Threshold Effects in Synaptosomal and Nonsynaptic Mitochondria from Hippocampal CA1 and Paramedian Neocortex Brain Regions

Abstract: After a brief period of global ischemia, the hippocampal CA1 region is more susceptible to irreversible damage than the paramedian neocortex. To test whether primary differences in bioenergetic parameters may be present between these regions, respiration rates and respiratory control activities were measured. In synaptosomal and nonsynaptic mitochondria isolated from the hippocampal CA1 region, state 3 respiration rates and complex IV activities were significantly lower than those present in synaptosomal and nonsynaptic mitochondria from the paramedian neocortex. These results suggest that mitochondria from the CA1 hippocampal area differ in some properties of metabolism compared with the neocortex area, which may render them more susceptible to a toxic insult such as that of ischemia. In addition, when complex I and IV activities were titrated with specific inhibitors, thresholds in ATP synthesis and oxygen respiration became apparent. Complex I and IV activities were decreased by 60% in nonsynaptic mitochondria from the hippocampal CA1 region and paramedian neocortex before oxidative phosphorylation was severely compromised; however, in synaptosomes from these regions, complex I activities had a threshold of 25%, indicating heterogenous behaviour for brain mitochondria. Reduced complex I thresholds in mitochondria, in association with other constitutive defects in energy metabolism, may induce a decreased ATP supply in the synaptic region. The implications of these findings are discussed in relation to delayed neuronal death and processes of neurodegeneration.     

Circadian Rhythms of Oxidative Phosphorylation: Effects of Rotenone and Melatonin on Isolated Rat Brain Mitochondria

Mitochondrial experiments are of increasing interest in different fields of research. Inhibition of mitochondrian activities seems to play a role in Parkinson's disease and in this regard several animal models have used inhibitors of mitochondrial respiration such as rotenone or MPTP. Most of these experiments were done during the daytime. However, there is no reason for mitochondrial respiration to be constant during the 24h. This study investigated the circadian variation of oxidative phosphorylation in isolated rat brain mitochondria and the administration-time-dependent effect of rotenone and melatonin. The respiratory control ratio, state 3 and state 4, displayed a circadian fluctuation. The highest respiratory control ratio value (3.01) occurred at 04:00h, and the lowest value (2.63) at 08:00h. The highest value of state 3 and state 4 oxidative respiration occurred at 12:00h and the lowest one at 20:00h. The 24h mean decrease in the respiratory control ratio following incubation with melatonin and rotenone was 7 and 32%, respectively; however, the exact amount of the inhibition exerted by these agents varied according to the time of the mitochondria isolation. Our results show the time of mitochondrial isolation could lead to interindividual variability. When studies require mitochondrial isolation from several animals, the time between animal experiments has to be minimized. In oxidative phosphorylation studies, the time of mitochondria isolation must be taken into account, or at least specified in the methods section.     

Decreased Brain Protein Levels of Cytochrome Oxidase Subunits in Alzheimer's Disease and in Hereditary Spinocerebella Ataxia Disorders

Abstract : Controversy exists as to the clinical importance, cause, and disease specificity of the cytochrome oxidase (CO) activity reduction observed in some patients with Alzheimer's disease (AD). Although it is assumed that the enzyme is present in normal amount in AD, no direct measurements of specific CO protein subunits have been conducted. We measured protein levels of CO subunits encoded by mitochondrial (COX I, COX II) and nuclear (COX IV, COX VIc) DNA in autopsied brain of patients with AD whom we previously reported had decreased cerebral cortical CO activity. To assess disease specificity, groups of patients with spinocerebellar ataxia type I and Friedreich's ataxia were also included. As compared with the controls, mean protein concentrations of all four CO subunits were significantly decreased (-19 to -47%) in temporal and parietal cortices in the AD group but were not significantly reduced (-12 to -17%) in occipital cortex. The magnitude of the reduction in protein levels of the CO subunits encoded by mitochondrial DNA (-42 to -47%) generally exceeded that encoded by nuclear DNA (-19 to -43%). In the spinocerebellar ataxia disorders, COX I and COX II levels were significantly decreased in cerebellar cortex (-22 to -32%) but were normal or close to normal in cerebral cortex, an area relatively unaffected by neurodegeneration. We conclude that protein levels of mitochondrial- and nuclear-encoded CO subunits are moderately reduced in degenerating but not in relatively spared brain areas in AD and that the decrease is not specific to this disorder. The simplest explanation for our findings is that CO is decreased in human brain disorders as a secondary event in brain areas having reduced neuronal activity or neuronal/synaptic elements consequent to the primary neurodegenerative process.     

