Address correspondence and reprint requests to Dr R. K. Porter, Department of Biochemistry, Trinity College Dublin, Dublin 2, Ireland. E-mail: email@example.com.
Synaptosomes, isolated from the whole brain of young (3 months) and old (24 months) rats were used to study the major bioenergetic systems of neuronal mitochondria in situ, within the synaptosome. Approximately 85% of the resting oxygen consumption of synaptosomes from both young and old rats was a result of proton leak (and possibly other ion cycling) across the mitochondrial inner membrane. There were no significant differences between synaptosomes from the young and old rats in the kinetic responses of the substrate oxidation system, the mitochondrial proton leak and the phosphorylation system to changes in the proton electrochemical gradient. Flux control coefficients of 0.71, 0.27 and 0.02 were calculated for substrate oxidation system, phosphorylation system and the proton leak, respectively, at maximal ATP producing capacity in synaptosomes from young animals. The corresponding values calculated for synaptosomes from old animals were 0.53, 0.43 and 0.05. Thus substrate oxidation had greatest control over oxygen consumption at maximal phosphorylating capacity for synaptosomes from whole brain, with proton leak, having little control under maximal ATP producing capacity. The uncoupled rate of oxygen consumption, in the presence of the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), was significantly lower (p = 0.0124) in synaptosomes from old rats (6.08 ± 0.42, n = 11) when compared with those from the young rats (7.87 ± 0.48, n = 8). Thus, there is an impaired flux through the substrate oxidation system is synaptosomes from old rats, as compared to synaptosomes from the young animals. These in situ results may have important implications for the interpretation of theories that age-dependent impairment of mitochondrial energy production may result in increased susceptibility to neurodegeneration.
and are the elasticities of the substrate oxidation (s), phosphorylation (p) and proton leak (L) systems to changes in ΔΨm
and are the flux control coefficients for substrate oxidation (s), phosphorylation (p) and proton leak (L) systems, respectively, over oxygen consumption at maximal phosphorylating capacity for synaptosomes from whole brain of young and old rats
The free radical theory of aging suggests that reactive oxygen-containing species (ROS) cause the progressive and irreversible damage and deterioration of cellular function (Harman 1956). The mitochondrial inner membrane has been suggested to be a significant site of constitutively produced ROS (Schulze-Osthoff et al. 1992). However, mitochondria are themselves a target for damage by ROS. Membrane polyunsaturated fatty acids and proteins, soluble proteins and mitochondrial DNA are all potentially vulnerable targets (Gutteridge et al. 1986; Ragusa et al. 1992; Bowling et al. 1993; Sohal and Dubey 1994; Wallace et al. 1995; Forsmark-Andree et al. 1997; Gardner 2002). For instance, the decline in the specific activities of the respiratory complexes with age has been correlated with the progressive accumulation of somatic mitochondrial DNA mutations. In particular, the aging process has been associated with a ∼4800 base pair deletion encoding four complex I subunits, two complex V subunits and one cytochrome oxidase subunit (Wallace et al. 1995; Schon 2000). It has also been suggested that postmitotic tissues, such as neurons, are more severely affected by reactive oxygen species than mitotic cells (Miquel and Fleming 1986).
Several studies have shown specific changes in brain mitochondrial function with age. There is a decrease in the ATPase activity of the F0F1 ATP synthase in sonicated submitochondrial particles isolated from 24-month-old rat brain compared to those isolated from 3- or 6-month-old rats (Guerrieri et al. 1992). The maximum amount of binding (Bmax) of cytochrome c to cytochrome oxidase is decreased in both synaptosomal and non-synaptic mitochondria isolated from cerebral cortex of 30-month-old rats when compared with 4-month-old rats (Curti et al. 1990). Significant decreases in the activities of complexes IV and V in synaptic mitochondria from old, compared with young, mice have been reported. However, age-dependent decreases in the activities of complexes I, II, III and IV were also found in non-synaptic mitochondria, which suggests a general mitochondrial dysfunction in brain aging (Ferrandiz et al. 1994). However, the significance of alterations in the activities of individual mitochondrial bioenergetic components cannot be fully assessed in terms of mitochondrial function without an assessment of the relative control strengths of those components (Harper et al. 1998).
