Rare genetic variation in fibronectin 1 (FN1) protects against APOEε4 in Alzheimer’s disease

The risk of developing Alzheimer’s disease (AD) significantly increases in individuals carrying the APOEε4 allele. Elderly cognitively healthy individuals with APOEε4 also exist, suggesting the presence of cellular mechanisms that counteract the pathological effects of APOEε4; however, these mechanisms are unknown. We hypothesized that APOEε4 carriers without dementia might carry genetic variations that could protect them from developing APOEε4-mediated AD pathology. To test this, we leveraged whole-genome sequencing (WGS) data in the National Institute on Aging Alzheimer's Disease Family Based Study (NIA-AD FBS), Washington Heights/Inwood Columbia Aging Project (WHICAP), and Estudio Familiar de Influencia Genetica en Alzheimer (EFIGA) cohorts and identified potentially protective variants segregating exclusively among unaffected APOEε4 carriers. In homozygous unaffected carriers above 70 years old, we identified 510 rare coding variants. Pathway analysis of the genes harboring these variants showed significant enrichment in extracellular matrix (ECM)-related processes, suggesting protective effects of functional modifications in ECM proteins. We prioritized two genes that were highly represented in the ECM-related gene ontology terms, (FN1) and collagen type VI alpha 2 chain (COL6A2) and are known to be expressed at the blood–brain barrier (BBB), for postmortem validation and in vivo functional studies. An independent analysis in a large cohort of 7185 APOEε4 homozygous carriers found that rs140926439 variant in FN1 was protective of AD (OR = 0.29; 95% CI [0.11, 0.78], P = 0.014) and delayed age at onset of disease by 3.37 years (95% CI [0.42, 6.32], P = 0.025). The FN1 and COL6A2 protein levels were increased at the BBB in APOEε4 carriers with AD. Brain expression of cognitively unaffected homozygous APOEε4 carriers had significantly lower FN1 deposition and less reactive gliosis compared to homozygous APOEε4 carriers with AD, suggesting that FN1 might be a downstream driver of APOEε4-mediated AD-related pathology and cognitive decline. To validate our findings, we used zebrafish models with loss-of-function (LOF) mutations in fn1b—the ortholog for human FN1. We found that fibronectin LOF reduced gliosis, enhanced gliovascular remodeling, and potentiated the microglial response, suggesting that pathological accumulation of FN1 could impair toxic protein clearance, which is ameliorated with FN1 LOF. Our study suggests that vascular deposition of FN1 is related to the pathogenicity of APOEε4, and LOF variants in FN1 may reduce APOEε4-related AD risk, providing novel clues to potential therapeutic interventions targeting the ECM to mitigate AD risk. Supplementary Information The online version contains supplementary material available at 10.1007/s00401-024-02721-1.


