Apolipoprotein E isoform-specific effects on lipoprotein receptor processing

Abstract

Recent findings indicate an isoform-specific role for apolipoprotein E (apoE) in the elimination of beta-amyloid (Aβ) from the brain. ApoE is closely associated with various lipoprotein receptors, which contribute to Aβ brain removal via metabolic clearance or transit across the blood–brain barrier (BBB). These receptors are subject to ectodomain shedding at the cell surface, which alters endocytic transport and mitigates Aβ elimination. To further understand the manner in which apoE influences Aβ brain clearance, these studies investigated the effect of apoE on lipoprotein receptor shedding. Consistent with prior reports, we observed an increased shedding of the low-density lipoprotein receptor (LDLR) and the LDLR-related protein 1 (LRP1) following Aβ exposure in human brain endothelial cells. When Aβ was co-treated with each apoE isoform, there was a reduction in Aβ-induced shedding with apoE2 and apoE3, while lipoprotein receptor shedding in the presence of apoE4 remained increased. Likewise, intracranial administration of Aβ to apoE-targeted replacement mice (expressing the human apoE isoforms) resulted in an isoform-dependent effect on lipoprotein receptor shedding in the brain (apoE4 > apoE3 > apoE2). Moreover, these results show a strong inverse correlation with our prior work in apoE transgenic mice in which apoE4 animals showed reduced Aβ clearance across the BBB compared to apoE3 animals. Based on these results, apoE4 appears less efficient than other apoE isoforms in regulating lipoprotein receptor shedding, which may explain the differential effects of these isoforms in removing Aβ from the brain.

Fig. 1

Appearance of extracellular soluble (A) LRP1 or (B) LDLR in human brain endothelial cells (HBMEC) upon treatment with Aβ (1–42). HBMEC were exposed to various concentrations (0.1, 0.2, 1, 2, and 10 µM) of human Aβ(1–42) for 48 hours at 37°C. Following the treatment period, the extracellular media was collected and analyzed for LRP1 or LDLR content by ELISA. Values represent mean ± SEM (n = 3) and are expressed as ng of LRP1 or LDLR per ml of media. *P < 0.05 compared to control as determined by ANOVA and Bonferroni post-hoc test.

Fig. 1

Appearance of extracellular soluble (A) LRP1 or (B) LDLR in human brain endothelial cells (HBMEC) upon treatment with Aβ (1–42). HBMEC were exposed to various concentrations (0.1, 0.2, 1, 2, and 10 µM) of human Aβ(1–42) for 48 hours at 37°C. Following the treatment period, the extracellular media was collected and analyzed for LRP1 or LDLR content by ELISA. Values represent mean ± SEM (n = 3) and are expressed as ng of LRP1 or LDLR per ml of media. *P < 0.05 compared to control as determined by ANOVA and Bonferroni post-hoc test.

Fig. 2

Appearance of extracellular soluble (A) LRP1 or (B) LDLR in human brain endothelial cells (HBMEC) in the presence of Aβ(1–42), apoE isoforms, or combinations thereof. HBMEC were treated with human Aβ(1–42) (2µM) and/or each apoE isoform (25ng/ml) for 48 hours at 37°C. Following the treatment period, the extracellular media was collected and analyzed for LRP1 or LDLR content by ELISA. Values represent mean ± SEM (n = 3) and are expressed as ng of LRP1 or LDLR per ml of media. *P < 0.05 compared to control. *P < 0.05 comparing apoE4 in the presence of Aβ versus apoE2 or apoE3 in the presence of Aβ, as indicated on the graph. Statistics determined by ANOVA and Bonferroni post-hoc test.

Fig. 2

Appearance of extracellular soluble (A) LRP1 or (B) LDLR in human brain endothelial cells (HBMEC) in the presence of Aβ(1–42), apoE isoforms, or combinations thereof. HBMEC were treated with human Aβ(1–42) (2µM) and/or each apoE isoform (25ng/ml) for 48 hours at 37°C. Following the treatment period, the extracellular media was collected and analyzed for LRP1 or LDLR content by ELISA. Values represent mean ± SEM (n = 3) and are expressed as ng of LRP1 or LDLR per ml of media. *P < 0.05 compared to control. *P < 0.05 comparing apoE4 in the presence of Aβ versus apoE2 or apoE3 in the presence of Aβ, as indicated on the graph. Statistics determined by ANOVA and Bonferroni post-hoc test.

