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Cited by 161Alzheimer's disease research and treatmentsNeuroinflammation and Neurodegeneration MechanismsBarrier Structure and Function Studies

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Opposing effects of viral mediated brain expression of apolipoprotein E2 (apoE2) and apoE4 on apoE lipidation and Aβ metabolism in apoE4-targeted replacement mice

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Hu, Jin, Liu, Chia-Chen, Chen, Xiao-Fen, Zhang, Yun-wu, Xu, Huaxi, Bu, Guojun·2015·Molecular Neurodegeneration
DOI 10.1186/s13024-015-0001-3PMC PMC4356137
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Abstract

BACKGROUND: Human apolipoprotein E (apoE) exists in three major isoforms: apoE2, apoE3 and apoE4. In the brain, apoE is produced mostly by astrocytes and transports cholesterol to neurons via apoE receptors. Among the gene alleles encoding the three isoforms, the APOE4 allele is the strongest genetic risk factor for late-onset Alzheimer's disease (AD), whereas APOE2 is protective. ApoE4 confers a gain of toxic function, a loss of neuroprotective function or a combination of both in AD pathogenesis. Given that therapeutic impacts of modulating apoE expression may be isoform-dependent, we sought to investigate the relationship between overexpressing apoE isoform and apoE-related functions in apoE-targeted replacement (TR) mice. Specifically, apoE isoform expression driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter was built into an adeno-associated virus serotype 8 (AAV8) vector and injected into the ventricles of postnatal day 2 (P2) apoE3-TR or apoE4-TR mice. Upon confirmation of apoE isoform expression, effects on apoE lipidation and the levels of amyloid-β (Aβ) in the brain were assessed. RESULTS: AAV8-GFAP-apoE isoforms were specifically expressed in astrocytes throughout all brain regions, which led to overall increased apoE levels in the brain. Viral mediated overexpression of apoE4 in the apoE4-TR background increased poorly-lipidated apoE lipoprotein particles and decreased apoE-associated cholesterol in apoE4-TR mice. Conversely, apoE2 overexpression in apoE4-TR mice enhanced apoE lipidation and associated cholesterol. Furthermore, overexpression of apoE4 elevated the levels of endogenous Aβ, whereas apoE2 overexpression trended to lower endogenous Aβ. CONCLUSIONS: Overexpression of apoE isoforms induces differential effects in the apoE4-TR background: apoE4 decreases apoE lipidation and enhances Aβ accumulation, whereas apoE2 has the opposite effects. Our findings suggest that increasing apoE2 in APOE4 carriers is a beneficial strategy to treat AD, whereas increasing apoE4 in APOE4 carriers is likely harmful. We have also established novel methods to express apoE isoforms in mouse brain to study apoE-related pathways in AD and related dementia.

