Regulation of beta-amyloid production in neurons by astrocyte-derived cholesterol

Abstract

Alzheimer's disease (AD) is characterized by the presence of amyloid β (Aβ) plaques, tau tangles, inflammation, and loss of cognitive function. Genetic variation in a cholesterol transport protein, apolipoprotein E (apoE), is the most common genetic risk factor for sporadic AD. In vitro evidence suggests that apoE links to Aβ production through nanoscale lipid compartments (lipid clusters), but its regulation in vivo is unclear. Here, we use superresolution imaging in the mouse brain to show that apoE utilizes astrocyte-derived cholesterol to specifically traffic neuronal amyloid precursor protein (APP) in and out of lipid clusters, where it interacts with β- and γ-secretases to generate Aβ-peptide. We find that the targeted deletion of astrocyte cholesterol synthesis robustly reduces amyloid and tau burden in a mouse model of AD. Treatment with cholesterol-free apoE or knockdown of cholesterol synthesis in astrocytes decreases cholesterol levels in cultured neurons and causes APP to traffic out of lipid clusters, where it interacts with α-secretase and gives rise to soluble APP-α (sAPP-α), a neuronal protective product of APP. Changes in cellular cholesterol have no effect on α-, β-, and γ-secretase trafficking, suggesting that the ratio of Aβ to sAPP-α is regulated by the trafficking of the substrate, not the enzymes. We conclude that cholesterol is kept low in neurons, which inhibits Aβ accumulation and enables the astrocyte regulation of Aβ accumulation by cholesterol signaling.

Keywords: Alzheimer’s; apoE; cholesterol; lipids; neurodegeneration.

Fig. 1.

Astrocyte cholesterol signals to membrane nanostructure in neurons. (A) dSTORM superresolution imaging on WT brain slices showing lipid cluster nanostructure in cell membrane. (Scale Bar, 2 µm.) (B) Comparison of apparent cluster length in N2a cells; primary neurons cultured with astrocytes with and without (SREBP^−/−) cholesterol synthesis and brain slices. Data are expressed as mean ± SEM and n = 1,748 to 6,779 clusters from >3 images. (C) Mass spectrometry analysis showing that the majority of lipids extracted by apoE (a cholesterol transport protein) from N2a cells are cholesterol. Some cluster-associated lipids are also transported by apoE. Abbreviations for lipids: FC, free cholesterol; CE, cholesterol ester; SM, sphingomyelin; dhSM, dihydrosphingomyelin; and Pep, plasmalogen phosphatidylethanolamine. (D) Quantitation of cluster lengths in N2a cells indicates changes in cluster structure after apoE treatment with and without a source of cholesterol (± chol). Data are expressed as mean ± SEM and n = 1,911 to 6,779 clusters from four to seven cells. (E) Quantitation of GM1 clusters in N2a cells shows that apoE decreased the number of clusters under low-cholesterol conditions, while apoE treatment with high, environmental cholesterol doesn’t affect cluster number. Data are expressed as mean ± SEM and one-way ANOVA. (F) Exposure of N2a cells to apoE removes cholesterol from cellular membranes, as measured by a fluorescent-based live cell cholesterol assay. Cholesterol loading by apoE increases cellular cholesterol level. Data are expressed as mean ± SEM, n = 4 to 7, one-way ANOVA, *P < 0.05, ***P < 0.001, and ****P < 0.0001.

Fig. 2.

APP is regulated by cholesterol, not its hydrolytic enzymes. (A) Workflow for dSTORM superresolution. Cultured cells (blue) are depicted in a dish. Cells were exposed to apoE with and without cholesterol supplementation and fixed. GM1 lipids and amyloid proteins were fluorescently labeled (CTxB and antibody, respectively) and imaged with superresolution (dSTORM). The proximity of the two labels (shown as red and green circles) was then determined by cluster analysis. Idealized pair correlations are shown for objects that strongly colocalize (Top) and weakly colocalize (Bottom) at a given radius. Pair correlations are unitless with 1 being little to no correlation and >5 a value typically significant in our experimental conditions. (B) α-secretase sits in the disordered region (low correlation with GM1 lipid clusters), while APP, β-, and γ-secretases are GM1 associated in N2a cells. (C and D) Pair correlation analysis showing that APP moving in (C) and out (D) of the GM1 clusters under high- and low-cholesterol conditions, respectively. (E) Under low-cholesterol conditions (−Chol), APP colocalization with GM1 clusters decreases markedly after apoE treatment. Under high-cholesterol conditions (+Chol), apoE-mediated APP colocalization with GM1 clusters increases (i.e., apoE induces APP to cluster with GM1 lipids). α-, β-, and γ-secretase localization do not respond to apoE-mediated GM1 clustering. (F) ELISA in N2a cells showing a shift from Aβ to sAPP-α production after apoE treatment under low-cholesterol condition and increased Aβ production under high-cholesterol condition. Data are expressed as mean ± SEM, n = 3 to 10. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. (not significant), and two-sided Student’s t test. (G and H) Because of cluster disruption mediated by apoE, APP is dissociated from GM1 clusters under low-cholesterol conditions, exposing it to α-secretase to be cleaved into nonamyloidogenic sAPP-α. With high, environmental cholesterol, apoE-induced cluster stabilization mediates more APP to be translocated into lipid clusters and cleaved by β- and γ-secretase, producing amyloidogenic Aβ peptides.

Fig. 3.

