δ-Catenin Is Genetically and Biologically Associated with Cortical Cataract and Future Alzheimer-Related Structural and Functional Brain Changes

<div><p>Multiple lines of evidence suggest that specific subtypes of age-related cataract (ARC) and Alzheimer disease (AD) are related etiologically. To identify shared genetic factors for ARC and AD, we estimated co-heritability of quantitative measures of cataract subtypes with AD-related brain MRI traits among 1,249 members of the Framingham Eye Study who had a brain MRI scan approximately ten years after the eye exam. Cortical cataract (CC) was found to be co-heritable with future development of AD and with several MRI traits, especially temporal horn volume (THV, ρ = 0.24, P<10⁻⁴). A genome-wide association study using 187,657 single nucleotide polymorphisms (SNPs) for the bivariate outcome of CC and THV identified genome-wide significant association with <em>CTNND2</em> SNPs rs17183619, rs13155993 and rs13170756 (P<2.6×10⁻⁷). These SNPs were also significantly associated with bivariate outcomes of CC and scores on several highly heritable neuropsychological tests (5.7×10⁻⁹≤P<3.7×10⁻⁶). Statistical interaction was demonstrated between rs17183619 and <em>APP</em> SNP rs2096488 on CC (P = 0.0015) and CC-THV (P = 0.038). A rare <em>CTNND2</em> missense mutation (G810R) 249 base pairs from rs17183619 altered δ-catenin localization and increased secreted amyloid-β_1–42 in neuronal cell culture. Immunohistopathological analysis of lens tissue obtained from two autopsy-confirmed AD subjects and two non-AD controls revealed elevated expression of δ-catenin in epithelial and cortical regions of lenses from AD subjects compared to controls. Our findings suggest that genetic variation in delta catenin may underlie both cortical lens opacities in mid-life and subsequent MRI and cognitive changes that presage the development of AD.</p> </div

Effect of the <i>CTNND2</i> G810R mutation on intracellular distribution of δ-catenin and Aβ secretion in HEK293 cells stably expressing human APP with the Swedish mutation (APP_sw).

<p><b>A.</b> HEK293 APP_sw cells were transfected with empty vector, wild type delta-catenin or G810R mutant delta-catenin (n = 3 each). Effect of G810R on cell morphology. In contrast to wild type δ-catenin, mutant δ-catenin is predominantly located at/under the cell surface and G810R mutant cells show a more elaborate network of protrusions (white arrows), suggesting that it may be altering interactions of the plasma membrane with the underlying actin cytoskeleton. Lower panels show detail for apical segments (red rectangles) at a higher magnification. <b>B.</b> Semi-quantitative analysis shows significantly more protrusions extending from apical segments in cells with G810R mutation (p = 0.006). Error bars represent the standard deviation. <b>C.</b> Effect of G810R on Aβ secretion. Conditioned media was collected at 16 hours and assayed for Aβ_1–40 and Aβ_1–42. Aβ concentrations were corrected for total protein levels. Error bars represent the standard deviation. When compared to empty vector controls, wild type δ-catenin had no effect on secreted Aβ levels. In contrast, cells expressing mutant δ-catenin displayed a significant and specific increase in the secretion of Aβ_1–42 (p = 0.02).</p

Summary of association results for bivariate analysis of cortical cataract (CC) and temporal horn volume (THV).

<p>P-values are expressed as –log₁₀(P) (y-axis) for every tested SNP ordered by chromosomal location (x-axis). Genome-wide significance level is shown as a dotted line at P = 2.6×10⁻⁷. Genomic position was determined using the NCBI database (Build 37.1). (<b>A</b>) Manhattan plot showing results for the entire genome. Genotyped and imputed <i>CTNND2</i> SNPs are indicated with red dots. (<b>B</b>) Regional association plot for the <i>CTNND2</i> region on chromosome 5 including the top SNP rs17183619 shown by a red circle. Computed estimates of linkage disequilibrium (r²) of SNPs in this region with rs17183619 are shown as orange circles for r²≥0.8, yellow circles for 0.5≤r²<0.8, light blue circles for 0.2≤r²<0.5, and blue circles for r²<0.2. The gene structure and reading frame are shown with the red arrow. Exons are denoted with vertical bars on the arrow. The region demarcated by a light blue dotted box includes the most significant SNPs flanking exon 14. (<b>C</b>) Expanded view of the region in the light blue box. The three genome-wide significant SNPs indicated in red encompass a non-synonymous variant, rs61754599, indicated in blue that is an amino acid change from glycine to arginine at residue 810. Genomic positions of these SNPs are shown at the top of the panel.</p

