Non-synaptic Mitochondria Research Articles

Impairment of mitochondrial oxidative phosphorylation in the brain of aged mice

To elucidate the role of mitochondrial oxidative phosphorylation in neuronal aging, we have studied the activity of the respiratory complexes in the brain of young, adult and old mice. In synaptic mitochondria, we found a significant decrease in complexes IV (29%, P < 0.001) and V (21%, P < 0.01) in old as compared with adult mice. Nonsynaptic mitochondria also showed a senescent decrease in complexes I (15%, P < 0.01), II + III (34%, P < 0.01) and IV (17%, P < 0.01) activities. These findings suggest a dysfunction in mitochondrial oxidative phosphorylation in brain aging.

Hyperammonemia and hepatic encephalopathy stimulate rat cerebral synaptic mitochondrial glutamate dehydrogenase activity specifically in the direction of glutamate oxidation

The effects of hepatic encephalopathy (HE) due to thioacetamide (TAA)-induced liver failure and hyperammonemia (HA) produced by repeated i.p. administration of ammonium acetate on the activity of glutamate dehydrogenase (GIDH) in the direction of glutamate (Glu) synthesis from — (GIDH-NADH) or its oxidation to α-ketoglutarate (α-KG) (GlDH-NAD), respectively, were examined in non-synaptic and synaptic mitochondria from rat cerebral hemispheres. In non-synaptic mitochondria, HE and HA stimulated the GlDH-NADH activity by, respectively, 33% and 49%, but neither condition affected the GlDH-NAD activity. In synaptic mitochondria, HE and HA decreased the GlDH-NADH activity by, respectively, 31% and 28%, but stimulated the GlDH-NAD activity by as much as 90% (HE) and 100% (HA). Kinetic assays revealed that HA increased the V max of the synaptic mitochondrial GLDH-NAD by 105%, without affecting the Km for Glu. The stimulation of GlDH-NAD favors the oxidation of synaptic Glu to α-KG, and may represent an adaptive response serving to counteract hyperammonemia-induced decrease of cerebral α-KG production in other metabolic pathways.

Changes in the cytoplasmic (lactate dehydrogenase) and plasma membrane (acetylcholinesterase) marker enzymes in the synaptic and nonsynaptic mitochondria derived from rats with moderate hyperammonemia.

The activities of the cytoplasmic and plasma membrane marker enzymes: lactate dehydrogenase (LDH) and acetylcholinesterase (AChE), respectively, were measured in the cerebral homogenates, in the synaptic and nonsynaptic mitochondrial fractions, and in the postmitochondrial supernatants derived from rats in which a 3-d, moderately hyperammonemic condition (no more than 120% increases in blood ammonia) was produced by repeated administration of ammonium acetate (simple hyperammonemia, SHA) or a hepatotoxin, thioacetamide (TAA) (hepatic encephalopathy, HE). As measured in the homogenate and postmitochondrial supernatants, neither of the enzyme activities was affected by SHA or HE. SHA and HE increased the synaptic mitochondrial LDH activity by respectively 53 and 24%, but reduced this enzyme activity in nonsynaptic mitochondria by 19%. Both conditions stimulated the synaptic and nonsynaptic mitochondrial AChE activity by 30-40%. By contrast, the only significant change produced in these fractions by in vitro treatment with a toxic (3 mM) concentration of ammonium chloride was a slight decrease of LDH activity in nonsynaptic mitochondria and postmitochondrial supernatants. It is concluded that moderate hyperammonemia modifies subsequent separation of both cerebral classes of mitochondria from the cytosolic and plasma membrane components. This modification is likely to reflect subtle hyperammonemia-related changes in the physicochemical properties of the two mitochondrial classes and/or other subcellular components.

Effect of disulfiram (DS) on mitochondria from rat hippocampus: Metabolic compartmentation of DS neurotoxicity

This experiment was designed to study the acute effects of disulfiram on mitochondrial enzymes in nonsynaptic and synaptic mitochondria from rat hippocampus. Cytochrome c oxidase, monoamine oxidase-B, glycerolphosphate acyltransferase and beta-hydroxybutyrate dehydrogenase were studied. Differences in enzyme activity were seen in controls. Cytochrome c oxidase activity was higher in synaptic mitochondria whereas glycerolphosphate acyltransferase activity was higher in nonsynaptic mitochondria. Mitochondria from disulfiram treated rats, particularly synaptic mitochondria, exhibited lower specific activities of cytochrome c oxidase and monoamine oxidase-B. These alterations were not limited to either the inner or outer mitochondrial membrane. Transmission electron microscopy revealed that mitochondria from disulfiram treated rats were severely altered in isolated preparations as well as in those from whole tissue. This study shows that disulfiram exerts a differential effect on mitochondrial subpopulations.

