Atomic force microscopy reveals new biophysical markers for monitoring subcellular changes in oxidative injury: Neuroprotective effects of quercetin at the nanoscale

Oxidative stress has been recognised as an important pathological mechanism underlying the development of neurodegenerative diseases. The biomarkers for assessing the degree of oxidative stress have been attracting much interest because of their potential clinical relevance in understanding the cellular effects of free radicals and evaluation of the efficacy of drug treatment. Here, an interdisciplinary approach using atomic force microscopy (AFM) and cellular and biological molecular methods were used to investigate oxidative damage in P19 neurons and to reveal the underlying mechanism of protective action of quercetin. Biological methods demonstrated the oxidative damage of P19 neurons and showed that quercetin improved neuronal survival by preventing H2O2- induced p53 and Bcl-2 down-regulation and modulated Akt and ERK1/2 signalling pathways. For the first time, AFM was employed to evaluate morphologically (roughness, height, Feret dimension) and nanomechanical (elasticity) properties in H2O2-induced neuronal damage. The AFM analysis revealed that quercetin suppressed H2O2-provoked changes in cell membrane elasticity and morphological properties, thus confirming its neuroprotective activity. The obtained results indicate the potential of AFM-measured parameters as a biophysical markers of oxidative stress-induced neurodegeneration. In general, our study suggests that AFM can be used as a highly valuable tool in other biomedical applications aimed at screening and monitoring of drug- induced effects at cellular level

The interactions of p53 with tau and Aß as potential therapeutic targets for Alzheimer’s disease

Alzheimer's disease (AD), the most common progressive neurodegenerative disorder, is characterized by severe cognitive decline and personality changes as a result of synaptic and neuronal loss. The defining clinicopathological hallmarks of the disease are deposits of amyloid precursor protein (APP)-derived amyloid-β peptides (Aβ) in the brain parenchyma, and intracellular aggregates of truncated and hyperphosphorylated tau protein in neurofibrillary tangles (NFT). At the cellular and molecular levels, many intertwined pathological mechanisms that relate Aβ and tau pathology with a transcription factor p53 have been revealed. p53 is activated in response to various stressors that threaten genomic stability. Depending on damage severity, it promotes neuronal death or survival, predominantly via transcription-dependent mechanisms that affect expression of apoptosis-related target genes. Levels of p53 are enhanced in the AD brain and maintain sustained tau hyperphosphorylation, whereas intracellular Aβ directly contributes to p53 pool and promotes downstream p53 effects. The review summarizes the role of p53 in neuronal function, discusses the interactions of p53, tau, and Aβ in the normal brain and during the progression of AD pathology, and considers the impact of the most prominent hereditary risk factors of AD on p53/tau/Aβ interactions. A better understanding of this intricate interplay would provide deeper insight into AD pathology and might offer some novel therapeutic targets for the improvement of treatment options. In this regard, drugs and natural compounds targeting the p53 pathway are of growing interest in neuroprotection as they may represent promising therapeutic approaches in the prevention of oxidative stress-dependent pathological processes underlying AD

PI3K/Akt and ERK1/2 Signalling Are Involved in Quercetin-Mediated Neuroprotection against Copper-Induced Injury

Copper, a transition metal with essential cellular functions, exerts neurotoxic effects when present in excess by promoting production of reactive oxygen species (ROS). The aim of the present study was to investigate potential benefits of flavonoid quercetin against copper-induced toxicity. Results obtained with MTT assay indicate that the effects of quercetin are determined by the severity of the toxic insult. In moderately injured P19 neuronal cells, concomitant treatment with 150 μM quercetin improved viability by preventing ROS formation, caspase-3 activation, and chromatin condensation. Western blot analysis revealed that quercetin reduced copper-induced increase in p53 upregulated modulator of apoptosis (PUMA) expression and promoted upregulation of nucleoside diphosphate kinase NME1. Levels of p53 and Bax proteins were not affected by both copper and quercetin. UO126 and wortmannin, inhibitors of ERK1/2 and PI3K/Akt signalling pathways, respectively, prevented neuroprotective effects of quercetin. In severely injured neurons, 30 μM quercetin exerted strong prooxidative action and exacerbated cytotoxic effects of copper, whereas 150 μM quercetin failed to affect neuronal survival. These results demonstrate the dual nature of quercetin action in copper-related neurodegeneration. Hence, they are relevant in the context of considering quercetin as a possible therapeutic for neuroprotection and imply that detailed pharmacological and toxicological studies must be carried out for natural compounds capable of acting both as antioxidants and prooxidants