Response of Isolated Rat Liver Mitochondria to Variation of External Osmolarity in KCl Medium: Regulation of Matrix Volume and Oxidative Phosphorylation

When isolated rat liver mitochondria are incubated in KCl medium, matrix volume, flux, and forces in both hypo- and hyperosmolarity are time-dependent. In hypoosmotic KCl medium, matrix volume is regulated via the K⁺/H⁺ exchanger. In hyperosmotic medium, the volume is regulated in such a manner that at steady state, which is reached within 4 min, it is maintained whatever the hyperosmolarity. This regulation is Pi- and -dependent, indicating Pi-K salt entry into the matrix. Under steady state, hyperosmolarity has no effect on isolated rat liver mitochondria energetic parameters such as respiratory rate, proton electrochemical potential difference, and oxidative phosphorylation yield. Hypoosmolarity decreases the NADH/NAD⁺ ratio, state 3 respiratory rate, and , while oxidative phosphorylation yield is not significantly modified. This indicates kinetic control upstream the respiratory chain. This study points out the key role of potassium on the regulation of matrix volume, flux, and forces. Indeed, while matrix volume is regulated in NaCl hyperosmotic medium, flux and force restoration in hyperosmotic medium occurs only in the presence of external potassium.     

Control of oxidative phosphorylation by Complex I in rat liver mitochondria: implications for aging

We compared NAD-dependent state 4 and state 3 respiration, NADH oxidation and Complex I specific activity in liver mitochondria from 4- and 30-month-old rats. All the activities examined were significantly decreased with aging. In both groups of animals, the flux control coefficients measured by rotenone titration indicated that Complex I is largely rate controlling upon NADH aerobic oxidation while, in state 3 respiration, it shares the control with other steps in the pathway. Moreover, we observed a trend wherein flux control coefficients of Complex I became higher with age. This indication was strengthened by examining the rotenone inhibition thresholds showing that Complex I becomes more rate controlling, over all the examined activities, during aging. Our results point out that age-related alterations of the mitochondrial functions are also present in tissues considered less prone to accumulate mitochondrial DNA mutations.     

Metabolic consequences of the cytochrome c oxidase deficiency in brain of copper-deficient Mo(vbr) mice

Biochemical micromethods were used for the investigation of changes in mitochondrial oxidative phosphorylation associated with cytochrome c oxidase deficiency in brain cortex from Mo(vbr) (mottled viable brindled) mice, an animal model of Menkes' copper deficiency syndrome. Enzymatic analysis of cortex homogenates from Mo(vbr) mice showed an approximately twofold decrease in cytochrome c oxidase and a 1.4-fold decrease in NADH:cytochrome c reductase activities as compared with controls. Assessment of mitochondrial respiratory function was performed using digitonin-treated homogenates of the cortex, which exhibited the main characteristics of isolated brain mitochondria. Despite the substantial changes in respiratory chain enzyme activities, no significant differences were found in maximal pyruvate or succinate oxidation rates of brain cortex homogenates from Mo(vbr) and control mice. Inhibitor titrations were used to determine flux control coefficients of NADH:CoQ oxidoreductase and cytochrome c oxidase on the rate of mitochondrial respiration. Application of amobarbital to titrate the activity of NADH:CoQ oxidoreductase showed very similar flux control coefficients for control and mutant animals. Alternately, titration of respiration with azide revealed for Mo(vbr) mice significantly sharper inhibition curves than for controls, indicating a more than twofold elevated flux control coefficient of cytochrome c oxidase. Owing to the reserve capacity of respiratory chain enzymes, the reported changes in activities do not seem to affect whole-brain high-energy phosphates, as observed in a previous study using 31P NMR.     

