There is growing recognition regarding the role of intracellular amyloid beta (Aβ) in the Alzheimer's disease process, which has been linked with aberrant signaling and the disruption of protein degradation mechanisms. Most notably, intraneuronal Aβ likely underlies the oxidative stress and mitochondrial dysfunction that have been identified as key elements of disease progression. In this study, we employed fluorescence imaging to explore the ability of a bifunctional small molecule to reduce aggregates of intracellular Aβ and attenuate oxidative stress. Structurally, this small molecule is comprised of a nitroxide spin label linked to an amyloidophilic fluorene and is known as spin-labeled fluorene (SLF). The effect of the SLF on intracellular Aβ accumulation and oxidative stress was measured in MC65 cells, a human neuronal cell line with inducible expression of the amyloid precursor protein and in the N2a neuronal cell line treated with exogenous Aβ. Super-resolution microscopy imaging showed SLF decreases the accumulation of intracellular Aβ. Confocal microscopy imaging of MC65 cells treated with a reactive oxygen species (ROS)-sensitive dye demonstrated SLF significantly reduces the intracellular Aβ-induced ROS signal. In order to determine the contributions of the separate SLF moieties to these protective activities, experiments were also carried out on cells with nitroxides lacking the Aβ targeting domain or fluorene derivatives lacking the nitroxide functionality. The findings support a synergistic effect of SLF in counteracting both the conformational toxicity of both endogenous and exogenous Aβ, its promotion of ROS, and Aβ metabolism. Furthermore, these studies demonstrate an intimate link between ROS production and Aβ oligomer formation.
Keywords: Alzheimer’s disease; Aβ oligomer; amyloid beta; bifunctional drug; intracellular Aβ; intraneuronal Aβ; nitroxide antioxidant; oxidative stress; spin label.