Astrocyte cholesterol regulates APP processing in neurons. (A) Strategy for conditional knockout of SREBP2 in astrocytes using Cre-Lox recombination system. SREBP2 flox mice were crossed to ALDH1L1-specific Cre transgenic mice, which express Cre recombinase specifically in astrocytes when presented 4-hydroxytamoxifen (4-HT). Cre promotes SREBP2 knockout and blocks cholesterol synthesis in astrocytes. (B–D) Cortical cells were isolated from embryonic day 17 mice and cultured for dSTORM imaging. (B) ApoE translocates APP from lipid clusters into disordered regions in a pure, neuronal population sorted with cell surface neuronal marker Thy1.2. (C) ApoE increases APP’s cluster localization in primary neurons cultured in mixed population with glia cells, including astrocytes. (D) Astrocyte-specific cholesterol depletion completely disrupts APP cluster localization in neurons in cultured cells. (E) Astrocyte cholesterol depletion disrupts APP cluster localization in vivo, as demonstrated in SREBP2^fl/fl GFAP–Cre^+/− mouse brain slices. Data are expressed as mean ± SEM, n = 3 to 10, *P < 0.05, **P < 0.01, one-way ANOVA (D), or two-sided Student’s t test (B, C, and E).

Fig. 4.

Loss of cholesterol synthesis in astrocytes blocks Aβ plaque formation and Tau phosphorylation in vivo. 3xTg-AD mice (AD) were crossed to SREBP2^fl/fl GFAP–Cre^+/− mice (AD × SB2^−/−) and aged to 60 wk. (A) Hippocampus were isolated and soluble (RIPA extracted) and insoluble (guanidine extracted) Aβ40 and Aβ42 species were measured by ELISA. (B) 60-wk-old brain slices stained for human Aβ (red) and DAPI (blue) in AD × SB2flox mice (Left) and AD × SB2^−/− mice (Right). (C) pTau levels were measured in 60-wk-old hippocampus lysate by ELISA. (D) 60-wk-old brain slices were immune stained for pTau (green) and Aβ (red), and the subiculum region of the hippocampus was imaged. Data are expressed as mean ± SEM, n = 4 to 7, one-way ANOVA with Tukey’s post hoc analysis, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 5.

Astrocyte cholesterol regulates APP and Tau processing, not protein expression. Primary embryonic cultures of mixed neurons and astrocytes were grown by crossing 3xTg SREBP2^fl/fl mice with 3xTg SREBP2^fl/fl GFAP–Cre mice. Each culture was grown independently from a single embryo, and Cre genotype was confirmed by PCR. (A) Human sAPP-α levels (from transgenic APP) were measured in conditioned media from mixed cultures. (B) Protein in conditioned media was precipitated, and sAPP-β, an APP fragment produced by β-secretase cleavage, was measured by Western blot. (C) Mixed cultures were lysed, and protein content was examined by Western blot. Data are expressed as mean ± SEM and n = 4 per genotype. *P < 0.05 and ***P < 0.001.

Fig. 6.

Astrocytic apoE is required for cholesterol transportation from astrocytes to neurons. (A) Cartoon diagram showing the experimental setup for neuron culture with ACM. Neurons from SREBP2 GFAP–Cre mice were used to eliminate transport of cholesterol from contaminating astrocytes in the culture. Neurons were cultured with ACM from either WT or APOE^−/− astrocytes. (B) dSTORM superresolution imaging shows that astrocytic apoE is required for regulating APP’s GM1 clustering in neurons. Neurons cultured with ACM-containing apoE increased APP’s clustering with GM1 lipids, while ACM from APOE^−/− animals fail to increase APP’s cluster association. (C) APP in APOE4 brains (E4F) are more clustered with GM1 lipids, compared with E3F. Aβ overexpression (APP/PS1) also robustly increases APP’s raft association. APP/PS1/E4F has slightly higher APP-GM1 pair correlation than APP/PS1/E3F. (D) Aβ overexpression (APP/PS1) increases brain cholesterol level robustly. In both control and APP/PS1 animals, E4F has a slightly higher cholesterol level in brain tissue compared with E3F. Data are expressed as mean ± SEM, n = 3 to 10, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and one-way ANOVA. (E) N2a cells were treated with purified apoE3 or apoE4 proteins to compare their abilities to load and unload cholesterol (^± Chol) from the plasma membrane. (F) Under low-cholesterol condition, apoE extracts cholesterol from the cells, decreasing APP’s GM1 clustering. There is no significant difference between apoE3 and apoE4. (G) Under high-cholesterol condition, apoE loads cholesterol into the cells, increasing APP’s GM1 clustering. There is no significant difference between apoE3 and apoE4. (H) Exposure of N2a cells to apoE removes cholesterol from cellular membranes, as measured by a fluorescent-based live cell cholesterol assay. There is no significant difference between apoE3 and apoE4.

Fig. 7.

Astrocyte cholesterol-dependent regulation of amyloid processing in neurons through apoE. A model for amyloid production in the Alzheimer’s brain. Cholesterol is synthesized in astrocytes and shuttled to neurons in apoE lipoprotein particles. Adding recombinant, cholesterol-containing apoE enriches neuronal membrane cholesterol levels, while delipidated apoE reduces membrane cholesterol. Cholesterol loading of the neuronal membranes regulates Aβ production by increasing APP interactions with β- and γ-secretase. In low-cholesterol membranes, APP interacts with α-secretase, generating sAPPα. When neuronal membranes are loaded with cholesterol, APP increasingly interacts with β- and γ-secretases generating Aβ peptides, resulting in brain plaque formation over time.