Effect of Aging on the Metabolism of Pyruvate and 3-Hydroxybutyrate in Nonsynaptic and Synaptic Mitochondria from Rat Brain

Abstract: Age-dependent changes in the oxidative metabolism in nonsynaptic and synaptic mitochondria from brains of 3, 12, and 24-month-old rats were investigated. When pyruvate and malate were used in conjunction as substrates, a significant reduction in State 3 respiration was observed in both mitochondrial populations from 12-and 24-month-old rats compared with 3-month-old animals. A similar age-dependent reduction in the oxidation of [1-¹¹C]pyruvate was also observed in nonsynaptic and synaptic mitochondria from senescent rats. Pyruvate dehydrogenase complex activity (both active and total) was, however, not decreased in the two mitochondrial populations from brains of 3, 12, and 24-month-old rats. When DL-3-hydroxybutyrate plus malate were used as substrates, a decrease in State 3 respiration was observed only in synaptic mitochondria from 24-month-old rats compared with 3- month-old animals. Similarly, an age-dependent reduction in the oxidation of 3-hydroxy[3-¹¹C]butyrate was also observed only in synaptic mitochondria from 12-and 24-month-old rats. However, a significant reduction in the activities of ketone body-metabolizing enzymes, namely, 3-hydroxybutyrate dehydrogenase, 3-ketoacid CoA transferase, and acetoacetyl-CoA thiolase was observed in both mitochondrlal populations from 12- and 24-month-old rats compared with 3 month-old animals. These findings show that specific alterations in oxidative metabolism occur in nonsynaptic and synaptic mitochondria from aging rats. The data also suggest that in addition to alterations in enzyme activities, permeability of anions (e.g. pyruvate) across the inner mitochondrial membrane may be altered in nonsynaptic and synaptic mitochondria from senescent animals.     

Effects of Aluminum and Calcium on Acetyl-CoA Metabolism in Rat Brain Mitochondria

Abstract: Al complexes are known to accumulate in extra- and intracellular compartments of the brain in the course of different encephalopathies. In this study possible effects of Al accumulation in the cytoplasmic compartment on mitochondrial metabolism were investigated. Al, like Ca, inhibited pyruvate utilization as well as citrate and oxoglutarate accumulation by whole brain mitochondria. Potencies of Ca²⁺_total effects were 10–20 times stronger than those of Al. Al decreased mitochondrial acetyl-CoA content in a concentration-dependent manner, along with an equivalent rise of free CoA level, whereas Ca caused loss of both intermediates from mitochondria. In the absence of P_i in the medium, Ca had no effect on mitochondrial metabolism, whereas Al lost its ability to suppress pyruvate utilization and acetyl-CoA content in Ca-free conditions. Verapamil potentiated, whereas ruthenium red reversed, Ca-evoked suppression of mitochondrial metabolism. On the other hand, in Ca-supplemented medium, Al partially overcame the inhibitory influence of verapamil. Accordingly, verapamil increased mitochondrial Ca levels much more strongly than Al. However, Al partially reversed the verapamil-evoked rise of Ca²⁺_total level. These data indicate that Al accumulated in cytoplasm in the form of the Al(PO₄)OH⁻ complex may inhibit mitochondrial functions by an increase of intramitochondrial [Ca²⁺]_total resulting from the Al-evoked rise of cytoplasmic [Ca²⁺]_free, as well as from inhibitory interference with the verapamil binding site on the Na⁺/Ca²⁺ antiporter.     

Localization at Complex I and Mechanism of the Higher Free Radical Production of Brain Nonsynaptic Mitochondria in the Short-Lived Rat Than in the Longevous Pigeon

Free radical production and leak of brain nonsynaptic mitochondria were higher with pyruvate/malate than with succinate in rats and pigeons. Rotenone, antimycin A, and myxothiazol maximally stimulated free radical production with pyruvate/malate but not with succinate. Simultaneous treatment with myxothiazol plus antimycin A did not decrease the stimulated rate of free radical production brought about independently by any of these two inhibitors with pyruvate/malate. Thenoyltrifluoroacetone did not increase free radical production with succinate. No free radical production was detected at Complex IV. Free radical production and leak with pyruvate/malate were higher in the rat (maximum longevity 4 years) than in the pigeon (maximum longevity 35 years). These differences between species disappeared in the presence of rotenone. The results localize the main free radical production site of nonsynaptic brain mitochondria at Complex I. They also suggest that the low free radical production of pigeon brain mitochondria is due to a low degree of reduction of Complex I in the steady state in this highly longevous species.     

