APOE genotype regulates pathology and disease progression in synucleinopathy

Abstract

Apolipoprotein E (APOE) ε4 genotype is associated with increased risk of dementia in Parkinson's disease (PD), but the mechanism is not clear, because patients often have a mixture of α-synuclein (αSyn), amyloid-β (Aβ), and tau pathologies. APOE ε4 exacerbates brain Aβ pathology, as well as tau pathology, but it is not clear whether APOE genotype independently regulates αSyn pathology. In this study, we generated A53T αSyn transgenic mice (A53T) on Apoe knockout (A53T/EKO) or human APOE knockin backgrounds (A53T/E2, E3, and E4). At 12 months of age, A53T/E4 mice accumulated higher amounts of brainstem detergent-insoluble phosphorylated αSyn compared to A53T/EKO and A53T/E3; detergent-insoluble αSyn in A53T/E2 mice was undetectable. By immunohistochemistry, A53T/E4 mice displayed a higher burden of phosphorylated αSyn and reactive gliosis compared to A53T/E2 mice. A53T/E2 mice exhibited increased survival and improved motor performance compared to other APOE genotypes. In a complementary model of αSyn spreading, striatal injection of αSyn preformed fibrils induced greater accumulation of αSyn pathology in the substantia nigra of A53T/E4 mice compared to A53T/E2 and A53T/EKO mice. In two separate cohorts of human patients with PD, APOE ε4/ε4 individuals showed the fastest rate of cognitive decline over time. Our results demonstrate that APOE genotype directly regulates αSyn pathology independent of its established effects on Aβ and tau, corroborate the finding that APOE ε4 exacerbates pathology, and suggest that APOE ε2 may protect against αSyn aggregation and neurodegeneration in synucleinopathies.

Fig 1.. APOE genotypes differentially regulate αSyn aggregation and phosphorylation in A53T mice.

(A-D) Total αSyn concentration was measured by ELISA in RAB, RAB+TX-100, RIPA, and SDS fractions from the brainstem of 12-month old A53T mice. A53T/EKO, n=10; A53T/E2, n=6; A53T/E3, n=9; A53T/E4, n=10. Closed symbols indicate asymptomatic mice; open symbols indicate symptomatic mice with endstage paralysis. Symbols shown in black in (D) indicate A53T/EKO and A53T/E3 samples below the limit of detection. (E-F) Phospho-αSyn concentration was measured by ELISA in RIPA and SDS fractions. Data expressed as mean ± SEM, one-way ANOVA with Tukey’s multiple comparisons test (A, B, E, F) or Kruskal-Wallis test with Dunn’s multiple comparisons test (C). *p<0.05, **p<0.01, ***p<0.001.

Fig 2.. APOE genotype relates to pSyn pathology and astrogliosis in A53T mice.

(A) Representative images showing pSyn pathology (b81A) and astrogliosis (GFAP) in the brainstem of 9–12 month old A53T mice. Images represent maximum-intensity projections of z stacks. Scale bar, 50 μm. Quantitation of the percent area covered by (B) pSyn and (C) GFAP staining in the brainstem of A53T/EKO (n=12), A53T/E2 (n=9), A53T/E3 (n=8), and A53T/E4 (n=19) mice. Closed symbols indicate asymptomatic mice; open symbols indicate symptomatic mice with endstage paralysis. Each data point represents the average of 2 adjacent regions of interest from 3 brain sections spaced 300 μm apart. Data expressed as mean ± SEM, Kruskal-Wallis test with Dunn’s multiple comparisons test. *p<0.05, **p<0.01, ***p<0.001. (D) Stratification of pSyn percent area by symptomatic vs asymptomatic status of A53T/EKO (n=12) and A53T/E4 (n=19). Data expressed as mean ± SEM, multiple t-tests, ***p<0.001. (E) Stratification of GFAP percent area by symptomatic vs asymptomatic status of A53T/EKO (n=12) and A53T/E4 (n=19). Data expressed as mean ± SEM, multiple t-tests, **p<0.01, ***p<0.001. (F) Correlation between pSyn and GFAP staining in the brainstem of A53T mice (n=12 A53T/EKO, n=9 A53T/E2, n=8 A53T/E3, n=19 A53T/E4; r2=0.8510, p<0.0001).

Fig 3.. Inflammatory gene expression in A53T mice correlates with pSyn pathology but not APOE genotype.

Volcano plot showing differences in gene expression in the midbrain of A53T mice stratified by (A) presence of pSyn pathology in corresponding immunohistochemical analysis, (B) EKO vs. E2 as baseline, (C) E4 vs. E2 as baseline. For each plot, significance is plotted against fold-change. Red symbols denote genes with adjusted significance of p<0.01.

Fig 4.. Gene co-expression analysis in A53T mice defines modules associated with pSyn and APOE.

(A) WGCNA dendrogram groups genes measured in the midbrain of 12 month-old A53T mice into distinct modules defined by dendrogram branch clustering, enriched for gene ontologies linked to specific cell type or cellular function. (B) Module-trait analysis between gene modules defined by WGCNA and APOE genotype or pSyn IHC. Data are shown as correlation co-efficient (p-value). (C) Heatmap of relative expression of turquoise module genes in A53T mice stratified by APOE genotype and endstage paralysis (denoted with *). (D) Eigengene analysis for turquoise module by APOE genotype. Open symbols indicate mice with endstage paralysis. Data are expressed as mean ± SEM, Kruskal-Wallis test. (E) Linear regression between pSyn IHC % area and turquoise module eigenvalue among A53T/E4 mice (n=10; r2=0.9045, p<0.0001). (F) Heatmap of relative expression of green module genes stratified by APOE genotype and endstage paralysis (denoted with *). (G) Eigengene analysis for green module by APOE genotype. Open symbols indicate mice with endstage paralysis. Data are expressed as mean ± SEM, Kruskal-Wallis test with Dunn’s multiple comparisons test. *p<0.05

Fig 5.. APOE2 genotype protects against motor deficits and prolongs survival in A53T mice.

(A) Assessment of motor function in A53T mice. Latency to fall in the inverted wire screen test was measured for A53T/EKO (n=24), A53T/E2 (n=28), A53T/E3 (n=8), and A53T/E4 (n=22) mice. (B) Kaplan-Meier survival analysis of A53T mice by APOE genotype for A53T/EKO (n=10, median survival 11.6 months), A53T/E2 (n=7, median survival 18.4 months), A53T/E3 (n=5, median survival 12.7 months), A53T/E4 (n=18, median survival 11.7 months) mice. Overall log-rank (Mantel-Cox) p=0.0030.

Fig 6.. APOE4 exacerbates spreading of αSyn pathology.

(A) Representative images showing pSyn pathology within the SNpc three months after unilateral injection of αSyn PFFs into the striatum of EKO (n=6), E2 (n=9), E3 (n=11), and E4 (n=9) mice. Scale bar, 250 μm; inset scale bar, 50 μm. (B) Quantitation of the percent area covered by pSyn staining in the SNpc. Data are expressed as mean ± SEM, two-way ANOVA with Tukey’s multiple comparisons test *p<0.05, **p<0.01. (C) Cell counts of TH-positive neurons from 4 sections spaced 150 μm apart. Data are expressed as mean ± SEM, multiple t-tests with correction for multiple comparisons using the Holm-Sidak method.