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Regulation of Amyloid Processing in Neurons by Astrocyte-Derived Cholesterol

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Regulation of Amyloid Processing in Neurons by Astrocyte-Derived Cholesterol bioRxiv preprint doi: https://doi.org/10.1101/2020.06.18.159632; this version posted June 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Regulation of amyloid processing in neurons by astrocyte-derived cholesterol Hao Wang1,2,3,6, Joshua A. Kulas4,5,6, Heather A. Ferris4,5*, Scott B. Hansen1,2* 1Department of Molecular Medicine, 2Department of Neuroscience, 3Skaggs Graduate School of Chemical and Biological Sciences, The Scripps Research Institute, Jupiter, FL 33458, USA 4Division of Endocrinology and Metabolism, 5Department of Neuroscience, University of Virginia, Charlottesville, VA 22908, USA; 6these authors contributed eQually *Correspondence: [email protected], [email protected] ABSTRACT 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 cholesterol- dependent 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 α- secretase 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. soluble APP fragment (sAPPα) which, in healthy INTRODUCTION individuals, helps set neuronal excitability5. α- Alzheimer’s disease (AD), the most prevalent secretase-mediated APP cleavage and prevents neurodegenerative disorder, is characterized by Aβ production, and thus is protective and progressive loss of cognitive function and the considered the non-amyloidogenic pathway, accumulation of amyloid β (Aβ) peptide1. The loss whereas sequential β- and g-secretase-mediated of cognitive function is thought to involve APP cleavage is called the amyloidogenic aggregates of Aβ peptide, a small hydrophobic pathway6. Both pathways are finely regulated by protein excised from the transmembrane domain of cholesterol7(Figure S1b). amyloid precursor protein (APP) by proteases In cellular membranes, cholesterol regulates the known as gamma- (g-) and beta- (b-) secretases formation of lipid domains (also Known as lipid (Figure S1a). In high concentration Aβ peptide is rafts) and the affinity of proteins to lipid rafts8 toxic and aggregates of Aβ form plaques. In 9–11 healthy individuals, Aβ peptide cycles daily with including b-secretase and g-secretase . α- sleep2,3 and appears to have antimicrobial activity4. Secretase does not reside in lipid rafts, rather α- A third enzyme, alpha- (α-) secretase generates a secretase is thought to reside in a region made up of disordered polyunsaturated lipids, separated 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.18.159632; this version posted June 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. from rafts12. The location of APP is less clear, in monosialotetrahexosylganglioside1 (GM1) lipids in detergent resistant membranes (DRMs) studies it mouse brain slices. GM1 lipids reside in cholesterol primarily associates with lipid from the disordered dependent lipid rafts and bind cholera toxin B region, although not exclusively9,11,13,14. (CTxB) with high affinity29,30. These lipid rafts or Endocytosis is thought to bring APP in proximity to GM1 domains are separate from b-secretase and g-secretase and this correlates phosphatidylinositol 4,5 bisphosphate (PIP2) with Ab production. Cross linking of APP with b- domains, which are polyunsaturated and secretase on the plasma membrane also increases cholesterol independent17,31,32. We labeled GM1 Ab production, leading to a hypothesis that raft domains (i.e. lipid rafts) from cortical slices with localization contributes to APP processing12,15 Alexa Fluor 647 conjugated fluorescent CTxB, and (Figure S1a). Testing this hypothesis in vivo has imaged with confocal and super-resolution direct been hampered by the small size of lipid rafts stochastical optical reconstruction microscopy 33–36 (often <100 nm), which is below the resolution of (dSTORM) . dSTORM is capable of visualizing light microscopy. nanoscale arrangements (i.e. sub-100 nm diameter lipid domain structures37) in intact cellular Recently super resolution imaging has emerged as membranes. a complimentary technique to DRM’s, with the potential to interrogate raft affinity more directly in CTxB appeared to label most cell types in cortical a native cellular environment16. We recently brain slices (Figure 1a, green shading). By employed super resolution imaging to directly zooming in on neurons, labeled with antibody demonstrate a process called substrate against neurofilament medium chain (NFM), a presentation17. In substrate presentation, neuronal specific protein, CTxB can be seen increases of cholesterol causes lipid rafts to outlining the plasma membrane (outside of the cell) sequester the enzyme away from its substrate, as opposed to NFM which labels throughout the removal of cholesterol then releases and activates cells (Figure 1a right panels). the enzyme by giving it access to its substrate We used dSTORM (an ~10-fold increase in 8,17 (Figure S1c) . A similar mechanism has been resolution compared to confocal) to characterize proposed to regulate the exposure of APP to its the relative size and number of GM1-domains in 12,15,18–20 cutting enzymes . mammalian brain tissue (Figure 1c). Cortical tissue Neurons are believed to be the major source of Aβ from mouse averaged ~141 nm in apparent in normal and AD brains21,22. In the adult brain, the diameter, a diameter slightly smaller than the ability of neurons to produce cholesterol is apparent size in primary neurons (~150 nm impaired during development23. Instead, diameter) (Figure 1c). N2a cells exhibited the astrocytes make cholesterol and transport it to smallest rafts by far, on average only 100 nm in neurons with apolipoprotein E (apoE)24–26. apparent diameter. All of the mammalian cells had Interestingly, apoE, specifically the e4 subtype domains larger than what we saw in intact fly brain 38 (apoE4), is the strongest genetic risK factor which had an apparent diameter of ~90 nm . Lipid associated with sporadic AD27,28. This led to the rafts were also measured in neurons with the theory that apoE may be controlling Aβ formation SREBP2 gene ablated in cocultured astrocytes. through regulation of lipid raft function12,15,18. Here SREBP2 is an essential regulator of cholesterol we show that astrocyte-derived cholesterol synthesis enzymes39 and was specifically knocked controls Aβ formation in vivo and links out in astrocytes using an ALDH1L1 promoter inflammatory cytoKines, apoE, Aβ, and plaque driven Cre recombinase40. The raft size of primary formation to a single molecular pathway. neurons cultured with SREBP2-/- astrocytes was significantly reduced (130 nm), suggesting that astrocyte derived cholesterol contributes to RESULTS cholesterol content of neuronal membranes. Characterization of lipid raft prevalence in Since GM1 domains are cholesterol dependent, to brain tissue. confirm that apoE is regulating GM1 domain To establish a role for Aβ regulation by lipid rafts in formation in vivo, we added apoE to primary vivo, we first labeled and imaged 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.18.159632; this version posted June 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Figure 1. ApoE inserts and sequesters cholesterol to modulate lipid raft characteristics.
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