Timothy E. Bates

Inhibition of N-acetylaspartate production: implications for 1H MRS studies in vivo

The effect of specific irreversible inhibitors of complexes I, III, IV and V of the mitochondrial respiratory chain, (rotenone, myxothiazol, cyanide and oligomycin, respectively) on mitochondrial N-acetylaspartate production, and its relationship to oxidative phosphorylation (ATP production and oxygen consumption) were investigated in isolated rat brain mitochondria. Mitochondrial N-acetylaspartate production, ATP production and oxygen consumption were all significantly decreased in the presence of each of the inhibitors used compared with control incubations, and correlated positively with each other. It is postulated that decreased N-acetylaspartate levels seen in disease states by 1H NMR spectroscopy in vivo may reflect primarily an impaired mitochondrial energy production rather than neuronal cell loss.

NO synthase and NO-dependent signal pathways in brain aging and neurodegenerative disorders: the role of oxidant/antioxidant balance.

Nitric oxide and other reactive nitrogen species appear to play several crucial roles in the brain. These include physiological processes such as neuromodulation, neurotransmission and synaptic plasticity, and pathological processes such as neurodegeneration and neuroinflammation. There is increasing evidence that glial cells in the central nervous system can produce nitric oxide in vivo in response to stimulation by cytokines and that this production is mediated by the inducible isoform of nitric oxide synthase. Although the etiology and pathogenesis of the major neurodegenerative and neuroinflammatory disorders (Alzheimers disease, amyothrophic lateral sclerosis, Parkinsons disease, Huntingtons disease and multiple sclerosis) are unknown, numerous recent studies strongly suggest that reactive nitrogen species play an important role. Furthermore, these species are probably involved in brain damage following ischemia and reperfusion, Downs syndrome and mitochondrial encephalopathies. Recent evidence also indicates the importance of cytoprotective proteins such as heat shock proteins (HSPs) which appear to be critically involved in protection from nitrosative and oxidative stress. In this review, evidence for the involvement of nitrosative stress in the pathogenesis of the major neurodegenerative/ neuroinflammatory diseases and the mechanisms operating in brain as a response to imbalance in the oxidant/antioxidant status are discussed.

Mitochondrial involvement in brain function and dysfunction: relevance to aging, neurodegenerative disorders and longevity.

It is becoming increasingly evident that the mitochondrial genome may play a key role in neurodegenerative diseases. Mitochondrial dysfunction is characteristic of several neurodegenerative disorders, and evidence for mitochondria being a site of damage in neurodegenerative disorders is partially based on decreases in respiratory chain complex activities in Parkinsons disease, Alzheimers disease, and Huntingtons disease. Such defects in respiratory complex activities, possibly associated with oxidant/antioxidant balance perturbation, are thought to underlie defects in energy metabolism and induce cellular degeneration. Efficient functioning of maintenance and repair process seems to be crucial for both survival and physical quality of life. This is accomplished by a complex network of the so-called longevity assurance processes, which are composed of genes termed vitagenes. A promising approach for the identification of critical gerontogenic processes is represented by the hormesis-like positive effect of stress. In the present review, we discuss the role of energy thresholds in brain mitochondria and their implications in neurodegeneration. We then review the evidence for the role of oxidative stress in modulating the effects of mitochondrial DNA mutations on brain age-related disorders and also discuss new approaches for investigating the mechanisms of lifetime survival and longevity.

Cellular stress response: a novel target for chemoprevention and nutritional neuroprotection in aging, neurodegenerative disorders and longevity

The predominant molecular symptom of aging is the accumulation of altered gene products. Moreover, several conditions including protein, lipid or glucose oxidation disrupt redox homeostasis and lead to accumulation of unfolded or misfolded proteins in the aging brain. Alzheimer’s and Parkinson’s diseases or Friedreich ataxia are neurological diseases sharing, as a common denominator, production of abnormal proteins, mitochondrial dysfunction and oxidative stress, which contribute to the pathogenesis of these so called “protein conformational diseases”. The central nervous system has evolved the conserved mechanism of unfolded protein response to cope with the accumulation of misfolded proteins. As one of the main intracellular redox systems involved in neuroprotection, the vitagene system is emerging as a neurohormetic potential target for novel cytoprotective interventions. Vitagenes encode for cytoprotective heat shock proteins (Hsp) Hsp70 and heme oxygenase-1, as well as thioredoxin reductase and sirtuins. Nutritional studies show that ageing in animals can be significantly influenced by dietary restriction. Thus, the impact of dietary factors on health and longevity is an increasingly appreciated area of research. Reducing energy intake by controlled caloric restriction or intermittent fasting increases lifespan and protects various tissues against disease. Genetics has revealed that ageing may be controlled by changes in intracellular NAD/NADH ratio regulating sirtuin, a group of proteins linked to aging, metabolism and stress tolerance in several organisms. Recent findings suggest that several phytochemicals exhibit biphasic dose responses on cells with low doses activating signaling pathways that result in increased expression of vitagenes encoding survival proteins, as in the case of the Keap1/Nrf2/ARE pathway activated by curcumin and NAD/NADH-sirtuin-1 activated by resveratrol. Consistently, the neuroprotective roles of dietary antioxidants including curcumin, acetyl-l-carnitine and carnosine have been demonstrated through the activation of these redox-sensitive intracellular pathways. Although the notion that stress proteins are neuroprotective is broadly accepted, still much work needs to be done in order to associate neuroprotection with specific pattern of stress responses. In this review the importance of vitagenes in the cellular stress response and the potential use of dietary antioxidants in the prevention and treatment of neurodegenerative disorders is discussed.

