Research ArticleNEURODEGENERATIVE DISEASES

APOE genotype regulates pathology and disease progression in synucleinopathy

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Science Translational Medicine  05 Feb 2020:
Vol. 12, Issue 529, eaay3069
DOI: 10.1126/scitranslmed.aay3069
  • Fig. 1 APOE genotypes differentially regulate αSyn aggregation and phosphorylation in A53T mice.

    (A to D) Total αSyn concentration was measured by ELISA in RAB, RAB + Triton X-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 end-stage paralysis. Symbols shown in black in (D) indicate A53T/EKO and A53T/E3 samples below the limit of detection. (E and F) Phospho-αSyn concentration was measured by ELISA in RIPA and SDS fractions. Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, one-way ANOVA with Tukey’s multiple comparisons test (A, B, E, and F) or Kruskal-Wallis test with Dunn’s multiple comparisons test (C).

  • 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- to 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 end-stage paralysis. Each data point represents the average of two adjacent regions of interest from three brain sections spaced 300 μm apart. Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, Kruskal-Wallis test with Dunn’s multiple comparisons test. (D) Stratification of pSyn percent area by symptomatic versus asymptomatic status of A53T/EKO (n = 12) and A53T/E4 (n = 19). Data are expressed as means ± SEM. ***P < 0.001, multiple t tests. (E) Stratification of GFAP percent area by symptomatic versus asymptomatic status of A53T/EKO (n = 12) and A53T/E4 (n = 19). Data are expressed as means ± SEM. **P < 0.01 and ***P < 0.001, multiple t tests. (F) Correlation between pSyn and GFAP staining in the brainstem of A53T mice (A53T/EKO, n = 12; A53T/E2, n = 9; A53T/E3, n = 8; A53T/E4, n = 19; 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) the presence of pSyn pathology in corresponding immunohistochemical analysis, (B) EKO versus E2 as baseline, and (C) E4 versus 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 coexpression analysis in A53T mice defines modules associated with pSyn and APOE.

    (A) WGCNA dendrogram group genes measured in the midbrain of 12-month-old A53T mice into distinct modules defined by dendrogram branch clustering, enriched for GOs 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 coefficient (P value). (C) Heatmap of relative expression of turquoise module genes in A53T mice stratified by APOE genotype and end-stage paralysis (denoted with asterisks). (D) Eigengene analysis for turquoise module by APOE genotype. Open symbols indicate mice with end-stage paralysis. Data are expressed as means ± SEM. Kruskal-Wallis test. n.s., not significant. (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 end-stage paralysis (denoted with asterisk). (G) Eigengene analysis for green module by APOE genotype. Open symbols indicate mice with end-stage paralysis. Data are expressed as means ± SEM. *P < 0.05, Kruskal-Wallis test with Dunn’s multiple comparisons test.

  • 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), and 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 3 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 means ± SEM. *P < 0.05 and **P < 0.01, two-way ANOVA with Tukey’s multiple comparisons test. (C) Cell counts of TH-positive neurons from four sections spaced 150 μm apart. Data are expressed as means ± SEM. Multiple t tests with correction for multiple comparisons using the Holm-Sidak method.

  • Table 1 Multivariate regression analysis of rate of change of MoCA scores over time in PPMI participants.

    Covariates included age at onset of PD (Age at onset); age at baseline MoCA test (Age at baseline); sex; years of education (Education); baseline MoCA score (Baseline MoCA); principal components analysis of genetic ancestry (PC1 to PC4); APOE genotype; last available CSF Aβ42, tau, and αSyn concentrations; and the time interval between MoCA assessments. Interaction of covariates with the rate of change of MoCA score over time is listed as “Covariate: Time.” Data are presented as β coefficients with 95% confidence interval (CI) and P value, after accounting for the effect of covariates.

