Spare respiratory capacity rather than oxidative stress regulates glutamate excitotoxicity after partial respiratory inhibition of mitochondrial complex I with rotenone

Abstract

Partial inhibition of mitochondrial respiratory complex I by rotenone reproduces aspects of Parkinson's disease in rodents. The hypothesis that rotenone enhancement of neuronal cell death is attributable to oxidative stress was tested in an acute glutamate excitotoxicity model using primary cultures of rat cerebellar granule neurons. As little as 5 nM rotenone increased mitochondrial superoxide (O2*-) levels and potentiated glutamate-induced cytoplasmic Ca2+ deregulation, the first irreversible stage of necrotic cell death. However, the potent cell-permeant O2*- trap manganese tetrakis (N-ethylpyridinium-2yl) porphyrin failed to prevent the effects of the inhibitor. The bioenergetic consequences of rotenone addition were quantified by monitoring cell respiration. Glutamate activation of NMDA receptors used the full respiratory capacity of the in situ mitochondria, and >80% of the glutamate-stimulated respiration was attributable to increased cellular ATP demand. Rotenone at 20 nM inhibited basal and carbonyl cyanide p-trifluoromethoxyphenylhydrazone-stimulated cell respiration and caused respiratory failure in the presence of glutamate. ATP synthase inhibition by oligomycin was also toxic in the presence of glutamate. We conclude that the cell vulnerability in the rotenone model of partial complex I deficiency under these specific conditions is primarily determined by spare respiratory capacity rather than oxidative stress.

Figure 1.

Rotenone-induced mitochondrial O₂^·− levels in intact granule neurons. Mitochondrial O₂^·− levels were quantified from the rate of MitoSOX oxidation. A, Rotenone (0–20 nm) was added when indicated. B, The experiment was repeated with CGNs treated with 25 μm MnTE-2-PyP for 45 min before and during imaging. C, Relative levels of matrix O₂^·− were calculated from the slopes of the fluorescence time courses after rotenone addition, normalized to their corresponding initial slopes. Results show mean and SD of nine independent experiments. Time courses in A and B are representative of nine independent experiments. Reactive oxygen species levels (slopes) in A at all concentrations of rotenone are significantly different from control (p < 0.001). Pooled data for 5 and 10 nm are different from 20 nm. p < 0.05.

Figure 2.

Rotenone-facilitated DCD and failure of MnTE-2-PyP to protect. A, Changes in TMRM⁺ (red) and Fluo-5F (green) fluorescence before (Control), 6 min after addition of 100 μm glutamate plus 10 μm glycine (+Glut), and 30 min after addition of 10 μm MK-801 after 60 min exposure to glutamate (+Glut + MK801). In the lower sequence, 20 nm rotenone was present throughout. B, Cells were treated with 25 μm MnTE-2-PyP for 45 min before and during imaging. C, The extent of deregulation in the absence of 25 μm Mn-TE-2-PyP as a function of rotenone concentration. Cells in which Fluo-5F fluorescence did not return to basal level after MK-801 treatment were considered deregulated. D, Effect of 20 nm rotenone on the extent of cell death 24 h after 60 min exposure to 100 μm glutamate/10 μm glycine in the presence (filled bars) or absence (open bars) of 25 μm Mn-TE-2-PyP. Results show mean and SD of four independent experiments.

Figure 3.

Quantitation of the respiratory demand imposed by NMDA receptor activation. A, CGNs were superfused with incubation medium in the respirometer. When indicated the medium was supplemented with FCCP (3 μm F₃ or 4 μm F₄) for 30 min. Note that 3 μm FCCP is sufficient to attain uncontrolled respiration. B, C, CGNs were superfused with medium containing 100 μm glutamate plus 10 μm glycine (G) or 3 μm FCCP (F₃) for the periods indicated. D, Quantitative analysis of the glutamate-stimulated respiration. Respiration rates were normalized relative to the FCCP-stimulated rate and are the mean and SD of four independent experiments. The x-axis is labeled in the same order as the perfusions were made during respirometry in trace C.

Figure 4.

Rotenone titration of basal and FCCP-stimulated respiration. A, Representative respirometry trace showing inhibition of cellular respiration by successive perfusions with buffer containing 20, 40, and 60 nm rotenone (R₂₀, R₄₀, and R₆₀), respectively. Arrows indicate the times at which perfusion with the indicated buffer was started. Note that respiration in the absence of additions remains stable for the duration of the experiment (broken line). B, C, Representative respirometer traces quantifying the inhibition of uncontrolled respiration by 10 (R₁₀) or 20 nm rotenone (R₂₀) in the presence of 3 μm FCCP (F₃). D, Quantitation of the rotenone inhibition in the presence (triangles) or absence (diamonds) of 3 μm FCCP. One-way ANOVA with Tukey's post hoc test for multiple comparisons was used to determine which means differed significantly from the basal respiration rate (for 5–10 nm, n = 6; for 20 nm, n = 7). **p < 0.01; ***p < 0.001.

Figure 5.

CGN respiration fails rapidly after NMDA receptor activation in the presence of 20 nm rotenone. A, Representative respiratory stimulation by successive glutamate (“G”) exposures. B, Quantitative analysis of the relative respiration rates obtained from replicate respirometer traces as in A. C, A second cycle of NMDA receptor stimulation in the presence of 20 nm rotenone (G+R₂₀) shows decreased maximal stimulation and rapid respiratory failure. D, Quantitative analysis of the relative respiration rates obtained from replicate respirometer traces as in C. Respirations rates are compared with the glutamate-stimulated rate (black bars). ***p < 0.001 first addition versus second addition of glutamate.

Figure 6.

The ATP-independent component of NMDA receptor-dependent respiratory stimulation. A, Glutamate addition simultaneous with 0.2 μg/ml oligomycin (G+O); the numbers correspond to the images shown in B. B, Simultaneous imaging of TMRM⁺ and Fluo-5F fluorescence at the time points shown in A. Note the extensive deregulation of the cells after glutamate in the presence of oligomycin (i.e., high [Ca²⁺]_c, low membrane potentials). C, Oligomycin addition (O) before glutamate. Note the respiratory inhibition with oligomycin showing the residual proton leak and the small stimulation with subsequent glutamate.