Nobiletin attenuates neurotoxic mitochondrial calcium overload through K+ influx and Delta psi(m) across mitochondrial inner membrane

Abstract

Mitochondrial calcium overload is a crucial event in determining the fate of neuronal cell survival and death, implicated in pathogenesis of neurodegenerative diseases. One of the driving forces of calcium influx into mitochondria is mitochondria membrane potential (Delta psi(m)). Therefore, pharmacological manipulation of Delta psi(m) can be a promising strategy to prevent neuronal cell death against brain insults. Based on these issues, we investigated here whether nobiletin, a Citrus polymethoxylated flavone, prevents neurotoxic neuronal calcium overload and cell death via regulating basal Delta psi(m) against neuronal insult in primary cortical neurons and pure brain mitochondria isolated from rat cortices. Results demonstrated that nobiletin treatment significantly increased cell viability against glutamate toxicity (100 mu M, 20 min) in primary cortical neurons. Real-time imaging-based fluorometry data reveal that nobiletin evokes partial mitochondrial depolarization in these neurons. Nobiletin markedly attenuated mitochondrial calcium overload and reactive oxygen species (ROS) generation in glutamate (100 mu M)-stimulated cortical neurons and isolated pure mitochondria exposed to high concentration of Ca2+ (5 mu M). Nobiletininduced partial mitochondrial depolarization in intact neurons was confirmed in isolated brain mitochondria using a fluorescence microplate reader. Nobiletin effects on basal Delta psi m were completely abolished in K+-free medium on pure isolated mitochondria. Taken together, results demonstrate that K+ influx into mitochondria is critically involved in partial mitochondrial depolarization-related neuroprotective effect of nobiletin. Nobiletin-induced mitochondrial K+ influx is probably mediated, at least in part, by activation of mitochondrial K+ channels. However, further detailed studies should be conducted to determine exact molecular targets of nobiletin in mitochondria.