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Figures

Figure 1 Specific expression of AAV8-GFAP-GFP and AAV8-GFAP-apoE isoforms in astrocytes in vivo. Newborn postnatal day 2 (P2) apoE3-TR mice were injected intracerebroventricularly with viruses carrying AAV8-GFAP-GFP or AAV8-GFAP-apoE isoforms. Expression of GFP or apoE isoforms was evaluated 3 months after viral injection. (A) GFP expression was detected by green fluorescence and the images from different brain regions are shown. (B-E) Mouse brain sections transduced with AAV8-GFAP-GFP (B), AAV8-GFAP-apoE2 (C), AAV8-GFAP-apoE3 (D) or AAV8-GFAP-apoE4 (E) were co-immunostained with HA antibody for apoE expression and antibodies for cell type-specific markers (GFAP: astrocyte; Iba1: microglia; NeuN: neuron). Note that apoE (HA immunoreactivity) is specifically expressed in GFAP-positive astrocytes.
Figure 1 Specific expression of AAV8-GFAP-GFP and AAV8-GFAP-apoE isoforms in astrocytes in vivo. Newborn postnatal day 2 (P2) apoE3-TR mice were injected intracerebroventricularly with viruses carrying AAV8-GFAP-GFP or AAV8-GFAP-apoE isoforms. Expression of GFP or apoE isoforms was evaluated 3 months after viral injection. (A) GFP expression was detected by green fluorescence and the images from different brain regions are shown. (B-E) Mouse brain sections transduced with AAV8-GFAP-GFP (B), AAV8-GFAP-apoE2 (C), AAV8-GFAP-apoE3 (D) or AAV8-GFAP-apoE4 (E) were co-immunostained with HA antibody for apoE expression and antibodies for cell type-specific markers (GFAP: astrocyte; Iba1: microglia; NeuN: neuron). Note that apoE (HA immunoreactivity) is specifically expressed in GFAP-positive astrocytes.CC BY · PubMed Central
Figure 2 Increased apoE expression in apoE-TR mice following AAV viral mediated expression of apoE isoforms in neonatal mice. Neonatal P2 apoE3-TR or apoE4-TR mice were injected intracerebroventricularly with viruses carrying AAV8-GFAP-GFP or AAV8-GFAP-apoE isoforms, and mouse brains were harvested 3 months after viral injection. (A and B) Levels of apoE mRNA (A) and protein (B) in the cortices of apoE3-TR mice transduced with viruses encoding GFP or apoE isoforms were analyzed by real-time PCR or apoE-specific ELISA, respectively. (C and D) Same as in A and B, except apoE4-TR mice were used. Data are expressed as mean ± SEM (n = 7-8/group). *,p < 0.05; **,p < 0.01; ***,p < 0.001.
Figure 2 Increased apoE expression in apoE-TR mice following AAV viral mediated expression of apoE isoforms in neonatal mice. Neonatal P2 apoE3-TR or apoE4-TR mice were injected intracerebroventricularly with viruses carrying AAV8-GFAP-GFP or AAV8-GFAP-apoE isoforms, and mouse brains were harvested 3 months after viral injection. (A and B) Levels of apoE mRNA (A) and protein (B) in the cortices of apoE3-TR mice transduced with viruses encoding GFP or apoE isoforms were analyzed by real-time PCR or apoE-specific ELISA, respectively. (C and D) Same as in A and B, except apoE4-TR mice were used. Data are expressed as mean ± SEM (n = 7-8/group). *,p < 0.05; **,p < 0.01; ***,p < 0.001.CC BY · PubMed Central
Figure 3 Viral mediated expression of apoE4 in apoE4-TR mice shifts apoE particles to smaller sizes. Neonatal P2 apoE4-TR mice were injected with AAV viruses and brains were harvested as in Figure 2. (A) ApoE-associated particles in the TBS fractions of cortical brain tissues were analyzed by native gel electrophoresis. ApoE particle-immunoreactivity was detected at approximately between 242 kDa and 1048 kDa, and defined as three categories of particle sizes: Large particles (>720 kDa); Medium particles (480–720 kDa) and Small particles (<480 kDa). Cortical lysates from age-matched Apoe knockout (KO) mice was used as a negative control for apoE immunoreactivity. (B) The percentages of apoE particles in different size categories were quantified. Data are expressed as mean ± SEM (n = 3-5). *,p < 0.05; **,p < 0.01; ***,p < 0.001.
Figure 3 Viral mediated expression of apoE4 in apoE4-TR mice shifts apoE particles to smaller sizes. Neonatal P2 apoE4-TR mice were injected with AAV viruses and brains were harvested as in Figure 2. (A) ApoE-associated particles in the TBS fractions of cortical brain tissues were analyzed by native gel electrophoresis. ApoE particle-immunoreactivity was detected at approximately between 242 kDa and 1048 kDa, and defined as three categories of particle sizes: Large particles (>720 kDa); Medium particles (480–720 kDa) and Small particles (<480 kDa). Cortical lysates from age-matched Apoe knockout (KO) mice was used as a negative control for apoE immunoreactivity. (B) The percentages of apoE particles in different size categories were quantified. Data are expressed as mean ± SEM (n = 3-5). *,p < 0.05; **,p < 0.01; ***,p < 0.001.CC BY · PubMed Central
Figure 4 Viral mediated expression of apoE4 in apoE4-TR mice leads to reduced levels of apoE-associated cholesterol. Neonatal P2 apoE4-TR mice were injected with AAV viruses and brains were harvested as in Figure 2. Mouse cortical brain tissues were lysed in TBS and the apoE-associated particles were isolated by immunoprecipitation using a biotinylated-apoE specific antibody. Mice injected with GFP viruses treated with beads only were used as a negative control. (A) The immunoprecipitated apoE under different conditions was detected by Western blot. (B) ApoE-associated cholesterol was analyzed by Amplex Red Cholesterol Assay. (C) The ratios of cholesterol-to-apoE under each condition were calculated. Data are expressed as mean ± SEM (n = 3-5/group). *,p < 0.05; **,p < 0.01; ***,p < 0.001.
Figure 4 Viral mediated expression of apoE4 in apoE4-TR mice leads to reduced levels of apoE-associated cholesterol. Neonatal P2 apoE4-TR mice were injected with AAV viruses and brains were harvested as in Figure 2. Mouse cortical brain tissues were lysed in TBS and the apoE-associated particles were isolated by immunoprecipitation using a biotinylated-apoE specific antibody. Mice injected with GFP viruses treated with beads only were used as a negative control. (A) The immunoprecipitated apoE under different conditions was detected by Western blot. (B) ApoE-associated cholesterol was analyzed by Amplex Red Cholesterol Assay. (C) The ratios of cholesterol-to-apoE under each condition were calculated. Data are expressed as mean ± SEM (n = 3-5/group). *,p < 0.05; **,p < 0.01; ***,p < 0.001.CC BY · PubMed Central
Figure 5 Viral mediated expression of apoE4 in apoE4-TR mice leads to increases of mouse endogenous Aβ. Neonatal P2 apoE3-TR or apoE4-TR mice were injected with AAV viruses and brains were harvested as in Figure 2. (A and B) Levels of mouse endogenous Aβ in the cortices of apoE3-TR mice (A) or apoE4-TR mice (B) transduced with AAV viruses encoding GFP or apoE isoforms were quantified by mouse Aβ-specific ELISA. Data are expressed as mean ± SEM (n = 7-8/group). N.S., Not significant; *,p < 0.05; **,p < 0.01.
Figure 5 Viral mediated expression of apoE4 in apoE4-TR mice leads to increases of mouse endogenous Aβ. Neonatal P2 apoE3-TR or apoE4-TR mice were injected with AAV viruses and brains were harvested as in Figure 2. (A and B) Levels of mouse endogenous Aβ in the cortices of apoE3-TR mice (A) or apoE4-TR mice (B) transduced with AAV viruses encoding GFP or apoE isoforms were quantified by mouse Aβ-specific ELISA. Data are expressed as mean ± SEM (n = 7-8/group). N.S., Not significant; *,p < 0.05; **,p < 0.01.CC BY · PubMed Central
Figure 6 Positive correlations between mouse endogenous Aβ and apoE in apoE4-TR mice transduced with AAV8-GFAP-apoE4 viruses. Three months after intracranial delivery of viruses carrying AAV8-GFAP-GFP or AAV8-GFAP-apoE isoforms, the levels of mouse endogenous Aβ40 and apoE in the cortices of apoE4-TR mice were examined by Aβ40-specific ELISA and apoE-specific ELISA, respectively. (A-D) The correlation between levels of Aβ40 and apoE in the cortex of apoE4-TR mice transduced with GFP (A), apoE2 (B), apoE3 (C) or apoE4 (D) were analyzed and plotted. The Pearson correlation coefficient (r) and p values are shown.
Figure 6 Positive correlations between mouse endogenous Aβ and apoE in apoE4-TR mice transduced with AAV8-GFAP-apoE4 viruses. Three months after intracranial delivery of viruses carrying AAV8-GFAP-GFP or AAV8-GFAP-apoE isoforms, the levels of mouse endogenous Aβ40 and apoE in the cortices of apoE4-TR mice were examined by Aβ40-specific ELISA and apoE-specific ELISA, respectively. (A-D) The correlation between levels of Aβ40 and apoE in the cortex of apoE4-TR mice transduced with GFP (A), apoE2 (B), apoE3 (C) or apoE4 (D) were analyzed and plotted. The Pearson correlation coefficient (r) and p values are shown.CC BY · PubMed Central

