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Journal of Alzheimer’s Disease 46 (2015) 365–374 DOI 10.3233/JAD-142184 IOS Press
APOE Genotype Alters Immunoglobulin Subtypes in Knock-In Mice Ye Zhoua,1 , Wenjuan Zhaob,1 , Nour Al-muhtasibc and G. William Rebeckd,∗ a University
of Florida, Gainesville, FL, USA of Pharmacy, Shanghai Jiao Tong University, Shanghai, China c Department of Pharmacology, Georgetown University, Washington, DC, USA d Department of Neuroscience, Georgetown University, Washington, DC, USA b School
Handling Associate Editor: Jose Abisambra
Accepted 17 February 2015
Abstract. Apolipoprotein E (APOE) alleles are strongly related to the risk of Alzheimer’s disease (AD). APOE genotype also affects inflammatory processes in response to damage. We tested whether APOE genotype affected the levels of specific immunoglobulins in healthy, uninfected APOE knock-in mice. We measured specific immunoglobulins in brain, spleen, and plasma. Levels of total IgG in brain and spleen were highest in APOE-3 mice, significantly higher than in APOE-2 and APOE-4 mice; no differences were observed for levels of total IgG in plasma. We also measured specific subtypes of IgG. IgG1 was only detectable in plasma and did not differ by APOE genotype. IgG3 was detectable in plasma and spleen, and also did not differ by APOE genotype. IgG2b showed the same pattern as levels of total IgG by APOE genotype, with the highest levels of IgG2b in brain, spleen, and plasma of APOE-3 mice. IgG2a showed an entirely different pattern, with significantly higher levels in spleen and plasma of APOE-4 mice compared to APOE-2 and APOE-3 mice. We also measured IgM and IgA in spleens and plasma of these mice. In spleen, APOE-4 mice had the lowest IgA levels and the highest levels of IgM; both being significantly different from APOE-2 mice. In total, murine IgG2a and IgM were highest in APOE-4 mice, while total IgG and Ig2b were highest in APOE-3 mice. These dramatically different distributions of immunoglobulins could allow for human AD risk biomarkers based on specific immunoglobulin subtypes. Keywords: Apolipoprotein E, brain, immunoglobulin, inflammation, plasma, spleen
INTRODUCTION Polymorphisms in the gene for apolipoprotein E (APOE) greatly affect the risk of late-onset Alzheimer’s disease (AD) [1]. There are three common human APOE alleles (APOE-2, APOE-3, and APOE-4), encoding ApoE isoforms that differ from each other by single amino acids [2]. Individuals homozygous for APOE-4 are 16 times more susceptible in AD than APOE-3 homozygotes, while 1 These
authors contributed equally to this work. to: G. William Rebeck, Department of Neuroscience, Georgetown University, 3970 Reservoir Rd, NW, Washington DC 20007, USA. Tel.: +1 202 687 1534; Fax: +1 202 687 0617; E-mail: [email protected]. ∗ Correspondence
those who have inherited APOE-2 have a significantly reduced risk of AD [3–5]. The strong association of APOE-4 with risk of AD makes it possible to identify individuals at high risk for AD well before the onset of symptoms. The mechanism of APOE increasing AD risk alters how APOE-directed therapies are developed. ApoE is a lipid transport molecule, associating with lipoproteins and promoting their endocytosis in various tissues [6]. ApoE affects metabolism of amyloid- (A) due to its partially hydrophobic nature [7], either by preventing A clearance from the brain interstitial fluid [6] or promoting glial clearance [8, 9]. However, the effects of APOE on AD pathogenesis may also be due in part to its role in regulation of inflammatory
ISSN 1387-2877/15/$35.00 © 2015 – IOS Press and the authors. All rights reserved
366
Y. Zhou et al. / APOE Alters Levels of Immunoglobulin Subtypes
responses [10]. APOE knock-out [11] and APOE4 knock-in mice [12] have increased neuroinflammation in response to various agents [13–16], including the A protein [17, 18]. Microglia of APOE4 knock-in mice have a more active immune reaction in brain after infection compared to APOE3 mice, including increased proliferation and pro-inflammatory cytokine release [19, 20]. In vitro studies also demonstrate the anti-inflammatory effects of ApoE, with ApoE4 being deficient compared to ApoE2 or ApoE3 [13, 21]. The effects of APOE on inflammation are also supported by the observations that APOE knock-out mice have higher IgM levels, more T cell proliferation and more reactive nitric oxide production after antigen stimulation [22–24]. We hypothesize that APOE genotype may also affect neuroinflammation even in the absence of a proinflammatory stimulus. In this study, we address the role of APOE as an immune modulator by examining the immunoglobulin levels in healthy APOE knock-in mice. We found that APOE4 mice had a higher level of IgG2a and IgM but lower levels of IgA; this being consistent with the role of apoE4 promoting a more inflammatory status in vivo. Furthermore, these findings identify several specific blood proteins that could be evaluated as biomarkers for APOE-associated risk of AD.
