Ca2+ and mitochondria as substrates for deficits in synaptic plasticity in normal brain ageing

Normal brain ageing is associated with a degree of functional impairment of neuronal activity that results in a reduction in memory and cognitive functions. One mechanism proposed to explain the age-dependent changes was the "Ca(2+) hypothesis of ageing" but data accumulated in the last decade revealed a number of inconsistencies. Two important questions were raised: (a) which are, if any, the most reliable age-associated change in neuronal Ca(2+) homeostasis and (b) are these changes primary, and thus determinant of the ageing phenotype, or are they secondary to other changes in the physiology of the aged neurones. After a brief review of the evidence accumulated for the age-induced changes in synaptic plasticity, we assess the proposal that these changes are, ultimately, determined by changes in the metabolic state of the aged neurones, that are manifest particularly after neuronal stimulation. In this context, it appears that the changes in mitochondrial status and function are of primary importance.     

Age-related structural and functional changes of brain mitochondria

Normal ageing is associated with a gradual decline in the capacity of various cell types, including neurones, to respond to metabolic stress and return to the resting state. An important factor in the decrease of this 'homeostatic reserve' is the gradual, age-dependent impairment of mitochondrial function. In this article we review some of the major structural and functional changes in mitochondria associated with ageing. Apart from the increased mutations in mitochondrial DNA and the evidence for increased oxidative stress with ageing, we also discuss, in some detail, the importance of the mitochondrial membrane structure and composition (in particular lipid composition) for mitochondrial function in general and during ageing. Although some of the neurodegenerative diseases are also associated with some degree of mitochondrial dysfunction, it is not yet clear if these changes are due to the underlining process of normal, physiological ageing or due to the specific pathophysiologic agents responsible for the neurodegenerative processes. Furthermore, we are proposing that there are important differences between normal ageing and neurodegeneration.     

Neuronal ageing from an intraneuronal perspective: roles of endoplasmic reticulum and mitochondria

The nature of brain ageing and the age-dependent decline in cognitive functions remains poorly understood. Physiological brain ageing is characterised by mild mental dysfunctions, whereas age-dependent neurodegeneration, as illustrated by Alzheimer disease (AD), results rapidly in severe dementia. These two states of the aged brain, the physiological and the pathological, are fundamentally different as the latter stems from significant neuronal loss, whereas the former develops without significant neuronal demise. In this paper, we review the changes in neuronal Ca(2+) homeostasis that occur during brain ageing, and conclude that normal, physiological ageing is characterised mainly by a decrease of neuronal homeostatic reserve, defined as the capacity to respond effectively to functional and metabolic stressors, but does not reach the trigger required to induce neuronal death. In contrast, during neurodegenerative states, Ca(2+) homeostasis is affected early during the pathological process and result in significant neuronal demise. We also review recent evidence suggesting that the endoplasmic reticulum (ER) might play an important role in controlling the balance between healthy and pathological neuronal ageing.     

Modulation of the matrix redox signaling by mitochondrial Ca~(2+)

Mitochondria sense,shape and integrate signals,and thus function as central players in cellular signal transduction. Ca2+ waves and redox reactions are two such intracellular signals modulated by mitochondria. Mitochondrial Ca2+ transport is of utmost physio-pathological relevance with a strong impact on metabolism and cell fate. Despite its importance,the molecular nature of the proteins involvedin mitochondrial Ca2+ transport has been revealed only recently. Mitochondrial Ca2+ promotes energy metabolism through the activation of matrix dehydrogenases and downstream stimulation of the respiratory chain. These changes also alter the mitochondrial NAD(P)H/NAD(P)+ ratio,but at the same time will increase reactive oxygen species(ROS) production. Reducing equivalents and ROS are having opposite effects on the mitochondrial redox state,which are hard to dissect. With the recent development of genetically encoded mitochondrial-targeted redoxsensitive sensors,real-time monitoring of matrix thiol redox dynamics has become possible. The discoveries of the molecular nature of mitochondrial transporters of Ca2+ combined with the utilization of the novel redox sensors is shedding light on the complex relation between mitochondrial Ca2+ and redox signals and their impact on cell function. In this review,we describe mitochondrial Ca2+ handling,focusing on a number of newly identified proteins involved in mitochondrial Ca2+ uptake and release. We further discuss our recent findings,revealing how mitochondrial Ca2+ influences the matrix redox state. As a result,mitochondrial Ca2+ is able to modulate the many mitochondrial redox-regulated processes linked to normal physiology and disease.     

