Successful development of effective therapeutics for Alzheimer’s disease (AD) requires a deep understanding of the mechanisms of disease pathogenesis. Recent human induced pluripotent stem cell (hiPSC)-derived models have shown it is possible to recapitulate the complex genetic diversity of a patient and more completely model AD pathology. While most work has focused on neuronal AD phenotypes, there is mounting evidence that failure to regulate cholesterol homeostasis by neighboring nonneuronal support cells may be involved in AD pathogenesis. Amyloid precursor protein (APP) or multiple fragments generated by APP proteolytic processing 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 the brain’s de novo cholesterol biosynthesis and regulation, remains unclear. To address this, we independently validated and characterized a novel in vitro differentiation protocol to generate human astrocytes from hiPSC. We found that cells derived by this method recapitulated native astrocytes in their form and function. We also utilized CRISPR/Cas9 genome editing to generate isogenic APP knockout (KO) hiPSCs. We found that APP KO astrocytes have reduced cholesterol, elevated levels of sterol regulatory element-binding protein (SREBP)-target gene transcripts, and increased low-density lipoprotein (LDL) receptor protein, all downstream consequences of reduced lipoprotein endocytosis. To elucidate which APP fragments regulate cholesterol homeostasis and examine whether familial AD mutations in APP affect lipoprotein metabolism, we analyzed an isogenic allelic series using the APP Swedish and APP V717F mutations. Only astrocytes homozygous for the APP Swedish (APPSwe/Swe) mutation, which had reduced full-length APP (FL APP) due to aggressive beta-secretase cleavage, recapitulated the APP KO phenotypes. Further, astrocytic internalization of amyloid-beta (A-beta), another ligand of LDL receptors, was impaired in APP KO and APPSwe/Swe astrocytes. Given that FL APP is known to bind to multiple LDL receptors, we propose that FL APP acts as a co-receptor for known LDL receptor ligands and is required for proper cholesterol homeostasis and A-beta clearance in human astrocytes.

Overexpression and/or abnormal cleavage of amyloid precursor protein (APP) are linked to Alzheimer's disease (AD) development and progression. However, the molecular mechanisms regulating cellular levels of APP or its processing, and the physiological and pathological consequences of altered processing are not well understood. Here, using mouse and human cells, we found that neuronal damage induced by UV irradiation leads to specific APP, APLP1, and APLP2 decline by accelerating their secretase-dependent processing. Pharmacological inhibition of endosomal/lysosomal activity partially protects UV-induced APP processing implying contribution of the endosomal and/or lysosomal compartments in this process. We found that a biological consequence of UV-induced γ-secretase processing of APP is impairment of APP axonal transport. To probe the functional consequences of impaired APP axonal transport, we isolated and analyzed presumptive APP-containing axonal transport vesicles from mouse cortical synaptosomes using electron microscopy, biochemical, and mass spectrometry analyses. We identified a population of morphologically heterogeneous organelles that contains APP, the secretase machinery, molecular motors, and previously proposed and new residents of APP vesicles. These possible cargoes are enriched in proteins whose dysfunction could contribute to neuronal malfunction and diseases of the nervous system including AD. Together, these results suggest that damage-induced APP processing might impair APP axonal transport, which could result in failure of synaptic maintenance and neuronal dysfunction.