Unravelling the Connection Between Hypercholesterolemia and Alzheimer’s Disease: Insights into Oxidative Stress and Signalling Pathways

Abstract

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the leading cause of dementia worldwide, characterised by amyloid beta (Aβ) plaques, neurofibrillary tau tangles and overall cognitive decline. Despite extensive research, the complex and multifactorial nature of AD has left its aetiology largely unknown, but emerging evidence identified hypercholesterolemia as a potential risk factor for AD. Hypercholesterolemia is characterised by elevated levels of low-density lipoprotein (LDL) in patients, commonly due to mutations in the LDL receptor (LDLR) gene and treated using statin therapy. Common cellular markers between these diseases, such as mitochondrial dysfunction, and the influence of cholesterol levels on Aβ pathogenesis, highlight a possible link between them. However, the exact cellular mechanisms constituting this link remain unclear, hence, this study aimed to assess the effects of hypercholesterolemia on Alzheimer’s disease hallmarks and signalling pathways. Proteomic analysis of SWATH-MS data from familial hypercholesterolemia (FH) patient plasma samples revealed 28 differentially expressed proteins (DEPs) involved in cytoskeletal dynamics and regulation, oxidative stress, antioxidant defence, inflammation, immune regulation, and metabolism, with most of these processes also being associated with AD pathogenesis. Furthermore, proteomic analysis of the plasma samples of FH patients on statin therapy indicated a reversible effect of statin therapy on 13 of the DEPs. Based on the proteomic analysis, an in vitro investigation was done whereby a squalene synthase inhibitor (SSI) was employed to upregulate LDLR expression and inhibit cholesterol biosynthesis in HEK293 and SH-SY5Y cell lines, which were treated with Aβ, LDL, and hydrogen peroxide (H2O2) to create an early-stage hypercholesterolemic-AD cell model. After validating SSI-induced LDLR upregulation via western blot analysis, the MTT and Alamar Blue® assays revealed that SSI had limited effects on rescuing metabolic activity in the cell model. In contrast, a fluorescence-based assay measuring reactive oxygen species (ROS) production indicated an antioxidant effect of SSI against the excess ROS produced by H2O2 treatment. Additionally, quantitative PCR analysis of mitochondrial DNA copy number (mtDNA-CN) found distinct effects of the hypercholesterolemic-AD conditions on mtDNA-CN levels where SSI treatment increased the levels across all conditions. Overall, the results of this study provided both in vitro and systemic insights into the complexity of cholesterol modulation and the potential molecular pathways that may link hypercholesterolemia and AD.