Introduction
Alzheimer's disease (AD) is typically characterized clinically by progressive memory impairment and decline in other cognitive domains; however, there is a long pre-symptomatic period without clinical manifestations [74].At death, pathological hallmarks in the brain include extracellular β-amyloid protein in diffuse and neuritic plaques and neurofibrillary tangles made of hyper-phosphorylated tau protein.AD, a progressive neurodegenerative disorder, is currently unpreventable, and, with available drugs only marginally affecting disease severity and progression, remains effectively untreatable.A critical barrier to lessening the impact of late-onset AD (LOAD) is the slow development of drugs that prevent or treat AD due, in part, to an incomplete characterization of the basic pathologic mechanisms.Determining which genes and gene networks contribute to AD could reveal the biological pathways for drug development and inform the development of genetic testing methods for identifying those at greatest risk for AD.
The presence of the APOEε4 allele is among the most prominent genetic risk factors for AD in White, non-Hispanic populations [21], but the associated risks observed in African-Americans and Hispanics are somewhat lower [82].Relative risk of AD associated with a single copy of APOEε4 is 2.5-to 3.5-fold in Caucasians compared to 1.0-2.4 and 1-1.9 in African-Americans and Hispanics, respectively [9,82].However, in every population, homozygosity for the APOEε4 allele is associated with increased risk and nearly complete penetrance [7,64,91].APOE, a critical player in lipid metabolism and transport, has been extensively studied for its role in Alzheimer's disease (AD) and other neurodegenerative disorders [14,55,56].The APOEε4 allele is a well-established risk factor for late-onset AD, with carriers of this allele exhibiting an increased susceptibility to cognitive decline and dementia and earlier age at onset of clinical symptoms.However, within the population of APOEε4 carriers, there is variability in age of onset and severity of AD symptoms.Some "resilient" or "cognitively normal, unaffected" individuals who carry the ε4 allele do not develop AD or experience a delayed onset of symptoms.Several potential factors might contribute to the variability in AD risk and presentation among APOEε4 carriers.Genetic modifier mutations outside of the APOE gene might interact with APOEε4 to influence the risk of AD.APOEε4 carriers might also be influenced by other risk factors for AD, such as vascular health, inflammation, and metabolic conditions.Interactions between APOEε4 and these factors could modify the course of the disease.Certain rare protective variants in other genes could offset the risk posed by APOEε4.
Amid the well-documented association between APOEε4 and AD risk, a growing body of evidence suggests intriguing nuances in the effects of this allele, particularly in certain subsets of individuals who defy the expected trajectory of cognitive decline and remain remarkably resilient to neurodegenerative diseases.Notably, heterozygosity of APOEε4 has incomplete penetrance [31], and the polygenic risk of the rest of the genome could stratify APOEε4 carriers into high-and low-risk strata.In this study, we aimed to identify putative protective mechanisms, influenced by genetic modifiers that might counteract the detrimental effects of the APOEε4 allele.We sought to identify "protective" genetic factors that can modify or reduce the effect of APOEε4 on AD risk and to identify new pathogenic mechanisms, proteins, and pathways that inform development of therapeutic targets and diagnostics.

Whole-genome sequencing identifies putative protective variants in cognitively unaffected elderly APOEε4 carriers
We accessed whole-genome sequencing data in 3,578 individuals from over 700 non-Hispanic White and Caribbean Hispanic families multiply affected by AD (Table 1).After harmonization and QC of the WGS data, we identified rare (MAF < 1% in gnomAD) coding variants in the healthy elderly APOEε4 homozygous (over the age of 70) and heterozygous (over the age of 80) carriers that were absent in non-carriers (Fig. 1).We further prioritized exon coding variants in healthy APOEε4 carriers that bear the potential to be damaging to the resulting protein product.Supplementary Tables 1-3 provide the lists of candidate variants that were identified in cognitively unaffected elderly APOEε4 carriers.
Our strategy and analysis pipeline are summarized in Fig. 2. We found 510 variants in 476 genes that were present in at least 1% of APOEε4 unaffected homozygous carriers (388 in EFIGA/WHICAP and 130 in NIA-AD FBS and 8 variants found in both datasets) (Supplementary Table 1 and 2).Two variants (rs116558455 and rs140926439) in the FN1 gene (fibronectin-1) were found in healthy elderly ε4 homozygous carriers in EFIGA/WHICAP and NIA-AD FBS cohorts with MAF = 1.85% and 3.33%, respectively (Table 2).In Hispanics, rs116558455 was absent in all APOEε4 carriers with AD.In non-Hispanic Whites rs140926439 was absent in homozygous APOEε4 AD patients, but found in 1% of heterozygous patients.Pathway analysis of the genes harboring variants segregating in APOEε4 carriers identified several biological pathways and molecular functions such as "actin binding", "microtubule binding", and "extracellular matrix structural constituent" (Fig. 3).These results suggested a strong correlation with cellular morphologies and the architectural organization of those cells.