heart puncture, treated to 10 mM EDTA, and spun at 2,000 rpm for 20 min; plasma was stored at −20◦ C. Animal perfusion was performed using ice-cold saline (PBS, pH 7.4). Brains and spleens were rapidly removed (brains were then hemisected), frozen on dry ice, weighed and stored at -80◦ C. The spleens and left hemispheres of the brains were homogenized with a 7 ml dounce with ice-cold Tris-buffered saline (TBS) buffer (50 mM Tris-HCl, 150 mM NaCl, 1x protease inhibitor and phosphatase inhibitor cocktails, pH 7.4). The homogenates were centrifuged in a Beckman Coulter Le-80 Ultracentrifuge (Brea, CA, USA, rotor sw55Ti) at 33,400 rpm for 45 min at 4◦ C, and the supernatants were removed and labeled as the TBS (soluble) fraction. The remaining pellet was sonicated in ice-cold TBS-X buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1x protease inhibitor and phosphatase inhibitor cocktails, pH 7.4). The resuspended pellet was centrifuged at 33,400 rpm for 45 min at 4◦ C, and the supernatants were removed and labeled as the TBS-X (membrane) fraction. Total protein concentration was determined by BCA Protein Assay Kit (Pierce, Rockford, IL, USA) and absorbance was analyzed by Origin 8.0 (OriginLab, Northampton, MA, USA). Total protein levels were adjusted to load equivalent amounts for western blot analysis.
MATERIALS AND METHODS
Western blots
Mice
All samples were reduced with 2-mercaptoethanol and heated at 95◦ C for 5 min before being subjected to SDS-PAGE: For brain and spleen samples, 20 g of protein was analyzed by electrophoresis; for plasma, 3 l was similarly analyzed. Separated proteins were transferred onto a nitrocellulose membrane. After blocking with 5% non-fat dried milk in TBS-T (TBS with 0.05% Tween 20) for 1 h, the membranes were incubated with antibody diluted in TBS-T with 5% milk for at least 2 h. The following antibodies were used: Horseradish peroxidase (HRP)conjugated goat anti-mouse IgG, (Sigma-Aldrich, St. Louis, MO, USA); HRP-conjugated goat anti-mouse IgM and HRP-conjugated goat anti-mouse IgA (SouthernBiotech, Birmingham, AL, USA); HRP-conjugated goat anti-mouse IgG1 (Abcam Inc, Cambridge, MA, USA); HRP-conjugated goat anti-mouse IgG2a (Santa Cruz Biotechnology, Santa Cruz, CA, USA); HRPconjugated goat anti-mouse IgG2b (Life Technologies, Carlsbad, CA, USA) and HRP-conjugated goat antimouse IgG3 (Abcam Inc). Antibodies were diluted 1000 to 10,000 fold. After washing with TBS-T five times for 3 min, membranes were developed by
APOE2, APOE3, and APOE4 knock-in mice on a C57B16/J background each express human APOE isoforms regulated by the endogenous murine APOE promoter [25]. All mice were maintained in a constant room temperature and humidity under a 12-h light/dark cycle at Georgetown University Medical Center, and freely provided food and water. Experiments were performed on age-matched 9-month-old female mice (APOE2, n = 4; APOE3, n = 6; APOE4, n = 6). This age was chosen to allow analysis of immunoglobulins in adult animals but without alterations that may occur in aged mice; female mice were chosen for the sake of consistency. All experiments were performed in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Georgetown University Animal Care and Use Committee (protocol number 12-044). Mouse tissue collection and preparation Mice were fasted overnight before euthanasia by carbon dioxide asphyxiation. Blood was taken via
Y. Zhou et al. / APOE Alters Levels of Immunoglobulin Subtypes
SuperSignal West PICO (Pierce) for 5 min and subjected to X-ray film development. The X-ray film was scanned and the density of bands was quantified using ImageJ software (National Institutes of Health). For loading control experiments in spleen and brain samples, rabbit anti-tubulin and HRP-conjugated goat anti-rabbit IgG (Sigma-Aldrich) were used. For plasma samples, nitrocellulose blots were stained with Ponceau to determine that there was no large variation in total protein levels. Statistical analysis Statistical analysis was performed by one-way ANOVA or T-test using GraphPad Prism (GraphPad, San Diego, CA, USA). Tukey’s post hoc tests were applied to detect statistical differences among groups. Data are expressed as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 compared to APOE3 mice; # p < 0.05, ##
Journal of Alzheimer’s Disease 46 (2015) 365–374 DOI 10.3233/JAD-142184 IOS Press
APOE Genotype Alters Immunoglobulin Subtypes in Knock-In Mice Ye Zhoua,1 , Wenjuan Zhaob,1 , Nour Al-muhtasibc and G. William Rebeckd,∗ a University