This paper reports the results of a study of the kinetic responses (elasticities) of the mitochondrial proton leak, substrate oxidation and phosphorylation in response to changes in the mitochondrial membrane potential. Flux-control coefficients were then derived from these elasticities. These kinetic responses to alterations of the mitochondrial membrane potential were compared in synaptosomes prepared from the brains of young (3 month) and old (24 month) rats, in order to gain insights into the possible age-dependent changes in mitochondrial function.
Materials and methods
Hepes, calcium chloride, glucose, sodium hydroxide, Percoll, sodium potassium tartrate, copper sulfate pentahydrate, tetraphenylboron, veratridine, FCCP, myxothiazol, valinomycin and oligomycin were all obtained from Sigma, Poole, Dorset, UK. Sucrose, magnesium chloride hexahydrate, sodium dihydrogen orthophosphate, hydrochloric acid, sodium carbonate, sodium dodecyl sulfate, Dow Corning 550 fluid and perchloric acid were obtained from BDH Laboratory Supplies, Poole, Dorset, UK. Sodium chloride was from Merck, Darmstadt, Germany. [3H]TPMP+, 86Rb+, [14C]methoxyinulin, 3H2O were obtained from New England Nuclear, Dreieich, Germany. Ecoscint scintillation fluid was obtained from National Diagnostics, Atlanta, GA, USA.
Preparation of whole rat brain synaptosomes. Whole brain synaptosomes were prepared from young (3 month) or old (24 month) male Wistar rats as described by Gordon-Weeks (1987). For each preparation four rats were killed by cervical dislocation and their brains were rapidly removed and placed into ice-cold solution A (0.32 m sucrose, 5 mm Hepes/NaOH buffer, pH 7.4). The brains were chopped into small pieces using a backed razor blade and homogenized by hand in a Potter-Elvehjem homogenizer using 12 passes of the 6.6 mm (loose) clearance pestle followed by homogenization by hand with six passes using a pestle of 3 mm (tight) clearance. The homogenate was placed in two 50 mL centrifuge tubes, adjusted to 40 mL with Solution A and centrifuged at 3000 r.p.m. (1000 × g) in a Sorval SS34 rotor for 5 min at 4°C. The supernatants were decanted and stored on ice while the pellets were resuspended, homogenized and centrifuged as before. The two supernatants were pooled and centrifuged at 10 000 r.p.m. (12 000 × g) for 20 min at 4°C. This step was repeated twice more. The combined pellets, crude synaptosomes, were resuspended in approximately 10 mL of Solution A and adjusted to 7.5% (v/v) Percoll with 90% (v/v) Percoll solution (one volume of 3.2 m sucrose added to nine volumes Percoll). The total volume of the suspension was adjusted to approximately 36 mL with solution A. The Percoll density gradients were made by placing 4 mL of 10% (v/v) Percoll above 4 mL of 16% (v/v) Percoll in 17 mL plastic centrifuge tubes. The 7.5% Percoll-synaptosomal suspension was then layered onto the gradient.
The Percoll/synaptosomal gradients were centrifuged at 11 000 r.p.m. (15 000 × g) in a Sorval SM24 rotor for 20 min at 4°C. The synaptosomes formed a band at the 10%/16% Percoll–gradient interface and were removed using a plastic Pasteur pipette. The volume of the synaptosomal suspension was adjusted to 40 mL with Krebs–Henseleit buffer (145 mm NaCl, 1.3 mm MgCl2·6H2O, 5 mm KCl, 1.2 mm NaH2PO4, 10 mm glucose, 1.2 mm CaCl2, 20 mm Hepes, pH 7.4) and the synaptosomes were collected by centrifugation at 15 000 × g for 20 min at 4°C. The synaptosomal pellet was resuspended in Krebs–Henseleit buffer to a final volume of approximately 3 mL. The protein concentration of the solution was measured using the method of Markwell et al. (1978) with bovine serum albumin as the standard. All synaptosomal mitochondria were considered to be of neuronal origin (Whittaker 1993).