Ca2+-induced oxidative stress in brain mitochondria treated with the respiratory chain inhibitor rotenone

In this study we show that micromolar Ca(2+) concentrations (>10 microM) strongly stimulate the release of reactive oxygen species (ROS) in rotenone-treated isolated rat forebrain mitochondria. Ca(2+)-stimulated mitochondrial ROS release was associated with membrane lipid peroxidation and was directly correlated with the degree of complex I inhibition by rotenone. On the other hand, Ca(2+) did not increase mitochondrial ROS release in the presence of the complex I inhibitor 1-methyl-4-phenylpyridinium. Cyclosporin A had no effect on Ca(2+)-stimulated mitochondrial ROS release in the presence of rotenone, indicating that mitochondrial permeability transition is not involved in this process. We hypothesized that Ca(2+)-induced mitochondrial oxidative stress associated with partial inhibition of complex I may be an important factor in neuronal cell death observed in the neurodegenerative disorder Parkinson's disease.     

Mitochondria and the redox control of development in cnidarians

Mitochondria are the product of an ancient symbiosis between bacteria and host cells. While mitochondria function primarily in energy conversion, increasing amounts of evidence indicate that mitochondrial metabolic state can influence various emergent features of eukaryotic cells. Important intermediaries in such redox signaling include by-products of metabolism, particularly reactive oxygen species (ROS). This review uses cnidarians, a group of basally branching animals, to illustrate the many and varied effects of ROS on development. ROS from both mitochondria and algal symbionts are considered. Because some applications of ROS may lack specificity, the signaling demands of mitochondria and algae may to some extent conflict. An extensive algal symbiosis may thus be incompatible with a well-developed capacity for mitochondrial signaling.     

Changes in astrocyte mitochondrial function with stress: effects of Bcl-2 family proteins

Mitochondria are central to both apoptotic and necrotic cell death, as well as to normal physiological function. Astrocytes are crucial for neuronal metabolic, antioxidant, and trophic support, as well as normal synaptic function. In the setting of stress, such as during cerebral ischemia, astrocyte dysfunction may compromise the ability of neurons to survive. Despite their central importance, the response of astrocyte mitochondria to stress has not been extensively studied. Limited data already suggest clear differences in the response of neuronal and astrocytic mitochondria to oxygen-glucose deprivation (GD). Prominent mitochondrial alterations during stress that can contribute to cell death include changes in production of reactive oxygen species (ROS) and release of death regulatory and signaling molecules from the intermembrane space. In response to stress mitochondrial respiratory function and membrane potential also change, and these changes appear to depend on cell type. Bcl-2 family proteins are the best studied regulators of cell death, especially apoptosis, and mitochondria are a major site of action for these proteins. Although much data supports the role of Bcl-2 family proteins in the regulation of some of these mitochondrial alterations, this remains an area of active investigation. This mini-review summarizes current knowledge regarding mitochondrial control of cell survival and death in astrocytes and the effects of anti-apoptotic Bcl-2 proteins on astrocyte mitochondrial function.     

Mitochondrial energy metabolism and redox state in dyslipidemias

Changes in mitochondrial function are intimately associated with metabolic diseases. Here, we review recent evidence relating alterations in mitochondrial energy metabolism, ion transport and redox state in hypercholesterolemia and hypertriglyceridemia. We focus mainly on changes in mitochondrial respiration, K(+) and Ca(2+) transport, reactive oxygen species generation and susceptibility to mitochondrial permeability transition.     

