844610, OR = 1.24, 95% CI = 1.16-1.32, P = 4.21 × 10−10), APOE (rs429358, OR = 4.01, 95% CI = 3.66-4.38, P = 4.54 × 10−201), and a newly identified brain amyloidosis-associated variant on chromosome 17 (rs35635959, OR = 1.18, 95% CI = 1.12-1.25, P = 1.47 × 10−8). SuSiE fine-mapping identified a single credible set of 15 putative causal variants with rs35635959 as the lead variant. Subsequent eQTL and SuSiE-based colocalization analyses prioritized rs35635959 as a strong eQTL for TUBG2, encoding tubulin gamma 2, which is involved in microtubule organization and synaptic plasticity. Further cell-type-specific characterization of this gene in neurons from dorsolateral prefrontal cortex tissue indicated that decreased TUBG2 expression was associated with increased Aβ burden and AD case status (PFDR < 0.045). Furthermore, our study is the first to report a modest genetic covariance (covariance=0.17, P < 6.54 × 10−8) between the genetic architecture of amyloid burden captured by different modalities. While APOE showed a strong association with both amyloid endophenotypes, the observed genetic covariance was not substantially attenuated after excluding variants within the APOE region (covariance=0.16, P < 1.32 × 10−7). Our results highlight the benefits of leveraging compatible, harmonized AD endophenotypes to increase power to uncover new molecular insights into the etiology of AD neuropathology. Wang et al. present the largest GWAS of brain amyloidosis to date, analysing 13,555 individuals using amyloid PET and postmortem Aβ measures. They identify a novel amyloidosis-associated variant on chromosome 17, demonstrate genetic covariance between modalities, and highlight complex traits sharing genetic architecture with Aβ burden.

Abstract 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.

AbstractBackgroundAlzheimer’s disease (AD) missing heritability remains extensive despite numerous genetic risk loci identified by genome‐wide association or sequencing studies. This has been attributed, at least partially, to mechanisms not currently investigated by traditional single‐marker/gene approaches. Polygenic Risk Scores (PRS) aggregate sparse genetic information across the genome to identify individual genetic risk profiles for disease prediction and patient risk stratification. Recent advancements have pivoted on innovative approaches utilizing OMICS data to construct such risk scores.MethodWe employed a random forest algorithm to identify a list of gene candidates from bulk RNA sequencing data in prefrontal cortex from 565 AD brain samples (non‐Hispanic Whites, n = 399; Hispanics, n = 113; African American, n = 12) across six U.S. brain banks. Subsequently, we calculated their effect size on Braak staging using regression models to construct a polytranscriptomic risk score (PTRS). We employed two distinct models: “Ethnicity‐Agnostic” Model (randomly assigning samples to training and testing samples) and “Ethnicity‐Aware” Model (assigning NHW samples to training and Hispanics to testing sample). Analysis of variance and the receiver operating characteristics area under the curve (ROC AUC) was used to evaluate PTRS’s classification performances. We validated findings using the Religious Orders Study/Memory and Aging Project study (ROS/MAP, n = 1,095).ResultWe found a significant difference in PTRS between samples with low vs. high Braak stages (≤4 vs. ≥5, p = 1*E‐04; Figure 1 upper panel). AUC was found to be 79‐81%, consistently in both Ethnicity‐Agnostic and Ethnicity‐Aware models (Figure 1 lower panel). Finally, the PTRS in ROS/MAP yielded a similar classification performance (p = 2*E‐04, AUC = 77%).Conclusioncontrary to prior studies, we developed a PTRS with optimal transferability across ethnicities. This underscores the importance of developing novel tools to stratify and harmonize large brain repositories for AD.