Oxygen consumption measurements. The rate of synaptosomal oxygen consumption was measured in a thermostated Clark-type oxygen electrode (Rank Brothers, Bottisham, Cambridge, UK). The total volume of the electrode chamber was 1 mL. All experiments were performed in a final volume of 0.5 mL at a temperature of 37°C. A Philips (PM 8252) dual pen chart recorder, with the maximum full-scale deflection set to 1 mV, was used to record oxygen consumption rates. Oxygen consumption rates were measured in duplicate by preincubating synaptosomes (1.2 mg protein) in 0.6 mL Krebs–Henseleit buffer containing 10 mm glucose in 5 mL plastic stoppered vials for 10 min in a shaking water bath at 37°C. 0.5 mL of this synaptosomal incubation was then transferred to the oxygen electrode at 37°C. The suspension also contained TPMP+ (1.3 µm) and TPB– (3 µm).
Measurement of mitochondrial membrane potential, plasma membrane potential and synaptosomal volume. Synaptosomal mitochondrial membrane potentials were measured using radiolabelled methyltriphenylphosphonium (TPMP+) which equilibrates with the ΔΨ across membranes, as described for guinea-pig synaptosomes by Scott and Nicholls (1980). Synaptosomes (0.5 mg protein) were preincubated in 0.5 mL Krebs–Henseleit buffer containing 1.3 µm[3H]TPMP+(0.2 µCi/mmol), 3 µm tetraphenylboron (TPB–) and 86Rb+ (50 µm, 0.5 µCi) in stoppered polycarbonate flasks in a shaking water bath at 37°C. After the incubation period, 0.4 mL of the synaptosomal suspension was layered onto 0.4 mL of a mixture of Dow Corning 550 Fluid–dinonyl phthalate (60 : 40, v/v) layered above 0.15 mL perchloric acid (1 m) and centrifuged in a bench-top centrifuge for 3 min at 15 000 × g. Aliquots of the supernatant (0.2 mL) and infranatant (0.1 mL) were removed and placed into separate scintillation vials containing 10 mL of Ecoscint scintillation fluid. The solutions were mixed and counted for radioactivity. The mitochondrial membrane potential was calculated according to the following equation, described in Scott and Nicholls (1980):
where ΔΨm is the mitochondrial membrane potential, Vs is the synaptosomal volume, Vm is the mitochondrial volume, ae, ac and am are the extracellular medium, cytosolic and mitochondrial matrix binding correction factors for TPMP+, respectively. [Rb+]e/[Rb+]s is a measure of the plasma membrane potential. The TPMP+ binding correction values, which were assumed to be the same for synaptosomes from young and old animals, are from Scott and Nicholls (1980): ae, 1; ac, 0.6; am, 0.4. The assumption that TPMP activity parameters were similar in synaptosomes from young and old rats is based on the following: (i) the volumes of the synaptosomes from young and old animals are not significantly different; (ii) the proportion of the volume of synaptosomes that contain mitochondria is the same from synaptosomes from young and old animals; (iii) Vitorica et al. (1985) have indicated that there is no age-dependent difference in TPMP binding; and (iv) the activity coefficient for the presence of BSA would not be different for synaptosomes from young and old animals. ΔpH cannot be determined without measuring the intrasynaptosomal pH. However, studies with rat liver mitochondria (Hafner et al. 1990) have shown that it may be assumed that ΔpH represents a small component of Δp and that it will remain small during the course of titrations of the type used here. Thus, as ΔΨm decreases so does ΔpH and, ultimately, ΔΨm provides a good estimate of Δp.
