Research ArticleNEURODEGENERATIVE DISEASES

APOE4 exacerbates α-synuclein pathology and related toxicity independent of amyloid

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Science Translational Medicine  05 Feb 2020:
Vol. 12, Issue 529, eaay1809
DOI: 10.1126/scitranslmed.aay1809
  • Fig. 1 Increased α-synuclein pathology in αSyn-APOE4 mice.

    Brain sections were prepared from αSyn-APOE mice at 9 months of age. The conformationally changed pathogenic α-synuclein was determined by immunohistochemical staining with 5G4 antibody. (A) Representative images are shown for the deposition of 5G4-positive pathogenic α-synuclein in the brain regions of cerebral cortex, CA1 subfield of the hippocampus, amygdala, and thalamus from αSyn-APOE2, αSyn-APOE3, and αSyn-APOE4 mice. Scale bar, 100 μm. (B) The immunoreactivity of 5G4 staining from different brain regions and the overall immunoreactivity from all these regions were evaluated and quantified by Aperio ImageScope (n = 14 to 21 mice per group, mixed gender). Data represent means ± SEM relative to the αSyn-APOE2 mice. Kruskal-Wallis tests with Dunn’s multiple comparison tests were used. *P < 0.05; **P < 0.01; ***P < 0.001; N.S., not significant.

  • Fig. 2 Impaired behavioral performances in αSyn-APOE4 mice.

    Behavioral performance was assessed in Ctrl-APOE and αSyn-APOE mice (n = 10 to 18 mice per group, mixed gender) at 9 months of age. (A) Exploratory behavior was evaluated in the EPM, and the ratios of the time spent in open arms to close arms are shown. (B and C) Fear conditioning test was used to examine associative memory. The percentage of time with freezing behavior in response to stimulus during contextual and cued tests is shown. (D) The hindlimb clasping test was performed to examine the motor coordination. The clasping scores are shown. (E and F) Hangwire tests were performed to evaluate muscle function and coordination. The latency of the first fall off and numbers of falls within 2 minutes were determined. (G) Rotarod performance tests were used to evaluate motor coordination and balance. The latency to fall off was assessed. Data are expressed as means ± SEM relative to their own Ctrl-APOE mice. Mann-Whitney U tests followed by Bonferroni correction for multiple comparisons were used. P values of <0.0167 were considered statistically significant. *P < 0.0167; **P < 0.01; ***P < 0.001; ****P < 0.0001.

  • Fig. 3 Neuronal and synaptic loss in αSyn-APOE4 mice.

    Brain sections and RIPA lysates from Ctrl-APOE and αSyn-APOE mice at 9 months of age were prepared. (A) Representative images are shown for the NeuN immunohistochemical staining. Scale bar, 2 mm. (B) The immunoreactivity of NeuN staining was evaluated and quantified by Aperio ImageScope (n = 12 to 21 mice per group, mixed gender). (C) The postsynaptic markers in the cortical RIPA lysate from Ctrl-APOE and αSyn-APOE mice were evaluated by Western blotting at 9 months of age (n = 6 mice per group, mixed gender). The amount of PSD95 (D), GluR2 (E), and NR2A (F) was quantified. Results were normalized to β-actin expression. Data represent means ± SEM relative to their own Ctrl-APOE mice. Mann-Whitney U tests (B) and Student’s t tests (D to F) followed by Bonferroni correction for multiple comparisons were used. P values of <0.0167 were considered statistically significant. *P < 0.0167; **P < 0.01; ***P < 0.001.

  • Fig. 4 Astrogliosis in αSyn-APOE4 mice.

    Brain sections, RNA, and RIPA lysates were prepared from Ctrl-APOE and αSyn-APOE mice at 9 months of age. (A) Representative images are shown for the GFAP immunohistochemical staining. Scale bar, 2 mm. (B) The immunoreactivity of GFAP staining was evaluated by Aperio ImageScope (n = 12 to 21 mice per group, mixed gender). (C) The mRNA expression of Gfap was determined by qPCR (n = 6 mice per group, mixed gender). (D and E) The GFAP expression in RIPA lysates was assessed by Western blot (n = 6 mice per group, mixed gender). The immunoblotting results were normalized to β-actin expression. Data represent means ± SEM relative to Ctrl-APOE2 mice. Mann-Whitney U tests (B) and Student’s t tests (C to E) followed by Bonferroni correction for multiple comparisons were used. P values of <0.0167 were considered statistically significant. *P < 0.0167.