Endogenous Protein Phosphorylation in Rat Brain Mitochondria: Occurrence of a Novel ATP-Dependent Form of the Autophosphorylated Enzyme Succinyl-CoA Synthetase

Abstract: When rat brain mitochondria are incubated with [γ-³²P]ATP, there is a rapid (10 s) phosphorylation of proteins designated E, and F of M.W. 42,000 and 32,000, respectively. Although [γ-³²P]ATP was the preferred substrate for protein F, a small amount of labeling did occur with [γ-³²P]GTP. Phosphorylation of E₁ was absolutely ATP-dependent. On the other hand, a 32,000 M.W. protein from rat liver mitoplasts (mitochondria devoid of an outer membrane) was highly phosphorylated when [γ-³²P]GTP was used but not at all phosphorylated within short time periods with [γ-³²P]ATP. Both the ATP-labeled brain phosphoprotein F and GTP-labeled liver protein migrated to identical positions on high-resolution two-dimensional polyacrylamide gels, and both contained acid-labile phosphoryl groups. Furthermore, both phosphoproteins were identified as the autophosphorylated subunit of succinyl-CoA synthetase (SCS, EC 6.2.1.4) by using antibody directed against purified GTP-dependent porcine SCS. However, immunotitration experiments with anti-porcine SCS revealed that ATP- and GTP-labeled protein F in brain differed in their interactions with antibody, suggesting that in rat brain mitochondria two different forms of the enzyme exist that are immunologically distinct and differ in substrate specificity. When mitochondrial preparations enriched in particular brain cell or subcellular types were examined, an unequal distribution of E₁ and the two forms of protein F were observed. A brain subfraction containing neuronal cell body and glial mitochondria (CM) was found to contain E₁ and approximately equal amounts of the ATP- and GTP-dependent forms of protein F. Light synaptic mitochondria(SM₁) contained ATP-dependent protein F almost exclusively and were depleted in E₁. Dense synaptic mitochondria (SM₂) are rich in the ATP form of SCS but also contain low amounts of the GTP enzyme.     

The Involvement of Protein Phosphorylation/Dephosphorylation in the Redox Control of Translation in Cereal Mitochondria

A comparative analysis of a dependence of protein synthesis in mitochondria of cultivated (Zea mays) and wild (Elymus sibiricus) cereals on redox conditions showed that the addition of oxidized glutathione enhanced and the addition of reduced glutathione suppressed translation in organello. Inhibitors of protein kinases and protein phosphatases modified substantially the effects of redox agents on protein synthesis in mitochondria. It is supposed that protein phosphorylation in mitochondria may be a mechanism mediating the interrelation between the redox state of the respiratory chain and the activity of mitochondrial translation.     

Effects of 1-Methyl-4-Phenylpyridinium on Isolated Rat Brain Mitochondria: Evidence for a Primary Involvement of Energy Depletion

Abstract: The effects of 1-methyl-4-phenylpyridinium (MPP⁺) on the oxygen consumption, ATP production, H₂O₂ production, and mitochondrial NADH-CoQ₁ reductase (complex I) activity of isolated rat brain mitochondria were investigated. Using glutamate and malate as substrates, concentrations of 10–100 µ M MPP⁺ had no effect on state 4 (−ADP) respiration but decreased state 3 (+ADP) respiration and ATP production. Incubating mitochondria with ADP for 30 min after loading with varying concentrations of MPP⁺ produced a concentration-dependent decrease in H₂O₂ production. Incubation of mitochondria with ADP for 60 min after loading with 100 µ M MPP⁺ caused no loss of complex I activity after washing of MPP⁺ from the mitochondrial membranes. These data are consistent with MPP⁺ initially binding specifically to complex I and inhibiting both the flow of reducing equivalents and the production of H₂O₂ by the mitochondrial respiratory chain, without irreversibly damaging complex I. However, mitochondria incubated with H₂O₂ in the presence of Cu²⁺ ions showed decreased complex I activity. This study provides additional evidence that cellular damage initiated by MPP⁺ is due primarily to energy depletion caused by specific binding to complex I, any increased damage due to free radical production by mitochondria being a secondary effect.     