Age-related changes of enzyme activities of non-synaptic and synaptic mitochondria of cerebral cortex and CDP-choline

Age-related changes of enzyme activities of non-synaptic and synaptic mitochondria of cerebral cortex and CDP-choline

In vivo effect of acetyl- l-carnitine on succinate oxidation, adenine nucleotide pool and lipid composition of synaptic and non-synaptic mitochondria from cerebral hemispheres of senescent rats

The effect of acetyl- l-carnitine on succinate oxidation, adenine nucleotide pool and lipid composition of synaptic and ‘free’, non-synaptic, mitochondria in cerebral hemispheres of senescent rats has been studied. Fisher rats (24- or 28-month-old) were treated with acetyl- l-carnitine (300 mg/kg body wt., intraperitoneally (i.p.)) 3 h before being killed. Oxygen consumption was measured using succinate as a substrate; adenine nucleotides and lipids were analyzed by high performance liquid chromatography (HPLC). Acetyl- l-carnitine reverses, in synaptic mitochondria, the age-related decrease in the respiratory control ratio due to a higher state 4 respiration rate. Administration of acetyl- l-carnitine to senescent rats does not affect the total adenine nucleotide pool of synaptic and non-synaptic mitochondria which was unchanged with age. Finally, pretreatment of senescent rats with acetyl- l-carnitine brings the cholesterol and phospholipid contents of synaptic mitochondria, reduced in senescent rats, to the adult level; pretreatment of adult rats has no such effect. Altogether these results suggest that acetyl- l-carnitine is able to reverse age-related deficits of brain mitochondria.

Aspartate aminotransferase, malate dehydrogenase, and pyruvate carboxylase activities in rat cerebral synaptic and nonsynaptic mitochondria: Effects ofin vitro treatment with ammonia, hyperammonemia and hepatic encephalopathy

The effects of in vitro treatment with ammonium chloride, hepatic encephalopathy (HE) due to thioacetamide (TAA) induced liver failure and chronic hyperammonemia produced by i.p. administration of ammonium acetate on the activity of the two malate-aspartate shuttle enzymes: aspartate aminotransferase (AAT), malate dehydrogenase (MDH), and on the pyruvate carboxylase (PC) activity were examined in synaptic and nonsynaptic mitochondria from rat brain. With regard to the shuttle enzymes the response to ammonium ions in vitro (3mM NH4Cl) was observed in nonsynaptic mitochondria only, and was manifested by a 27% decrease of AAT activity and a 16% decrease in MDH activity. By contrast, both in vivo conditions primarily affected the synaptic mitochondrial enzymes: TAA-induced HE produced a 26% decrease of synaptic mitochondrial AAT and a 50% decrease of synaptic mitochondrial MDH. Hyperammonemia inhibited synaptic mitochondrial AAT by 30% and synaptic mitochondrial MDH by 45%. HE produced no effect at all in nonsynaptic mitochondria while hyperammonemia produced a 30% increase in the AAT activity, but no changes in MDH. All the experimental conditions affected the nonsynaptic mitochondria PC: ammonium chloride in vitro produced a 20% decrease, TAA-induced HE--a 30% decrease, whereas hyperammonemia inhibited the enzyme by 53%. The PC activity in synaptic mitochondria was very low (about 2% of that measured in nonsynaptic mitochondria), which is consistent with the primarily astrocytic localization of the enzyme.

Action of L-acetylcarnitine on different cerebral mitochondrial populations from hippocampus and striatum during aging.

The maximum rates (Vmax) of some mitochondrial enzyme activities related to energy transduction (citrate synthase, malate dehydrogenase, NADH cytochrome c reductase, cytochrome oxidase) and amino acid metabolism (glutamate dehydrogenase) were evaluated in non-synaptic (free) and synaptic mitochondria from rat hippocampus and striatum. Three types of mitochondria were isolated from control rats aged 4, 8, 12, 16, 20 and 24 months and treated ones with L-acetylcarnitine (100 mg.kg-1, i.p., 60 min). Enzyme activities of non-synaptic and synaptic mitochondria are different in hippocampus and striatum, confirming that a different metabolic machinery exists in various types of brain mitochondria. During aging, enzyme activities behave quite similarly in both areas. In vivo administration of L-acetylcarnitine decreased the enzyme activities related to Krebs' cycle mainly of synaptic mitochondria, suggesting a specific subcellular trigger site of action. The drug increased cytochrome oxidase activity of synaptic and non-synaptic mitochondria, indicating the specificity of molecular interaction with this enzyme.