  • Fig. 5 Transcriptomic profiling of αSyn-APOE mice.

    RNA sequencing (RNA-seq) was performed using the cortical brain region from Ctrl-APOE and αSyn-APOE mice (n = 6 mice per group, mixed gender) at 9 months of age. (A to C) Volcano plots of differentially expressed genes (DEGs) identified between the Ctrl and αSyn mice in APOE2-TR (A), APOE3-TR (B), and APOE4-TR mice (C) backgrounds. The blue dots denote down-regulated DEGs, and the red dots denote up-regulated DEGs [Benjamini-Hochberg adjusted P < 0.05 and |fold change (FC)| ≥ 1.5]. The black circles denote the genes with significant P values (Benjamini-Hochberg adjusted P < 0.05), but |fold change| values are less than 1.5, and the gray dots denote the genes without marked differences (Benjamini-Hochberg adjusted P ≥ 0.05). The gray dotted lines are the reference threshold for P value (0.05) and fold change (±1.5). (D) Numbers of DEGs in Ctrl versus αSyn mice are shown. Blue or red bars represent significantly down- or up-regulated genes in each comparison. (E) Venn diagram shows the overlapped DEG in Ctrl versus αSyn comparison among APOE2-TR, APOE3-TR, and APOE4-TR mice. (F and G) Transcriptome-wide scatterplots demonstrate the correlation of fold change in APOE4-TR mice (Ctrl versus αSyn, x axis) and APOE2-TR mice [Ctrl versus αSyn, y axis in (F)], or APOE4-TR mice (Ctrl versus αSyn, x axis) and APOE3-TR mice [Ctrl versus αSyn, y axis in (G)] for all genes (each gene corresponds to one point). The blue dots denote genes changed in APOE4-TR mice but not in APOE2-TR or APOE3-TR mice after α-synuclein overexpression. (H to O) The key DEGs defined by RNA-seq were validated by qPCR. Data are expressed as means ± SEM relative to their own Ctrl-APOE mice. Student’s t tests followed by Bonferroni correction for multiple comparisons were used. P values of <0.0167 were considered statistically significant. *P < 0.0167; **P < 0.01; ***P < 0.001.

  • Fig. 6 WGCNA in αSyn-APOE4 mice.

    (A) Modules associated with the comparison of Ctrl versus αSyn in APOE4 mice (n = 6 mice per group). Numbers in the heatmap show the correlation coefficient. Modules with positive values (orange) indicate up-regulation in αSyn compared to Ctrl mice; modules with negative values (blue) indicate down-regulation. (B) Gene ontology (GO) term enrichment of the turquoise module using 4149 module genes. The orange dotted line indicates the threshold of significance (P = 0.05). (C) Network plot of the top 10 genes with the highest intramodular connectivity (hub genes) in the turquoise module. (D) Trajectory of the module eigengenes (MEs) in the turquoise module between Ctrl-APOE4 and αSyn-APOE4 mice. (E) GO term enrichment of the royalblue module using 147 module genes. (F) Network plot of the top 10 hub genes in the royalblue module. (G) Trajectory of the MEs in the royalblue module between Ctrl-APOE4 and αSyn-APOE4 mice. (H) GO term enrichment of the blue module using 4107 module genes. (I) Network plot of the top 10 hub genes in the blue module. (J) Trajectory of the MEs in the blue module between Ctrl-APOE4 and αSyn-APOE4 mice. Student’s t tests were used. **P < 0.01.

  • Fig. 7 Increased α-synuclein pathology in the human postmortem brains of APOE4 carriers with LBD and minimal AD type pathology.