of Florida, Gainesville, FL, USA of Pharmacy, Shanghai Jiao Tong University, Shanghai, China c Department of Pharmacology, Georgetown University, Washington, DC, USA d Department of Neuroscience, Georgetown University, Washington, DC, USA b School
Handling Associate Editor: Jose Abisambra
Accepted 17 February 2015
Abstract. Apolipoprotein E (APOE) alleles are strongly related to the risk of Alzheimer’s disease (AD). APOE genotype also affects inflammatory processes in response to damage. We tested whether APOE genotype affected the levels of specific immunoglobulins in healthy, uninfected APOE knock-in mice. We measured specific immunoglobulins in brain, spleen, and plasma. Levels of total IgG in brain and spleen were highest in APOE-3 mice, significantly higher than in APOE-2 and APOE-4 mice; no differences were observed for levels of total IgG in plasma. We also measured specific subtypes of IgG. IgG1 was only detectable in plasma and did not differ by APOE genotype. IgG3 was detectable in plasma and spleen, and also did not differ by APOE genotype. IgG2b showed the same pattern as levels of total IgG by APOE genotype, with the highest levels of IgG2b in brain, spleen, and plasma of APOE-3 mice. IgG2a showed an entirely different pattern, with significantly higher levels in spleen and plasma of APOE-4 mice compared to APOE-2 and APOE-3 mice. We also measured IgM and IgA in spleens and plasma of these mice. In spleen, APOE-4 mice had the lowest IgA levels and the highest levels of IgM; both being significantly different from APOE-2 mice. In total, murine IgG2a and IgM were highest in APOE-4 mice, while total IgG and Ig2b were highest in APOE-3 mice. These dramatically different distributions of immunoglobulins could allow for human AD risk biomarkers based on specific immunoglobulin subtypes. Keywords: Apolipoprotein E, brain, immunoglobulin, inflammation, plasma, spleen
INTRODUCTION Polymorphisms in the gene for apolipoprotein E (APOE) greatly affect the risk of late-onset Alzheimer’s disease (AD) [1]. There are three common human APOE alleles (APOE-2, APOE-3, and APOE-4), encoding ApoE isoforms that differ from each other by single amino acids [2]. Individuals homozygous for APOE-4 are 16 times more susceptible in AD than APOE-3 homozygotes, while 1 These
authors contributed equally to this work. to: G. William Rebeck, Department of Neuroscience, Georgetown University, 3970 Reservoir Rd, NW, Washington DC 20007, USA. Tel.: +1 202 687 1534; Fax: +1 202 687 0617; E-mail: [email protected]. ∗ Correspondence
those who have inherited APOE-2 have a significantly reduced risk of AD [3–5]. The strong association of APOE-4 with risk of AD makes it possible to identify individuals at high risk for AD well before the onset of symptoms. The mechanism of APOE increasing AD risk alters how APOE-directed therapies are developed. ApoE is a lipid transport molecule, associating with lipoproteins and promoting their endocytosis in various tissues [6]. ApoE affects metabolism of amyloid- (A) due to its partially hydrophobic nature [7], either by preventing A clearance from the brain interstitial fluid [6] or promoting glial clearance [8, 9]. However, the effects of APOE on AD pathogenesis may also be due in part to its role in regulation of inflammatory
ISSN 1387-2877/15/$35.00 © 2015 – IOS Press and the authors. All rights reserved
366
Y. Zhou et al. / APOE Alters Levels of Immunoglobulin Subtypes
responses [10]. APOE knock-out [11] and APOE4 knock-in mice [12] have increased neuroinflammation in response to various agents [13–16], including the A protein [17, 18]. Microglia of APOE4 knock-in mice have a more active immune reaction in brain after infection compared to APOE3 mice, including increased proliferation and pro-inflammatory cytokine release [19, 20]. In vitro studies also demonstrate the anti-inflammatory effects of ApoE, with ApoE4 being deficient compared to ApoE2 or ApoE3 [13, 21]. The effects of APOE on inflammation are also supported by the observations that APOE knock-out mice have higher IgM levels, more T cell proliferation and more reactive nitric oxide production after antigen stimulation [22–24]. We hypothesize that APOE genotype may also affect neuroinflammation even in the absence of a proinflammatory stimulus. In this study, we address the role of APOE as an immune modulator by examining the immunoglobulin levels in healthy APOE knock-in mice. We found that APOE4 mice had a higher level of IgG2a and IgM but lower levels of IgA; this being consistent with the role of apoE4 promoting a more inflammatory status in vivo. Furthermore, these findings identify several specific blood proteins that could be evaluated as biomarkers for APOE-associated risk of AD.
