Abstract
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.
Highlights
Oxidative stress represents a common mechanism of neuronal death in a variety of neuropathologies, including neurodegenerative diseases such as Alzheimer’s disease and Parkinson disease [1]
Inc. (Milwaukee WI, USA), wortmannin was purchased from Ascent Scientific (Princeton, NJ, USA) and hydrogen peroxide solutions were obtained from Kemika (Zagreb, Croatia) and Sigma-Aldrich Chemicals
In a concentration-dependent manner, exposure to H2O2 decreased the viability of P19 neurons (Fig 1A; F(5,8) = 169.8, P < 0.0001), whereas they tolerated relatively high concentrations of quercetin without a decrease in survival (Fig 1B)
Summary
Oxidative stress represents a common mechanism of neuronal death in a variety of neuropathologies, including neurodegenerative diseases such as Alzheimer’s disease and Parkinson disease [1]. At physiological levels ROS act as signalling molecules, but when present in excess they may induce an oxidative stress response and trigger cell death by modulating redox-sensitive signalling pathways and gene expression. At concentrations above the physiological threshold, H2O2 can change activities of different signalling cascades, such as pathways of mitogen-activated protein kinases (MAPK) and protein kinase B (PKB/Akt) This triggers specific nuclear or cytoplasmic response, often ending in cell death [5,6]. The aim of the present study was to combine capabilities of AFM with molecular biology tools to better understand cellular and molecular consequences of H2O2-induced oxidative injury in P19 neurons and to investigate molecular mechanisms of quercetin-mediated neuroprotection that falls beyond its antioxidant activity. We used AFM to screen subtle changes of neuronal membrane topography and nanomechanical properties induced by drug treatment during the oxidative insult
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