Potential protective alleles against APOEε4 enrich extracellular matrix components
To determine the molecular mechanisms enriched in the protective alleles that we identified, gene ontology review was performed with term analyses for biological processes, cellular compartments, and molecular functions (Fig. 3).We found a strong enrichment for extracellular matrix (ECM)related processes such as cell adhesion, ECM organization, integrin binding, and structural component of the ECM (Fig. 3).This suggested that functional alterations in the ECM composition could act as a protective mechanism in APOEε4 carriers, both heterozygotes and homozygotes without dementia.We hypothesized that APOEε4-related increase in ECM components could be counteracted by lossof-function (LOF) variants in those genes, leading to protection through rescue of pathological mechanisms that those ECM components partake.
To test our hypothesis, we selected two genes from the variant lists that were common in ECM-related gene ontology classes (Fig. 3), collagen type VI alpha 2 chain (COL6A2) and fibronectin 1 (FN1).These genes are well-known ECM components that harbor putatively protective variants in APOEε4 cognitively unaffected carriers.Additionally, prioritized variants in FN1 were, respectively, present in both Hispanic and non-Hispanic White cohorts (Supplementary     2).COL6A2 variation (rs777822883) generates a substitution of arginine at the 862nd residue to tryptophan, while FN1 variation (rs140926439) converts the glycine at the 357th position to glutamic acid.Since both alterations result in change in charged residues (loss in COL6A2, gain in FN1), we hypothesized that these variations could have detrimental effect on the protein function, as charged interactions are essential for matric proteins and their stability [20,61,94].Therefore, we analyzed the AlphaFold structures of these proteins in Ensembl (http:// www.ensem bl.org) and found that both variations are potentially detrimental according to SIFT, REVEL, and MetaL R predictions (Supplementary Fig. 1).Arginine in COL6A2 at the 862nd position may coordinate with valine 859 and glutamic acid 858 in the alpha helix structure, while glycine at the 357th position in FN1 may provide structural stability by coordinating with glutamic acid 358 and serine 355 (Supplementary  Fig. 1).Therefore, we categorized these variants as likely loss-of-function alleles based on loss of electrostatic interactions.

FN1 variant is protective in an independent cohort of APOEε4 carriers
We queried an independent collection of AD-related genetic cohorts from several sources: the Alzheimer's Disease Genetic Consortium (ADGC), the Alzheimer's Disease Sequencing Project (ADSP), and United Kingdom Biobank (UKB) resources, primarily consisting of non-Hispanic White individuals of European ancestry (EU) (Supplementary Table 4 and 5).A total of 465,669 NHW case-control individuals ages 60 and above were available after genetic and phenotypic quality control.Since rs116558455 is very rare in EU (gnomAD non-Finnish EU allele frequency = 0.016%), we focused on rs140926439 which is less rare in EU and thus provides sufficient allele counts to enable replication analyses (gnomAD non-Finnish EU allele frequency = 0.46%).We specifically focused analyses on APOEε4/4 carriers ages 60 and above (N = 7185), to interrogate whether there were any elevated frequencies for the rs140926439 minor allele (T) associated with reduced AD risk and delayed age at onset.The variant rs140926439 was associated with strongly reduced risk of AD in APOEε4/4 carriers (OR = 0.29; 95% CI [0.11, 0.78], P = 0.014; Table 3, Fig. 4a).Sensitivity analyses were conducted, which ensured that any overlapping samples with our discovery were excluded and corroborated primary findings (OR = 0.31; 95% CI [0.11, 0.87], P = 0.027; Supplementary Table 6, Fig. 4b).Secondary age at onset analyses further showed a significant protective effect in APOEε4/4 carriers, delaying age at onset by 3.4 years for a single copy of the minor allele (beta = 3.37; 95% CI [0.42, 6.32], P = 0.025; Fig. 4c).These analyses represent the largest-to-date genetic association tests in a sample of APOEε4/4 carriers at an age range relevant to AD.

