Alzheimer's disease risk gene CD33 inhibits microglial uptake of amyloid beta

Abstract

The transmembrane protein CD33 is a sialic acid-binding immunoglobulin-like lectin that regulates innate immunity but has no known functions in the brain. We have previously shown that the CD33 gene is a risk factor for Alzheimer's disease (AD). Here, we observed increased expression of CD33 in microglial cells in AD brain. The minor allele of the CD33 SNP rs3865444, which confers protection against AD, was associated with reductions in both CD33 expression and insoluble amyloid beta 42 (Aβ42) levels in AD brain. Furthermore, the numbers of CD33-immunoreactive microglia were positively correlated with insoluble Aβ42 levels and plaque burden in AD brain. CD33 inhibited uptake and clearance of Aβ42 in microglial cell cultures. Finally, brain levels of insoluble Aβ42 as well as amyloid plaque burden were markedly reduced in APP(Swe)/PS1(ΔE9)/CD33(-/-) mice. Therefore, CD33 inactivation mitigates Aβ pathology and CD33 inhibition could represent a novel therapy for AD.

Figure 1. Increased CD33 Expression in AD

(A) Quantitative real-time PCR analysis of CD33 expression in the frontal cortex reveals increased CD33 mRNA levels in AD relative to control subjects; CD33 mRNA levels have been normalized to GAPDH or β-Actin mRNA. (B) Western blotting detection of CD33 in the frontal cortex reveals a marked upregulation in AD cases relative to age-matched controls. A less pronounced increased expression is also seen for Iba1. GAPDH served as loading control. (C) The CD33/GAPDH and CD33/Iba1 protein ratios are increased in AD patients relative to age-matched controls. (D) The levels of CD33 protein are decreased in carriers of the protective minor (T) allele of the CD33 SNP rs3865444. Bar graph shows CD33 protein levels in individuals from the indicated groups (CTRL or AD) and genotypes (G/G versus G/T or T/T). For (A)–(D), *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t test. Data are represented as mean ± SEM. (E) Analysis based on general linear regression model reveals that the protective minor (T) allele of rs3865444 is associated with decreased CD33 protein, but not mRNA levels, in both controls and AD cases (p < 0.05 was considered statistically significant). See also Figure S1 and Table S1.

Figure 2. Increased Number of CD33-Immunoreactive Microglia in AD

(A–A″ and C–C″) Fluorescent immunolabeling reveals colocalization between CD33 (red) and the microglial marker Iba1 (green) in the frontal cortex of CTRL (A–A″) and AD (C–C″) subjects. (B and D) CD33 labeling using diaminobenzidine reveals numerous microglial cells that are positive for CD33. (E) Stereology-based quantification reveals increased numbers of CD33-positive microglial cells in the frontal cortex of AD cases (n = 28) relative to controls (n = 18), ***p < 0.001, Student’s t test. (F) CD33 protein levels normalized to Iba1 protein levels positively correlate with the numbers of CD33-immunoreactive microglia in the AD cases (n = 25; r = 0.525; p = 0.007, Pearson’s correlation test). (G and H) CD33 protein levels correlate with the levels of the microglial marker Iba1, both in controls (G; n = 15; r = 0.543; p = 0.03, Pearson’s correlation test) and AD (H; n = 25; r = 0.582; p = 0.002, Pearson’s correlation test) cases. (I–K) Carriers of the protective minor (T) allele of rs386544 exhibit decreased numbers of CD33-positive microglia. Shown are representative pictures from the frontal cortex of a G/G carrier (I), G/T carrier (J), and T/T AD carrier (K), stained for CD33. (L) Stereology-based quantifications reveal reduced numbers of CD33-positive microglia in carriers of two protective (T) alleles of rs3865444 (**p < 0.01 T/T carriers versus G/G carriers, one-way Kruskal-Wallis ANOVA, Dunn’s test). Data are represented as mean ± SEM. Scale bars represent 25 μm. (M) The protective (T) allele of rs3865444 is associated with decreased numbers of CD33-immunoreactive microglia in both controls and AD cases (general linear regression model, p < 0.05 was considered statistically significant). See also Figure S2.

Figure 3. CD33 Microglial Expression Positively Correlates with Formic Acid-Soluble Aβ42 Levels and Amyloid Plaque Burden in AD

(A) Formic acid (FA)-soluble Aβ42 levels are decreased in carriers of the rs3865444 minor (T) allele. ELISA analysis of Aβ40 and Aβ42 in FA-soluble fractions isolated from the frontal cortex of AD cases of the indicated genotypes (**p < 0.01, Student’s t test). Data are represented as mean ± SEM. (B) The protective rs3865444 (T) allele is associated with decreased levels of both FA-soluble Aβ42 and TBS-soluble Aβ40 in AD cases (n = 25 AD cases, n = 15 controls, general linear regression model, p < 0.05 was considered statistically significant). (C) The numbers of CD33-immunoreactive microglia positively correlate with the FA-soluble Aβ42 levels in AD cases (n = 25; r = 0.446; p = 0.02, Spearman’s correlation test). (D) The numbers of CD33-positive microglia positively correlate with amyloid plaque burden in AD brain (n = 25; r = 0.471; p = 0.017; Spearman’s correlation test). See also Figure S3.

