Apolipoprotein E4 Reduction with Antisense Oligonucleotides Decreases Neurodegeneration in a Tauopathy Model

Abstract

Objective: Apolipoprotein E (ApoE) genotype is the strongest genetic risk factor for late-onset Alzheimer's disease, with the ε4 allele increasing risk in a dose-dependent fashion. In addition to ApoE4 playing a crucial role in amyloid-β deposition, recent evidence suggests that it also plays an important role in tau pathology and tau-mediated neurodegeneration. It is not known, however, whether therapeutic reduction of ApoE4 would exert protective effects on tau-mediated neurodegeneration.

Methods: Herein, we used antisense oligonucleotides (ASOs) against human APOE to reduce ApoE4 levels in the P301S/ApoE4 mouse model of tauopathy. We treated P301S/ApoE4 mice with ApoE or control ASOs via intracerebroventricular injection at 6 and 7.5 months of age and performed brain pathological assessments at 9 months of age.

Results: Our results indicate that treatment with ApoE ASOs reduced ApoE4 protein levels by ~50%, significantly protected against tau pathology and associated neurodegeneration, decreased neuroinflammation, and preserved synaptic density. These data were also corroborated by a significant reduction in levels of neurofilament light chain (NfL) protein in plasma of ASO-treated mice.

Interpretation: We conclude that reducing ApoE4 levels should be explored further as a therapeutic approach for APOE4 carriers with tauopathy including Alzheimer's disease. ANN NEUROL 2021;89:952-966.

Figure 1.. Anti-ApoE ASO treatment reduces ApoE mRNA and protein levels in astrocytes and microglia of aged P301S/ApoE4 mice.

(A) Timeline of the treatment: 350 μg of anti-ApoE ASOs or control (c) ASOs were injected into right lateral ventricle of P301S/ApoE4 animals via i.c.v. injection at 6 and 7.5 months of age; assessment of pathology was performed at 9 months. Image created with BioRender Software. (B) Human ApoE protein levels in hippocampus and cortex measured by ApoE ELISA. Results from RAB-soluble fraction. N=12/group. Student`s t-test. **p<0.01. *p<0.05. (C, D) Representative ApoE immunostaining (C) and quantification of area covered by ApoE staining in hippocampus and cortex (D) of cASO and anti-ApoE ASO-treated mice. N=12/group. Scale bar: 0.5 mm. Student`s t-test. ***p<0.001. (E) Representative immunofluorescent staining of ApoE mRNA foci in GFAP-positive astrocytes and Iba1-positive microglia from CA3 area of hippocampus of 9 month old P301S/ApoE4 mice treated with cASOs or anti-ApoE ASOs (top panel) and its 3D-rendering by IMARIS software (bottom panel). Scale bar: 5 μm. (F) Average percent ratio of ApoE mRNA foci in hippocampal astrocytes or microglia of mice treated with anti-ApoE ASOs or cASOs. N=6 mice/group; n=50 cells/animal, Student`s t-test. *p<0.05; **p<0.01. (G) Average percent ratio of ApoE mRNA foci in cortical astrocytes or microglia of mice treated with anti-ApoE ASOs or cASOs. N=6 mice/group; n=50 cells/animal Student`s t-test. **p<0.01; ***p<0.001.

Figure 2.. Anti-ApoE ASO treatment markedly reduces neurodegeneration in 9 month old P301S/ApoE4 mice.

(A) Representative images of brain sections from 9-month-old cASO and anti-ApoE ASO-treated P301S/ApoE4 mice brain sections stained with cresyl violet. Scale bar: 0.5 mm. (B) Volumes of piriform/entorhinal cortex, hippocampus and lateral ventricle (LV) in 9-month-old mice. N=12/group. Student`s t-test. *p<0.05. (C) Representative images of the dentate gyrus (DG) area of control and ASO-treated animals. Scale bar: 100 μm. (D) Volume (left) and thickness (right) of dentate gyrus granule cell layer of 9-month-old mice. N=12/group. Student`s t-test. **p<0.01; ***p<0.001. (E) Correlation between granule cell layer thickness and hippocampal volume in 9-month-old P301S/ApoE4 mice. N=24 independent animals. Pearson`s correlation test, R²=0.3605, p=0.002. All data represented as mean ± SEM.

Figure 3.. Anti-ApoE ASO does not affect ApoE or lipids in liver and plasma.

(A-B) Levels of human ApoE in liver (A) and plasma (B) of 9 month old cASO and anti-ApoE ASO-treated mice. N=12/group. Student`s t-test. N.S. – non-significant. (C-D) Total (C) and HDL cholesterol (D) levels in plasma of 9 month old cASO and anti-ApoE ASO-treated mice. N=12/group. Student`s t-test. N.S. – non-significant. All data represented as mean ± SEM.

Figure 4.. Anti-ApoE ASO treatment decreases tau pathology in the 9 month old P301S/ApoE4 mice.