Fibronectin loss-of-function zebrafish model enhances gliovascular endfeet retraction and microglial activity while reducing gliosis after amyloid toxicity
To determine whether fibronectin activity is related to cellular responses after amyloid toxicity, we used our established amyloid toxicity model in the adult zebrafish brain [11,13,22,42,47,72].Zebrafish has two fibronectin 1 genes: fn1a and fn1b [78].Our single-cell transcriptomics analyses in the zebrafish brain showed that fn1b, but not fn1a is expressed in the zebrafish forebrain (Fig. 7a).fn1b expression is predominantly detected in vascular smooth muscle cells and immune cells, while endothelia and astroglia express fn1b at considerably lower levels (Fig. 7b).Amyloid toxicity results in increased fn1b expression in immune cells and vascular smooth muscle cells (Fig. 7b), similar to what we observed in AD brains (Figs. 5, 6).To determine the effects of fibronectin function in amyloid-induced pathology, we used an fn1b full knockout zebrafish line (fn1b −/− ), which was previously functionally validated [33].After treating wild-type and fn1b −/− animals with Aβ42, we performed immunohistochemical stainings for astroglia (red, GS) and tight junctions that mark vascular structures (green, ZO-1) (Fig. 7c-f, Supplementary Dataset 4).Compared to wildtype animals treated with Aβ42, fn1b −/− animals with Aβ42 showed less colocalization of GS and ZO-1 (− 16.3%, P = 5.3E−09), suggesting that gliovascular interactions were reduced with fibronectin loss of function (LOF) (Fig. 7g).Based on our previous findings that reduced gliovascular contact upon amyloid toxicity is a protective mechanism through enhancing clearance of toxic protein aggregates and immune systems activity [47], our results suggest that fibronectin could negatively regulate amyloid beta clearance and therefore an LOF variant could be protective against disease pathology.By performing intensity measurements for astroglia with GS immunoreactivity, we observed that GS intensity reduces with fn1b LOF (− 24.7%, P = 4.7E−03; Fig. 7h, Supplementary Dataset 5), indicative of reduced gliotic response upon Aβ42.To determine the effect of fibronectin on synaptic density and the number and activation state of microglia, we performed immunostainings (Fig. 7i, j, Supplementary Dataset 6) and found that loss of fibronectin leads to increased numbers of total (41.5%,P = 8.7E−04) and activated microglia (64.3%, P = 2.9E−04).We did not observe change in the synaptic density when Aβ42-treated fn1b −/− was compared to Aβ42-treated wildtype animals (Fig. 7i-k, Supplementary Dataset 7).