Fig. 3

Expression of (A) LRP1 and (B) LDLR in cerebrovasculature isolated from apoE transgenic mice. Human Aβ(1–42) or vehicle was intracranially administered to male mice (4–6 months old). Ten minutes after the intracerebral injection, the brains were collected and various brain fractions were isolated. LRP1 or LDLR levels in the cerebrovasculature were determined using an ELISA and normalized to total protein content using the BCA protein assay. Values represent mean ± SEM (n = 6 animals) and are expressed as ng of LRP1 or LDLR per mg protein. No comparisons reached statistical significance as determined by ANOVA and Bonferroni post-hoc test.

Fig. 3

Expression of (A) LRP1 and (B) LDLR in cerebrovasculature isolated from apoE transgenic mice. Human Aβ(1–42) or vehicle was intracranially administered to male mice (4–6 months old). Ten minutes after the intracerebral injection, the brains were collected and various brain fractions were isolated. LRP1 or LDLR levels in the cerebrovasculature were determined using an ELISA and normalized to total protein content using the BCA protein assay. Values represent mean ± SEM (n = 6 animals) and are expressed as ng of LRP1 or LDLR per mg protein. No comparisons reached statistical significance as determined by ANOVA and Bonferroni post-hoc test.

Fig. 4

Levels of (A) LRP1 and (B) LDLR in the soluble brain fraction of apoE transgenic mice. Human Aβ(1–42) or vehicle was intracranially administered to male mice (4–6 months old). Ten minutes after the intracerebral injection, the brains were collected and various brain fractions were isolated. LRP1 or LDLR levels in the soluble brain fraction were determined using an ELISA. Values represent mean ± SEM (n = 6 animals) and are expressed as ng of LRP1 or LDLR per ml of soluble brain material. *P < 0.05 comparing apoE4 to apoE2 or apoE3 for the respective vehicle and Aβ(1–42) groups as determined by ANOVA and Bonferroni post-hoc test.

Fig. 4

Levels of (A) LRP1 and (B) LDLR in the soluble brain fraction of apoE transgenic mice. Human Aβ(1–42) or vehicle was intracranially administered to male mice (4–6 months old). Ten minutes after the intracerebral injection, the brains were collected and various brain fractions were isolated. LRP1 or LDLR levels in the soluble brain fraction were determined using an ELISA. Values represent mean ± SEM (n = 6 animals) and are expressed as ng of LRP1 or LDLR per ml of soluble brain material. *P < 0.05 comparing apoE4 to apoE2 or apoE3 for the respective vehicle and Aβ(1–42) groups as determined by ANOVA and Bonferroni post-hoc test.

Fig. 5

Western blot analysis of brain fractions isolated from wild-type mice. Brain fractions from wild-type male mice (4–6 months old) were examined for the presence of LRP-85 (marker for the membrane-bound subunit of LRP1), laminin (brain blood vessel marker), synaptophysin (neuronal marker) and the housekeeping protein actin. Samples were collected from the brain fractions of three naïve (i.e., no intracerebral injection) wild-type mice and loaded into separate lanes of the gel.

Fig. 6

Correlation between brain lipoprotein receptor shedding and Aβ clearance across the BBB in apoE transgenic mice. LRP1 and LDLR levels in the soluble fraction of the brain were plotted versus the appearance of Aβ in the plasma (i.e., Aβ BBB clearance) following intracerebral Aβ administration (LRP1, R² = 0.94; LDLR, R² = 0.96). The Aβ BBB clearance data was derived from our previously published work (Bachmeier et al. 2013). Values represent the mean of 6 animals for each genotype. p < 0.05 for both LRP1 and LDLR as determined by Pearson correlation.