The plasma membrane potential was determined from the distribution of 86Rb+ across the plasma membrane using the Nernst equation as follows:
where ΔΨp is the plasma membrane potential and [Rb+]s and [Rb+]e are the concentrations of 86Rb+ in the synaptosome and extracellular medium, respectively.
The synaptosomal volume was measured using [14C]-methoxyinulin as an extrasynaptosomal space marker and 3H2O as the total space marker with 1 mg of synaptosomal protein. The total pellet volume and the extrasynaptosomal space were calculated as 3H2O space (µL/mg protein) minus the [14C]methoxyinulin (µL/mg protein) space. The following values were obtained under resting conditions: 4.45 ± 0.33 µL/mg (n = 16) for synaptosomes from whole brain of young rats and 3.64 ± 0.42 µL/mg (n = 7) for synaptosomes from whole brain of old rats. These values are not significantly different (p = 0.1719) and are similar to values obtained by other workers (Deutsch et al. 1981).
Determination of the purity and mitochondrial volume density of the synaptosomes. The purity of the synaptosomal preparations was assessed by transmission electron microscopy, and transmission electron microscopy and mitochondrial volume density were determined as described in Porter and Brand (1995). It was found that approximately 68% of the structures observed in preparations from either young or old rats were synaptosomes. The remaining 30% consisted of free mitochondria (about 5%) and other membranous structures (about 25%). It was also calculated that approximately 38% of the synaptosomes contained one or more mitochondria. Although the remainder appeared to contain only synaptic vesicles, some thin sections will miss mitochondria, so that the value of 38% will be a minimum.
The ratio of mitochondrial volume to synaptosomal volume (Vm/Vs) was also determined by electron microscopy. Values were calculated from all synaptosome images on 10 electron micrographs from each of three separate preparations. The ratio was not significantly different between the two groups of animals: 0.36 ± 0.02 (n = 3) for synaptosomes from old rats and 0.37 ± 0.02 (n = 3) for synaptosomes from young rats (p = 0.91).
Elasticity analysis. The control coefficients are described in terms of the schematic representation outlined in Fig. 1. They were calculated from the data in Figs 3, 4 and 5, as described in Hafner et al. (1990) for a branched system.
Values are given as means ± SEM with the sample number (n) in all cases. Significant differences were determined between two values using a Student's unpaired t-test for ‘n’ values, where significance is defined as less than 5% probability due to chance.
The analysis involved determination of the kinetic responses of the producers of ΔΨm (substrate oxidation) and the consumers of ΔΨm (proton leak and phosphorylation system) to changes in ΔΨm, the intermediate in the branched pathway shown schematically in Fig. 1. Flux through substrate oxidation, proton leak and the phosphorylation system were measured indirectly by measuring oxygen consumption at different values of ΔΨm. ΔΨm was modulated in situ using (i) an inhibitor of the mitochondrial electron transport chain (myxothiazol); (ii) an inhibitor of the ATPsynthase (oligomycin); and (iii) a plasma membrane sodium ionophore (veratridine). The titration curves that might be expected for the measurement of oxygen consumption by in situ synaptosomal mitochondria as a function of the magnitude of ΔΨm during the course of these titrations are depicted in the schematic diagram (Fig. 2).
