Dottorato in BIOLOGIA UMANA E GENETICA MEDICA

Titolo della tesi: Epigenetic Modulation of Amyloid β Clearance: Insights from LRP1 and PICALM

Late-Onset Alzheimer Disease (LOAD) is a multifactorial disease where genetic susceptibility combines with nutritional, environmental, and lifestyle factors to increase disease risk. Epigenetic mechanisms are proposed as potential links between this complex genetic background and the modifiable risk factors associated with LOAD. One such factor, homocysteine (Hcy), a metabolite in One-Carbon Metabolism, is associated with AD through its role in hyperhomocysteinemia (HHcy), a significant and modifiable risk factor for AD. Additionally, SAM, another metabolite of the One-Carbon Metabolism and the primary methyl donor in transmethylation reactions, is decreased in AD patients. One-Carbon Metabolism is closely connected with multiple epigenetic processes, including DNA methylation, histone modifications, and microRNA regulation. This metabolic pathway has been extensively demonstrated to influence the production of amyloid β (Aβ) peptides, whose accumulation is a hallmark of AD. Although various mechanisms have been proposed to explain the detrimental effects of HHcy on the central nervous system, including the upregulation of the amyloidogenic pathway, the interaction between HHcy and the clearance of Aβ has not been thoroughly investigated. LRP1, PICALM, and P-gp are key proteins that act coordinated in the uptake and clearance of Aβ in astrocytes and in the blood-brain-barrier (BBB). This study employed nutritional protocols involving B-vitamin (B6, B9, and B12) deficiency and SAM supplementation to explore how One-Carbon Metabolism modulates key proteins in the LRP1-PICALM-P-gp axis, investigating these effects across various cell types and an in vitro BBB model. The results underscore the essential role of One-Carbon Metabolism in regulating BBB function and Aβ clearance through epigenetic mechanisms, specifically showing that PICALM expression is influenced by promoter DNA methylation and LRP1 expression is modulated through both promoter methylation and miRNA regulation. The findings also reveal that One-Carbon Metabolism affects transcription factors EGR1 and SP1 in human astrocytoma U87-MG cells, which may influence PICALM and LRP1 expression. Additionally, this study demonstrates that HHcy induces the downregulation of tight junction proteins, Claudin-5 and ZO-1, thereby increasing paracellular diffusion and compromising BBB integrity. In the BBB model, One-Carbon Metabolism further regulates LRP1, PICALM, and P-gp expression. Importantly, SAM supplementation enhances Aβ42 clearance in a co-culture model of human neuroblastoma SK-N-BE cells and murine brain endothelial bEnd.3 cells, emphasizing its potential role in Aβ clearance and AD pathology. In conclusion, this study sheds light on epigenetic mechanisms that regulate key proteins involved in Aβ clearance and BBB integrity, highlighting the therapeutic potential of One-Carbon Metabolism modulators like SAM for AD. Further studies are needed to assess the implications of these findings in neurodegenerative diseases and explore potential therapeutic applications.