AbstractBackgroundGenome‐wide association studies (GWAS) in Alzheimer’s disease (AD) leveraging endophenotypes beyond case/control diagnosis, such as brain amyloid β pathology, have shown promise in identifying novel variants and understanding their potential functional impact. In this study, we leverage two brain amyloid β pathology measurement modalities, PET imaging and neuropathology, to address sample size limitations and to discover novel genetic drivers of disease.MethodWe conducted a meta‐analysis on an amyloid PET imaging GWAS (N = 7,036, 35% amyloid positive, 53.67% female, age = 71) and an autopsy GWAS of brain amyloidosis (N = 6,519, 63.08% amyloid positive, 51.34% female, age at death = 83), both adjusted for covariates including age, sex and principal components of genetic ancestry. All participants in both GWAS were unrelated individuals of European descent. We defined amyloid positivity as moderate or frequent neuritic plaques using the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) staging scores within each autopsy cohort. A Gaussian mixture model (GMM) was applied to each amyloid PET imaging cohort to identify the cohort and tracer‐specific cut‐offs that differentiate amyloid positive and negative populations.ResultThe genome‐wide significant results from the meta‐analysis identified three known AD loci (APOE, CR1, and BIN1) and a novel chromosome 17 locus (rs35635959, intergenic, MAF = 0.27, OR = 1.18, p = 1.47 × 10‐8). The MAGMA gene‐level analysis excluding the APOE region suggests significant associations between rs35635959 and COASY, PLEKHH3, and TUBG2 on chromosome 17, implying the potential roles of these genes on amyloidosis. Functional annotations of rs35635959 leveraging brain eQTL databases indicate its association with differential gene expression of COASY and TUBG2 in AD brains. ROSMAP bulk and single‐nucleus RNAseq analyses link TUBG2 downregulation to the higher amyloid burden and AD diagnosis while suggesting such observations are enriched in excitatory and inhibitory neurons, reaching FDR significance.ConclusionThis study is the most extensive European GWAS of brain amyloidosis, and our findings replicate known AD loci and identify a possible novel locus (index SNP rs35635959) on chromosome 17. Functional annotations of the novel variant indicate TUBG2, implicating in microtubule organization, warrants further assessment. Ongoing efforts aim to validate these novel effects using independent datasets.

AbstractBackgroundAPOEε4 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.MethodsWhole 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.ResultWGS 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.ConclusionThe 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.

AbstractBackgroundAlzheimer’s Disease (AD) presents complex molecular heterogeneity, influenced by a variety of factors including heterogeneous phenotypic, genetic, and neuropathologic presentations. Regulation of gene expression mechanisms is a primary interest of investigations aiming to uncover the underlying disease mechanisms and progression.MethodWe generated bulk RNA‐sequencing in prefrontal cortex from 565 AD brain samples (non‐Hispanic Whites, n = 399; Hispanics, n = 113; African American, n = 12) across six U.S. brain banks, and conducted differential gene expression and enrichment analyses. We sought to identify cross‐ancestry and ancestry‐specific differentially expressed genes (DEG) and pathways across Braak stages, adjusting for sex, age at death, and RNA quality metrics. We validated our findings using the Religious Orders Study/Memory and Aging Project study (ROS/MAP, n = 1,095). Lastly, we validated top DEG using publically‐available human single‐nucleus RNA sequencing (snRNAseq) data.ResultAD‐known genes VGF (LFC = ‐0.661, padj = 3.78) and ADAMTS2 (padj = 1.21) were consistently differentially expressed across statistical models, ethnic groups, and replicated in ROS/MAP (Figure 1). Genes from the heat shock protein (HSP) family, e.g. HSPB7 (padj = 3.78), were the top DEG, also replicated in ROS/MAP. Ethnic‐stratified analyses prioritized TNFSF14 and SPOCD1 as top DEG in Hispanic samples. Gene set enrichment analysis highlighted several significantly pathways, including “TYROBP causal network in microglia” (WP3945; padj = 1.68) and “Alzheimer Disease” (WP5124; padj = 4.24). snRNAseq validated several DEG, including VGF downregulated in neurons (padj = 1.1).ConclusionTo our knowledge, this is the largest diverse transcriptome study for AD in post‐mortem tissue. We identified perturbated genes and pathways resulting in cross‐ethnic and ethnic‐specific findings, ultimately highlighting the importance of diversity in AD investigations.

AbstractBackgroundRecent studies increased the number of genes that are associated with the co‐existence of vascular pathologies and Alzheimer’s disease (AD). However, the biological functions and the mechanism of action by which they contribute to the disease pathology are still to be further elucidated. Therefore, animal models that allow streamlined large‐throughput functional screens in a biologically relevant manner are essential. Towards this goal, we generated adult zebrafish models of amyloid toxicity to functionally analyze candidate genes and how they relate to disease perturbations.MethodThrough cerebroventricular injection of human amyloid‐beta42 monomers into the cerebrospinal fluid of the adult zebrafish brain, we inflict acute amyloid toxicity. By performing immunohistological analyses, single cell transcriptomics, gene editing for generating gene variants or full knockouts, transient functional knockdown of candidate genes, pharmacological intervention of selected signalling pathways and comparing the findings to human AD brain transcriptomics datasets, we determined the similarities and differences of the effects of amyloid pathology on the vasculature in zebrafish in relation to the human conditions.ResultsWe found that amyloid pathology in zebrafish brain shows histological and molecular similarities to human brains. Integration of single cell transcriptomics showed remarkable similarities in neurons to amyloid in humans and zebrafish. We identified several molecular mechanisms that underlie this pathology‐induced cellular response and translated to mammalian models including rodent brains. By performing genome‐wide association in AD patients, we identified that the FMNL2 links cerebrovascular disease and AD, by regulating the astroglial and blood vessel interactions and controlling the efficient clearance of toxic proteins from the brain. Our new single cell transcriptomics analyses of the gliovascular niche cells of the zebrafish brain revealed potential cellular interactions and the pathways perturbed in vascular disease and AD.ConclusionWe propose zebrafish as a useful model for functional investigation of vascular pathology and AD‐related genes identified in clinical studies by providing in vivo biological knowledge on disease mechanisms, on which novel drug development strategies can be based.

