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.     

14,15-EET Suppresses Neuronal Apoptosis in Ischemia–Reperfusion Through the Mitochondrial Pathway

Neuronal apoptosis mediated by the mitochondrial apoptosis pathway is an important pathological process in cerebral ischemia–reperfusion injury. 14,15-EET, an intermediate metabolite of arachidonic acid, can promote cell survival during ischemia/reperfusion. However, whether the mitochondrial apoptotic pathway is involved this survival mechanism is not fully understood. In this study, we observed that infarct size in ischemia–reperfusion injury was reduced in sEH gene knockout mice. In addition, Caspase 3 activation, cytochrome C release and AIF nuclear translocation were also inhibited. In this study, 14,15-EET pretreatment reduced neuronal apoptosis in the oxygen–glucose deprivation and re-oxygenation group in vitro. The mitochondrial apoptosis pathway was also inhibited, as evidenced by AIF translocation from the mitochondria to nucleus and the reduction in the expressions of cleaved-caspase 3 and cytochrome C in the cytoplasm. 14,15-EET could reduce neuronal apoptosis through upregulation of the ratio of Bcl-2 (anti-apoptotic protein) to Bax (apoptosis protein) and inhibition of Bax aggregation onto mitochondria. PI3K/AKT pathway is also probably involved in the reduction of neuronal apoptosis by EET. Our study suggests that 14,15-EET could suppress neuronal apoptosis and reduce infarct volume through the mitochondrial apoptotic pathway. Furthermore, the PI3K/AKT pathway also appears to be involved in the neuroprotection against ischemia–reperfusion by 14,15-EET.     

Protein kinase Cepsilon: the mitochondria-mediated signaling pathway

Mitochondria, which are the cellular energy plants, also act as the integration center of cellular signaling pathways. Apoptosis is a well-known pathway in which mitochondria are involved. Protein kinase Cepsilon has been classified as a novel type of protein kinase C and is involved in many cellular events regulating mitochondrial function. Much evidence has accumulated regarding the relationships between mitochondria-mediated apoptosis and protein kinase Cepsilon. Therefore, by focusing on these relationships, in particular the anti-apoptotic effects of protein kinase Cepsilon on mitochondrial function, we highlight the importance and significance of protein kinase Cepsilon in cell survival and death.     

Induction of mitochondrial dysfunction by poly(ADP-ribose) polymer: Implication for neuronal cell death

Poly(ADP-ribose) polymerase-1 (PARP-1) mediates neuronal cell death in a variety of pathological conditions involving severe DNA damage. Poly(ADP-ribose) (PAR) polymer is a product synthesized by PARP-1. Previous studies suggest that PAR polymer heralds mitochondrial apoptosis-inducing factor (AIF) release and thereby, signals neuronal cell death. However, the details of the effects of PAR polymer on mitochondria remain to be elucidated. Here we report the effects of PAR polymer on mitochondria in cells in situ and isolated brain mitochondria in vitro. We found that PAR polymer causes depolarization of mitochondrial membrane potential and opening of the mitochondrial permeability transition pore early after injury. Furthermore, PAR polymer specifically induces AIF release, but not cytochrome c from isolated brain mitochondria. These data suggest PAR polymer as an endogenous mitochondrial toxin and will further our understanding of the PARP-1-dependent neuronal cell death paradigm.     