The role of an astrocytic NADPH oxidase in the neurotoxicity of amyloid beta peptides

Amyloid beta peptide (Abeta) accumulates in the CNS in Alzheimer's disease. Both the full peptide (1-42) or the 25-35 fragment are toxic to neurons in culture. We have used fluorescence imaging technology to explore the mechanism of neurotoxicity in mixed asytrocyte/neuronal cultures prepared from rat or mouse cortex or hippocampus, and have found that Abeta acts preferentially on astrocytes but causes neuronal death. Abeta causes sporadic transient increases in [Ca2+]c in astrocytes, associated with a calcium dependent increased generation of reactive oxygen species (ROS) and glutathione depletion. This caused a slow dissipation of mitochondrial potential on which abrupt calcium dependent transient depolarizations were superimposed. The mitochondrial depolarization was reversed by mitochondrial substrates glutamate, pyruvate or methyl succinate, and by NADPH oxidase (NOX) inhibitors, suggesting that it reflects oxidative damage to metabolic pathways upstream of mitochondrial complex I. The Abeta induced increase in ROS and the mitochondrial depolarization were absent in cells cultured from transgenic mice lacking the NOX component, gp91phox. Neuronal death after 24 h of Abeta exposure was dramatically reduced both by NOX inhibitors and in gp91phox knockout mice. Thus, by raising [Ca2+]c in astrocytes, Abeta activates NOX, generating oxidative stress that is transmitted to neurons, causing neuronal death.     

Ageing free radicals and cellular stress

A number of theories have attempted to account for ageing processes in various species. Following the < rate of living > theory of Pearl, Harman suggested fifty years ago that the accumulation of oxidants could explain the alteration of physical and cognitive functions with ageing. Oxygen metabolism leads to reactive species, including free radicals, which tend to oxidize surrounding molecules such as DNA, proteins and lipids. As a consequence various functions of cells and tissues can be altered, leading to DNA instability, protein denaturation and accumulation of lipid byproducts. Oxidative stress is an adaptive process which is triggered upon oxidant accumulation and which comprises the induction of protective and survival functions. Experimental evidence suggests that the ageing organism is in a state of oxidative stress, which supports the free radical theory. A number of other theories have been proposed ; some of these are actually compatible with the free radical theory. Caloric restriction is among the best models to increase life span in many species. While the relationship between caloric restriction and corrected metabolic rate is controversial, the decrease in ROS production by mitochondria appears to be experimentally supported. The ROS and mitochondrial theories of ageing appear to be compatible. Genetic models of increased life span, particularly those affecting the Foxo pathway, are usually accompanied by an increased resistance to oxidative insult. The free radical theory is not consistent with programmed senescence theories involving the cell division dependent decrease in telomere length ; however, oxidants are known to alter telomere structure. An appealing view of the role of oxidative stress in ageing is the trade-off principle which states that a phenotypic trait can be evolutionarily conserved because of its positive effects on development, growth or fertility, and despite its negative effect on somatic functions and ageing. It is likely that most cellular stresses which comprise adaptive and toxic functions follow such a rule.     

2-Deoxyglucose and NMDA inhibit protein synthesis in neurons and regulate phosphorylation of elongation factor-2 by distinct mechanisms