Values for the resting oxygen consumption rates (Table 1), the resting in situ mitochondrial membrane potential (Table 2) and the resting plasma membrane potential (Table 2) did not differ significantly between synaptosomes isolated from young and old rats. The values for resting oxygen consumption rates and plasma membrane potentials are consistent with those of other workers (Scott and Nicholls 1980; Ereciñska et al. 1996). Despite this consistency, the plasma membrane potentials (as measured by potassium diffusion potentials) are lower than might be expected from knowledge of intraneuronal concentrations of potassium ions. Microelectrode measurements show the intraneuronal potassium concentration to be 80–90 mm, whereas intrasynaptosomal potassium concentrations of between 45 and 50 mm have been reported for synaptosomes prepared in the same way as used here (McMahon and Nicholls 1991; Ereciñska et al. 1996). Thus it appears that potassium loss is inherent in the generation of synaptosomes. In order to determine the oxygen consumption rate by synaptosomes that is not due to oxidative phosphorylation, it was necessary to inhibit oxygen consumption by the mitochondrial electron transport chain. This was achieved by addition of excess myxothiazol (2 µm). In order to determine the accumulation of the lipophilic cation, TPMP+, at zero mitochondrial membrane potential, excess oligomycin (0.4 µg/mL), FCCP (20 µm) and valinomycin (3 µm) were added to the incubation medium containing synaptosomes in addition to myxothiazol (2 µm). Figure 3 shows (i) the amount of oxygen consumption that is not due to oxidative phosphorylation for the synaptosomes from the young (Fig. 3a) and old (Fig. 3b) animals in conjunction with the measured overall kinetic responses of (ii) the substrate oxidation system, (iii) the proton leak and (iv) the phosphorylation system (uncorrected for proton leak) to changes in ΔΨm.
Table 1. Oxygen consumption values in whole-brain synaptosomes from young and old rats. Values represent means ± SEM, with the number of determinations in parenthesis
*Significant difference p = 0.0002. **Significant difference p = 0.0124. Unpaired student t-test, two-tailed p-value, comparing synaptosomes from young and old rats. [Oligomycin] = 0.4 µg/mL incubation medium, [Veratridine] = 50 µm for old and 100 µm for young, [FCCP] = 240 nm.
Oxygen consumption not due to oxidative phosphorylation
0.54 ± 0.05 (6)
0.47 ± 0.10 (6)
Table 2. The in situ mitochondrial membrane potentials and plasma membrane potentials in synaptosomes from young and old rats. Values represent means ± SEM, with the number of determinations in parenthesis
Membrane potential (mV)
[Oligomycin] = 0.4 µg/mL incubation medium, [Veratridine] = 50 µm for old and 100 µm for young rats, [FCCP] = 240 nm.
There was no significant difference between the oxygen consumption rates not due to oxidative phosphorylation (i.e. when mitochondrial function is inhibited) in synaptosomes from young (0.54 ± 0.05 nmolO2/min/mg; n = 6) and old (0.47 ± 0.10 nmolO2/min/mg; n = 6) rats. Subtraction of the oxygen consumption rates that were not due to oxidative phosphorylation from the total oxygen consumption rates by the synaptosomes gives the oxygen consumption due solely to the in situ mitochondria (Table 1). There was no significant difference between the resting mitochondrial oxygen consumption rates, expressed as nmolO2/min/mg synaptosomal protein (n = 6 in both cases), of synaptosomes isolated from young (3.61 ± 0.37) and old (2.75 ± 0.217) rats (see Table 1).
Figure 4 shows the mitochondrial proton leak values determined in situ, measured in the presence of optimal inhibitory amounts of oligomycin (0.4 µg/mL) and increasing concentrations of myxothiazol (0–0.5 µm). The data show the characteristic non-linear relationship between proton leak rate (or more correctly, the rate of oxygen consumption due to proton leak) and the driving force for that leak, ΔΨm. Curves fitted to data for synaptosomes from old and young rats are essentially superimposable, indicating the degree of mitochondrial proton leak to be similar. This is also apparent from the sem values for each pair of points defining the curve in Fig. 4, which indicate no significant differences.