heart puncture, treated to 10 mM EDTA, and spun at 2,000 rpm for 20 min; plasma was stored at −20◦ C. Animal perfusion was performed using ice-cold saline (PBS, pH 7.4). Brains and spleens were rapidly removed (brains were then hemisected), frozen on dry ice, weighed and stored at -80◦ C. The spleens and left hemispheres of the brains were homogenized with a 7 ml dounce with ice-cold Tris-buffered saline (TBS) buffer (50 mM Tris-HCl, 150 mM NaCl, 1x protease inhibitor and phosphatase inhibitor cocktails, pH 7.4). The homogenates were centrifuged in a Beckman Coulter Le-80 Ultracentrifuge (Brea, CA, USA, rotor sw55Ti) at 33,400 rpm for 45 min at 4◦ C, and the supernatants were removed and labeled as the TBS (soluble) fraction. The remaining pellet was sonicated in ice-cold TBS-X buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1x protease inhibitor and phosphatase inhibitor cocktails, pH 7.4). The resuspended pellet was centrifuged at 33,400 rpm for 45 min at 4◦ C, and the supernatants were removed and labeled as the TBS-X (membrane) fraction. Total protein concentration was determined by BCA Protein Assay Kit (Pierce, Rockford, IL, USA) and absorbance was analyzed by Origin 8.0 (OriginLab, Northampton, MA, USA). Total protein levels were adjusted to load equivalent amounts for western blot analysis.
MATERIALS AND METHODS
Western blots
Mice
All samples were reduced with 2-mercaptoethanol and heated at 95◦ C for 5 min before being subjected to SDS-PAGE: For brain and spleen samples, 20 g of protein was analyzed by electrophoresis; for plasma, 3 l was similarly analyzed. Separated proteins were transferred onto a nitrocellulose membrane. After blocking with 5% non-fat dried milk in TBS-T (TBS with 0.05% Tween 20) for 1 h, the membranes were incubated with antibody diluted in TBS-T with 5% milk for at least 2 h. The following antibodies were used: Horseradish peroxidase (HRP)conjugated goat anti-mouse IgG, (Sigma-Aldrich, St. Louis, MO, USA); HRP-conjugated goat anti-mouse IgM and HRP-conjugated goat anti-mouse IgA (SouthernBiotech, Birmingham, AL, USA); HRP-conjugated goat anti-mouse IgG1 (Abcam Inc, Cambridge, MA, USA); HRP-conjugated goat anti-mouse IgG2a (Santa Cruz Biotechnology, Santa Cruz, CA, USA); HRPconjugated goat anti-mouse IgG2b (Life Technologies, Carlsbad, CA, USA) and HRP-conjugated goat antimouse IgG3 (Abcam Inc). Antibodies were diluted 1000 to 10,000 fold. After washing with TBS-T five times for 3 min, membranes were developed by
APOE2, APOE3, and APOE4 knock-in mice on a C57B16/J background each express human APOE isoforms regulated by the endogenous murine APOE promoter [25]. All mice were maintained in a constant room temperature and humidity under a 12-h light/dark cycle at Georgetown University Medical Center, and freely provided food and water. Experiments were performed on age-matched 9-month-old female mice (APOE2, n = 4; APOE3, n = 6; APOE4, n = 6). This age was chosen to allow analysis of immunoglobulins in adult animals but without alterations that may occur in aged mice; female mice were chosen for the sake of consistency. All experiments were performed in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Georgetown University Animal Care and Use Committee (protocol number 12-044). Mouse tissue collection and preparation Mice were fasted overnight before euthanasia by carbon dioxide asphyxiation. Blood was taken via
Y. Zhou et al. / APOE Alters Levels of Immunoglobulin Subtypes
SuperSignal West PICO (Pierce) for 5 min and subjected to X-ray film development. The X-ray film was scanned and the density of bands was quantified using ImageJ software (National Institutes of Health). For loading control experiments in spleen and brain samples, rabbit anti-tubulin and HRP-conjugated goat anti-rabbit IgG (Sigma-Aldrich) were used. For plasma samples, nitrocellulose blots were stained with Ponceau to determine that there was no large variation in total protein levels. Statistical analysis Statistical analysis was performed by one-way ANOVA or T-test using GraphPad Prism (GraphPad, San Diego, CA, USA). Tukey’s post hoc tests were applied to detect statistical differences among groups. Data are expressed as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 compared to APOE3 mice; # p < 0.05, ##