Cerebral ischaemia is associated with brain damage and inhibition of neuronal protein synthesis. A deficit in neuronal metabolism and altered excitatory amino acid release may both contribute to those phenomena. In the present study, we demonstrate that both NMDA and metabolic impairment by 2-deoxyglucose or inhibitors of mitochondrial respiration inhibit protein synthesis in cortical neurons through the phosphorylation of eukaryotic elongation factor (eEF-2), without any change in phosphorylation of initiation factor eIF-2alpha. eEF-2 kinase may be activated both by Ca(2+)-independent AMP kinase or by an increase in cytosolic Ca2+. Although NMDA decreases ATP levels in neurons, only the effects of 2-deoxyglucose on protein synthesis and phosphorylation of elongation factor eEF-2 were reversed by Na(+) pyruvate. Protein synthesis inhibition by 2-deoxyglucose was not as a result of a secondary release of glutamate from cortical neurons as it was not prevented by the NMDA receptor antagonist 5-methyl-10,11-dihydro-5H-dibenzo-(a,d)-cyclohepten-5,10-imine hydrogen maleate (MK 801), nor to an increase in cytosolic-free Ca2+. Conversely, 2-deoxyglucose likely activates eEF-2 kinase through a process involving phosphorylation by AMP kinase. In conclusion, we provide evidence that protein synthesis can be inhibited by NMDA and metabolic deprivation by two distinct mechanisms involving, respectively, Ca(2+)-dependent and Ca(2+)-independent eEF-2 phosphorylation.     

Rethinking the excitotoxic ionic milieu: the emerging role of Zn(2+) in ischemic neuronal injury

Zn(2+) plays an important role in diverse physiological processes, but when released in excess amounts it is potently neurotoxic. In vivo trans-synaptic movement and subsequent post-synaptic accumulation of intracellular Zn(2+) contributes to the neuronal injury observed in some forms of cerebral ischemia. Zn(2+) may enter neurons through NMDA channels, voltage-sensitive calcium channels, Ca(2+)-permeable AMPA/kainate (Ca-A/K) channels, or Zn(2+)-sensitive membrane transporters. Furthermore, Zn(2+) is also released from intracellular sites such as metallothioneins and mitochondria. The mechanisms by which Zn(2+) exerts its potent neurotoxic effects involve many signaling pathways, including mitochondrial and extra-mitochondrial generation of reactive oxygen species (ROS) and disruption of metabolic enzyme activity, ultimately leading to activation of apoptotic and/or necrotic processes. As is the case with Ca(2+), neuronal mitochondria take up Zn(2+) as a way of modulating cellular Zn(2+) homeostasis. However, excessive mitochondrial Zn(2+) sequestration leads to a marked dysfunction of these organelles, characterized by prolonged ROS generation. Intriguingly, in direct comparison to Ca(2+), Zn(2+) appears to induce these changes with a considerably greater degree of potency. These effects are particularly evident upon large (i.e., micromolar) rises in intracellular Zn(2+) concentration ([Zn(2+)](i)), and likely hasten necrotic neuronal death. In contrast, sub-micromolar [Zn(2+)](i) increases promote release of pro-apoptotic factors, suggesting that different intensities of [Zn(2+)](i) load may activate distinct pathways of injury. Finally, Zn(2+) homeostasis seems particularly sensitive to the environmental changes observed in ischemia, such as acidosis and oxidative stress, indicating that alterations in [Zn(2+)](i) may play a very significant role in the development of ischemic neuronal damage.     

Skeletal muscle reactive oxygen species: A target of good cop/bad cop for exercise and disease

Abstract

Metabolic stresses associated with disease, ageing, and exercise increase the levels of reactive oxygen species (ROS) in skeletal muscle. These ROS have been linked mechanistically to adaptations in skeletal muscle that can be favourable (i.e. in response to exercise) or detrimental (i.e. in response to disease). The magnitude, duration (acute versus chronic), and cellular origin of the ROS are important underlying factors in determining the metabolic perturbations associated with the ROS produced in skeletal muscle. In particular, insulin resistance has been linked to excess ROS production in skeletal muscle mitochondria. A chronic excess of mitochondrial ROS can impair normal insulin signalling pathways and glucose disposal in skeletal muscle. In contrast, ROS produced in skeletal muscle in response to exercise has been linked to beneficial metabolic adaptations including mitochondrial biogenesis and muscle hypertrophy. Moreover, unlike insulin resistance, exercise-induced ROS appears to be primarily of non-mitochondrial origin. The present review summarizes the diverse ROS-targeted metabolic outcomes associated with insulin resistance versus exercise in skeletal muscle, thus, presenting two contrasting perspectives of pathologically harmful versus physiologically beneficial ROS. Here, we discuss the key sites of ROS production during exercise and the effect of ROS in skeletal muscle of people with type 2 diabetes.     

