Mitochondria: the Headquarters in Ischemia-Induced ...

Central Nervous System Agents in Medicinal Chemistry, 2011, 11, 000-000

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Mitochondria: the Headquarters in Ischemia-Induced Neuronal Death Joaquin Jordan1,*, Piet W. J. de Groot2 and Maria F. Galindo3 1

Grupo de Neurofarmacología, Dpto Ciencias Médicas, Fac de Medicina, Universidad de Castilla - La Mancha, Calle Almansa 14, Albacete 02006, Spain; 2Regional Center for Biomedical Research, Albacete Science & Technology Park, University of Castilla - La Mancha, Albacete, Spain; 3Unidad de Neuropsicofarmacología Translacional, Complejo Hospitalario Universitario de Albacete, Spain Abstract: Due to a lack of efficient treatments, searching for novel therapies against acute ischemic stroke represents one of the main fields in neuropharmacology. In this review we summarize and discuss the role of mitochondrial participation in ischemia-induced neuronal death. Mitochondria are regarded as the main link between cellular stress signals and the execution of programmed death of nerve cells. Since it was described that the release of mitochondrial proteins such as cytochrome c, apoptosis inducing factor and endonuclease G are key elements in cell death pathways, they have been the focus of cell death studies. Changes in the permeability of the mitochondrial outer membrane result in a non-reversible step in cell death processes. Cytochrome c released from mitochondria binds in the cytoplasm to Apaf-1 to initiate the formation of an apoptosome, which then binds pro-caspase-9. Active caspase-9 cleaves “executioner” caspases, which in turn proceed to cleave key substrates in the cell. Thus, the identification of new targets might enable establishment of novel strategies for therapeutic research, in this case based on the molecular mechanisms of mitochondrial pathways, to improve the development of compounds for treatment of ischemia.

Keywords: Apoptosis, neuroprotection, necrosis, mitochondrial permeability, pharmacological target, stroke, minocycline. INTRODUCTION Abrupt deprivation of oxygen and glucose to neuronal tissues elicits a series of pathological cascades, leading to spread of neuronal death. Currently, many efforts are focused on finding novel targets to avert this fatal cell fate. These studies focus mostly on the penumbra, a salvageable surrounding tissue of the core [1, 2]. Ischemia-induced neuronal death is generally considered to be a consequence of either necrosis or apoptosis. Although out of scope of this review, it is important to note that this binary distinction fails to account for cell death forms where markers of apoptosis and necrosis co-occur, including those in the brain during or after ischemia [3]. Thus, the two modes of cell death are not necessarily independent phenomena but may act synergistically. Neuronal cell body and dendrite swelling, both hallmarks of necrotic cell death, occurs when Na+ and Ca2+ enters the cell accompanied by the influx of Cl- and water [4]. The release of intracellular contents from dying necrotic cells is thought to provoke an inflammatory response, particularly directed against intracellular antigens and DNA. Apoptosis plays an essential role in development and maintenance of all mammalian tissues. The apoptotic program ensures that damaged, aged, or excessive cells are deleted in a regulated manner that is unharmful to the host. Three major apoptotic pathways originating from three separate subcellular compartments have been identified as (i) *Address correspondence to this author at the Grupo de Neurofarmacología, Facultad de Medicina, Universidad de Castilla - La Mancha, C/ Almansa, 14, 02006 Albacete. Spain; Tel: +34-967-599200; Fax:+34-967-599327; E-mail: [email protected] 1871-5249/11 $58.00+.00