Reduced synthesis of mtRNA in isolated mitochondria of senescent rat brain

A system for studying RNA synthesis in isolated mitochondria from rat brain was set up to investigate the mechanisms responsible for the age-dependent reduction of mtRNA content. In the presence of an appropriate incubation buffer both synaptic and non-synaptic mitochondria from cerebral hemispheres were able to synthesize and process mtRNA in a way quantitatively and qualitatively similar to the in vivo transcription. The comparison of the electrophoretic pattern of mtRNAs synthesized by adult and senescent rat showed, in the senescent rat, a 50% reduction in the mtRNA synthesis rate relative to the adult value. This indicates that the age-dependent decrease of the mtRNA content is linked to a lower efficiency of the mt transcription.

Different responses of rat cerebral mitochondrial 2-oxoglutarate dehydrogenase activity to ammonia and hepatic encephalopathy in synaptic and nonsynaptic mitochondria

Abstract The effects of in vitro treatment with ammonium chloride and acute hepatic encephalopathy (HE) induced by thioacetamide treatment (TAA), on the 2-oxoglutarate dehydrogenase (OGDH) activity in synaptic and nonsynaptic mitochondria from rat brain were examined. In control conditions, V max and K m for 2-oxoglutaric acid (2-OG) were higher in the synaptic than in nonsynaptic mitochondria by about 45 and 55%, respectively. A particularly high sensitivity of OGDH to ammonium ions in vitro was observed in nonsynaptic mitochondria, as manifested by a 30% decrease of V max and a 60% decrease of K m for 2-OG. Synaptic mitochondria showed a slight response to HE which was manifested by a 12% increase of V max . In nonsynaptic mitochondria a 19% decrease of K m for 2-OG was observed, but V max was unaffected. Nonsynaptic mitochondria from HE rats reacted to the addition of ammonium ions in vitro with a 30% inhibition of V max but with no alteration of K m for 2-OG. In synaptic mitochondria from HE rats there was a slight inhibition of V max , but an about 15% decrease of K m for 2-OG. Based on these results, the different responses of OGDH in two mitochondrial populations to HE and ammonium ions in vitro would appear to be due to intrinsic differences between the properties of the enzyme in the synaptic and nonsynaptic brain compartments.

Age-related modifications of cytochrome c oxidase activity in discrete brain regions

The apparent K m for cytochrome c cytochrome oxidase does not change but the V max decreases in synaptosomes and non-synaptic mitochonddria isolated from the cerebral cortex as a whole of 30-month-old rats compared with 4-month-old ones. When the subcellular organelles are submitted to stressfull conditions, namely incubation in media of altered osmolality, the percentage of cytochrome oxidase activity released is much higher in senescent rats. The activity of cytochrome oxidase evaluated in non-synaptic mitochondria and synaptosomes isolated from cortical and subcortical regions and cerebellum of rats aged 4 and 30 months shows a highly significant decrease ( P < 0.001) in the parietotemporal cortex of senescent rats (both in non-synaptic mitochondria and synaptosomes) and in the cerebellum (in synaptosomes).

Effect of age on kinetics and carbon monoxide binding to cytochrome oxidase in synaptic and non-synaptic brain mitochondria