Discussion
In our study, we found that two missense, potential lossof-function (LOF) variants in FN1 may protect against APOEε4-mediated AD pathology.We base our conclusions on four main observations: (1) FN1 coding variants were present in cognitively unaffected APOEε4 homozygous carriers, but not in affected carriers with clinically diagnosed AD (Supplementary Table 1), and the protective effect was independently replicated is a large cohort of APOEε4 homozygous carriers.(2) Deposition of FN1 at the BBB basement membrane increases with APOEε4 dosage (Fig. 5).( 3) Unaffected/resilient homozygous APOEε4 carriers above the age of 70 without AD have FN1 deposition levels similar to APOEε3 control individuals (Fig. 6).( 4) In the zebrafish brain, knockout of fn1b alleviates amyloid toxicity-related pathological changes (Fig. 7).These results suggest that the basement membrane thickening and remodeled ECM composition in the BBB may be a pathological contribution to APOEε4-mediated AD pathology that may be mitigated by variants in FN1 or other ECM genes (Fig. 8).This conclusion is supported by the presence of variants in other BBB-related ECM components, such as LAMA1, LAMA3, and HSPG2, in unaffected elderly APOEε4 carriers but not in carriers with AD (Supplementary Table 1).Therefore, our findings propose a new direction for potential therapeutic interventions reducing the impact of APOEε4mediated risk of AD by targeting the BBB basement membrane.Thus, we propose that fibronectin loss of function may be a protective mechanism for AD (Fig. 8).
APOEε4 has been associated with increased neuroinflammation and neurodegeneration, which can accelerate the progression of AD [63].Our results in zebrafish fn1b knockout model showed that reduced fibronectin 1 increased the gliovascular (GV) endfeet retraction and reduced gliosis.We previously showed that the relaxed GV contact was a beneficial response to amyloid toxicity [47] as it helps enhance the clearance of toxic aggregates through the bloodstream.Additionally, gliosis is an immediate response in astroglia to insult and prevents functional restoration of neuronal activity in disease [16,29,73,93].Independent reports showed that astrocytic removal of APOE protects against vascular pathology [89], and gliosis is a mediator of amyloid-dependent tauopathy in late AD [6].We propose that the relationship of fibronectin with these processes is pathogenic, and reduced fibronectin could be protective by allowing more efficient clearance through the bloodstream and reduced astrogliosis.The enhanced microglial activity supports this hypothesis, as acute activation of microglia is a beneficial response to toxic protein aggregation [36,62].
Our results are consistent with the previous findings on APOE-dependent vascular pathologies and their relationship to AD [38,45,46,56,67,79].Endothelial fibronectin induces disintegration of endothelial integrity and leads to atherosclerotic vascular pathologies [1,18,95], supporting our findings that reduced fibronectin 1 protects the blood-brain barrier integrity disrupted by APOEε4.Our findings are coherent with the previous observations, where AD-related changes in collagen and fibronectin around the blood-brain barrier (BBB) and alterations in the BBB's structure and function were documented [43,80,92].Additionally, the serum levels of fibronectin increase in AD patients in comparison to healthy individuals [15].Collagen and fibronectin can also be early pathological markers of AD [48], where the increase in the deposition and cross-linking of basement membrane around the cerebral blood vessels lead to a thickening of the basement membrane, potentially compromising its permeability and function [35,67,83,84,90].Fibronectin expression levels in brain vasculature increases in AD [22,41,49,79,88], where remodeling of the BM and replacing ECM with FN1 have been suggested to indicate hypoperfusion and atherosclerosis-prone state [1,46,54].Additionally, APOEε4 might regulate BM remodeling through inhibition of pericyte-mediated matrix proteinase expression [55].Pericyte degeneration, mural cell dysfunction, and alterations in cerebrospinal flow dynamics are long-term consequences of vascular pathologies in aging and AD and is accelerated with APOEε4 [5,25,27,34,38,66].Therefore, based on our findings, we propose that excess ECM deposition and BM thickening with collagen and fibronectin could promote the blood-brain barrier breakdown.Potential loss-of-function variants in ECM genes are likely to render ECM components non-functional, thus protecting against AD progression.Stronger instructive interactions of collagen and fibronectin with their receptors on various BBB cell types in AD [39,59,79,88] support this hypothesis.Consistently, FN1 provides attachment surface for immune cells, which-when becomes chronic-damages the vascular functions, contribute to BBB breakdown, and loss of synaptic integrity.
We found that despite their APOEε4/4 status, unaffected/ resilient individuals who do not develop cognitive decline have lower FN1 deposition and gliosis at the vascular basement membrane that are not different from APOEε3/3 control individuals, but significantly lower than those in APOEε4/4 AD patients (Fig. 6).This demonstrated that FN1 is a critical component of APOEε4-mediated development of AD, and a yet unknown protective mechanism against the effects of APOEε4/4 genotype suppresses FN1 deposition.We propose that FN1 is a critical downstream effector of APOEε4 and reduced FN1 levels, either through rare, protective genetic variations in FN1 or through other resilience mechanisms, promoting protection against AD.An interesting future research could investigate the other rare protective variants of APOE such as APOEε2 [28,31] and APOEε3 Christchurch [70] and their effects on the BBB basement membrane.
The strength of this study is the cross-species design with pathological and functional validation to show that ECM component fibronectin could be related to key pathological aspects of AD such as toxic protein clearance, blood-brain barrier integrity, and microglial activity.We present the first knockout zebrafish for fibronectin 1 in relation to amyloid toxicity and identified cellular changes that relate to fibronectin activity.
Further studies could address some limitations of our study.First, the mechanism by which APOEε4 enhances FN1 requires further investigations.Although in human and zebrafish brains, fibronectin is upregulated, the longitudinal relationship of amyloid aggregation to FN1 activity needs to be analyzed.Additionally, our genetic studies were conducted in clinically assessed individuals, and given the rarity of the FN1 mutation, we did not have neuropathological assessments of APOEε4/4 individuals with this rare protective mutation.Future studies in large-scale neuropathologic cohorts are necessary to demonstrate the pathological consequences of the rare FN1 variants.Finally, mechanistic studies of FN1 with and without the rare mutation are necessary to demonstrate the nuanced functional consequences.

