Efeitos do tratamento com Lipoproteínas de Baixa Densidade (LDL) na expressão da ADAM10 em células neuronais e endoteliais: uma investigação molecular dos fatores de risco modificáveis para demência

Abstract

Alzheimer’s disease (AD) is a progressive and multifactorial neurodegenerative disease whose pathophysiology involves neuronal, vascular, and metabolic alterations. Among the modifiable risk factors associated with dementia, lipid metabolism, particularly elevated levels of low-density lipoprotein (LDL), has received increasing attention due to its potential contribution to vascular and blood-brain barrier (BBB) dysfunction. However, the mechanisms through which this metabolic factor may interfere with neurodegenerative processes remain poorly understood. In this context, ADAM10, the major constitutive α-secretase in the central nervous system, participates in the non-amyloidogenic processing pathway of amyloid precursor protein (APP), reducing the formation of amyloid-β (Aβ) peptide. Considering that cholesterol contributes to the organization of cell membranes and may modulate APP processing, alterations in cholesterol homeostasis may influence ADAM10 activity and, consequently, the non-amyloidogenic pathway. This study aimed to establish and characterize an in vitro BBB platform using a triculture system composed of hCMEC/D3 endothelial cells, astrocytes, and SH-SY5Y cells differentiated into neuron-like cells, in order to investigate the effects of LDL exposure on ADAM10 gene expression. SH-SY5Y cells were differentiated using retinoic acid (10 µM), and the tri-culture was established in a Transwell system and characterized by transendothelial electrical resistance (TEER) and immunofluorescence analysis of Claudin-5 and GLUT-1. To standardize the treatment conditions, cells were exposed to 25–200 µg/mL LDL, and metabolic activity (AlamarBlue®) and cell density (sulforhodamine B, SRB) were evaluated. The tri-culture was subsequently exposed to 50 and 100 µg/mL LDL, and ADAM10 expression was quantified by RT-qPCR in SH-SY5Y cells (6 and 24 h) and endothelial cells (24 h). The tri-culture exhibited significantly higher TEER values than the hCMEC/D3 monoculture, with a progressive increase over 24 h, and the expression of Claudin-5 and GLUT-1 was confirmed in endothelial cells. The LDL concentrations tested did not significantly alter metabolic activity or cell density, nor did they significantly affect ADAM10 expression in any of the cell types or experimental time points evaluated. Thus, the hypothesis that LDL exposure would reduce ADAM10 expression was not confirmed. Nevertheless, this study established an initial in vitro BBB platform integrating endothelial, neuronal, and astrocytic components, providing a foundation for future investigations into modifiable metabolic risk factors in AD and their effects on BBB integrity and the neurovascular unit.