(A) Total tau levels in the hippocampus of 9 months old control and ASO-treated P301S/ApoE4 mice measured by ELISA of RAB, RIPA and 70% Formic acid (FA) fractions. N=12/group. Student`s t-test. N.S. – non-significant; *p<0.05. (B) Human phospho-tau Thr181/Ser202/Thr205 levels in the hippocampus of 9-month-old cASO and anti-ApoE ASO-treated P301S/ApoE4 mice measured by ELISA of RAB, RIPA and 70% Formic acid (FA) fractions. N=12/group. Student`s t-test. **p<0.01. (C) Correlation between human ApoE protein and total tau levels in the RAB fraction from hippocampal lysates. Pearson`s correlation for N=24 independent animals, R<sup>2</sup>=0.5004, p<0.0001. (D Correlation between human ApoE protein and total tau levels in the RIPA fraction from hippocampal lysates. Pearson`s correlation for N=24 independent animals, R²=0.4419, p=0.0004. (E) Correlation between human ApoE protein and total tau levels in the FA fraction from hippocampal lysates. Pearson`s correlation for N=24 independent animals, R<sup>2</sup>=0.09655, p=0.1394. (C-D) Representative AT8 immunostaining (C) in hippocampus of 9-month-old cASO and anti-ApoE ASO-treated mice with quantification of area covered by AT8 staining in hippocampus and cortex in (D). Scale bar: 100um. N=12/group. Student`s t-test. ***p<0.001. (E-F) Representative MC1 immunostaining (E) in hippocampus of 9-month-old cASO and anti-ApoE ASO-treated mice and quantification of area covered by MC1 staining in hippocampus and cortex in (F). Scale bar: 100 μm. N=12/group. Student`s t-test. ***p<0.001. All data is represented as mean ± SEM.

Figure 5.. ASO treatment modulates aspects of neuroinflammation in 9 months old P301S/ApoE4 mice.

(A-B) Representative GFAP immunofluorescence staining (A) and quantification of area covered by GFAP staining (B) in hippocampus of 9-month-old cASO and anti-ApoE ASO-treated mice. Scale bar: 100 μm. N=12/group. Student`s t-test. ***p<0.001. (C-D) Representative Iba1 immunofluorescence staining (C) and quantification of area covered by Iba-1 staining (D) in hippocampus of 9-month-old cASO and anti-ApoE ASO-treated mice. Scale bar: 100 μm. N=12/group. Student`s t-test. N.S. – non-significant (E-F) Representative CD68 immunofluorescence staining (E) and quantification of area covered by CD68 staining (F) in hippocampus of 9-month-old cASO and anti-ApoE ASO-treated mice. Scale bar: 50 μm. N=12/group. Student`s t-test. N.S. – non-significant. (G-I) Levels of IL1β (G), IL6 (H) and TNFα (I) in hippocampus as measured by ELISA, N=12/group. Student`s t-test. *p<0.05; **p<0.01; ***p<0.001. (J-K) Representative Clec7a immunostaining (J) in brain sections of cASO-treated and anti-ApoE ASO-treated mice and quantification of area covered by Clec7a staining in hippocampus and cortex (K). Scale bar (J-top): 0.5 mm. Scale bar (J-bottom): 100 μm. N=12/group. Student`s t-test. ***p<0.001. (L-M) Representative P2ry12 immunostaining (L) in brain sections of cASO-treated and anti-ApoE ASO-treated mice and quantification of area covered by P2ry12 staining in hippocampus and cortex (M). Scale bar (L-top): 0.5 mm. Scale bar (L-bottom): 100 μm. N=12/group. Student`s t-test. ***p<0.001. All data represented as mean ± SEM.

Figure 6.. Anti-ApoE ASO treatment reduces synapse loss in 9-month-old P301S/ApoE4 mice.

Representative synaptophysin (Syp) and Iba1 co-immunostaining (A) in the CA3 area of hippocampus of 9 months old cASO and anti-ApoE ASO-treated P301S/ApoE4 mice and quantification of area covered by synaptophysin in CA3 area of hippocampus (B). Scale bar: 50uM. N=12/group. Student`s t-test. ***p<0.001. (C) Correlation between Iba1-covered area in hippocampus and synaptophysin covered area in CA3 area of 9-month-old P301S/ApoE4 mice. n=24 independent animals. Pearson`s correlation test, R²=0.176, p=0.0413. (D) Representative high magnification image of co-localized synaptophysin (Syp) and PSD-95 synaptic puncta in the CA3 area of 9 month old mice. Scale bar: 5 μm. (E) Number of synaptophysin (Syp), PSD-95 and colocalized puncta in the CA3 area of control and ASO-treated mice. N=12/group. Student`s t-test. *p<0.05. (F) Representative high magnification image and its 3D rendering by IMARIS software of synaptophysin (Syp) and PSD95 signal inside Iba1-positive microglia of cASO and anti-ApoE ASO-treated mice. Scale bar: 5 μm. (G) Quantification of engulfed synaptophysin and PSD95 immunoreactive staining from (F). N=12/group. Student`s t-test. ***p<0.001. All data represented as mean ± SEM.

Figure 7.. Anti-ApoE ASO treatment reduces NfL levels in plasma of 9-month-old P301S/ApoE4 mice.

(A) Neurofilament Light Chain (NfL) levels in plasma of 9-month-old cASO and anti-ApoE ASO-treated mice. N=12/group. Student`s t-test. *p<0.05. (B-D) Correlation between NfL levels in plasma and hippocampal volume (B) (R<sup>2</sup>=0.1831, p=0.0598); cortical volume (C) (R<sup>2</sup>=0.07101, p=0.2561) and ventricular volume (C) (R<sup>2</sup>=0.2063, p=0.0443). N=24 independent animals. Pearson`s correlation test. All data represented as mean ± SEM.