death receptor-mediated pathway, (ii) the mitochondrial apoptotic pathway, and (iii) the reticulum pathway. From a pharmacological point of view, apoptosis but not necrosis is a modulatable process. Mitochondria are considered as the main link between cellular stress signals and the execution of programmed death of nerve cells [5, 6]. Mitochondria (from the Greek μ or mitos = thread + or chondrion = granule) consist of two well-defined compartments, the matrix and the intermembrane space, which are delimited by membranes. The inner and outer mitochondrial membranes have very different morphological and functional characteristics. For a long time it has been believed that the only important function of mitochondria is to serve as the main energy source for the cell. Later, it was recognized that it also plays other essential activities, in particular regulating the levels of second messengers such as calcium ions (Ca2+) and reactive oxygen species (ROS). In the mid 1990s it was discovered that mitochondria transduce death-inducing and/or survival signals across their membranes. The occurrence of harmful signals causes an increase in mitochondrial membrane permeability, allowing the release of >70 small soluble proteins with Mr of 15 kDa or less including cytochrome c, pro-caspase-9, endonuclease G, the SMAC/DIABLO enzyme, and apoptosis inducing factor [7]. This process seems to be the point-of-noreturn in the apoptotic signaling cascade because once released these proteins recruit other proteins and activate signaling pathways which irreversibly lead to cell death. For instance, cytochrome c becomes, as cofactor, part of a multiprotein complex called the apoptosome, which is required for activation of caspase-9. On the other hand, BID triggers condensation of chromatin and fragmentation of DNA, independently of members of the caspase family. However, there © 2011 Bentham Science Publishers Ltd.

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Fig. (1). Mitochondrial and calcium homeostasis.

Fig. (2). Regulation of mitochondrial outer membrane permeability and cellular fate.

is some dispute about the mechanisms involved in these processes, which may also depend on cell type and nature of the apoptotic stimulus [8]. Mitochondria play critical roles in regulating cellular viability and show selective vulnerability to injury. They are targets as well as important sources of ROS. Cumulative oxidative stress, disturbed mitochondrial respiration and oxi-

dative mitochondrial damage all are associated with and may promote cell deat [8-10]. Dysfunctioning of mitochondria affects proper cell functioning, causes DNA damage, and may initiate cell death. Furthermore, mitochondrial membrane permeabilization (MMP) is a critical event during apoptosis and represents the point-of-no-return of this lethal process [11]. Mitochondria can be considered as headquarters where the cell controls signaling pathways that under

Mitochondria: the Headquarters in Ischemia-Induced Neuronal Death

some circumstances can lead to cell death. Therefore, dysfunctioning of mitochondria is thought to play a crucial role in the pathogenesis of various neurological disorders, such as multiple sclerosis, Alzheimer's disease, Parkinson's disease and stroke [12]. In this review we will focus on the relevance of mitochondria in the activation of different pathways in the brain during stroke. We will discuss the role of mitochondria as mediators of death signals (source of energy, calcium regulation, ROS), the relevance of membrane permabilization, and effects on downstream signaling. MITOCHONDRIA AS A SOURCE OF ENERGY Maintenance of cellular energy is vital for neuronal cell survival. The brain has low storage levels of glycogen and is highly dependent on oxidative metabolism [13, 14]. Under homeostatic conditions, mitochondrial oxidative phosphorylation generates ATP to meet the cell’s energy demands. Oxidative phosphorylation for energy production depends almost exclusively on consumption of oxygen and glucose and is at a relatively high rate in brain tissue. Mitochondria use nearly 85% to 90% of the cell’s oxygen to support oxidative phosphorylation. A focal impairment of cerebral blood flow may restrict the delivery of substrates, particularly oxygen and glucose, and, as a consequence, impede the generation of sufficient energy to maintain ionic gradients. In response to occlusion of a cerebral artery, marked changes in ATP and related energy metabolites develop quickly, as expected when delivery of oxygen and glucose is limited [15]. Often these alterations are only partially reversed upon reperfusion despite improved substrate delivery. Thus, ischemia-induced impairments in the mitochondrial capacity for respiratory activity probably contribute to a continued affected energy metabolism during reperfusion, and possibly also to the multitude of changes seen during ischemia. Apart of energy depletion, neuronal damage can also be a consequence of excitotoxicity [16-18]. Under physiological conditions and normal synaptic functioning, activation of excitatory amino acid receptors is transitory and appears to be related to synaptic plasticity. However, if receptor activation becomes excessive or prolonged, the target neurons become damaged and eventually will die thus contributing to excessive activation of post-synaptic glutamate receptors [19]. The presence of glutamate in the synapse is regulated by active, ATP-dependent transporters in neurons and glia. For instance, in central nervous system ischemia, a reduction in ATP production would lead to an impairment of glutamate uptake. This is caused by a loss of membrane potential, and neurons and glia depolarize. Consequently, somatodendritic as well as presynaptic voltage-dependent Ca2+ channels become activated and excitatory amino acids are released into the extracellular space [20, 21]. At the same time, other energy-dependent processes, such as pre-synaptic re-uptake of excitatory amino acids, are impeded, which further increases the accumulation of glutamate in the extracellular space. In fact, therapeutic targets to mitigate such neurotoxicity include Ca2+-channel blockers to reduce excessive glutamate release and glutamate-receptor blockers (for a review see) [22-24].