Mitochondrial Ca(2+) and neurodegeneration

Mitochondria are essential for ensuring numerous fundamental physiological processes such as cellular energy, redox balance, modulation of Ca(2+) signaling and important biosynthetic pathways. They also govern the cell fate by participating in the apoptosis pathway. The mitochondrial shape, volume, number and distribution within the cells are strictly controlled. The regulation of these parameters has an impact on mitochondrial function, especially in the central nervous system, where trafficking of mitochondria is critical to their strategic intracellular distribution, presumably according to local energy demands. Thus, the maintenance of a healthy mitochondrial population is essential to avoid the impairment of the processes they regulate: for this purpose, cells have developed mechanisms involving a complex system of quality control to remove damaged mitochondria, or to renew them. Defects of these processes impair mitochondrial function and lead to disordered cell function, i.e., to a disease condition. Given the standard role of mitochondria in all cells, it might be expected that their dysfunction would give rise to similar defects in all tissues. However, damaged mitochondrial function has pleiotropic effects in multicellular organisms, resulting in diverse pathological conditions, ranging from cardiac and brain ischemia, to skeletal muscle myopathies to neurodegenerative diseases. In this review, we will focus on the relationship between mitochondrial (and cellular) derangements and Ca(2+) dysregulation in neurodegenerative diseases, emphasizing the evidence obtained in genetic models. Common patterns, that recognize the derangement of Ca(2+) and energy control as a causative factor, have been identified: advances in the understanding of the molecular regulation of Ca(2+) homeostasis, and on the ways in which it could become perturbed in neurological disorders, may lead to the development of therapeutic strategies that modulate neuronal Ca(2+) signaling.     

The mitochondrial inner membrane GTPase, optic atrophy 1 (Opa1), restores mitochondrial morphology and promotes neuronal survival following excitotoxicity

Mitochondrial dynamics have been extensively studied in the context of classical cell death models involving Bax-mediated cytochrome c release. Excitotoxic neuronal loss is a non-classical death signaling pathway that occurs following overactivation of glutamate receptors independent of Bax activation. Presently, the role of mitochondrial dynamics in the regulation of excitotoxicity remains largely unknown. Here, we report that NMDA-induced excitotoxicity results in defects in mitochondrial morphology as evident by the presence of excessive fragmented mitochondria, cessation of mitochondrial fusion, and cristae dilation. Up-regulation of the mitochondrial inner membrane GTPase, Opa1, is able to restore mitochondrial morphology and protect neurons against excitotoxic injury. Opa1 functions downstream of the calcium-dependent protease, calpain. Inhibition of calpain activity by calpastatin, an endogenous calpain inhibitor, significantly rescued mitochondrial defects and maintained neuronal survival. Opa1 was required for calpastatin-mediated neuroprotection because the enhanced survival found following NMDA-induced toxicity was significantly reduced upon loss of Opa1. Our results define a mechanism whereby breakdown of the mitochondrial network mediated through loss of Opa1 function contributes to neuronal death following excitotoxic neuronal injury. These studies suggest Opa1 as a potential therapeutic target to promote neuronal survival following acute brain damage and neurodegenerative diseases.     

Mitochondria and Neuronal Death/Survival Signaling Pathways in Cerebral Ischemia

Apoptotic cell death pathways have been implicated in acute brain injuries, including cerebral ischemia, brain trauma, and spinal cord injury, and in chronic neurodegenerative diseases. Experimental ischemia and reperfusion models, such as transient focal/global ischemia in rodents, have been thoroughly studied and suggest the involvement of mitochondria and the cell survival/death signaling pathways in cell death/survival cascades. Recent studies have implicated mitochondria-dependent apoptosis involving pro- and anti-apoptotic protein binding, the release of cytochrome c and second mitochondria-derived activator of caspase, the activation of downstream caspases-9 and –3, and DNA fragmentation. Reactive oxygen species are known to be significantly generated in the mitochondrial electron transport chain in the dysfunctional mitochondria during reperfusion after ischemia, and are also implicated in the survival signaling pathway that involves phosphatidylinositol-3-kinase (PI3-K), Akt, and downstream signaling molecules, like Bad, 14-3-3, and the proline-rich Akt substrate (PRAS), and their bindings. Further studies of these survival pathways may provide novel therapeutic strategies for clinical stroke.Special issue dedicated to Lawrence F. Eng.     