The kinetic parameters of cytochrome oxidase activity in synaptic and non-synaptic brain mitochondria from 3- and 30-month-old rats were determined at room temperature. The value of K m for cytochrome c increased only 12–13% with age. The maximal velocity did not change with age, but the value of V max in synaptic mitochondria is twice that observed in non-synaptic mitochondrial cytochrome oxidase. The kinetics of CO binding to cytochrome oxidase at temperatures from 183 to 225K were also studied in synaptic and non-synaptic mitochondria from 3- and 30-month-old rat forebrains. Age-dependent differences were observed only in mitochondria of synaptic origin. Following flash photolysis at low temperatures, CO migration to the iron requires crossing two free energy barriers separating two intermediate regions from the iron. In 3-month-old synaptic mitochondria, CO must migrate across a 10.3 kca/mol barrier separating two intermedite regions, I 2 and I; a 4.7 kcal/mol barrier separates the innermost region I from the iron. Each intermediate region in 3-month-old cytochrome oxidase can hold only one CO molecule. In 30-month-old synaptic mitochondria, 10.3 kcal/mol barriers separate the two intermediate regions as well as region I and the iron; each intermediate region can hold two CO molecules. Region I 2 in non-synaptic cytochrome oxidase at either age can hold two CO molecules and the innermost region I holds only one CO molecule; energy barriers of approximately 10.3 kcal/moIe separate regions I 2, I, and the iron. These age-dependent changes may reflect age-dependent conformational changes in cytochrome oxidase.

Effect of Ca2+-homopantothenate and mild hypoxia on some enzyme activities evaluated in subcellular fractions from different rat brain regions

The effect of Ca2+-homopantothenate (HOPA) treatment (250 mg/kg for 5 d) has been studied by evaluating the specific activity of enzymes related to: glycolytic pathway (hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase), tricarboxylic acid cycle (citrate synthase, malate dehydrogenase), mitochondrial electron transfer chain (succinate dehydrogenase, cytochrome oxidase), NADH redox state (NADH cytochrome c reductase), acetylcholine metabolism (acetylcholinesterase), and glutamate metabolism (glutamate dehydrogenase). The enzymatic activity assays were performed on homogenate in toto, nonsynaptic mitochondria and synaptosomes isolated from: cerebral cortex, hippocampus, striatum, hypothalamus, medulla oblongata, and cerebellum of normoxic rats and rats submitted to intermittent normobaric hypoxia (90:10, N2:O2). In normoxic rats, HOPA was unable to induce any modification. Hypoxia per se induced a decrease in the activity of synaptosomal cytochrome oxidase in cerebral cortex, hippocampus, and cerebellum.

A critique on the preparation and enzymatic characterization of synaptic and nonsynaptic mitochondria from hippocampus.

1. In literature two interesting methods are described to obtain from whole pooled brains or areas three types of mitochondria, namely, those of perikaryal origin and those contained in synaptosomes. 2. However, for many types of studies, such "preparative" preparations are not useful; for example, in pharmacological studies only data from a single n number of animals may be of statistical usefulness and may be correctly analyzed by statistical tests. 3. Thus a method is described by which it was possible to characterize by enzyme activities three populations from single rat brain hippocampus. 4. During preparative "analytical" procedure, it was noted that the 10% Ficoll gradients previously used in the literature were unable to separate purified mitochondria-free mitochondria. This gradient should be 12% Ficoll for single areas. 5. In addition, when results are compared using the more appropriate omega 2t for calculations of gravity forces to be applied instead of the maximum or average g for different rotors, enzymatic characterization differed considerably among the various mitochondrial populations. 6. The above considerations are also true when different pestle clearances and/or pestle rotations speeds are used during omogenizations; also lysis conditions are essential. 7. Results showed that selected experimental conditions are to be used when subcellular fractions are to be analyzed biochemically.

Effect of aging and acetyl- l-carnitine on energetic and cholinergic metabolism in rat brain regions

The effect of aging and subchronic treatment with acetyl- l-carnitine (50 mg/kg per day) was studied on mitochondrial bioenergetics and cholinergic metabolism in non-synaptic mitochondria and synaptosomes isolated from cerebral cortex, hippocampus and striatum of rats aged 4, 11 and 18 months. Respiratory activity and cytochrome oxidase specific activity were unaffected by aging in non-synaptic mitochondria. In synaptosomes, pyruvate dehydrogenase, choline acetyltransferase and acetylcholinesterase specific activity remained unchanged, but the high-affinity choline uptake decreased in cerebral cortex and striatum of 18-month-old rats. Acetyl- l-carnitine treatment increased the high-affinity choline uptake in cerebral cortex of 18-month-old rats. The treatment caused also an increase in cytochrome oxidase activity in all the three cerebral regions and in choline uptake in the hippocampus, parameters that were not directly affected by aging processes.

ADP-ribosylation of highly purified rat brain mitochondria.