Metabolic syndrome and mitochondrial function: molecular replacement and antioxidant supplements to prevent membrane peroxidation and restore mitochondrial function

Metabolic syndrome consists of a cluster of metabolic conditions, such as hypertriglyceridemia, hyper-low-density lipoproteins, hypo-high-density lipoproteins, insulin resistance, abnormal glucose tolerance and hypertension, that-in combination with genetic susceptibility and abdominal obesity-are risk factors for type 2 diabetes, vascular inflammation, atherosclerosis, and renal, liver and heart disease. One of the defects in metabolic syndrome and its associated diseases is excess cellular oxidative stress (mediated by reactive oxygen and nitrogen species, ROS/RNS) and oxidative damage to mitochondrial components, resulting in reduced efficiency of the electron transport chain. Recent evidence indicates that reduced mitochondrial function caused by ROS/RNS membrane oxidation is related to fatigue, a common complaint of MS patients. Lipid replacement therapy (LRT) administered as a nutritional supplement with antioxidants can prevent excess oxidative membrane damage, restore mitochondrial and other cellular membrane functions and reduce fatigue. Recent clinical trials have shown the benefit of LRT plus antioxidants in restoring mitochondrial electron transport function and reducing moderate to severe chronic fatigue. Thus LRT plus antioxidant supplements should be considered for metabolic syndrome patients who suffer to various degrees from fatigue.     

Age-dependent changes in Ca2+ homeostasis in peripheral neurones: implications for changes in function

Calcium ions represent universal second messengers within neuronal cells integrating multiple cellular functions, such as release of neurotransmitters, gene expression, proliferation, excitability, and regulation of cell death or apoptotic pathways. The magnitude, duration and shape of stimulation-evoked intracellular calcium ([Ca2+]i) transients are determined by a complex interplay of mechanisms that modulate stimulation-evoked rises in [Ca2+]i that occur with normal neuronal function. Disruption of any of these mechanisms may have implications for the function and health of peripheral neurones during the aging process. This review focuses on the impact of advancing age on the overall function of peripheral adrenergic neurones and how these changes in function may be linked to age-related changes in modulation of [Ca2+]i regulation. The data in this review suggest that normal aging in peripheral autonomic neurones is a subtle process and does not always result in dramatic deterioration in their function. We present studies that support the idea that in order to maintain cell viability peripheral neurones are able to compensate for an age-related decline in the function of at least one of the neuronal calcium-buffering systems, smooth endoplasmic reticulum calcium ATPases, by increased function of other calcium-buffering systems, namely, the mitochondria and plasmalemma calcium extrusion. Increased mitochondrial calcium uptake may represent a 'weak point' in cellular compensation as this over time may contribute to cell death. In addition, we present more recent studies on [Ca2+]i regulation in the form of the modulation of release of calcium from smooth endoplasmic reticulum calcium stores. These studies suggest that the contribution of the release of calcium from smooth endoplasmic reticulum calcium stores is altered with age through a combination of altered ryanodine receptor levels and modulation of these receptors by neuronal nitric oxide containing neurones.     

The effect of permeability transition pore opening on reactive oxygen species production in rat brain mitochondria