The Activities of Some Energy-Metabolising Enzymes in Nonsynaptic (Free) and Synaptic Mitochondria Derived from Selected Brain Regions

Abstract: The enzyme complement of two different mitochondrial preparations from adult rat brain has been studied. One population of mitochondria (synaptic) is prepared by the lysis of synaptosomes, the other (nonsynaptic or free) by separation from homogenates. These populations have been prepared from distinct regions of the brain: cortex, striatum, and pons and medulla oblongata. The following enzymes have been measured: pyruvate dehydrogenase (EC 1.2.4.1), citrate synthase (EC 4.1.3.7), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41), NADP-linked isocitrate dehydrogenase (EC 1.1.1.42), fumarase (EC 4.2.1.2), NAD-linked malate dehydrogenase (EC 1.1.1.37), D-3-hydroxybutyrate dehydrogenase (EC 1.1.1.30), and mitochondrially bound hexokinase (EC 2.7.1.1) and creatine kinase (EC 2.7.3.2). The nonsynaptic (free) mitochondria show higher enzyme specific activities in the regions studied than the corresponding values recorded for the synaptic mitochondria. The significance of these observations is discussed in the light of the different metabolic activities of the two populations of mitochondria and the compartmentation of the metabolic activities of the brain.     

Age-related Oxidative Modifications of Proteins and Lipids in Rat Brain

Oxidants have been shown to play a major role in ageing and ageing-related neurodegenerative diseases. In the present study, we investigated the effect of ageing on oxidative damage to lipids and proteins in brain homogenate, mitochondria and synaptosomes of adult (6-month-old), old (15-month-old), and senescent (26-month-old) Wistar rats. There was a significant increase in thiobarbituric acid-reactive substances and conjugated dienes in homogenates, which indicate increased lipid peroxidation (LPO). Oxidative modifications of homogenate proteins were demonstrated by a loss of sulfhydryl content, accumulation of dityrosines and formation of protein conjugates with LPO-end products. Increase in protein conjugates with LPO-end products and a decrease in SH groups were observed also in mitochondria and synaptosomes, but dityrosine content was elevated only in synaptosomes. Protein surface hydrophobicity, measured by fluorescent probe 1-anilino-8-naphthalenesulfonate (ANS), was increased only in homogenate. These results suggest that besides mitochondria and synaptosomes other cellular compartments are oxidatively modified during brain ageing.     

Normal Mitochondrial Genome in Brain from Patients with Parkinson''s Disease and Complex I Defect

The mitochondrial genome codes for 13 proteins which are located in the respiratory chain. In postmortem brain of patients with Parkinson's disease, decreased activity of complex I of the respiratory chain could be demonstrated. Because seven subunits of complex I are coded by the mitochondrial genome, we analyzed the mitochondrial DNA of human postmortem substantia nigra, putamen, and frontal cortex by the Southern blot technique. No deletions of the mitochondrial genome could be demonstrated, thus indicating that either subunits which are encoded by the nuclear genome are decreased or enzyme activity is diminished by metabolites, toxins, or increase of Fe3+.     

Effects of Vitamin A and Its Analogs on Nonenzymatic Lipid Peroxidation in Rat Brain Mitochondria

Vitamin A (retinol) and some of its analogs exhibited varying degrees of inhibition on induced iron and ascorbic acid lipid peroxidation of rat brain mitochondria. Malonyldialdehyde production was used as an index of the extent of in vitro lipid peroxidation. The fat-soluble vitamins retinol, retinol acetate, retinoic acid, retinol palmitate, and retinal at concentrations between 0.1 and 10.0 mmol/L inhibited brain lipid peroxidation. Retinol and retinol acetate were the most effective inhibitors. It is concluded from this study that retinol and its analogs can be considered as potential antioxidant factors, more potent than some of the well-known antioxidants such as alpha-tocopherol and butylated hydroxytoluene.     