Bioinformatic evaluation of the <i>CTNND2</i> non-synonymous coding SNP rs61754599 located 249 base pairs from the top-ranked GWAS result.

<p><b>A.</b> Rs61754599 is a missense mutation changing glycine to arginine at residue 810 and was predicted using the PolyPhen2 program (v2.1.0r364) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043728#pone.0043728-Adzhubei1" target="_blank">[59]</a> to have a damaging effect on the protein structure. <b>B.</b> Comparison of the δ-catenin protein sequence in human, zebra fish, dog, chicken, zebra fish, Japanese puffer fish, green puffer, and European seabass. Columns shaded with the same color denote identity of the same domain across species as determined by phylogenetic clustering of protein sequences. The residue glycine (G) at position 810 (orange column bracketed by the black box) is highly conserved across species.</p

Correlations of cortical cataract with temporal horn volume and lateral ventricular volume.

*<p>Trait values transformed to normalized residuals.</p>†<p>Adjusted for age and sex.</p>‡<p>Calculated by averaging the correlations of cortical cataract in sib 1 with MRI trait in sib 2, and cortical cataract in sib 2 with MRI trait in sib 1.</p

Immunohistochemistry of δ-catenin in the human lens in Alzheimer disease (AD) from a 68 year-old male and 71 year-old female with neuropathologically-confirmed AD and from two normal male controls ages 68 and 70.

<p>Staining was observed in the anterior (<b>Panels A–D</b>), equatorial (<b>Panels E–H</b>) and supranuclear (<b>Panels I–L</b>) regions. In the anterior region, intense δ-catenin immunoreactivity was observed in the epithelial cell layer with a striking basolaminar distribution in the AD cases (<b>Panels B and D</b>) compared to age-matched controls (<b>Panels A and C</b>). Dense punctate δ-catenin immunoreactive deposits were observed in the epithelium (white arrowheads in <b>Panels B and D</b>) with laminar preponderance in the subjacent cortex and in the subequatorial and deep cortex of AD lenses (black arrowheads in <b>Panels B and D</b>). Dense punctate δ-catenin immunoreactive staining was also observed in the supranuclear region in the AD cases (black arrowheads in <b>Panels J and L</b>).</p

Protein levels of CCL11 are elevated in the DLFC in CTE but not AD.

<p>CCL11 protein levels were measured using ELISA. CCL11 fold change is shown for non-exposed control, CTE, and AD subjects. Bar graphs shows mean ± SEM, *p < 0.05; One-way ANOVA.</p

Demographic and exposure characteristics of subject groups.

<p>Demographic and exposure characteristics of subject groups.</p

CCL11 is elevated in cases with more than 16 years of exposure to American football.

<p>CCL11 protein fold changes are shown for non-exposed controls (n = 18), cases with less than 16 years of exposure (n = 10), and cases with more than or equal to 16 years of exposure (n = 13). Bar graphs shows mean ± SEM, *p < 0.05, ***p < 0.001; One-way ANOVA.</p

CCL11 is elevated in the CSF during CTE.

<p>(A) Quantitation of CCL11 fold change in the CSF is shown for control (n = 4), CTE (n = 7), and AD (n = 4) subjects. (B) Receiver operatic characteristic (ROC) curve for CSF CCL11 predicting CTE. Red line denotes CCL11 while the black line is the reference, AUC = 0.839, 95% CI 0.62–1.058, p = 0.028. Bar graphs shows mean ± SEM, One-way ANOVA.</p