    Human postmortem brain sections from LBD cases were prepared as described in Materials and Methods. The p-S129 phosphorylated α-synuclein and conformationally changed pathogenic α-synuclein were determined by immunohistochemical staining with pSyn#63 and 5G4 antibodies, respectively. Representative images are shown for the deposition of p-S129–positive (A) and 5G4-positive (B) pathogenic α-synuclein in the superior temporal cortex of APOE4 carriers and noncarriers. Scale bars, 600 μm (left) and 50 μm (right) in both (A) and (B). The immunoreactivities of p-S129 staining (C) and 5G4 staining (D) were evaluated and quantified by Aperio ImageScope (n = 22 cases per group). Data represent means ± SEM relative to non-APOE4 carriers. Mann-Whitney U tests were used. *P < 0.05. (E) The correlation between p-S129 and 5G4 immunoreactivities in all the cases was determined by Spearman correlation tests. The black and purple circles represent non-APOE4 carriers and APOE4 carriers, respectively.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/529/eaay1809/DC1

    Fig. S1. Widespread expression of human α-synuclein protein in the brain of αSyn-APOE mice.

    Fig. S2. Widespread expression of GFP in the brain of Ctrl-APOE mice.

    Fig. S3. Expression of human and mouse α-synuclein in the brain of Ctrl-APOE and αSyn-APOE mice.

    Fig. S4. α-Synuclein pathology in the substantia nigra region of αSyn-APOE4 mice.

    Fig. S5. Increased phosphorylated α-synuclein in αSyn-APOE4 mice.

    Fig. S6. Solubility of human α-synuclein in the brain of Ctrl-APOE and αSyn-APOE mice.

    Fig. S7. Solubility of total α-synuclein in the brain of Ctrl-APOE and αSyn-APOE mice.

    Fig. S8. Behavioral abnormalities in αSyn-APOE mice.

    Fig. S9. Correlation of behavioral performances and α-synuclein pathologies in αSyn-APOE mice.

    Fig. S10. Correlation of neurodegeneration and astrogliosis with α-synuclein pathologies in αSyn-APOE mice.

    Fig. S11. Microglial marker expression in αSyn-APOE mice.

    Fig. S12. DEGs and related pathways between Ctrl-APOE and αSyn-APOE mice in different APOE genetic backgrounds identified by transcriptomic profiling.

    Fig. S13. Pathways specifically affected in APOE4-TR mice upon human α-synuclein expression.

    Fig. S14. Glia marker expression in human LBD brain.

    Table S1. Patient characteristics for LBD cohort.

    Data file S1. Raw data for all the figures where n < 20.

  • The PDF file includes:

    • Fig. S1. Widespread expression of human α-synuclein protein in the brain of αSyn-APOE mice.
    • Fig. S2. Widespread expression of GFP in the brain of Ctrl-APOE mice.
    • Fig. S3. Expression of human and mouse α-synuclein in the brain of Ctrl-APOE and αSyn-APOE mice.
    • Fig. S4. α-Synuclein pathology in the substantia nigra region of αSyn-APOE4 mice.
    • Fig. S5. Increased phosphorylated α-synuclein in αSyn-APOE4 mice.
    • Fig. S6. Solubility of human α-synuclein in the brain of Ctrl-APOE and αSyn-APOE mice.
    • Fig. S7. Solubility of total α-synuclein in the brain of Ctrl-APOE and αSyn-APOE mice.
    • Fig. S8. Behavioral abnormalities in αSyn-APOE mice.
    • Fig. S9. Correlation of behavioral performances and α-synuclein pathologies in αSyn-APOE mice.
    • Fig. S10. Correlation of neurodegeneration and astrogliosis with α-synuclein pathologies in αSyn-APOE mice.
    • Fig. S11. Microglial marker expression in αSyn-APOE mice.
    • Fig. S12. DEGs and related pathways between Ctrl-APOE and αSyn-APOE mice in different APOE genetic backgrounds identified by transcriptomic profiling.
    • Fig. S13. Pathways specifically affected in APOE4-TR mice upon human α-synuclein expression.
    • Fig. S14. Glia marker expression in human LBD brain.
    • Table S1. Patient characteristics for LBD cohort.
    • Legend for data file S1

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Raw data for all the figures where n < 20.

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