    Variableβ95% CIP
    LowerUpper
    Age at onset−5.22 × 10−2−1.46 × 10−14.15 × 10−22.79 × 10−1
    Age at baseline−1.20 × 10−2−8.28 × 10−21.07 × 10−18.06 × 10−1
    Sex3.81 × 10−15.04 × 10−27.07 × 10−12.39 × 10−2
    Education1.45 × 10−2−4.02 × 10−26.98 × 10−26.07 × 10−1
    Baseline MoCA6.29 × 10−15.11 × 10−17.49 × 10−11.20 × 10−2
    PC1−1.45 × 102−4.35 × 1021.46 × 1023.33 × 10−1
    PC2−1.07 × 101−1.87 × 1021.62 × 1029.04 × 10−1
    PC3−9.10 × 100−2.47 × 1016.52 × 1002.57 × 10−1
    PC41.18 × 101−2.26 × 1014.62 × 1015.05 × 10−1
    APOE ε24.34 × 10−2−3.92 × 10−14.84 × 10−18.47 × 10−1
    APOE ε4−3.51 × 10−2−4.07 × 10−13.32 × 10−18.53 × 10−1
    CSF Aβ42−6.70 × 10−5−5.76 × 10−44.45 × 10−47.98 × 10−1
    CSF pTau2.23 × 10−3−3.18 × 10−23.58 × 10−28.98 × 10−1
    CSF αSyn−6.61 × 10−2−3.27 × 10−11.89 × 10−16.18 × 10−1
    Time interval5.78 × 10−2−2.03 × 1002.15 × 1009.58 × 10−1
    Age at onset: Time1.63 × 10−2−2.75 × 10−26.06 × 10−24.80 × 10−1
    Age at baseline: Time−2.91 × 10−2−7.39 × 10−21.52 × 10−22.14 × 10−1
    Sex: Time−8.07 × 10−2−2.30 × 10−17.21 × 10−23.09 × 10−1
    Education: Time2.99 × 10−24.60 × 10−35.48 × 10−22.40 × 10−2
    Baseline MoCA: Time−3.24 × 10−2−8.69 × 10−22.11 × 10−22.54 × 10−1
    PC1: Time−1.05 × 102−2.36 × 1022.52 × 1011.27 × 10−1
    PC2: Time−7.63 × 100−8.61 × 1017.28 × 1018.54 × 10−1
    PC3: Time−5.18 × 100−1.20 × 1011.64 × 1001.51 × 10−1
    PC4: Time4.55 × 100−1.09 × 1012.01 × 1015.78 × 10−1
    APOE ε2: Time−4.02 × 10−2−2.41 × 10−11.57 × 10−17.00 × 10−1
    APOE ε4: Time−2.26 × 10−1−3.95 × 10−1−5.43 × 10−21.19 × 10−2
    CSF Aβ42: Time5.19 × 10−42.81 × 10−47.57 × 10−44.77 × 10−5
    CSF pTau: Time−3.85 × 10−2−5.43 × 10−2−2.27 × 10−25.87 × 10−6
    CSF αSyn: Time−4.42 × 10−2−1.63 × 10−17.69 × 10−24.82 × 10−1
    APOE2 count012
    Number of individuals217313
    APOE4 count012
    Number of individuals182618
  • Table 2 Multivariate regression analysis of rate of change of MMSE scores over time in WUSTLPD participants.

    Covariates included age at onset of PD (Age at onset), age at baseline MMSE test (Age at baseline), sex, baseline MMSE score (Baseline MMSE), principal components analysis of genetic ancestry (PC1 to PC4), APOE genotype, and the time interval between MMSE assessments. Interaction of covariates with the rate of change of MMSE score over time is listed as Covariate: Time. Data are presented as β coefficients with 95% confidence interval and P value, after accounting for the effect of covariates.

