Abstract
Mounting evidence suggests that alterations in cholesterol homeostasis are involved in Alzheimer's disease (AD) pathogenesis. Amyloid precursor protein (APP) or multiple fragments generated by proteolytic processing of APP have previously been implicated in the regulation of cholesterol metabolism. However, the physiological function of APP in regulating lipoprotein homeostasis in astrocytes, which are responsible for de novo cholesterol biosynthesis and regulation in the brain, remains unclear. To address this, here we used CRISPR/Cas9 genome editing to generate isogenic APP-knockout (KO) human induced pluripotent stem cells (hiPSCs) and differentiated them into human astrocytes. We found that APP-KO astrocytes have reduced cholesterol and elevated levels of sterol regulatory element-binding protein (SREBP) target gene transcripts and proteins, which were both downstream consequences of reduced lipoprotein endocytosis. To elucidate which APP fragments regulate cholesterol homeostasis and to examine whether familial AD mutations in APP affect lipoprotein metabolism, we analyzed an isogenic allelic series harboring the APP Swedish and APP V717F variants. Only astrocytes homozygous for the APP Swedish (APPSwe/Swe) mutation, which had reduced full-length APP (FL APP) due to increased β-secretase cleavage, recapitulated the APP-KO phenotypes. Astrocytic internalization of β-amyloid (Aβ), another ligand for low-density lipoprotein (LDL) receptors, was also impaired in APP-KO and APPSwe/Swe astrocytes. Finally, impairing cleavage of FL APP through β-secretase inhibition in APPSwe/Swe astrocytes reversed the LDL and Aβ endocytosis defects. In conclusion, FL APP is involved in the endocytosis of LDL receptor ligands and is required for proper cholesterol homeostasis and Aβ clearance in human astrocytes.
Highlights
Mounting evidence suggests that alterations in cholesterol homeostasis are involved in Alzheimer’s disease (AD) pathogenesis
Regulation of LDL receptor mRNA and protein is relevant to mechanisms of A clearance in the brain because LDL receptors like low-density lipoprotein receptor (LDLR) and lipoprotein receptor-related protein 1 (LRP1) mediate the internalization of A by binding to A directly or via apoE [17,18,19,20,21]
To determine whether reduced lipoprotein endocytosis in familial Alzheimer’s disease (FAD) astrocytes with reduced FL amyloid precursor protein (APP) levels led to downstream alterations in cholesterol metabolism, we looked at the expression of multiple proteins involved in lipoprotein regulation (Fig. 6C)
Summary
Mounting evidence suggests that alterations in cholesterol homeostasis are involved in Alzheimer’s disease (AD) pathogenesis. Amyloid precursor protein (APP) or multiple fragments generated by proteolytic processing of APP have previously been implicated in the regulation of cholesterol metabolism. To elucidate which APP fragments regulate cholesterol homeostasis and to examine whether familial AD mutations in APP affect lipoprotein metabolism, we analyzed an isogenic allelic series harboring the APP Swedish and APP V717F variants. Astrocytic internalization of -amyloid (A), another ligand for low-density lipoprotein (LDL) receptors, was impaired in APP-KO and APPSwe/Swe astrocytes. FL APP is involved in the endocytosis of LDL receptor ligands and is required for proper cholesterol homeostasis and A clearance in human astrocytes. APP or various proteolytic fragments of APP have recently been implicated in the control of brain cholesterol metabolism via regulation of LDL receptor-family mRNA and protein [11,12,13,14,15]. Regulation of LDL receptor mRNA and protein is relevant to mechanisms of A clearance in the brain because LDL receptors like low-density lipoprotein receptor (LDLR) and lipoprotein receptor-related protein 1 (LRP1) mediate the internalization of A by binding to A directly or via apoE [17,18,19,20,21]
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