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.

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Background Purified microglia cultures are useful tools to study microglial behavior in vitro. Microglial cell lines serve as an attractive alternative to primary microglia culture, circumventing the costly and lengthy preparation of the latter. However, immortalization by genetic or pharmacologic manipulations may show altered physiology from primary microglia. New Method A novel microglial cell line was isolated from a primary glial culture of postnatal murine cerebral cortices. The culture contained a population of spontaneously transformed microglia that continued to divide without genetic or pharmacological manipulations. After several clones were isolated, one particular clone, SIM-A9, was analyzed for its microglial characteristics. Results SIM-A9 cells expressed macrophage/microglia-specific proteins, CD68 and Iba1. SIM-A9 cells were responsive to exogenous inflammatory stimulation with lipopolysaccharide and β-amyloid, triggering tyrosine kinase-based and NFκB signaling cascades as well as TNFα secretion. SIM-A9 cells also exhibited phagocytic uptake of fluorescent labeled β-amyloid and bacterial bioparticles. Furthermore, lipopolysaccharide increased the levels of inducible nitric oxide synthase and cyclooxygenase-2, whereas IL-4 stimulation increased arginase-1 levels demonstrating that SIM-A9 cells are capable of switching their profiles to pro- or anti-inflammatory phenotypes, respectively. Comparison with Existing Methods The use of SIM-A9 cells avoids expensive and lengthy procedures required for the preparation of primary microglia. Spontaneously immortalized SIM-A9 cells are expected to behave more comparably to primary microglia than virally transformed or pharmacologically induced microglial cell lines. Conclusions SIM-A9 cells exhibit key characteristics of cultured primary microglia and may serve as a valuable model system for the investigation of microglial behavior in vitro.

Alzheimer's Disease (AD), a common and burdensome neurodegenerative disorder, is characterized by the presence of β-Amyloid (Aβ) plaques, inflammation, and loss of cognitive function. A cholesteroldependent process sorts Aβ-producing enzymes into nanoscale lipid compartments (also called lipid rafts). Genetic variation in a cholesterol transport protein, apolipoprotein E (apoE), is the most common genetic marker for sporadic AD. Evidence suggests apoE links to Aβ production through lipid rafts, but so far there has been little scientific validation of this link in vivo. Here we use super-resolution imaging to show apoE utilizes astrocyte-derived cholesterol to specifically traffic amyloid precursor protein (APP) into lipid rafts where it interacts with β-and g-secretases to generate Aβ-peptide. We find that targeted deletion of astrocyte cholesterol synthesis robustly reduces amyloid 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 rafts where it interacts with αsecretase and gives rise to soluble APPα (sAPPα), a neuronal protective product of APP. Changes in cellular cholesterol have no effect on α-, β-, and g-secretase trafficking, suggesting the ratio of Aβ to sAPPα is regulated by the trafficking of the substrate, not the enzymes. Treatment of astrocytes with inflammatory cytokines IL-1β, IL-6 and TNF-α upregulates the synthesis of cholesterol in the astrocytes. We conclude that cholesterol is a signaling molecule kept low in neurons to inhibit raft function, decrease Aβ formation, and enable astrocyte regulation of APP by cholesterol control of substrate presentation. Highlights:ApoE regulates amyloid precursor protein localization to rafts and its exposure to α-vs. β-secretase. α-, β-, and g-Secretases are activated by substrate presentation. ApoE specifically transports astrocyte cholesterol to neurons. Astrocyte cholesterol synthesis disruption prevents Alzheimer's-associated amyloid pathology in mice.

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