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Depending on the availability of intracellular ATP, the cell-death pathway may switch from apoptosis to necrosis. At least a part of necrotic cell death, which has been viewed traditionally as a form of passive cell death, may be executed through a mechanism termed necroptosis or programmed necrosis [25]. Interestingly, necroptosis may be activated upon stimulation by TNF, FasL, and TRAIL, the same ligand that can activate apoptosis. Given the central role of death-receptor signaling in cerebral ischemia, it is plausible that necroptosis contributes to cell death after ischemiareperfusion injury. The mitochondrion is well known for its function in apoptotic cell death and has also been implicated downstream of RIP1. A role for adenine nucleotide translocase (ANT) and cyclophilin D (CypD) in the mitochondrial permeability transition has been proposed in necroptosis. TNF and zVAD.fmk treatment results in a mitochondrial defect in ADP transport through ANT, which is dependent on RIP1 [26]. While apoptosis is the predominant cell-death pathway in the presence of sufficient ATP, a drastic depletion of ATP may result in necrotic cell death [27]. ATP depletion in cells undergoing apoptosis leads to necrosis, whereas ATP replenishment to prevent necrosis can in turn induce apoptosis. Cytochrome c released from mitochondria induces Apaf-1 and procaspase-9 to assemble into haptomeric apoptosomes. Procaspase-9 is converted into active caspase-9, which then activates caspase-3 to initiate the final executioner phase of apoptosis, in an ATP or dATP-dependent fashion. When ATP is depleted, apoptosis is blocked and instead the same upstream pro-apoptotic signals induce a form of necrotic cell death. It has been proposed that the major deficits in mitochondrial ATP production in severely ischemic core tissue create conditions that ensure the development of necrotic death in all cells within a few hours of stroke onset. This tissue provides poor prospects for cell survival unless there is early reperfusion. The milder metabolic deficits in the penumbra allow longer survival of the cells and provide more opportunities for protective interventions. However, in the absence of reperfusion or other treatments, limited availability of substrates for oxidative metabolism and progressive deterioration of mitochondrial function may cause metabolic changes that contribute to the demise of cells, also in this region. MITOCHONDRIA AND CALCIUM HOMEOSTASIS Calcium is an extraordinary versatile signaling ion, mediating cellular responses to a wide variety of external stimuli. The cytosolic free-calcium concentration ([Ca2+]cyt) is involved in the control of a large number of cellular and physiological processes, including neuronal excitability and synaptic plasticity, and it regulates neuronal plasticity, which underlies learning and memory capability, and gene transcription [28, 29]. Mitochondria play an important role in the regulation of [Ca2+]cyt. Mitochondria buffer variations in [Ca2+]cyt by accumulating Ca2+ when and where the [Ca2+]cyt is passing a threshold level above which the mitochondrial Ca2+ uniporter is activated. But, this can not account for all Ca2+ uptake. Alternatively, mitochondria can accumulate enormous amounts of