Ethics statement
All human samples were de-identified and the researchers could not infer or obtain personal information of the donors.Institutional Review Board approval from Columbia University Irving Medical Center and Mayo Clinic was taken before clinical data generation.Human cohorts and their characteristics are provided below.Animal experiments were carried out in accordance with the animal experimentation permits of the Institutional Animal Care and Use Committee (IACUC) at Columbia University (protocol number AC-AABN3554).Animals were maintained according to the Institutional Animal Care and Use Committee (IACUC) standards of the Institute of Comparative Medicine at the Columbia University Irving Medical Center and the accepted guidelines [2,30,44,77].The animal care and use program at Columbia University is accredited by the AAALAC International and maintains an Animal Welfare Assurance with the Public Health Service (PHS), Assurance number D16-00003 (A3007-01).Animal experiments were approved by the IACUC at Columbia University (protocol number AC-AABN3554).For zebrafish studies, 8-to 10-month-old wild-type AB strains or fn1b −/− homozygous knockout fish lines of both genders were used.In every experimental set, animals from the same fish clutch were randomly distributed for each experimental condition.

Human cohort information
NIA-AD Family Based Study (NIA-AD FBS): This study recruited multiplex families across the USA.Families were included if at least one member had a diagnosis of definite or probable Alzheimer's disease [40,51] with onset after age 60 and a sibling with definite, probable, or possible disease with a similar age at onset.Demographic information, diagnosis, age at onset for patients with Alzheimer's disease, method of diagnosis, Clinical Dementia Rating Scale [37], and the presence of other relevant health problems were available for each individual.The age at onset for patients was the age at which the family first observed signs of impaired cognition.For unaffected family members, we used their age at the time of their latest examination without impairment.Each recruitment site used standard research criteria for the diagnosis of Alzheimer's disease [51].For deceased family members who had undergone autopsy, the results were used to determine the diagnosis.For analyses, clinical Alzheimer's disease was defined as any individual meeting NINCDS-ADRDA criteria for probable or possible Alzheimer's disease [51] and definite Alzheimer's disease when CERAD pathological criteria [53] were met postmortem.
Washington Heights/Inwood Columbia Aging Project (WHICAP): WHICAP is a multiethnic, community-based, prospective cohort study of clinical and genetic risk factors for dementia.Three waves of individuals were recruited in 1992, 1999, and 2009 in WHICAP, all using similar study procedures [32,60].Briefly, participants were recruited as representatives of individuals living in the communities of northern Manhattan who were 65 years and older.At the study entry, each person underwent a structured interview of general health and function, followed by a comprehensive assessment including medical, neurological, and psychiatric histories, and standardized physical, neurological, and neuropsychological examinations.Individuals were followed every 18-24 months, repeating examinations that were similar to baseline.All diagnoses were made in a diagnostic consensus conferences attended by a panel consisting of at least one neurologist and one neuropsychologist with expertise in dementia diagnosis, using results from the neuropsychological battery and evidence of impairment in social or occupational function.All-cause dementia which was determined based on Diagnostic and Statistical Manual of Mental Disorders, 4th Edition criteria [4].Furthermore, we used the criteria from the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer Disease and Related Disorders Association to diagnose probable or possible AD [51].
Estudio Familiar de Influencia Genetica en Alzheimer (EFIGA): We used families from a different ethnic group to identify protective alleles in APOEε4 healthy individuals.This cohort comprises participants from a group of families from the Dominican Republic, Puerto Rico, and New York.Recruitment, study design, adjudication, and clinical assessment of this cohort have been previously described [86] as were details of genome-wide SNP data, quality control, and imputation procedures of the GWAS data [68,85].Participants were followed every 2 years and evaluated using a neuropsychological battery [76], a structured medical and neurological examination, and an assessment of depression [65,75].The Clinical Dementia Rating Scale (CDR) [57,58] and functional status were done and the clinical diagnosis of Alzheimer's disease was based on the NINCDS-ADRDA criteria [10,50].

Whole-genome sequencing and quality control
The demographics of the individuals selected for sequencing is shown in Table 1.WGS was performed at the New York Genome Center (NYGC) using 1 µg of DNA, an Illumina PCR-free library protocol, and sequencing on the Illumina HiSeq platform.We harmonized the WGS and the EFIGA families (n = 307), and jointly called variants to create a uniform, analysis set.Genomes were sequenced to a mean coverage of 30×.Sequence data analysis was performed using the NYGC automated analysis pipeline which matches the CCDG and TOPMed-recommended best practices [3].Briefly, sequencing reads were aligned to the human reference, hs38DH, using BWA-MEM v0.7.15.Variant calling was performed using the GATK best practices.Variant filtration was performed using variant quality score recalibration (VQSR at tranche 99.6%) which identified annotation profiles of variants that were likely to be real and assigns a score (VQSLOD) to each variant.