Highly purified synaptic and nonsynaptic mitochondria were prepared from rat brain, and their ADP-ribosyl transferase and NAD glycohydrolase activities were investigated. Data show that there is no significant difference in ADP-ribosyl transferase activity between these two types of subcellular preparations. However, NAD glycohydrolase activity appeared to be much higher in nonsynaptic mitochondria. The specific activity of both enzymes was investigated in the presence of the inhibitor nicotinamide or its analogue 3-aminobenzamide or other adenine nucleotides, such as ATP or ADP-ribose. The inhibitory effect of nicotinamide or 3-aminobenzamide on ADP-ribosyl transferase appears rather weak compared with their effect on NAD glycohydrolase activity. However, ADP-ribose and ATP appeared more effective in inhibiting ADP-ribosyl transferase. Our results provide evidence for the existence of ADP-ribosyl transferase activity in rat brain mitochondria. When NAD glycohydrolase was inhibited totally by nicotinamide, the transfer of ADP-ribose from NAD to mitochondrial proteins still occurred. The chain length determinations show that the linkage of ADP-ribose to mitochondrial proteins is oligomeric.

Effect of nimodipine on mitochondrial respiration in different rat brain areas after subarachnoid haemorrhage.

The mitochondrial respiration was evaluated in three different rat brain areas (cerebral cortex, hippocampus and brain stem) after experimental subarachnoid haemorrhage (SAH). The haemorrhage was induced by injecting 0.35 ml of autologous arterial blood into cisterna magna. Intravenous administration of Nimodipine (2 micrograms/kg/min for 30 minutes) was started immediately after the haemorrhage induction. At the set time (1 hour after SAH procedure), animals were sacrificed and non-synaptic mitochondria from the above mentioned areas were isolated. The following respiratory parameters were evaluated utilizing glutamate plus malate and succinate plus rotenone as substrates: state 3, state 4, uncoupled state, respiratory control ratio (RCR) and ADP/O ratio. SAH significantly influences respiratory parameters, mainly RCR; the cerebral cortex and brain stem seem to be more sensitive during the acute phase of vasospasm which follows SAH procedure. Nimodipine treatment significantly ameliorates mitochondrial respiratory conditions.

Brain mitochondrial cytochrome P-450scc: Spectral and catalytic properties

The cytochrome P-450-dependent cholesterol side chain cleavage system of the brain has been studied using nonsynaptic mitochondria as the source of enzymatic activity. The system has been found to bind cholesterol and 11-deoxycorticosterone, producing type I difference spectra, whereas the binding of pregnenolone induced a reverse type I difference spectrum. Inhibitors of cytochrome P-450-linked monooxygenase activities produced type II spectra. The formation of labeled pregnenolone after incubation of brain mitochondria with [4- 14C]cholesterol has been obtained, and this formation was inhibited by glutethimide, a specific inhibitor of cytochrome P-450scc. The functional significance of this enzymatic activity is discussed.

Age-dependent changes in rat brain mitochondria of synaptic and non-synaptic origins

Synaptic and non-synaptic mitochondria were isolated from the brains of 3- 12- and 28–30-month-old female Fisher 344 rats. Total oxygen consumption and oxygen consumption due to mitochondrial respiration decreases 83% with increasing age in synaptic mitochondria using malate plus glutamate as substrate, but only 33% in nonsynaptic mitochondria; succinate-driven activity is not affected. Succinate-driven superoxide generation decreases over 90% in both fractions; malate plus glutamate-driven superoxide generation decreases 50% in synaptic mitochondria only. The amount of c- and a-type cytochromes decreases approximately 50% in synaptic mitochondria. The absorbance wavelength maximum of cytochrome b decrease 2.6 nm is synaptic mitochondria from senescent brains but only 1.6 nm in non-synaptic mitochondria.

Preferential glutamine uptake in rat brain synaptic mitochondria

Glutamine uptake has been studied in purified rat brain mitochondria of synaptic or non-synaptic origin. It was taken up by an active saturable transport mechanism, with an affinity two-times higher in synaptic than in non-synaptic mitochondria ( K m = 0.45 and 0.94 mM, respectively), V max of uptake was 7-times higher in synaptic mitochondria ( V max = 9.2 and 1.3 nmol min per mg protein, respectively). Glutamine transport was found to be inhibited by L-glutamate (IC 50 = 0.64 mM) as well as thiol reagents (mersalyl, N-ethylmaleimide). It is suggested that differential uptake of glutamine in mitochondria of synaptic or nonsynaptic origin may be a major mechanism in the regulation of the synthesis of the neurotransmitter glutamate.