The influence of mitochondrial permeability transition pore (MPTP) opening on reactive oxygen species (ROS) production in the rat brain mitochondria was studied. It was shown that ROS production is regulated differently by the rate of oxygen consumption and membrane potential, dependent on steady-state or non-equilibrium conditions. Under steady-state conditions, at constant rate of Ca2+-cycling and oxygen consumption, ROS production is potential-dependent and decreases with the inhibition of respiration and mitochondrial depolarization. The constant rate of ROS release is in accord with proportional dependence of the rate of ROS formation on that of oxygen consumption. On the contrary, transition to non-equilibrium state, due to the release of cytochrome c from mitochondria and progressive respiration inhibition, results in the loss of proportionality in the rate of ROS production on the rate of respiration and an exponential rise of ROS production with time, independent of membrane potential. Independent of steady-state or non-equilibrium conditions, the rate of ROS formation is controlled by the rate of potential-dependent uptake of Ca2+ which is the rate-limiting step in ROS production. It was shown that MPTP opening differently regulates ROS production, dependent on Ca2+ concentration. At low calcium MPTP opening results in the decrease in ROS production because of partial mitochondrial depolarization, in spite of sustained increase in oxygen consumption rate by a cyclosporine A-sensitive component due to simultaneous work of Ca2+-uniporter and MPTP as Ca2+-influx and efflux pathways. The effect of MPTP opening at low Ca2+ concentrations is similar to that of Ca2+-ionophore, A-23187. At high calcium MPTP opening results in the increase of ROS release due to the rapid transition to non-equilibrium state because of cytochrome c loss and progressive gating of electron flow in respiratory chain. Thus, under physiological conditions MPTP opening at low intracellular calcium could attenuate oxidative damage and the impairment of neuronal functions by diminishing ROS formation in mitochondria.     

Mitochondria-targeted antioxidants and metabolic modulators as pharmacological interventions to slow ageing

Populations in many nations today are rapidly ageing. This unprecedented demographic change represents one of the main challenges of our time. A defining property of the ageing process is a marked increase in the risk of mortality and morbidity with age. The incidence of cancer, cardiovascular and neurodegenerative diseases increases non-linearly, sometimes exponentially with age. One of the most important tasks in biogerontology is to develop interventions leading to an increase in healthy lifespan (health span), and a better understanding of basic mechanisms underlying the ageing process itself may lead to interventions able to delay or prevent many or even all age-dependent conditions. One of the putative basic mechanisms of ageing is age-dependent mitochondrial deterioration, closely associated with damage mediated by reactive oxygen species (ROS). Given the central role that mitochondria and mitochondrial dysfunction play not only in ageing but also in apoptosis, cancer, neurodegeneration and other age-related diseases there is great interest in approaches to protect mitochondria from ROS-mediated damage. In this review, we explore strategies of targeting mitochondria to reduce mitochondrial oxidative damage with the aim of preventing or delaying age-dependent decline in mitochondrial function and some of the resulting pathologies. We discuss mitochondria-targeted and -localized antioxidants (e.g.: MitoQ, SkQ, ergothioneine), mitochondrial metabolic modulators (e.g. dichloroacetic acid), and uncouplers (e.g.: uncoupling proteins, dinitrophenol) as well as some alternative future approaches for targeting compounds to the mitochondria, including advances from nanotechnology.     

Mitochondrial calcium and oxidative stress as mediators of ischemic brain injury

Acute ischemic and brain injury is triggered by excitotoxic elevation of intraneuronal Ca2+ followed by reoxygenation-dependent oxidative stress, metabolic failure, and cell death. Studies performed in vitro with neurons exposed to excitotoxic concentrations of glutamate demonstrate an initial rise in cytosolic [Ca2+], followed by a reduction to a normal, albeit slightly elevated concentration. This reduction in cytosolic [Ca2+] is due partially to active, respiration-dependent mitochondrial Ca2+ sequestration. Within minutes to an hour following the initial Ca2+ transient, most neurons undergo delayed Ca2+ deregulation characterized by a dramatic rise in cytosolic Ca2+. This prelethal secondary rise in Ca2+ is due to influx across the plasma membrane but is dependent on the initial mitochondrial Ca2+ uptake and associated oxidative stress. Mitochondrial Ca2+ uptake can stimulate the net production of reactive oxygen species (ROS) through activation of the membrane permeability transition, release of cytochrome c, respiratory inhibition, release of pyridine nucleotides, and loss of intramitochondrial glutathione necessary for detoxification of peroxides. Targets of mitochondrially derived ROS may include plasma membrane Ca2+ channels that mediate excitotoxic delayed Ca2+ deregulation.