Enhanced Oxidative Stress and Altered Antioxidants in Brains of Bcl-2-Deficient Mice

Abstract: Bcl-2 is an antiapoptotic protein located in the outer mitochondrial membrane. Cellular perturbations associated with programmed cell death may be the consequence of disrupted mitochondrial function as well as excessive production of reactive oxygen species (ROS). Numerous studies indicate that Bcl-2 is involved in opposing cell death induced by oxidative stimuli, but its mode of action is uncertain. We reexamined the role of Bcl-2 by using a loss-of-function model, Bcl-2 knockout mice. Brains from Bcl-2 -deficient mice had a 43% higher content of oxidized proteins and 27% lower number of cells in the cerebellum relative to wild-type mice. Incubation of cerebellar neurons from Bcl-2 +/+ brains with 0.5 m M dopamine caused 25% cell death, whereas in Bcl-2 -deficient cells, it resulted in 52% death; glial cells provided protection in both cultures. Splenocytes from Bcl-2 -deficient mice were also killed more effectively by dopamine as well as paraquat. Bcl-2 -deficient mice did not survive intraperitoneal injection of MPTP, which caused a decrease in dopamine level in the striatum of Bcl-2 +/− brains, which was more significant than in wild-type mice. When compared with Bcl-2 +/+ brains, brains of 8-day-old Bcl-2 -deficient mice had higher activities of the antioxidant enzymes GSH reductase (192%) and GSH transferase (142%), whereas at the age of 30 days, GSH peroxidase was significantly lower (66%). Activities of GSH transferase and GSH reductase increased significantly (158 and 262%, respectively) from day 8 to day 30 in Bcl-2 +/+ mice, whereas GSH peroxidase decreased (31%) significantly in Bcl-2 −/− animals. In summary, our results demonstrated enhanced oxidative stress and susceptibility to oxidants as well as altered levels of antioxidant enzymes in brains of Bcl-2 -deficient mice. It is concluded that Bcl-2 affects cellular levels of ROS, which may be due to an effect either on their production or on antioxidant pathways.     

Estimating Hydroxyl Radical Content in Rat Brain Using Systemic and Intraventricular Salicylate: Impact of Methamphetamine

Abstract: Free radicals have been implicated in the etiology of many neurodegenerative conditions. Yet, because these species are highly reactive and thus short-lived it has been difficult to test these hypotheses. We adapted a method in which hydroxyl radicals are trapped by salicylate in vivo, resulting in the stable and quantifiable products, 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA. After systemic (100 mg/kg i.p.) or intraventricular (4 µmol) administration of salicylate, the amount of DHBA in striatal tissue correlated with tissue levels of salicylate. After systemic salicylate, the ratio of total DHBA to salicylate in neostriatum was at least 10-fold higher than that observed after central salicylate. In addition, systemic salicylate resulted in considerably higher concentrations of 2,3- and 2,5-DHBA in plasma than in brain. Therefore, a large portion of the DHBA present in brain after systemic salicylate may have been formed in the periphery. A neurotoxic regimen of methamphetamine increased the concentration of DHBA in neostriatum after either central or systemic administration of salicylate. The increase in 2,3-DHBA after the central administration of salicylate was significant at 2 h, but not at 4 h, after the last dose of methamphetamine. These results suggest that (1) when assessing specific events in brain, it is preferable to administer salicylate centrally, and (2) neurotoxic doses of methamphetamine increase the hydroxyl radical content in brain in a time-dependent manner.     

Cholinergic Muscarinic Receptors in Human Fetal Brain: Ontogeny of [3H]Quinuclidinyl Benzilate Binding Sites in Corpus Striatum, Brainstem, and Cerebellum

This study investigated mitochondrial respiratory activity in Huntington's disease (HD) brain. Mitochondrial membranes from caudate and cortex of HD and non-HD autopsied brains were assayed for succinate oxidation, cytochrome oxidase activity, and cytochromes b, cc1, and aa3. There was a significant decrease in HD caudate mitochondrial respiration, cytochrome oxidase activity, and cytochrome aa3, whereas cytochromes b and cc1 were normal. These findings are consistent with the hypothesis that mitochondrial dysfunction may contribute to the localized hypometabolism and progressive atrophy of the HD caudate.