Functional Significance of the Mitochondrial Membrane Potential

The electrical polarization of the inner mitochondrial membrane largely determines the electrochemical potential of hydrogen ifons, being thereby a significant factor in the energy transformation during oxidation of respiratory substrates and its accumulation in the form of newly synthesized ATP. However, the gradient of the electric potential on the inner mitochondrial membrane (ΔΨm) performs a number of functions not related to energy production. Even under hypoxic conditions, precluding the formation of ATP in mitochondria through oxidative phosphorylation, mitochondria maintain their ΔΨm at the expense of the hydrolysis of cellular ATP, which indicates the exceptional importance of ΔΨm for non-energetic functions of mitochondria. Among these functions, the mitochondrial inward transport of metal cations and proteins carrying a positively charged amino acid sequence and export of anions including nucleic acids possibly providing retrograde signaling, seem very important and essential for maintaining mitochondrial structure and metabolism. ΔΨm is a powerful regulator of mitochondrial generation of reactive oxygen species that perform physiological and pathological functions. And finally, ΔΨm is a critical element in the mechanism of disposal of dysfunctional mitochondria, the so-called quality control machinery of mitochondria. The disturbance of this mechanism leads to increase of heterogeneity in the population of mitochondria in the cell, and the degree of heterogeneity can be considered as an indicator of the pathological cellular phenotype. Correlation between Ψm and cell functions is difficult to identify without adequate quantitative estimates of the magnitude of ΔΨm, which are complicated due to several cellular and mitochondrial processes that affect the experimentally obtained values. Recommendations for assessing the contribution of these processes and avoiding artifacts in the measurements of ΔΨm by standard methods are given.     

Mitochondrial permeability transition in neuronal damage promoted by Ca2+ and respiratory chain complex II inhibition

Changes in mitochondrial integrity, reactive oxygen species release and Ca2+ handling are proposed to be involved in the pathogenesis of many neurological disorders including methylmalonic acidaemia and Huntington's disease, which exhibit partial mitochondrial respiratory inhibition. In this report, we studied the mechanisms by which the respiratory chain complex II inhibitors malonate, methylmalonate and 3-nitropropionate affect rat brain mitochondrial function and neuronal survival. All three compounds, at concentrations which inhibit respiration by 50%, induced mitochondrial inner membrane permeabilization when in the presence of micromolar Ca2+ concentrations. ADP, cyclosporin A and catalase prevented or delayed this effect, indicating it is mediated by reactive oxygen species and mitochondrial permeability transition (PT). PT induced by malonate was also present in mitochondria isolated from liver and kidney, but required more significant respiratory inhibition. In brain, PT promoted by complex II inhibition was stimulated by increasing Ca2+ cycling and absent when mitochondria were pre-loaded with Ca2+ or when Ca2+ uptake was prevented. In addition to isolated mitochondria, we determined the effect of methylmalonate on cultured PC12 cells and freshly prepared rat brain slices. Methylmalonate promoted cell death in striatal slices and PC12 cells, in a manner attenuated by cyclosporin A and bongkrekate, and unrelated to impairment of energy metabolism. We propose that under conditions in which mitochondrial complex II is partially inhibited in the CNS, neuronal cell death involves the induction of PT.     

Uncoupling protein-2 prevents neuronal death and diminishes brain dysfunction after stroke and brain trauma

Whereas uncoupling protein 1 (UCP-1) is clearly involved in thermogenesis, the role of UCP-2 is less clear. Using hybridization, cloning techniques and cDNA array analysis to identify inducible neuroprotective genes, we found that neuronal survival correlates with increased expression of Ucp2. In mice overexpressing human UCP-2, brain damage was diminished after experimental stroke and traumatic brain injury, and neurological recovery was enhanced. In cultured cortical neurons, UCP-2 reduced cell death and inhibited caspase-3 activation induced by oxygen and glucose deprivation. Mild mitochondrial uncoupling by 2,4-dinitrophenol (DNP) reduced neuronal death, and UCP-2 activity was enhanced by palmitic acid in isolated mitochondria. Also in isolated mitochondria, UCP-2 shifted the release of reactive oxygen species from the mitochondrial matrix to the extramitochondrial space. We propose that UCP-2 is an inducible protein that is neuroprotective by activating cellular redox signaling or by inducing mild mitochondrial uncoupling that prevents the release of apoptogenic proteins.     