Identification of variants segregating in healthy APOEε4 individuals
First, we filtered high-quality rare (MAF < 0.01 in gno-mAD) variants with genotype quality (GQ) ≥ 20 and depth (DP) ≥ 10.We then excluded any variant observed in APOE ε4 non-carriers.Within variants that segregated in APOEε4 carriers, we prioritized those that were observed in at least 1% of APOEε4 homozygous healthy elderly (≥ 70 years) and had additional support in healthy elderly (≥ 80 years) heterozygous carriers.We further prioritized variants that were absent in AD patients carrying an APOEε4 allele.A simplified pipeline is provided in Fig. 2.

Genotyping, amyloid administration, and tissue preparation
A previously generated fn1b knockout line using CRISPR-Cas9 gene editing [33] was used in homozygous form.The full deletion was genotyped as described [33].Amyloid-β42 was administered to the adult zebrafish brain through cerebroventricular microinjection into the cerebral ventricle [13].Euthanasia and tissue preparation were performed as per institutional ethics committee approval and international guidelines [13,44].12-µm-thick cryo-sections were prepared from these brain samples using a cryostat and collected onto glass slides which were then stored at − 20 °C.

Replication of FN1 variant
An in-depth overview of the methodology and analyses of replication datasets is provided in the Supplementary Text.The current study followed STREGA reporting guidelines.Participants or their caregivers provided written informed consent in the original studies.The current study protocol was granted an exemption by the Stanford Institutional Review Board because the analyses were carried out on "deidentified, off-the-shelf" data; therefore, additional informed consent was not required.Case-control, family-based, and longitudinal AD genetic cohorts were available through public repositories, with genetic data from high-density singlenucleotide polymorphism microarrays, exome microarrays, whole-exome (WES) and whole-genome sequencing (WGS) (Supplementary .These data pertained to cohorts belonging to the ADGC and the ADSP R3.We additionally used population-based data from the UKB, where we had access to health record information to derive case-control diagnoses [17]. Genetic quality control procedures for the UKB are detailed elsewhere.ADGC and ADSP genetic data underwent extensive quality control, imputation to the TOPMed reference panel (for ADGC array-based samples) [26,81], and ancestry determination (SNPweights v2.1) [19].Duplicated individuals were identified and their clinical, diagnostic, and pathological data, as well as age at onset of cognitive symptoms, age at examination for clinical diagnosis, age at last examination, age at death, sex, race, ethnicity, and APOE genotype, were cross-referenced across cohorts.Duplicate entries with irreconcilable phenotypes were excluded.APOE genotypes were adjudicated using state-of-the-art APOE prioritization approaches, filtering out samples where APOE genotypes lacked robustness (prioritizing APOE genotypes from sequencing data and cross-referencing APOE genotypes from high-quality imputation with those provided in study demographics through various protein-based and DNA-based methods) [8].
Finally, in all datasets, samples were filtered to ages 60 years and above, cases or controls, belonging to non-Hispanic White ethnicity and European ancestry, and retaining only a single individual per cryptic relatedness cluster (determined down to third-degree relatedness).Secondary analyses evaluated associations with AD age at onset.Significant discoveries were considered at P < 0.05.All statistical analyses were conducted using R (v.4.2.1).