DEPLETION OF BRAIN GLUTATHIONE IS ACCOMPANIED BY IMPAIRED MITOCHONDRIAL-FUNCTION AND DECREASED N-ACETYL ASPARTATE CONCENTRATION

The effect of depletion of reduced glutathione (GSH) on brain mitochondrial function and N-acetyl aspartate concentration has been investigated. Using pre-weanling rats, GSH was depleted by L-buthionine sulfoximine administration for up to 10 days. In both whole brain homogenates and purified mitochondrial preparations complex IV (cytochrome c oxidase) activity was decreased, by up to 27%, as a result of this treatment. In addition, after 10 days of GSH depletion, citrate synthase activity was significantly reduced, by 18%, in the purified mitochondrial preparations, but not in whole brain homogenates, suggesting increased leakiness of the mitochondrial membrane. The whole brain N-acetyl aspartate concentration was also significantly depleted at this time point, by 11%. It is concluded that brain GSH is important for the maintenance of optimum mitochondrial function and that prolonged depletion leads also to loss of neuronal integrity. The relevance of these findings to Parkinsons disease and the inborn errors of glutathione mtabolism are also discussed.

β‐Amyloid fragment 25–35 selectively decreases complex IV activity in isolated mitochondria

Defects in mitochondrial oxidative metabolism, in particular decreased activity of cytochrome c oxidase, have been demonstrated in Alzheimers disease, and after the expression of the amyloid precursor protein (APP) in cultured cells, suggesting that mitochondria might be involved in β‐amyloid toxicity. Recent evidence suggests that the proteolysis of APP to generate β‐amyloid is at least in part intracellular, preceding the deposition of extracellular fibrils. We have therefore investigated the effect of incubation of isolated rat brain mitochondria with the β‐amyloid fragment 25–35 (100 μM) on the activities of the mitochondrial respiratory chain complexes I, II–III, IV (cytochrome c oxidase) and citrate synthase. The peptide caused a rapid, dose‐dependent decrease in the activity of complex IV, while it had no effect on the activities on any of the other enzymes tested. The reverse sequence peptide (35–25) had no effect on any of the activities measured. We conclude that inhibition of mitochondrial complex IV might be a contributing factor to the pathogenesis of Alzheimers disease.

Interpretation of the breath hydrogen profile obtained after ingesting a solid meal containing unabsorbable carbohydrate.

The extent to which monitoring breath hydrogen excretion provides information concerning the entry of the residues of a solid test meal into the colon was investigated in 89 normal subjects, and 11 patients with the irritable bowel syndrome. The profile of breath hydrogen concentration showed an early peak, that occurred soon after ingesting the test meal in 89% subjects. This was followed by a later more prolonged rise in breath hydrogen concentration. The early peak occurred well before a radioactive marker, incorporated in the test meal, reached the caecum and the data suggest it was predominantly caused by the emptying of the remnants of the previous meal from the ileum into the colon. This hypothesis was supported by direct measurements of the rate of delivery of ileostomy effluent in 12 subjects with terminal ileostomies. Fermentation of carbohydrate in the mouth may, however, contribute to the initial peak, but this contribution may be avoided by collecting gas samples from the nares. The secondary rise in breath hydrogen excretion was closely correlated with the arrival of the radioactive marker in the caecum (r = 0.91), p less than 0.001), though the time, at which the secondary peak of breath hydrogen excretion occurred was poorly correlated with the time that all the radioactive test meal had entered the colon. When lactulose was infused directly into the colon, as little as 0.5 g produced a discernible hydrogen response, which occurred within two minutes of the infusion. Increasing the rate of colonic infusion of a 50 ml solution of 10% lactulose from 0.02 to 0.15 g/min in five subjects significantly increased the breath hydrogen concentration. At infusion rates below 0.075 g lactulose/minute, the peak breath hydrogen response preceded the end ot the infusion, while at higher rates of infusion, the peak hydrogen response occurred after the end of the infusion. Although these results confirmed that monitoring breath hydrogen concentration usefully signalled the time taken for a meal containing unabsorbed carbohydrate to reach the colon, it did not reliably indicate the time when all of the meal had entered the colon. Finally, the use of the maximum increase in breath hydrogen concentration as an index of the degree of carbohydrate malabsorption assumes uniform rates of entry into the colon.