Figure 4. CD33 Inactivation Leads to Increased Microglial Uptake of Aβ42

(A–A‴ and B–B‴) Primary microglial cell cultures were incubated with fluorescently labeled Aβ42 for 3 hr and then subjected to immunofluorescent labeling for CD33 (A and B) and Iba1 (A‴ and B‴). Microglial cultures derived from CD33^−/− mice, at postnatal day 1, exhibited a markedly increased Aβ42 uptake (compare A′ and B′). Scale bar represents 25 μM. (C) Quantification of Aβ42 average signal intensity in individual cells reveals a strong increase in Aβ42 signal in CD33^−/− cells relative to WT cells (at least 30 cells were scored per genotype, **p < 0.01, Student’s t test). (D and E) Microglial cell cultures were treated with unlabeled Aβ42 and incubated for 3 hr. After incubation, cells were either collected for ELISA analysis (t = 0) or washed and incubated for an additional 3 hr in Aβ-free medium, followed by ELISA analysis (t = 3 hr). (D) CD33^−/− microglia exhibit increased Aβ42 uptake levels relative to WT microglia (t = 0) (results were derived from four independent experiments; ***p < 0.001, Student’s t test). (E) Similar rates of Aβ42 degradation in CD33^−/− and WT microglial cells incubated in the absence of Aβ42 for an additional 3 hr (the percentage of remaining Aβ42 was the ratio of Aβ42 remaining at t = 3 hr to the total amount of Aβ42 internalized at t = 0). Data are represented as mean ± SEM. See also Figure S4.

Figure 5. CD33 Inhibits Microglial Uptake of Aβ42

(A–D‴) BV2 microglial cells were incubated with fluorescently labeled Aβ42 for 3 hr and then subjected to immunofluorescent labeling for CD33 (A–D) and Iba1 (A‴–D‴). Overexpression of WT-CD33 decreases the amount of internalized Aβ42 (compare A′ and B′). Expression of an ubiquitylation-defective CD33 variant (K7R-CD33) further decreases the amount of internalized Aβ42 (compare B′ and C′). A CD33 variant lacking the sialic acid-binding V-type immunoglobulin-like extracellular domain (ΔV-Ig-CD33) no longer inhibits Aβ42 uptake (compare B′, C′, with D′). Scale bar represents 25 μM. (E) Quantification of Aβ42 signal intensity in individual cells transfected with the indicated constructs or with an empty vector; at least 30 cells were scored per condition (*p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA, Tukey’s test). (F and G) BV2 cells were treated with unlabeled Aβ42 and incubated for 3 hr. After incubation, cells were either collected for ELISA analysis (t = 0) or washed and incubated for an additional 3 hr in Aβ-free medium, followed by ELISA analysis (t = 3 hr). (F) WT-CD33 inhibits microglial uptake of Aβ42. ELISA quantifications of Aβ42 levels in BV2 cells transfected with the indicated constructs or with empty vector (results were obtained from four independent experiments; *p < 0.05, **p < 0.01, one-way ANOVA, Tukey’s test). (G) CD33 overexpression does not affect the rate of Aβ42 degradation by BV2 cells (the percentage of remaining Aβ42 was the ratio of Aβ42 remaining at t = 3 hr to the total amount of Aβ42 internalized at t = 0). Both empty vector and a GFP vector served as controls. Data are represented as mean ± SEM.

Figure 6. CD33 Deletion Decreases Formic Acid-Soluble Aβ42 Levels in APP/PS1 Mice

(A) Western blotting analysis of cortical extracts reveals increased levels of APP and APP C-terminal fragments (CTFs) in 4-month-old APP/PS1 mice in comparison to controls. However, APP and APP-CTFs levels are similar in APP/PS1 and APP/PS1/CD33^−/− mice. β-Actin served as loading control. (B) Quantification of APP, α-CTF, and β-CTF levels in APP/PS1 and APP/PS1/CD33^−/− mice (n = 9 male mice were analyzed per group). (C–F) ELISA analysis of Aβ40 (C and E) and Aβ42 (D and F) in TBS-soluble (C and D) or formic acid (FA)-soluble (E and F) fractions isolated from the cortex of 4-month-old male mice of the indicated genotypes. APP/PS1 mice exhibit increased levels of Aβ40 and Aβ42 relative to WT and CD33^−/− mice, as expected. No differences in Aβ40 levels and TBS-soluble Aβ42 levels were seen in APP/PS1 and APP/PS1/CD33^−/− mice. However, the FA-soluble Aβ42 levels were markedly decreased in APP/PS1/CD33^−/− relative to APP/PS1 mice (n = 9–12 mice were analyzed per group, *p < 0.05, one-way Kruskal-Wallis ANOVA, Dunn’s test). Data are represented as mean ± SEM. See also Figure S5.

Figure 7. CD33 Deletion Mitigates Aβ Plaque Pathology in APP/PS1 Mice

(A–D) Photomicrographs of selected cortical (A and B) and hippocampal (C and D) fields from 6- to 7-month-old APP/PS1 (A and C) and APP/PS1/CD33^−/− (B and D) brains were stained with the anti-Aβ antibody 3D6 to reveal Aβ plaques. The Aβ plaque burden is decreased in APP/PS1/CD33^−/− brains relative to APP/PS1 brains (compare B with A and D with C). (E and F) Quantification of amyloid plaque burden in the cortex (E) and hippocampus (F) of 6- to 7-month-old APP/PS1 and APP/PS1/CD33^−/− brains (n = 9–11 male mice were analyzed per group, *p < 0.05, **p < 0.01, Student’s t test). Data are represented as mean ± SEM. Scale bar represents 50 μm.