Image acquisition, quantification, and statistical analyses
Five random illumination field images per patient from the immunostained slides were acquired using Zeiss LSM800 confocal microscope equipped with ZEN software (version blue edition, v3.2, Carl Zeiss, Jena, Germany).Based on vascular markers, coronally sectioned blood vessels were delineated with the selection tool of ZEN software.Fluorescence intensity measures, diameter, and area was calculated.Acquisitions were performed in a blinded fashion (sample IDs, neuropathology details, and genotypes were revealed after the acquisition) and no vessels were specifically left out unless their diameters were larger than 50 μm.GraphPad Prism software version 9.2.0.was used for the statistical analyses.For multiple comparisons, oneway Brown-Forsythe and Welch ANOVA test with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli comparison with individual calculation of variances was used.For non-Gaussian distributions, non-parametric Kruskal-Wallis test with Dunn's multiple comparison test was performed.For correlation of vessel diameter to fluorescent intensity, simple linear regression model and second-order polynomial robust regression with no weighting was used.Significance is indicated by * (P < 0.0332), * * (P < 0.0021), * * * (P < 0.002), ****(P < 0.0001).No asterisks indicate non-significance.No sample set was excluded from the analyses unless the histological sections were damaged severely during the acquisition of the sections (constitutes less than 3% of all sections analyzed).
For zebrafish studies, the effect sizes for animal groups were calculated using G-Power, and the sample size was estimated with n-Query.Four zebrafish from both sexes were used per group.For quantification of SV2-positive synapses, 3D object counter module of ImageJ software was used with the same standard cutoff threshold for every image.For quantification of activated/resting l-plastin-positive microglial cells, two different microglial states were classified based on their cellular morphology: slender and branched as resting microglia; round and regular as active microglia.Six images each from telencephalon sections were analyzed per animal.For colocalization studies, vascular fields were determined using ZO-1 staining on sections (20 for every group), and colocalization with glial endfeet labeled with GS stainings was performed using ImageJ software (v.2.1.0/1.53c) with its colocalization test.Data acquisition was randomized with Fay (x, y, z translation) to acquire in total 1670 data points from two experimental groups.R(and) correlation values from wild-type and fn1b −/− animals were compared using GraphPad Prism (v.9.2.0).Intensity values for individual fluorescent channels were obtained with modal gray values and integrated density measurements using ImageJ.Comparison of 40 sections from two experimental groups was performed.An unpaired non-parametric Kolmogorov-Smirnov t test was performed to test the statistical significance for all analyses.

In silico structure prediction
Protein structures, interspecies similarities, and the deleterious effects of variants were analyzed by SWISS-MODEL protein structure homology-modeling server through Expasy web server (https:// swiss model.expasy.org).SWISS-MODEL repository entries for respective proteins were retrieved and compared to desired protein orthologs using the superposition function.Deleterious mutation prediction was performed using Ensembl-integrated AlphaFold prediction model with SIFT, MetaLR, and REVEL modules for prediction of deleteriousness.

Amyloid toxicity and single-cell sequencing
Amyloid toxicity was induced as described [13,47] in the adult telencephalon; the brains were dissected and singlecell suspensions were generated as previously described [23,24].Chromium Single-Cell 3' Gel Bead and Library Kit v3.1 (10X Genomics, 120,237) was used to generate singlecell cDNA libraries.Generated libraries were sequenced via Illumina NovaSeq 6000 as described [12,13,23,24,71].The cell clusters were identified using a resolution of 1.In total, 34 clusters were identified.The main cell types were identified by using s100b and gfap for astroglia; sv2a, nrgna, grin1a, grin1b for neurons; pdgfrb and kcne4 for pericytes; cd74a and apoc1 for microglia; mbpa and mpz for oligodendrocytes; myh11a and tagln2 for vascular smooth muscle cells, kdrl for endothelial cells.

Fig. 1
Fig. 1 Study design.Comparison of the genomes of elderly APOEε4 carriers with non-carriers

Fig. 2
Fig. 2 Schematic analytical pipeline for this study

Fig. 3
Fig. 3 Pathway analysis of variants segregating in APOEε4 carriers

Fig. 4
Fig. 4 Replication analyses.a Forest plot showing the association of rs140926439 with Alzheimer's disease risk in APOEε4/4 carriers.Significance was considered at P < 0.05.Results across the datasets were combined using fixed-effects inverse-variance weighted metaanalysis.Cochran's Q test indicated no significant heterogeneity.OR, odds ratio; CI, confidence interval.b Case-control regression sensitivity analyses for rs140926439 in APOEε4/4 carriers.To ensure an independent replication of discovery findings, in ADGC, samples from NIA-AD FBS cohort were excluded and ADSP whole-genome sequencing data was fully excluded.Results across the datasets were

Fig. 8
Fig. 8 Schematic abstract for the protective effect of FN1 variants

Table 2
FN1 minor allele frequencies *Elderly APOEε4 homozygous are over 70 years old and heterozygous are over 80 years old

Table 3
Replication of FN1 variant in the ADSP and UKBB cohorts