The Psi(m) depolarization that accompanies mitochondrial Ca2+ uptake is greater in mutant SOD1 than in wild-type mouse motor terminals

Abstract

The electrical gradient across the mitochondrial inner membrane (Psi(m)) is established by electron transport chain (ETC) activity and permits mitochondrial Ca(2+) sequestration. Using rhodamine-123, we determined how repetitive nerve stimulation (100 Hz) affects Psi(m) in motor terminals innervating mouse levator auris muscles. Stimulation-induced Psi(m) depolarizations in wild-type (WT) terminals were small (<5 mV at 30 degrees C) and reversible. These depolarizations depended on Ca(2+) influx into motor terminals, as they were inhibited when P/Q-type Ca(2+) channels were blocked with omega-agatoxin. Stimulation-induced Psi(m) depolarization and elevation of cytosolic [Ca(2+)] both increased when complex I of the ETC was partially inhibited by low concentrations of rotenone (25-50 nmol/l). This finding is consistent with the hypothesis that acceleration of ETC proton extrusion normally limits the magnitude of Psi(m) depolarization during mitochondrial Ca(2+) uptake, thereby permitting continued Ca(2+) uptake. Compared with WT, stimulation-induced increases in rhodamine-123 fluorescence were approximately 5 times larger in motor terminals from presymptomatic mice expressing mutations of human superoxide dismutase I (SOD1) that cause familial amyotrophic lateral sclerosis (SOD1-G85R, which lacks dismutase activity; SOD1-G93A, which retains dismutase activity). Psi(m) depolarizations were not significantly altered by expression of WT human SOD1 or knockout of SOD1 or by inhibiting opening of the mitochondrial permeability transition pore with cyclosporin A. We suggest that an early functional consequence of the association of SOD1-G85R or SOD1-G93A with motoneuronal mitochondria is reduced capacity of the ETC to limit Ca(2+)-induced Psi(m) depolarization, and that this impairment contributes to disease progression in mutant SOD1 motor terminals.

Fig. 1.

Stimulation at 100 Hz increases cytosolic and mitochondrial [Ca²⁺] and depolarizes Ψ_m in WT mouse motor terminals at 30 °C. (A) Upper trace: Elevation of cytosolic [Ca²⁺] in response to three stimulus trains at 100 Hz, monitored as normalized increases (F/F_rest) in the fluorescence of intra-axonally injected OG-5N (vertical lines indicate duration of stimulation). The average increase in cytosolic [Ca²⁺] is 1.0 μmol/l above an assumed resting value of 0.1 μmol/l (64). Second trace: Elevations of mitochondrial matrix [Ca²⁺], monitored as increases in the fluorescence of mitochondrially-loaded X-rhod-1 (mean of five traces). The average increase in mitochondrial [Ca²⁺] is ≈1–2 μmol/l above an assumed resting value of 0.05–0.1 μmol/l (13, 40, 41). Third trace: Depolarization of Ψ_m, monitored as increases in the fluorescence of Rh-123. The line is a smoothed moving bin average of three neighboring points. Lower trace shows (using a different vertical scale) these same data, along with the much larger Ψ_m depolarization produced in this terminal by the proton carrier CCCP (2 μmol/l). Cytosolic [Ca²⁺], mitochondrial [Ca²⁺], and Ψ_m were recorded from different preparations, aged 64–88 days.

Fig. 2.

The stimulation-induced Ψ_m depolarization increases with cooling or addition of 3,4-diaminopyridine (3,4-DAP) and requires Ca²⁺ entry through plasma membrane Ca²⁺ channels. (A) The Ψ_m depolarization produced by 100 Hz stimulation at 30 °C (left) was increased by cooling to 18 °C (upper right) or by prolonging the action potential with 20 μmol/l 3,4-DAP (lower right). (B) Ψ_m depolarizations (the magnitudes of which were enhanced by both cooling to 20 °C and 3,4-DAP, open circles) were reduced by omitting Ca²⁺ from the bath (filled circles, left) or (in a different terminal) by adding 0.6 μmol/l ω-agatoxin-TK (filled circles, right). The effects of low bath [Ca²⁺] were readily reversible; reversal of agatoxin effects was slow and incomplete. Each record in (A) and (B) is the mean of two to nine traces. The effects of cooling, 3,4-DAP, and removal of bath Ca²⁺ were observed in 10, 5, and 4 additional terminals, respectively. Exposures to 3,4-DAP and agatoxin were 7–62 minutes and 60 minutes, respectively.

Fig. 3.

A low concentration of rotenone increases stimulation-induced elevations of cytosolic [Ca²⁺] and Ψ_m depolarizations. Cytosolic [Ca²⁺] (A) and Ψ_m depolarizations (B) produced by three trains at 100 Hz before and after addition of rotenone (50 nM). [Ca²⁺] and Ψ_m traces came from different terminals. (C) Paired data from eight terminals studied before and after rotenone exposure show that in mice expressing normal SOD1 (WT or human) rotenone increased the average change in Rh-123 fluorescence from 0.96% ± 0.22% (SEM) in control medium to 4.13% ± 1.18% in rotenone (* P < 0.05). Only measurements from the initial 100 Hz train were included in the averages. The duration of rotenone exposure was 17–30 minutes. Mice were 50–375 days old.

Fig. 4.

Stimulation-induced Ψ_m depolarizations are increased in presymptomatic SOD1-G85R and SOD1-G93A mice. (A) Phase (left) and Rh-123 fluorescence (middle) images show a resting motor terminal in a 121-day-old SOD1-G85R mouse. At right is a difference image of the same region, calculated by subtracting the resting fluorescence from the fluorescence during 100 Hz stimulation. (B) Representative stimulation-induced Ψ_m depolarizations evoked by repeated brief 100 Hz trains in a WT mouse (mean of two traces), a mouse lacking SOD1 (SOD1-KO), an “overexpressor” mouse with both normal mouse and normal human SOD1 (SOD1-OX), the SOD1-G85R terminal in (A), and an SOD1-G93A terminal. Bars shows mean peak amplitude (±SEM) for the first 100-Hz train for each of these groups. The Ψ_m depolarizations in SOD1-G85R (P < 0.01) and SOD1-G93A (P < 0.05) mice were significantly different from WT at 30 °C (analysis of variance followed by Dunnett's multiple comparison test). These differences were not significant at lower temperatures, where mitochondrial Ca²⁺ uptake may be reduced (64). Data were averaged from 47 terminals in 16 WT mice 50–145 days old; comparable values for SOD-KO mice were 19, 3, and 253–355; for SOD1-OX 5, 4, and 89–375; for SOD1-G85R mice 60, 6, and 121–161; for SOD1-G93A 13, 6, and 43–85. At these ages, all endplates were fully innervated. Additional details in SI Text, Item #3.

Fig. 5.

Stimulation-induced Ψ_m depolarizations in SOD1-G85R mice are not reduced by cyclosporin A. (A) Representative traces show the Rh-123 fluorescence response evoked by a 100-Hz stimulus train before and 56 minutes after exposure to 5 μmol/l cyclosporin. (B) Graph plots peak amplitudes (normalized to resting fluorescence) before and during drug exposure for this terminal. Similar experiments on three additional SOD1-G85R mice (130–150 days) also showed no significant change in Ψ_m depolarizations in cyclosporin A (paired Wilcoxon signed rank test, P > 0.50). Similar experiments in additional mouse types (e.g., SOD1 knockout treated with 50 μmol/l 3,4-diaminopyridine) confirmed the lack of effect of 8–10 μmol/l cyclosporin A.