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Metadata

Authors
Hu, J., Liu, C.-C., Chen, X.-F., Zhang, Y.-W., Xu, H., & Bu, G.
Affiliations
  • Xiamen University (CN)
  • Mayo Clinic (US)
Corresponding author
Bu, Guojun
ORCID
Venue
Molecular Neurodegeneration
Publisher
Springer Science and Business Media LLC
Published
2015-03-05
Volume
10
Issue
1
Pages
6-6
ISSN
1750-1326 (ISSN-L)
DOI
10.1186/s13024-015-0001-3
PMC
PMC4356137
OpenAlex
W2111723622
Cited by
161
References
51
Citations by year
2026: 7 · 2025: 24 · 2024: 25 · 2023: 13 · 2022: 14 · 2021: 17 · 2020: 16 · 2019: 13 · 2018: 7 · 2017: 12 · 2016: 12 · 2015: 1
FWCI
7.86
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Neurodegeneration (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Rare & Special e-Zone (The Hong Kong University of Science and Technology) (submittedVersion)
Funders
  • Alzheimer's Association
  • Hope Center for Neurological Disorders
  • National Institutes of Health
  • National Institute on Aging (P01AG030128, R01AG027924, RF1AG046205, R01AG046205, R01AG035355, P50AG016574)
  • National Institute of Neurological Disorders and Stroke (P30NS057105, P01NS074969)
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
Keywords
apolipoprotein E isoforms, APOE2, APOE ε4, amyloid-beta metabolism, AAV8 vector, glial fibrillary acidic protein promoter, Alzheimer's disease, APOE ε4 carriers
MeSH terms
Dependovirus, Alzheimer Disease, Animals, Astrocytes, Brain, Disease Models, Animal, Humans, Mice, Transgenic, Neurons, Amyloid beta-Peptides, Apolipoprotein E4, Apolipoprotein E2

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