Figure 5 shows the kinetic response of the substrate oxidation system to changes in ΔΨm. This kinetic response was achieved by first titrating the resting (non-stimulated, non-inhibited) oxygen consumption rate of synaptosomal mitochondria to a non-phosphorylating state (state 4) with oligomycin. The values in Table 1 show that there was no significant difference between the resting oxygen consumption rates and the non-phosphorylating oxygen consumption rates with synaptosomes from the young and old rats. Thus, oligomycin had no significant effect on oxygen consumption rates (Table 1). Similarly oligomycin had no significant effects on ΔΨm when the resting state and the non-phosphorylating state for synaptosomes from young and old rats were compared (Table 2).
The kinetic response of the substrate oxidation system was investigated under conditions of stimulated ATP turnover in the synaptosome, which in turn stimulates the synaptosomal mitochondria to synthesize more ATP; thus driving the mitochondria to the maximal phosphorylation state (state 3). This was achieved by titration with a plasma membrane Na+ ionophore, veratridine. This stimulates the plasma membrane Na+/K+-ATPase to work harder to re-establish the plasma membrane Na+ gradient, thus increasing the rate of ATP turnover. The in situ oxygen consumption rates by mitochondria at maximal phosphorylation were significantly greater than the resting oxygen consumption rates for synaptosomes from both young and old rats. However, as shown in Table 1, there was no significant difference between the rates of oxygen consumption in synaptosomes from young and old rats in either in the non-phosphorylating or maximal phosphorylating state. Furthermore, there were no significant differences between young and old rats in the ΔΨm values for the non-phosphorylating and maximal phosphorylating states (Table 2).
Maximal substrate oxidation flux (as opposed to maximal phosphorylation flux) was measured by titration with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (up to 240 nm), an uncoupler of mitochondrial oxidative phosphorylation. Under these conditions it was found that there was a significantly (p = 0.0124) reduced oxygen consumption rate in synaptosomes from old rats (6.08 ± 0.42 nmolO2/min/mg synaptosomal protein, n = 11) when compared with synaptosomes from young rats (7.87 ± 0.48 nmolO2/min/mg synaptosomal protein, n = 8) (see Table 1). However, ΔΨm at maximal substrate oxidation flux, i.e. in the presence of excess FCCP, was found not to be significantly different between synaptosomes from young and old animals (Table 2). In addition, it was found that there was no significant difference in oxygen consumption rates in synaptosomes from old animals in a comparison of maximal substrate oxidation flux (i.e. in the presence of excess FCCP)(6.08 ± 0.42 nmolO2/min/mg synaptosomal protein, n = 11) and maximal phosphorylation flux (6.77 ± 0.18 nmolO2/min/mg synaptosomal protein, n = 4). Nor was there a significant difference in a similar comparison in synaptosomes from young animals (7.87 ± 0.48 nmolO2/min/mg synaptosomal protein, n = 8 vs. (6.92 ± 0.91 nmolO2/min/mg synaptosomal protein, n = 5, respectively).
Figure 6 shows the kinetic response of the phosphorylation system at maximal phosphorylating capacity, corrected for proton leak. This was achieved by titrating the in situ mitochondrial oxygen consumption with an electron transport chain inhibitor (myxothiazol) while the mitochondria were under maximal phosphorylating conditions. As some proton leak is occurring in this phosphorylating-state titration, one can only get a true measure of the phosphorylation system kinetics when proton leak, at any given Δp, is subtracted. The data show that the kinetic responses of the phosphorylation systems for in situ mitochondria of synaptosomes from young and old animals were not significantly different, as may be seen from the overlapping error bars of the pairs of points defining the line in Fig. 6.