Ca2+ via a Ruthenium red-sensitive mitochondrial Ca2+ uniporter. The affinity of this uniporter for Ca2+ is low (K m 10-20 μM) and the rate at which Ca2+ is transported is considered to be too slow [30, 31]. Later, when the [Ca2+]cyt drops to below the threshold level again, mitochondria slowly release Ca2+ back into the cytosol [32]. Ca2+ is extruded from the mitochondria by two different mechanisms [33], (i) based on exchange of the cation with Na+ and/or H +, and (ii) through the induction of the mitochondria permeability transition pore (MPTP) [29, 34]. Mitochondria are dependent on the uptake of Ca2+ for the production of ATP to meet the neuronal energy demands. Mitochondrial Ca2+ activates key dehydrogenases of the tricarboxylic acid cycle (isocitrate dehydrogenase, ketoglutarate dehydrogenase and pyruvate dehydrogenase), which provide reduction equivalents to the electron transport chain (ETC). This accelerates the production of NADH, thereby creating a driving force in the ETC to increase the proton motive force and to maintain ATP production from the F1F0-ATPase complex (also known as Complex V) [3537]. During this process, some of the electrons passing through the ETC leak out and react with molecular oxygen. This initiates a series of reduction reactions and the production of ROS, which may be lethal to the cells. In addition, physiological Ca2+ can also switch to a death signal when the [Ca2+]cyt increases dramatically. This is related to neuronal damage in cytotoxic events, including ischemia, excitotoxicity, and depolarization (for reviews see) [17, 18]. Mitochondrial Ca2+ accumulation is an early event in excitotoxic neuronal death [38, 39]. Mitochondria are involved in the control of neuronal Ca2+ homeostasis, neuronal Ca2+ signaling, and Ca2+-dependent exocytosis [32, 38, 40, 41]. Mitochondrial Ca2+ overload and dysfunction, due to excessive Ca2+ entry through over-activated glutamate receptors, is a crucial early event in the excitotoxic cascade that follows ischemic or traumatic brain injury [for reviews see [38, 42]. On the other hand, an important consequence of excitotoxic stimulation is delayed Ca2+ deregulation (DCD), originally described by Manev and colleagues [43], and further characterized by the groups of Thayer [44] and Tator [45]. DCD is closely associated with mitochondrial dysfunction [46, 47], and refers to the latent loss of Ca2+ homeostasis in cultured neurons upon exposure to glutamate, which precedes neuronal cell death [45, 48-50] DCD reliably predicts the necrotic death of cultured neurons [45]. Because mitochondria may accumulate a considerable amount of Ca2+ during neurotoxic exposure, a reasonable hypothesis is that DCD represents the ultimate consequence of mitochondrial Ca2+ overload. The Ca2+ accumulation is limited to a pool of mitochondria close to the sites of Ca2+ entry and release [51]. Thus, maintaining Ca2+ homeostasis is essential to life. REACTIVE OXYGEN SPECIES AND MITOCHONDRIA ROS are major players in the signaling cascade following cellular injury occurring both during ischemia and reperfusion. The mitochondrial respiratory chain on the inner mito-

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chondrial membrane is a major intracellular source of ROS. Oxygen normally serves as the ultimate electron acceptor and is reduced to water, however, electron leakage to oxygen through complexes I and III can generate oxygen radicals (O2-) [52]. Persistent increases in ROS are associated with pathological dysfunctioning [53-55]. Considerable evidence exists suggesting that ROS, which are generated when blood flow returns to ischemic brain areas, are mainly responsible for the reperfusion injury. Increased ROS and/or cytokines that are produced as a result of inflammation promote oxidative modification of fatty acids of membrane phospholipids. The oxidation of fatty acids and phospholipids causes detrimental effects on cell signaling and may result in cell and tissue injury [56]. The spontaneous reaction between nitric oxide (NO) and superoxide generates peroxynitrite [57], which is known to irreversibly inhibit complexes I and III of the OxPhos chain [58, 59]. NO itself can reversibly inhibit complex IV [60]. There is evidence that some anti-oxidant treatments can attenuate or delay disease progression in animal models of neurodegenerative diseases. Unfortunately, none of the available anti-oxidants specifically targets mitochondria, let alone the mitochondrial inner membrane (MIM). In addition, most of the anti-oxidants are poorly cell-permeable. Compounds with free-radical scavenging activity (tirilazad, ebselen), iron chelator (deferoxamine) or free-radical trapping properties (NXY-059) have been examined in experimental models of stroke but trials did not confirm those as neuroprotective agents [22, 24]. Coenzyme Q-10 (CoQ10) acts both as an antioxidant and as an electron acceptor at the level of the mitochondria. In several animal models of neurodegenerative diseases including amyotrophic lateral sclerosis, Huntington’s disease, and Parkinson’s disease, CoQ10 has been shown to have beneficial effects [61]. A neuroprotective effect of CoQ10 has also been found in various animal models of stroke and this has been attributed to its role as a potent antioxidant and oxygenderived free-radical scavenger [62, 63]. However, Li et al. found that immediate treatment with CoQ10 via intraperitoneal injection did not prevent neuronal injuries following global or focal ischemia [64]. REGULATION OF MITOCHONDRIAL MEMBRANE PERMEABILITY