AbstractBackgroundInter‐cellular communication within the gliovascular unit (GVU) is critical for cerebral blood flow regulation, and maintenance of the blood‐brain‐barrier (BBB) properties. The breakdown of BBB in Alzheimer’s disease (AD) is well‐established, but precise underlying molecular changes remain unclear. Additionally, whether GVU molecular alterations observed in AD brains are also detected in blood from living patients is unknown. Further, these GVU molecular perturbations require further investigation in different model systems to identify both human brain‐specific and cross‐species conserved alterations. In this study, we investigated prioritized GVU molecules altered in AD brains for their conservation in blood and cross‐species model systems.MethodsWe performed single nucleus RNA sequencing (snRNAseq) of temporal cortex tissue in AD and control brains. We analyzed this data to detect cell‐specific GVU molecular perturbations and their interactions. We investigated molecular interactions between vascular and astrocyte clusters, the major cell types of the GVU of the BBB. To determine whether GVU transcriptional alterations detected in the brain are preserved in the blood, existing blood expression, genetic, and neuroimaging data from two longitudinal antemortem cohorts were analyzed. Using model systems, including mouse, drosophila, and zebrafish, we evaluated the cross‐species conservation of the top GVU alterations detected in AD brains.ResultsBrain snRNAseq revealed transcriptional profiles of 6,541 astrocytes and 2,210 vascular cells. The latter formed three distinct vascular clusters characterized as pericytes, endothelia and perivascular fibroblasts. We identified differentially expressed genes and their enriched pathways within these clusters and observed the highest levels of transcriptional changes within pericytes. Vascular targets that interact with astrocytic ligands have biological functions in cell signaling, angiogenesis, amyloid ß metabolism, and cytoskeletal architecture. We discovered that genetic variants influencing blood expression levels of some of the prioritized GVU genes were associated with neuroimaging burden of cerebrovascular disease in living human cohorts. Our ongoing studies in model systems revealed conservation of some of the top prioritized molecular perturbations across species.ConclusionOur findings prioritized by multiscale, cross‐tissue human data revealed GVU perturbations within interacting pericyte and astrocyte molecules, which are conserved across multiple cross‐species models. These results nominate new molecular targets and mechanistic insights for BBB disruptions in AD.

AbstractObjectiveTo compute penetrance and recurrence risk using a genome‐wide PRS (including and excluding the APOE region) in families with Alzheimer's disease.MethodsGenotypes from the National Institute on Aging Late‐Onset Alzheimer's Disease Family‐Based Study and a study of familial Alzheimer's disease in Caribbean Hispanics were used to compute PRS with and without variants in the 2 MB region flanking APOE. PRS was calculated in using clumping/thresholding and Bayesian methods and was assessed for association with Alzheimer's disease and age at onset. Penetrance and recurrence risk for carriers in highest and lowest PRS quintiles were compared separately within APOE‐ε4 carriers and non‐carriers.ResultsPRS excluding the APOE region was strongly associated with clinical and neuropathological diagnosis of AD. PRS association with AD was similar in participants who did not carry an APOE‐ε4 allele (OR = 1.74 [1.53–1.91]) compared with APOE‐ε4 carriers (1.53 [1.4–1.68]). Compared to the lowest quintile, the highest PRS quintile had a 10% higher penetrance at age 70 (p = 0.0006) and a 20% higher penetrance at age 80 (p < 10e‐05). Stratifying by APOE‐ε4 allele, PRS in the highest quintile was significantly more penetrant than the lowest quintile, both, within APOE‐ε4 carriers (14.5% higher at age 80, p = 0.002) and non‐carriers (26% higher at 80, p < 10e‐05). Recurrence risk for siblings conferred by a co‐sibling in the highest PRS quintile increased from 4% between the ages of 65–74 years to 39% at age 85 and older.InterpretationPRS can be used to estimate penetrance and recurrence risk in familial Alzheimer's disease among carriers and non‐carries of APOE‐ε4.