Nobiletin attenuates neurotoxic mitochondrial calcium overload through K+ influx and ΔΨm 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 (ΔΨ_m). Therefore, pharmacological manipulation of ΔΨ_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 ΔΨ_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 µM, 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 µM)-stimulated cortical neurons and isolated pure mitochondria exposed to high concentration of Ca²⁺ (5 µM). Nobiletin-induced partial mitochondrial depolarization in intact neurons was confirmed in isolated brain mitochondria using a fluorescence microplate reader. Nobiletin effects on basal ΔΨ_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.

Keywords: Calcium; Mitochondrial K+ channels; Mitochondrial calcium; Mitochondrial membrane potential; Nobiletin.

Fig. 1. Chemical skeletal structure of nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone).

Fig. 2. The effects of nobiletin on basal ΔΨ_m and cell viability against glutamate toxicity in primary cortical neurons.

(A, D) Recording traces of ΔΨ_m using real-time imaging-based fluorometry with TMRE (see ‘METHODS’ for the detailed description). Various concentrations of nobiletin and CPE were superfused over primary cortical neurons on a cover slip in a recording chamber from the arrow point. TMRE fluorescence values from individual cells were normalized to values before drug treatment shown as an arrow. (B, E) Quantification of ΔΨ_m at the end of experiment for panel A and D. (C, F) Effects of nobiletin and CPE on cell viability against glutamate toxicity (100 µM, 20 min) were investigated using MTT assay. Values are the mean±S.E.M. ^*p<0.05, ^**p<0.01, ^***p<0.001 as compared with the control group and ^#p<0.05 as compared with glutamate alone-treated group.

Fig. 3. The effects of nobiletin on glutamate-induced overload of cytosol and mitochondrial calcium in primary cortical neurons.

(A, C) Dual real-time imaging-based fluorometry of [Ca²⁺]c and [Ca²⁺]m were simultaneously conducted in the same neurons (see ‘METHODS’ for the detailed description). Fura-2 and Rohd-2 fluorescence values from individual cells were normalized to values before drug treatment. (B, D) Quantification of Fura-2 and Rohd-2 fluorescence values at the end of experiment for panel A and C. Values are the mean±S.E.M. ^*p<0.05, ^**p<0.01 as compared with the control group and ^#p<0.05 as compared with glutamate alone-treated group. N.S., not statistically significant.

Fig. 4. The effects of nobiletin on mitochondrial ROS generation in glutamate-stimulated cortical neurons and isolated brain mitochondria exposed to high concentration of Ca²⁺.

(A) Recording traces of mitochondrial superoxide using real-time imaging-based fluorometry with MitoSOX Red (see ‘METHODS’ for the detailed description). MitoSOX Red fluorescence values from individual cells were normalized to values before drug treatment shown as an arrow. (B) Quantification of MitoSOX Red fluorescence values at the end of experiment for panel A. (C) Effects of nobiletin on mitochondrial ROS generation were measured with DCF-DA indicator using a fluorescence microplate reader in an isolated brain mitochondrial model (See ‘METHODS’ for the detailed description). (D) Free radical scavenging activity of nobiletin was measured using DPPH assay. Values are the mean±S.E.M. ^**p<0.01, ^***p<0.001 as compared with untreated controls and, ^#p<0.05, ^##p<0.01, ^###p<0.001 as compared with glutamate or CaCl₂ (5 µM)-treated group.

Fig. 5. The effects of K⁺ influx on nobiletin-induced partial mitochondrial depolarization in isolated brain mitochondria.

(A) The effect of FCCP on ΔΨ_m was investigated with TMRE in an isolated brain mitochondrial model (see ‘METHODS’ for the detailed description) using a fluorescence microplate reader, as a positive control. (B, C). The effect of nobiletin on ΔΨ_m was measured with TMRE in the presence or absence of K+ in the medium using a fluorescence microplate reader in an isolated brain mitochondrial model. To remove K+ in the medium, KCl (100 mM) was replaced to CsCl (100 mM). Dose responses 10 min after nobiletin treatment (B) and time courses (C) were analyzed. Values are the mean±S.E.M. ^*p<0.05, ^**p<0.01, ^***p<0.001 as compared with the control group.