OUTER

The mitochondrial outer membrane (MOM) surrounds the entire organelle. Permeabilization of the MOM (MOMP) is a crucial step in apoptosis and necrosis. This phenomenon allows the release of mitochondrial death factors, which facilitate or trigger different signaling cascades ultimately causing the execution of the cell. MOMP is regulated either by the formation of the MPTP or by the insertion of Bcl-2 family members into the MOM. The MPTP is a non-specific large proteinaceous pore, which spans the inner and outer mitochondrial membranes, allowing the passage of ions and substrates. It is activated by Ca2+, inhibited by protons and ADP, and voltage-dependent. The exact composition of the molecules that form the MPTP is still elusive. However, the prevalent hypothesis is that the MPTP involves proteins from the outer membrane (voltagedependent anion channel (VDAC or porin), the inner mem-


brane (Adenine Nucleotide Transport, ANT), the peripheral benzodiazepine receptor, and cyclophilin D, a matrix protein with peptidyl-prolyl cis-trans isomerise activity. Physiologically, VDAC functions as the major channel allowing passage of metabolites between the intermembrane space of mitochondria and the cytoplasm. VDAC can exist in both open and closed conformations. In closed conformations, VDAC is impermeable to ATP and other metabolic anions [65, 66]. By maintaining VDAC in an open conformation, Bcl-2 family proteins would be able to maintain ATP/ADP exchange, thus preventing mitochondrial hyperpolarization, swelling and rupture. However, opposing arguments also exist since it has been shown that the BH4 domain of anti-apoptotic proteins closes VDAC and inhibits apoptotic mitochondrial changes [67]. Indeed, a protective effect of VDAC has been observed against H2O2 in exponentially growing S. cerevisiae cells [68], and pharmacological agents that target VDAC, ANT (in particular atractylate and bongkrekic acid), or cyclophilin D (in particular Cyclosporine A) can stimulate or inhibit MPTP formation in isolated mitochondria in vitro [69-71], as well as in vivo [72]. MPTP formation is now recognized as the major cause of necrotic cell death [73-76] and is known to occur particularly as a result of cellular ischemia- and reperfusion-induced injury. MPTP formation results in the mitochondrial release of cytochrome c and is accompanied by the loss of mitochondrial membrane potential (m). This leads to ATP depletion and energetic collapse, and hence contributes to cell death [77]. A rupture in the outer mitochondrial membrane, as a consequence of mitochondrial swelling due to the disturbed ionic homeostasis, has been proposed as an alternative cytochrome c-releasing mechanism in addition to MPTP formation [70]. Onset of a MPTP is such a severe perturbation of mitochondrial function that it basically assures cell death. If onset of the MPTP is rapid and synchronous in all the cell’s mitochondria, as occurs with stresses like ischemia/reperfusion, Ca2+ overload or exposure to ROS, a precipitous drop of ATP (and dATP) levels will occur. ROS induce promotion of Ca2+-dependent MPTP formation, with swelling of the mitochondrial matrix and rupture of the MOM. ROS may trigger the onset of a MPTP through oxidation of thiol groups on the adenine nucleotide translocator [78-80], a mechanism which is likely to occur during ischemia-reperfusion injury [81-84]. In our laboratory, we studied the role of ROS and Ca2+ on mitochondrial swelling. We observed that whereas O2-induced mitochondrial swelling was Ca2+-independent, ROS participated significantly in Ca2+-induced swelling. Neither Ca2+-free solution nor the presence of the Ca2+-uniporter blocker Ruthenium Red modified O2-induced mitochondrial swelling [85]. Involvement of the permeability transition in ischemic cell death was suggested originally by the ability of cyclosporine A to produce dramatic reductions in tissue infarction when given immediately after reperfusion [86-88]. The strongest evidence for a role of the MPTP in ischemic damage is provided by the much smaller infarcts that develop in mice lacking the protein cyclophilin D [89, 90].