Table 3 shows the flux control coefficients for the substrate oxidation system, the phosphorylation system and the proton leak over oxygen consumption at maximal phosphorylating capacity for whole-brain synaptosomes from young and old rats. The control coefficients are described in terms of the schematic representation outlined in Fig. 1 and these data were calculated from the data in Figs 3, 4 and 5, as described in Hafner et al. (1990) for a branched system. The elasticities to changes in ΔΨm at maximal phosphorylation capacity in synaptosomes from young rats were − 1.09 for the substrate oxidation system, 4.65 for the phosphorylation system and 2.76 for the proton leak (Table 3, top). The corresponding elasticities for the synaptosomes from the old animals were − 1.23, 1.93 and 1.30. The flux control coefficients over oxygen consumption were 0.71 for the substrate oxidation system, 0.27 for the phosphorylation system and 0.02 for the proton leak at maximal ATP producing capacity in synaptosomes from young animals (Table 3, bottom) and the corresponding values for the old rats were 0.53, 0.43 and 0.05.
Table 3. Control of oxygen consumption at maximal phosphorylating capacity. Elasticities and flux control coefficients for the substrate oxidation system, the phosphorylation system and the proton leak over oxygen consumption at maximal phosphorylating capacity for synaptosomes from whole brain of young and old rats
where , and are the elasticities of the substrate oxidation (s), phosphorylation (p) and proton leak (L) systems to changes in ΔΨm and , and are the flux control coefficients for substrate oxidation (s), phosphorylation (p) and proton leak (L) systems over oxygen consumption at maximal phosphorylating capacity for synaptosomes from whole brain of young and old rats.
Flux control coefficients
The investigation describes, for the first time, the pattern of control of mitochondrial energy metabolism in synaptosomes from rat brain as well as a comparison of the mitochondrial control in synaptosomes from old (24 months) and young (3 months) rats. There was no significant difference in the resting (non-stimulated) oxygen consumption rate, mitochondrial membrane potential and plasma membrane potential when comparing synaptosomes isolated from young and old rats. Thus, there was no difference in energy metabolism as manifest in these key parameters. However, the possibility remained that the distribution of control between substrate oxidation, proton leak and the phosphorylation may be different when comparing synaptosomes from young and old animals.
There was also no significant difference between the state 4 (non-phosphorylating) oxygen consumption rates or membrane potentials in synaptosomes from young and old rats, when to compared to their respective resting states, an observation noted previously by Scott and Nicholls (1980) for guinea pig synaptosomes. Clearly, there is insignificant ATP turnover via the mitochondria in non-stimulated synaptosomes and almost all the in situ mitochondrial oxygen consumption under resting conditions, in synaptosomes from both young and old rats, results from proton leak. In quantitative terms, about 85% of the resting oxygen consumption by synaptosomes from young and old animals is a result of mitochondrial proton leak. This observation also has implications for ROS production by the electron transport chain as one would expect ROS production to be higher the closer the mitochondria are poised towards state 4 (Rolfe and Brand 1997).
The in situ oxygen consumption rate due to proton leak in synaptosomes from young and old animals showed a typical non-ohmic relationship (see Fig. 3a,b). Similar non-ohmic relationships between proton leak and mitochondrial membrane potential have been observed in isolated mitochondria (Porter and Brand 1993) and in mitochondria in situ in hepatocytes (Porter and Brand 1995) and thymocytes (Krauss et al. 2002). There was no apparent difference between the mitochondrial proton leak curves in synaptosomes prepared from young and old rats. This suggests that, within the age range studied, there is no significant effect of age on the proton permeability of the inner membrane of neuronal mitochondria. These proton leak data are in good agreement with those of Vitorica et al. (1985) who found no difference in the proton leak rate in a comparison of mitochondria isolated from whole brain of young (2–3 months) and old (32 month) rats.
It was also found that there was no difference in the sensitivities of the substrate oxidation of in situ mitochondria in synaptosomes from old and young rats to changes in ΔΨm. The lack of any significant difference between the synaptosomes from young and older rats at maximal phosphorylating capacity indicates that the capacity of the substrate oxidation system to produce Δp for ATP synthesis is unaffected. Several investigators had found decreases in the specific activities of some of the respiratory complexes in mitochondria isolated from aged animals (Curti et al. 1990; Bowling et al. 1993; Ferrandiz et al. 1994). These might have been expected to decrease the mitochondrial Δp producing capacity. However, if there is a decreased specific activity of the enzyme complexes in synaptosomal mitochondria in situ, it is not manifest at the level of oxidative phosphorylation as measured in this study. A possible explanation for this may be the existence of threshold activities, related to the metabolic control strengths of the different components within the system, whereby a certain amount of a protein complex activity needs to be lost before oxidative phosphorylation is affected, as has been observed for isolated mitochondria from a variety of sources (Davey et al. 1998).
