Ketones prevent synaptic dysfunction induced by mitochondrial respiratory complex inhibitors

Abstract

Ketones have previously shown beneficial effects in models of neurodegenerative disorders, particularly against associated mitochondrial dysfunction and cognitive impairment. However, evidence of a synaptic protective effect of ketones remains lacking. We tested the effects of ketones on synaptic impairment induced by mitochondrial respiratory complex (MRC) inhibitors using electrophysiological, reactive oxygen species (ROS) imaging and biochemical techniques. MRC inhibitors dose-dependently suppressed both population spike (PS) and field potential amplitudes in the CA1 hippocampus. Pre-treatment with ketones strongly prevented changes in the PS, whereas partial protection was seen in the field potential. Rotenone (Rot; 100 nmol/L), a MRC I inhibitor, suppressed synaptic function without altering ROS levels and PS depression by Rot was unaffected by antioxidants. In contrast, antioxidant-induced PS recovery against the MRC II inhibitor 3-nitropropionic acid (3-NP; 1 mmol/L) was similar to the synaptic protective effects of ketones. Ketones also suppressed ROS generation induced by 3-NP. Finally, ketones reversed the decreases in ATP levels caused by Rot and 3-NP. In summary, our data demonstrate that ketones can preserve synaptic function in CA1 hippocampus induced by MRC dysfunction, likely through an antioxidant action and enhanced ATP generation.

Figure 1

Ketones suppress rotenone (Rot)-induced hippocampal synaptic suppression in a dose-dependent manner. (a) Time-course of the mean population spike (PS) amplitude after application of either 100 nM or 1 μM rotenone (Rot) with or without ketones. Either 100 nmol/L (•) or 1 μmol/L () Rot strongly depressed the PS amplitude during and after Rot application. Ketones alone , Each 1 mmol/L) did not alter the PS amplitude. Pre-treatment with ketones fully reversed the synaptic suppression seen with 100 nmol/L Rot (), but this was partially restored with 1 μmol/L Rot (□). In each condition where 0.5 mmol/L ketones were tested against 100 nmol/L Rot (), ketones induced partial protective effects compared to 1 mmol/L ketones. (b) Changes in the mean field potential amplitude before, during, and after either 100 nmol/L or 1 μmol/L Rot with or without ketones. Rot slightly suppressed the field potential amplitude compared to the change in PS amplitude after Rot treatment. Both 100 nmol/L and 1 μmol/L Rot-induced field potentials were partially restored by ketone application. Representative traces of PS or field potentials at respective time-points (i, ii, iii) are depicted in the traces above. The horizontal line in this and following figures indicates the drug infusion period. Data are expressed as mean ± SEM.

Figure 2

Ketones mitigate synaptic suppression induced by the mitochondrial complex II inhibitor [3-nitropropionic acid (3-NP)]. (a) Either 1 mmol/L (•) or 10 mmol/L () irreversibly suppressed the PS amplitude in CA1 hippocampus. Pre-treatment with ketones did not produce a synaptic protective effect against 10 mmol/L 3-NP (), whereas ketones significantly reduced PS suppression induced by 1 mmol/L 3-NP (), and then restored the PS amplitude to basal levels after a 20-min washout. Lower concentrations of ketones (0.5 mmol/L each) were partially protective (). (b) During the initial period of 1 mmol/L 3-NP treatment (○), the mean field potential amplitude was slightly increased. After 30 min, the field potential showed dramatic decay and then did not change during wash-out. While pre-treatment with ketones slowly decreased the field potential amplitude compared to 1 mmol/L 3-NP alone, no beneficial effect occurred after 60 min of co-application. However, ketones significantly enhanced the recovery of the field potential amplitude after wash-out.

Figure 3

Effects of antioxidants on hippocampal synaptic suppression by mitochondrial respiratory complex (MRC) inhibitors. (a) Neither 600 U/mL catalase (•) nor 200 μmol/L MnTBAP (○) blocked the synaptic suppression caused by 100 nmol/L Rot. Pre-treatment with cell-permeable 500 μmol/L GSHMEE () alone also had no effect on Rot-induced PS suppression. (b) Co-application of a cocktail of 600 U/mL catalase and 200 μmol/L MnTBAP with 1 mmol/L 3-NP resulted in recovery of the PS amplitude. Similarly, pre-treatment with GSHMEE also restored the PS amplitude against 3-NP application.

Figure 4

Ketones enhance catalase activity under conditions of oxidative stress. Calibration curve demonstrating an increase in arbitrary unit values as catalase dose increases. Similar to the effects seen with 2000 mU/mL of catalase alone, no differences were seen between physiological saline, ketones, and ketones plus 2 mmol/L H₂O₂ groups after 3 h of treatment. Conversely, H₂O₂ alone significantly decreased the level of catalase compared to the above three groups (**, p < 0.01). NS, not significant.

Figure 5

Changes in ROS levels after MRC inhibition. (a) No changes in DCF fluorescence intensity (FI) were seen in CA1 pyramidal neurons exposed to 100 nmol/L Rot over 30 min. (b) In the control group, pyramidal neurons were perfused with aCSF solution containing dithroethidium (DHE), and only a slight increase in DHE intensity was seen over time. Rot evoked a dose-dependent increase in DHE fluorescence intensity, but 100 nmol/L Rot did not evoke a significant change in DHE intensity compared to control. One mmol/L 3-NP prominently enhanced reactive oxygen species (ROS) among the three groups, whereas ketone application largely suppressed ROS generation induced by 3-NP. Values represent mean ± SEM. One way anova followed by Tukey test; *, p < 0.05; **, p < 0.01.

Figure 6

Ketones restore ATP levels depleted by oxidative stress and by inhibition of MRC function. ATP levels (represented as % of control) in micro-dissected CA1 tissue samples treated with H₂O₂, ketones plus H₂O₂, ketones, rotenone, ketones plus rotenone, 3-NP and ketones plus 3-NP are shown at various time-points. Control slices were perfused with physiological saline under similar experimental conditions. Values represent mean ± SEM. Paired t-tests were conducted in control vs. drug-treated groups; *, p < 0.05; **, p < 0.01: 2 mmol/L H₂O₂vs. ketones plus 2 mmol/L H₂O₂; ##, p < 0.05: 1 mmol/L 3-NP vs. ketones plus 1 mmol/L 3-NP; ††, p < 0.01. ANOVA followed by Tukey test; §§, p < 0.01.