Nitroxidative stress in human neural progenitor cells: In situ measurement of nitric oxide/peroxynitrite imbalance using metalloporphyrin nanosensors

Abstract

Nitric oxide (NO) is an essential inorganic signaling molecule produced by constitutive NO synthase (cNOS) in the neurological system. Under pathological conditions, NO rapidly reacts with superoxide (O₂^•−) to generate peroxynitrite (ONOO^¯). Elevated ONOO^¯ concentrations induce nitroxidative stress, potentially contributing to numerous pathological processes as observed in neurodegenerative diseases including Alzheimer's disease (AD). Metalloporphyrin nanosensors, (200$–$300 nm diameter), were applied to quantify the NO/ONOO^¯ balance produced by a single human neural progenitor cell (hNPC), in situ. These nanosensors, positioned in proximity of 4$–$5 ± 1 μm from the hNPCs membrane, enabled real-time measurement of NO and ONOO^¯ concentrations following calcium ionophore (CaI) stimulation. The ratio of NO to ONOO^¯ concentration ([NO]/[ONOO^¯]) was established for the purpose of quantifying nitroxidative stress levels. Normal hNPCs produced a maximum of 107 ± 1 nmol/L of NO and 451 ± 7 nmol/L of ONOO^¯, yielding a [NO]/[ONOO^¯] ratio of 0.25 ± 0.005. In contrast, the model of the dysfunctional hNPCs, for long-term (48 h) amyloid-beta 42 (Aβ₄₂) exposure significantly altered NO/ONOO^¯ production. The NO level decreased to 14 ± 0.1 nmol/L, while ONOO^¯ increased to 843 ± 0.8 nmol/L, resulting in a 94 % reduction of the [NO]/[ONOO^¯] ratio to 0.016 ± 0.0001. The [NO]/[ONOO^¯] ratio is determined by this work as a possible biomarker of nNOS efficiency and hNPC dysfunction, with implications for neurodegenerative disorders such as AD. Promising applications in the early medical diagnosis of neurological illnesses, electrochemical metalloporphyrin nanosensors demonstrate efficacy in real-time nitroxidative stress monitoring.