Multiple Defects in Energy Metabolism in Alzheimers Disease

Current Drug Targets, 2010, 11, 1193-1206 1193 Multiple Defects in Energy Metabolism in Alzheimer’s Disease I.L. Ferreira1, R. Resende1, E. Ferreiro1, A.C. Rego1,2 and C.F. Pereira*,1,2 1 Center for Neuroscience and Cell Biology; 2Faculty of Medicine, University of Coimbra, Portugal Abstract: Alzheimer’s disease (AD) is the most common form of dementia in old age. Cognitive impairment in AD may be partially due to overall hypometabolism. Indeed, AD is characterized by an early region-specific decline in glucose utilization and by mitochondrial dysfunction, which have deleterious consequences for neurons through increased production of reactive oxygen species (ROS), ATP depletion and activation of cell death processes. In this article, we provide an overview of the alterations on energetic metabolism occurring in AD. First, we resume the evidences that link the ‘metabolic syndrome’ with increased risk for developing AD and revisit the major changes occurring on both extramitochondrial and mitochondrial metabolic pathways, as revealed by imaging studies and biochemical analysis of brain and peripheral samples obtained from AD patients. We also cover the recent findings on cellular and animal models that highlight mitochondrial dysfunction as a fundamental mechanism in AD pathogenesis. Recent evidence posits that mitochondrial abnormalities in this neurodegenerative disorder are associated with changes in mitochondrial dynamics and can be induced by amyloid-beta (Aβ) that progressively accumulates within this organelle, acting as a direct toxin. Furthermore, Aβ induces activation of glutamate N-methyl-D-aspartate receptors (NMDARs) and/or excessive release of calcium from endoplasmic reticulum (ER) that may underlie mitochondrial calcium dyshomeostasis thereby disturbing organelle functioning and, ultimately, damaging neurons. Throughout the review, we further discuss several therapeutic strategies aimed to restore neuronal metabolic function in cellular and animal models of AD, some of which have reached the stage of clinical trials. Keywords: Alzheimer’s disease, glucose metabolism, mitochondria, glutamate receptors, endoplasmic reticulum, calcium. 1. INTRODUCTION Alzheimer’s disease (AD) is the most common agerelated neurodegenerative disease and cause of dementia in the elderly. Clinically, this disorder is characterized by global cognitive dysfunction, especially memory loss, behaviour and personality changes and impairments in the performance of activities of daily living. Neuropathologically, it is characterized by the presence of extracellular senile plaques mainly composed of fibrillar amyloid-beta peptide (Aβ), usually surrounded by reactive astrocytes, activated microglia and dystrophic neurites, and intracellular neurofibrillary tangles (NFTs) consisting of abnormally hyperphosphorylated tau protein [1,2]. These histopathological lesions are restricted to selective brain regions involved in memory and language, the hippocampus and the cerebral cortex, which are reduced in size in AD patients as the result of synaptic loss [3-5] and death of neurons [6, 7]. Regional hypometabolism in the brain, occurring as a consequence of multiple metabolic defects, is an early feature of AD (reviewed in [8, 9]). Thus, the comprehensive understanding of the genesis of metabolic changes leading to the pathogenesis of AD might be a key issue in the development of safe pharmacological therapies for this brain disorder. 2. ALZHEIMER’S SYNDROME DISEASE AND METABOLIC Several clinical and epidemiological studies have evidenced that ‘metabolic syndrome’, a constellation of risk *Address correspondence to this author at the Center for Neuroscience and Cell Biology, Largo Marquês de Pombal, University of Coimbra, 3004-517 Coimbra, Portugal; Tel: +351239820190; Fax: +351239822776; E-mail: [email protected] 1389-4501/10 $55.00+.00 factors of metabolic origin for cerebrovascular disease and insulin-resistant type 2 diabetes mellitus (T2DM) (e.g. hypertension, obesity, insulin resistance and dyslipidaemia), may also impact the onset and severity of AD [10]. Indeed, components of 'metabolic syndrome' are predictors of accelerated cognitive decline and dementia, particularly AD. Moreover, T2DM patients show significantly increased risk for developing AD and experimental induction of diabetes in mouse models of AD results in premature cognitive failure [9]. Concurrently, diabetic subjects are more prone to develop extense and earlier-than-usual white matter lesions and people with these lesions are at increased risk for cognitive impairment and dementia [11]. Enhanced cognitive decline in AD patients by ‘metabolic syndrome’ components seems to be linked to increased production of Aβ, the building blocks of senile plaques, one of the neuropathological hallmarks of AD. In this regard, a recent study found that, when transgenic AD mice were fed a high-fat diet that induces obesity and insulin resistance, hippocampal Aβ levels increased; this phenomenon was consistent with decreased cerebral insulin activity [12]. Therefore, modification of lifestyle factors such as nutrition may prove crucial to AD management. Recent studies have found that caloric restriction (CR) based on reduced amount of food intake over time is able to beneficially influence AD neuropathology [13]. In transgenic mice models of AD, CR attenuates AD-type amyloid neuropathology and ameliorates age-related behavioral deficits [14, 15]. The discovery of