    Variableβ95% CIP
    LowerUpper
    Age at onset1.89 × 10−2−2.02 × 10−25.84 × 10−23.48 × 10−1
    Age at baseline−4.33 × 10−2−9.05 × 10−23.56 × 10−37.31 × 10−2
    Sex1.49 × 10−1−2.73 × 10−15.71 × 10−14.91 × 10−1
    Baseline MMSE6.97 × 10−15.59 × 10−18.36 × 10−13.81 × 10−22
    PC13.22 × 101−1.74 × 1022.39 × 1027.61 × 10−1
    PC21.49 × 101−1.53 × 1021.84 × 1028.63 × 10−1
    PC3−3.28 × 100−1.21 × 10−15.59 × 1004.68 × 10−1
    PC4−1.24 × 100−2.61 × 1012.37 × 1019.22 × 10−1
    APOE ε23.90 × 10−1−3.51 × 10−11.12 × 1003.03 × 10−1
    APOE ε41.46 × 10−1−3.14 × 10−16.08 × 10−15.36 × 10−1
    Time interval1.51 × 100−1.13 × 1004.16 × 1002.77 × 10−1
    Age at onset: Time2.10 × 10−22.97 × 10−33.88 × 10−22.59 × 10−2
    Age at baseline: Time−4.29 × 10−2−6.39 × 10−2−2.14 × 10−21.68 × 10−4
    Sex: Time6.88 × 10−2−1.17 × 10−12.55 × 10−14.82 × 10−1
    Baseline MMSE: Time7.08 × 10−4−6.13 × 10−26.17 × 10−29.82 × 10−1
    PC1: Time−2.66 × 101−1.18 × 1026.46 × 1015.78 × 10−1
    PC2: Time6.99 × 10−1−7.19 × 1017.35 × 1019.85 × 10−1
    PC3: Time3.78 × 10−1−3.43 × 1004.18 × 1008.50 × 10−1
    PC4: Time−3.28 × 100−1.42 × 1017.67 × 1005.69 × 10−1
    APOE ε2: Time−1.82 × 10−1−4.93 × 1011.32 × 1012.69 × 10−1
    APOE ε4: Time−2.21 × 10−1−4.23 × 101−2.18 × 10−23.69 × 10−2
    APOE2 count012
    Number of individuals162150
    APOE4 count012
    Number of individuals127482

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/529/eaay3069/DC1

    Materials and Methods

    Fig. S1. Insoluble total αSyn correlates with end-stage paralysis in A53T mice.

    Fig. S2. Immunoblot analysis of total and pSyn in A53T mice.

    Fig. S3. pSyn pathology A53T mice localizes to neurons.

    Fig. S4. Microglial reactivity in A53T mice with pSyn pathology.

    Fig. S5. pSyn pathology in the cortex in A53T mice.

    Fig. S6. Sample variance of inflammatory gene expression correlates with pSyn pathology in A53T mice.

    Fig. S7. Gene expression modules correlate with end-stage paralysis in A53T mice.

    Fig. S8. Minimal colocalization of pSyn pathology and oligodendrocytes in A53T mice.

    Fig. S9. pSyn pathology in the striatum after αSyn PFF injection.

    Table S1. pSyn IHC differential expression data.

    Table S2. EKO versus E2 differential expression data.

    Table S3. E4 versus E2 differential expression data.

    Table S4. Gene module membership.

    Table S5. Gene-trait significance.

    Table S6. GO analysis for selected modules.

    Table S7. Linear regression analysis of MMSE scores in dbGaP participants.

    Data file S1. Primary data.

    Reference (75)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Insoluble total αSyn correlates with end-stage paralysis in A53T mice.
    • Fig. S2. Immunoblot analysis of total and pSyn in A53T mice.
    • Fig. S3. pSyn pathology A53T mice localizes to neurons.
    • Fig. S4. Microglial reactivity in A53T mice with pSyn pathology.
    • Fig. S5. pSyn pathology in the cortex in A53T mice.
    • Fig. S6. Sample variance of inflammatory gene expression correlates with pSyn pathology in A53T mice.
    • Fig. S7. Gene expression modules correlate with end-stage paralysis in A53T mice.
    • Fig. S8. Minimal colocalization of pSyn pathology and oligodendrocytes in A53T mice.
    • Fig. S9. pSyn pathology in the striatum after αSyn PFF injection.
    • Table S7. Linear regression analysis of MMSE scores in dbGaP participants.
    • Legends for tables S1 to S6
    • Legend for data file S1
    • Reference (75)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). pSyn IHC differential expression data.
    • Table S2 (Microsoft Excel format). EKO versus E2 differential expression data.
    • Table S3 (Microsoft Excel format). E4 versus E2 differential expression data.
    • Table S4 (Microsoft Excel format). Gene module membership.
    • Table S5 (Microsoft Excel format). Gene-trait significance.
    • Table S6 (Microsoft Excel format). GO analysis for selected modules.
    • Data file S1 (Microsoft Excel format). Primary data.

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