Activation of murine microglial cell lines by lipopolysaccharide and interferon-gamma causes NO-mediated decreases in mitochondrial and cellular function.

Activation of murine microglial and macrophage cell lines with lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ) resulted in the induction of the inducible form of nitric oxide synthase (NOS) and the release of micromolar amounts of NO into the surrounding medium. The synthesis of NO was associated with increased cellular membrane damage as assessed by trypan blue dye exclusion and the leakage of lactate dehydrogenase into the cell culture medium. However, the synthesis and release of cytokines was largely unaffected. NO‐mediated cell damage was also accompanied by a marked decrease in the intracellular levels of reduced glutathione and ATP. In addition, significant inhibition of mitochondrial respiratory chain enzyme activities was seen following cellular activation. However, citrate synthase activity (a mitochondrial matrix enzyme) was not detectable in the extracellular supernatants, suggesting preservation of the integrity of the mitochondrial inner membrane following activation. These effects were largely prevented by the addition of the NOS inhibitor, N‐guanidino monomethyl l‐arginine during the activation period. Our observations demonstrate that induction of NOS activity in microglia results in damage to the plasma membrane leading to a loss of glutathione, complex‐specific inhibition of the mitochondrial electron transport chain and depletion of cellular ATP. Our data suggest that pharmacological modulation of NOS activity in activated microglia in vivo may prevent cellular damage to bystander cells such as neurons, astrocytes and oligodendrocytes, as well as to microglia themselves.

Effect of Reperfusion Following Cerebral Ischaemia on the Activity of the Mitochondrial Respiratory Chain in the Gerbil Brain

Abstract: The effect of reperfusion following 30 min of cerebral ischaemia on brain mitochondrial respiratory chain activity has been studied in the gerbil. The state 3 respiration rates with both FAD‐ and NAD‐linked substrates were reduced after ischaemia. After 5 min of reperfusion, state 3 respiration with FAD‐linked substrates was restored, but levels of NAD‐linked substrates did not return to control values until 30 min of reperfusion. By 120 min of reperfusion state 3 respiration decreased relative to control values with all substrates studied. Measurement of the individual respiratory chain complexes showed that complex I, complex II–III, and complex V activities were reduced after ischaemia. By 5 min of reperfusion complex II–III activity was restored, but the activities of complexes I and V did not return to control values until 30 min of reperfusion. In contrast, complex IV activity was unaffected by ischaemia or 5 and 30 min of reperfusion but was significantly reduced after 120 min of reperfusion, possibly owing to free radical production and lipid peroxidation.

Characteristics of the calcium-triggered mitochondrial permeability transition in nonsynaptic brain mitochondria: effect of cyclosporin A and ubiquinone O

Abstract: The objective of the present study was to assess the capacity of nonsynaptic brain mitochondria to accumulate Ca2+ when subjected to repeated Ca2+ loads, and to explore under what conditions a mitochondrial permeability transition (MPT) pore is assembled. The effects of cyclosporin A (CsA) on Ca2+ accumulation and MPT pore assembly were compared with those obtained with ubiquinone 0 (Ub0), a quinone that is a stronger MPT blocker than CsA, when tested on muscle and liver mitochondria. When suspended in a solution containing phosphate (2 mM) and Mg2+ (1 mM), but no ATP or ADP, the brain mitochondria had a limited capacity to accumulate Ca2+ (210 nmol/mg of mitochondrial protein). Furthermore, when repeated Ca2+ pulses (40 nmol/mg of protein each) saturated the uptake system, the mitochondria failed to release the Ca2+ accumulated. However, in each instance, the first Ca2+ pulse was accompanied by a moderate release of Ca2+, a release that was not observed during the subsequent pulses. The initial release was accompanied by a relatively marked depolarization, and by swelling, as assessed by light‐scattering measurements. However, as the swelling was <50% of that observed following addition of alamethicin, it is concluded that the first Ca2+ pulse gives rise to an MPT in a subfraction of the mitochondrial population. CsA, an avid blocker of the MPT pore, only marginally increased the Ca2+‐sequestrating capacity of the mitochondria. However, CsA eliminated the Ca2+ release accompanying the first Ca2+ pulse. The effects of CsA were shared by Ub0, but when the concentration of Ub0 exceeded 20 μM, it proved toxic. The results thus suggest that brain mitochondria are different from those derived from a variety of other sources. The major difference is that a fraction of the brain mitochondria, studied presently, depolarized and showed signs of an MPT. This fraction, but not the remaining ones, contributed to the chemically and electron microscopically verified mitochondrial swelling.