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Alternatively, MOMP might be initiated by insertion of the proto-oncogen Bcl-2 family into the MOM (for a review see [91]). The Bcl-2 family is composed of about 25 key regulators of apoptotic processes, which present up to 4 regions with a high homology to Bcl-2 (regions BH 1 to 4). Among the members with pro-apoptotic properties are Bax (X Bcl-2-associated protein), Bak (Bcl-2-antagonist/killer), BIM, and BID. How these proteins modulate changes in mitochondrial permeability is still an enigma [91]. A common feature of this protein family is the presence of a hydrophobic domain at the C-terminal end, which allows them to be incorporated in the membrane of the endoplasmic reticulum, the mitochondrial outer membrane and the nuclear outer membrane [92, 93]. Another protein that migrates to mitochondria during processes of cell death is the Bcl-2 family member BID. This protein is activated by caspase-8 and gives rise to a spoofed form named t-bid, which is able to migrate to mitochondria, altering their permeability. How t-BID is able to produce alterations in the permeability of liposomes and allows release of cytochrome c is unknown. On the other hand, some members of the Bcl-2 family have been proposed as modulators of MPTP formation. One example is Bcl-2, which inhibits the formation of the MPTP, thereby stabilizing the mitochondrial membrane, increasing the buffering capacity of Ca2+ and protecting the m to various stimuli. Two theories have been postulated to account for such effects: the first is based on a direct interaction with a thiol residue of ANT, the second on the ability to occlude VDAC on its domain BH4. The protein Bcl-xL stimulates VDAC closure while Bax and Bak promote its opening. It is noteworthy that the cyclophilin D participation in MPTP activation is critical in adult ischemia, whereas in neonatal hypoxic-ischemic brain injury mitochondrial permeabilization appears to be primarily Bax-dependent [94]. MITOCHONDRIA AND ISCHEMIC TOLERANCE Mitochondria participate in the endogenous cell survival pathways involved in ischemic tolerance (pre-conditioning), a phenomenon in which the brain protects itself against future injury by adapting to low doses of noxious insults. In this sense, ischemic pre-conditioning and ischemic postconditioning represent two promising strategies in modulating ischemic damage. The mechanism underlying preconditioning are not fully understood, however, it is generally acknowledged that mitochondrial ATP-sensitive potassium channels (mitoKATP) play an important role in triggering protection in both the heart and the brain (e.g. [95, 96]). KATP channels couple cellular metabolism to electrical activity by opening as the intracellular ATP/ADP ratio decreases and by closing as the ratio increases. As reviewed recently by Obrenovitch [97] the features of ischemic tolerance partially resemble naturally occurring adaptive mechanisms, including alterations in cellular energy metabolism (metabolic depression). Although anoxic energy producing pathways cannot be increased during ischemia (limitation of substrate), preconditioning preserves mitochondrial function and membrane integrity [98, 99]. Fur-