Estrogen, mitochondria, and growth of cancer and non-cancer cells

In this review, we discuss estrogen actions on mitochondrial function and the possible implications on cell growth. Mitochondria are important targets of estrogen action. Therefore, an in-depth analysis of interaction between estrogen and mitochondria; and mitochondrial signaling to nucleus are pertinent to the development of new therapy strategies for the treatment of estrogen-dependent diseases related to mitochondrial disorders, including cancer.     

ROS, mitochondria and the regulation of autophagy

Accumulation of reactive oxygen species (ROS) is an oxidative stress to which cells respond by activating various defense mechanisms or, finally, by dying. At low levels, however, ROS act as signaling molecules in various intracellular processes. Autophagy, a process by which eukaryotic cells degrade and recycle macromolecules and organelles, has an important role in the cellular response to oxidative stress. Here, we review recent reports suggesting a regulatory role for ROS of mitochondrial origin as signaling molecules in autophagy, leading, under different circumstances, to either survival or cell death. We then discuss the relationship between mitochondria and autophagosomes and propose that mitochondria have an essential role in autophagosome biogenesis.     

The role of compartmentalized signaling pathways in the control of mitochondrial activities in cancer cells

Mitochondria are the powerhouse organelles present in all eukaryotic cells. They play a fundamental role in cell respiration, survival and metabolism. Stimulation of G-protein coupled receptors (GPCRs) by dedicated ligands and consequent activation of the cAMP·PKA pathway finely couple energy production and metabolism to cell growth and survival. Compartmentalization of PKA signaling at mitochondria by A-Kinase Anchor Proteins (AKAPs) ensures efficient transduction of signals generated at the cell membrane to the organelles, controlling important aspects of mitochondrial biology. Emerging evidence implicates mitochondria as essential bioenergetic elements of cancer cells that promote and support tumor growth and metastasis. In this context, mitochondria provide the building blocks for cellular organelles, cytoskeleton and membranes, and supply all the metabolic needs for the expansion and dissemination of actively replicating cancer cells. Functional interference with mitochondrial activity deeply impacts on cancer cell survival and proliferation. Therefore, mitochondria represent valuable targets of novel therapeutic approaches for the treatment of cancer patients. Understanding the biology of mitochondria, uncovering the molecular mechanisms regulating mitochondrial activity andmapping the relevant metabolic and signaling networks operating in cancer cells will undoubtly contribute to create a molecular platform to be used for the treatment of proliferative disorders.Here, we will highlight the emerging roles of signaling pathways acting downstream to GPCRs and their intersection with the ubiquitin proteasome system in the control of mitochondrial activity in different aspects of cancer cell biology.     

Mitochondrial Hexokinases: Guardians of the Mitochondria

There is accumulating evidence that cell survival and metabolism are inexorably linked. As a majormediator of both the metabolic and anti-apoptotic effects of growth factors, the serine/threonine kinaseAkt (also known as protein kinase B or PKB) is particularly well-suited to coordinate the regulation ofthese interrelated processes. Recent demonstrations that growth factors and Akt require glucose (Glc) toprevent apoptosis and promote cell survival are compatible with this contention, as is a positivecorrelation between Akt-regulated mitochondrial hexokinase (mtHK) association and apoptoticresistance. From a phylogenetic perspective, the ability of Akt to regulate cellular metabolismapparently preceded the capacity to control cell survival, suggesting an evolutionary basis for the Glcdependent anti-apoptotic effects of Akt. We speculate that, somewhere in the course of evolution, themetabolic regulatory function of Akt evolved into an adaptive sensing system involving mtHK thatensures mitochondrial homeostasis, thereby coupling metabolism to cell survival. We also propose thatthis “guardian” function of mtHK may be specifically exploited for therapeutic purposes.