Influência do tipo de lipídeo e peptídeo beta amiloide sobre a perturbação da membrana e consequências na toxicidade de células neuronais observadas pelas respostas luminescentes do complexo RuApy

Abstract

One of the hypotheses for the cause of Alzheimer's disease is the accumulation of amyloid-beta peptide (Aβ) aggregates in the brain that lead to severe synaptic and cognitive dysfunctions resulting in neurodegenerative dementia. Some studies indicate that the formation of amyloid plaques and Aβ peptide toxicity is associated with its interaction with the neuronal cell membrane. This interaction accelerates the aggregation process and generates pores in the cell membrane with the formation of oligomeric Aβ species. Studies in our laboratory have shown that the luminescent complex cis-[Ru(phen)2(3,4Apy)2]2+ (RuApy, phen = 1,10-phenanthroline, 3,4Apy = 3,4-diaminopyridine) protects cells PC12 against the toxicity of pore-like oligomeric species of Aβ1-40, changing the aggregation mechanism to a non-toxic pathway, leading to the formation of micelle-like structures. These results motivated the study of the influence of RuApy on the process of aggregation and toxicity of Aβ1-42 and to verify the possibility of involvement of RuApy in the mechanism of action of Aβ1-42 against the cell membrane. The studies carried out involved the spectroscopic techniques of absorption, steady-state emission and time-resolved in the UV-visible region, fluorescent image microscopy, circular dichroism and reflection-absorption spectroscopy in the infrared region with polarization modulation (PM-IRRAS). In this project, it was demonstrated that the RuApy complex has a protective effect for the human neuroblastoma cell line SH-SY5Y against toxic Aβ1-42 species, corroborating previous results that showed that RuApy alters the Aβ aggregation process leading to the formation of non-toxic fibrils. For the interaction studies between RuApy and the cell membrane, models of neutral lipid membranes, negatively charged, with cholesterol, marked with NBD and Texas Red organic dyes and in different media were used in order to elucidate how the complex interacts with the models of membrane. The interaction was evaluated by binding assays using fluorescence, supported lipid bilayers (SLB) and microfluidic devices. Using the Forster resonant energy transfer mechanism (FRET), the dissociation constant (Kd) of the interaction between RuApy and the lipid bilayer was determined and, according to the change in the constant, the influence of factors such as charge, fluidity of the membrane and hydrogen bonds in the interaction. The obtained results indicated that RuApy interacts with membrane models mainly through electrostatic interactions and hydrogen bonds. Molecular-level information on RuApy interactions with the lipid membrane was obtained in studies with Langmuir monolayers by superficial pressure versus area Isotherms and PM-IRRAS experiments and are consistent with the data obtained with SLBs in microfluidic devices. Altogether, the results indicate that the interaction of RuApy with the tested membrane models involves electrostatic and hydrogen bonding interactions, and that this interaction depends on the lipid composition. Also, that RuApy alters the aggregation and has an inhibitory effect on the cytotoxicity of Aβ1-42 against the SH-SY5Y cell line. Such conclusions suggest that the RuApy complex may play an important role in the interactions between the Aβ peptide and the cell membrane.