thermore, a preservation of mitochondrial membrane potential and possibly the opening of mitoKATP channels has been related to cytoprotection [99, 100]. Recently, we have shown that opening of mitoKATP channels prevents extasisinduced 5-HT depletions [101, 102]. On the other hand, in vitro studies suggested that the inhibition of MPTP opening and its signalling cascade represent crucial events responsible for cytoprotection observed in ischemic preconditioning [103, 104]. ROS and classical ligand stimuli play a role in post-conditioning with KATP channels and protein kinase C pathways acting as mediators. MINOCYCLINE AS A MITOCHONDRIAL-MODULATOR DRUG Oxidative phosphorylation involves a coupling of electron transport, through the ETC, with the active pumping of protons across the inner mitochondrial membrane. This generates an electrochemical gradient known as the proton motive force. The proton motive force drives protons through ATP synthase resulting in ATP formation. The uncoupling proteins (UCPs) are a family of mitochondrial anion-carrier proteins, which are located on the inner mitochondrial membrane (for review see [105]). The activation of UCPs allows protons to re-enter the mitochondrial matrix. This process reduces the driving force for ATP synthesis by modulating the proton flow through ATP synthase. This ability of neuronal UCPs to regulate the mitochondrial membrane potential underlies their ability to regulate neuronal calcium homeostasis. Neuronal UCPs are crucial for reducing the production of ROS and consequent oxidative stress. This also provides a plausible mechanistic explanation how neuronal UCPs act to reduce neurodegenerative pathology. An increase in mitochondrial membrane potential promotes ROS production [106] close to the inner mitochondrial membrane, as it increases ‘random’ single-electron transfer reactions from ETC components to molecular oxygen. Increased mitochondrial uncoupling by UCP2 decreases ROS production and oxidative stress, and therefore is neuroprotective in response to pharmacological and physical insults [107, 108]. Minocycline, a semi-synthetic derivate of tetracycline, is under debate as a potential therapeutic agent in neurological disease processes (for a review see) [22, 109]. We and others have postulated that mitochondria could be the pharmacological target affected by minocycline to govern cytoprotection [110-112]. When added to isolated mitochondria, minocycline is able to decrease the m, possibly preventing MPTP opening and the subsequent release of cytochrome c. Thus, small changes in m may contribute to the cytoprotection mechanisms. Mitochondrial depolarization, caused by low concentrations of mitochondrial uncouplers, has been shown to be protective against NMDA-induced neuronal death [111]. Recently, Fagans and colleagues [113] reported the study "Minocycline to Improve Neurologic Outcome in Stroke" (MINOS). This study had an open-label, dose-escalation design, where minocycline was administered intravenously within 6 hours of stroke-symptom onset in pre-set dose tiers of 3, 4.5, 6, or 10 mg/kg daily over 72 hours, and subjects were followed for 90 days. They found that minocycline was safe and well tolerated up to doses of 10 mg/kg when admin-

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istered intravenously alone and in combination with tissue plasminogen activator. The half-life of minocycline is approximately 24 hours, allowing dosing every 24-hours. The authors argued that minocycline may be an ideal agent to use in combination with tissue plasminogen activator. Previously, Lampl and co-workers [114] showed that patients with acute stroke had significantly better outcome with minocycline treatment as compared with placebo. The findings suggest a potential benefit of minocycline in acute ischemic stroke. ACKNOWLEDGEMENTS Due to space restrictions we were able to only cite a selection of the articles available on this topic and we apologize for any relevant omissions. We thank the members of the Neuropharmacology laboratory of UCLM and Neuropsicofarmacology unit of the CHUA for helpful comments during the preparation of this manuscript. This work was supported by the Ministerio de Sanidad y Consumo (04005-00), the Consejería de Sanidad from Junta de Comunidades de Castilla-La Mancha (PI2007/55) and grant SAF2008-05143C03-1 from CICYT to JJ. MFG was supported by “CCM Obra Social y Cultural-FISCAM” and “Incorporación de grupos emergentes” FIS CARLOS III, and PG by an INCRECyT fellowship from Parque Científico y Tecnológico de Albacete. ABBREVIATIONS [Ca2+]cyt = Cytosolic free-calcium concentration ANT

= Adenine Nucleotide Transport

CoQ10

= Coenzyme Q-10

DCD

= Delayed Ca2+ deregulation

ETC

= Electron transport chain

MMP

= Mitochondrial membrane permeabilization

MOM

= Mitochondrial outer membrane

MOMP

= Mitochondrial outer membrane permeabilization

MPTP

= Mitochondrial permeability transition pore

ROS

= Reactive oxygen species

UCPs

= Uncoupling proteins

VDAC

= Voltage-dependent anion channel

m

= Mitochondrial membrane potential

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