However there was a significantly decreased flux through the substrate oxidation system in synaptosomes from old rats, compared to those from young rats, when the mitochondria were uncoupled with FCCP. Thus an age-dependent effect is seen when the substrate oxidation system is forced to work hard in the absence of a substantial ΔΨm (Table 2). This is not inconsistent with the observation that there were no differences between the oxygen consumption rates in synaptosomes from young and old rats under conditions where mitochondria were producing ATP maximally (in situ state 3). In the uncoupled situation, the substrate oxidation system is running as fast as it can, the electrochemical gradient is abolished and oxygen consumption rate is not limited by the kinetics of the ATP synthase, the adenine nucleotide transporter or ATP usage. By contrast, under state 3 conditions, there is an electrochemical gradient and there are kinetic limitations associated with the ATP synthase and ATP usage. Thus a difference in the substrate oxidation flux is only seen when the limitations associated with the phosphorylating component and the ‘static head’ due to the proton electrochemical gradient are removed. It is possible that this age dependent effect may reflect alterations in the electron transport chain where, as already mentioned, there are reports of decreased electron transport chain activity with age in brain mitochondria. However, in the in situ system studied here, other components of the substrate oxidation system, such as substrate transport across the plasma membrane or mitochondrial inner membrane, glycolysis, the tricarboxylate cycle, might also contribute.
In contrast, there appears to be no significant difference between the kinetic responses of the phosphorylation systems in the synaptosomes from young and old animals to changes in ΔΨm. This observation appears inconsistent, it is worth noting, the report by Polosa and Attardi (1991), who showed a decrease in the level of the ATP synthase F0 component, A8, following in situ translation of mitochondrial DNA of synaptosomal mitochondria from 24-month-old rats compared to synaptosomal mitochondria from 2- to 3-month-old rats.
In conclusion, mitochondria in synaptosomes, in situ, from whole rat brain are not working very hard under resting conditions. Some 85% of the resting oxygen consumption of synaptosomes from both young and old rats, was a result of mitochondrial proton leak (and possibly other ion cycling) across the mitochondrial inner membrane. The majority of the energy requirements for maintaining resting synaptosomal function are met from other metabolic sources. There were no significant age differences in the kinetic responses of the substrate oxidation system, the mitochondrial proton leak and the phosphorylation system to changes in the proton electrochemical gradient.
Thus the age-dependent decrease in the amounts of certain respiratory-chain complexes, which have been reported to occur, do not significantly affect mitochondrial energy producing functions within the synaptosome. These results have implications for the theory that an age-dependent decline in mitochondrial function may underlie some neurodegenerative diseases. However, as synaptosomes from whole rat brain were used in these studies, it is possible that greater levels of impairment may occur in specific brain regions. Metabolic control analysis showed that the substrate oxidation had greatest control over oxygen consumption (i.e. it was more limiting), at maximal phosphorylating capacity for synaptosomes from whole brain, with proton leak, having little control under maximal ATP producing capacity. The pattern of control was similar in synaptosomes from young and old animals. However, there was a significantly lower rate of oxygen consumption in synaptosomes from the old rats in the presence of uncoupler, when compared with synaptosomes from young rats. These results demonstrate that the essential difference between old rats, when compared with those from young animals, is an impaired flux through the synaptosomal substrate oxidation system.
Funding for the project was provided by The Health Research Board of Ireland and BioResearch Ireland.