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. 2024 Apr 10;147(1):70.
doi: 10.1007/s00401-024-02721-1.

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

Prabesh Bhattarai #  1   2 , Tamil Iniyan Gunasekaran  1   3 , Michael E Belloy #  4   5   6 , Dolly Reyes-Dumeyer  1   3 , Dörthe Jülich  7 , Hüseyin Tayran  1   2 , Elanur Yilmaz  1   2 , Delaney Flaherty  2   8 , Bengisu Turgutalp  1   2 , Gauthaman Sukumar  9 , Camille Alba  9 , Elisa Martinez McGrath  9 , Daniel N Hupalo  9 , Dagmar Bacikova  9 , Yann Le Guen  4   10 , Rafael Lantigua  1   2   11 , Martin Medrano  12 , Diones Rivera  13   14 , Patricia Recio  13 , Tal Nuriel  2   8 , Nilüfer Ertekin-Taner  15   16 , Andrew F Teich  1   2   8 , Dennis W Dickson  15 , Scott Holley  7 , Michael Greicius  4 , Clifton L Dalgard  17   18 , Michael Zody  19 , Richard Mayeux #  1   2   3   20   21 , Caghan Kizil  22   23   24 , Badri N Vardarajan #  25   26   27
Affiliations

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

Prabesh Bhattarai et al. Acta Neuropathol. .

Abstract

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.

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Figures

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 meta-analysis. 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 combined using fixed-effects inverse-variance weighted meta-analysis. Cochran’s Q test indicated no significant heterogeneity. c Age at onset analyses for rs140926439 in APOEε4/4 carriers. The large confidence intervals in ADSP whole-genome sequencing (WGS) individuals reflect that there were only two case carriers and one of those had an age at onset at 60 years (an outlier compared to other case carriers). Results across the datasets were combined using fixed-effects inverse-variance weighted meta-analysis. Cochran’s Q test indicated no significant heterogeneity
Fig. 5
Fig. 5
Changes in FN1 and COL6A2 according to APOE genotype. a–c′ Double IFS for CD31 (green) and FN1 (red) with DAPI nuclear counterstain in APOEε3/3 (a, a′), APOEε3/4 (b, b′) and APOEε4/4 (c, c′). d FN1 and CD31 intensity comparisons in 2,044 blood vessels from 28 individuals. e Regression model for FN1 intensity with respect to blood vessel diameter in three APOE genotypes. f–h′ Double IFS for COL4 (green) and COL6A2 (red) with DAPI nuclear counterstain in APOEε3/3 (f, f′), APOEε3/4 (g, g′) and APOEε4/4 (h, h′). i COL4 and COL6A2 intensity comparisons in 1,816 blood vessels from 28 individuals. j Regression model for COL6A2 intensity with respect to blood vessel diameter in three APOE genotypes. Scale bars equal 100 μm
Fig. 6
Fig. 6
FN1 deposition and gliosis reduce to control levels in APOEε4/4 cognitively unaffected individuals, but not in APOEε4/4 AD patients. a–c Double IFS for FN1 (green) and GFAP (red) with DAPI nuclear counterstain in APOEε3/3 (a), APOEε4/4 AD (b), and APOEε4/4 cognitively unaffected individuals (c). Black–white images are individual fluorescent channels for FN1, GFAP, and DAPI. d–f Two blood vessels in every condition are shown in high magnification together with FN1 channel alone. g FN1 intensity comparisons (2 APOEε3/3 individuals without AD, 2 APOEε4/4 individuals with AD, and 6 APOEε4/4 individuals without AD). h GFAP intensity comparisons. Scale bars equal 50 μm (a-c) and 10 μm (d-f)
Fig. 7
Fig. 7
Fibronectin loss of function affects gliovascular interactions, gliosis, and microglial activity after amyloid toxicity in zebrafish brain. a Feature plots for fibronectin 1a (fn1a) and fibronectin 1b (fn1b) genes in zebrafish brain. b Violin plots in control and Aβ42-treated brains. fn1b is mainly expressed in vascular smooth muscle cells and immune cells and is upregulated with Aβ42. c, d Double IF for astroglia marker glutamine synthase (GS, red) and tight junction marker (ZO-1, green) in wild-type and fn1b−/− animals. Individual fluorescent channels in c′, c′′, d′, and d′′. e, f Individual GS channels. g Quantification for colocalization of ZO-1 and GS. h Comparison of intensity measurements for GS. i, j Double IF for synaptic marker SV2 (green) and microglial marker l-Plastin (red) in wild-type and fn1b−/− animals treated with Aβ42. Individual fluorescent channels in black–white channel. k Quantifications for synaptic density, total number of microglia, and activated microglia. Scale bars equal 25 µm
Fig. 8
Fig. 8
Schematic abstract for the protective effect of FN1 variants
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