future CR mimetics that attenuate or even reverse features of AD will help in the development of "lifestyle therapeutic strategies" for this neurodegenerative disorder. Insulin and insulin-like growth factors (IGFs) can directly modulate neuronal survival, energy metabolism and plasticity, which are required for learning and memory [16]. © 2010 Bentham Science Publishers Ltd. 1194 Current Drug Targets, 2010, Vol. 11, No. 10 Emerging data demonstrate pivotal roles for disturbances in insulin and IGFs signalling pathways in the periphery and in the brain as mediators of cognitive impairment and neurodegeneration in AD [17], leading to the proposal that AD is a "brain-type diabetes". Post-mortem brain studies showed that insulin levels are inversely proportional to the Braak stage of AD progression and revealed a markedly downregulated expression of insulin receptor (IR), IGF-1 receptor (IGF-1R), insulin receptor substrate (IRS)-1 and IRS-2 that progress with severity of neurodegeneration. Additionally, it has been recently shown that insulin and the insulin-sensitizing drug rosiglitazone improves cognitive performance in mouse models of AD and in patients with early AD [18, 19]. 3. IMPAIRED GLUCOSE METABOLISM AND BRAIN IMAGING STUDIES AD is characterized by a significant and pre-symptomatic reduction in brain glucose metabolism. Early studies by [18F]-fluorodeoxyglucose positron emission tomography (FDG-PET) revealed reduced cerebral metabolic rate for glucose (CMRglc) in senile demented AD patients, relatively to elderly normal subjects [20]. There is a significant decrease in CMRglc, more evident in regions of the precuneus and the posterior cingulate, parietotemporal, and frontal cortex. Furthermore, cerebral blood flow is also significantly decreased in the parietotemporal region, as analysed in AD patients by single-photon emission computed tomography [21]. This decrease in glucose metabolism was correlated with cognitive performance, suggesting its use as a marker of disease progression. Dementia severity correlates well with metabolic impairment especially in left hemisphere association areas that are typically affected in patients with AD [22]. Apathy in AD is also associated with reduced metabolic activity in the bilateral anterior cingulate gyrus and medial orbitofrontal cortex and may be associated with reduced activity in the medial thalamus [23]. Nevertheless, increasing evidence suggests that reduction of cerebral metabolic rate for glucose occurs at the preclinical stages of AD, as determined by FDG-PET analysis before the onset of the disease. The later includes individuals with mild cognitive impairment (MCI), presymptomatic individuals carrying mutations responsible for early-onset familial AD (FAD) such as pre-symptomatic carriers of a mutation in the presenilin 1 (PS1) gene, cognitively normal elderly individuals followed for several years until they declined to MCI and eventually to AD, or carriers of the apolipoprotein E (ApoE) ε4 allele (a genetic risk factor for late-onset AD) who express subjective memory complaints [8, 24-26]. Concordantly, the pattern of functional magnetic resonance imaging (fMRI) task-induced deactivation was recently demonstrated to be progressively disrupted from normal aging to MCI and to clinical defined AD, and more evident in elderly individuals at risk for AD due to the presence of the ApoE ε4 genotype, compared to non-carriers [27]. Although little work has been done in order to correlate metabolic dysfunction in AD with its neuropathological hallmarks, NFTs and senile plaques, evidence suggests that a reduction in CMRglc correlates moderately with regional Ferreira et al. densities of NFTs and not with senile plaque distribution [28]. Ceravolo and colleagues [29] reported a statistically significant correlation between levels of total and p-tau protein in cerebrospinal fluid (CSF) and relative metabolic indexes obtained from 18FDG-PET scans in parietal, temporal and occipital lobes bilaterally. These results indicate the existence of a correlation between impairment of cerebral metabolism, estimated through FDG-PET, and CSF tau protein levels. 4. CHANGES IN EXTRA-MITOCHONDRIAL METABOLISM Reduced glycolytic metabolism has been postulated in AD. Patients present a reduced cerebral glucose transport activity [30] and decreased levels of glucose transporters (GLUT1 and GLUT3) in their brains [31]. Decreased phosphofructokinase (which catalyzes the irreversible phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate) protein expression levels, accompanied by reduced enzyme activity, were previously observed in cerebral cortical tissue from 24-month-old transgenic Tg2576 mice overexpressing the Swedish mutation of human amyloid-beta precursor protein (APPsw), in comparison to non-transgenic littermates [32]. Apart from its function in glycolysis, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, which catalyzes the oxidation and phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate, producing NADH) was shown to interact with APP and was suggested to have a role in apoptosis, the latter requiring its translocation to the nucleus. Indeed, a high-molecular-weight GAPDH species was detected in nuclear fractions from AD fibroblasts, but not in nuclear fractions from age-matched controls [33]. GAPDH was also previously shown to undergo increased disulfide bonding in detergent-insoluble extracts from AD patient and transgenic AD mouse brain tissue, compared with age-matched controls, and in Aβtreated primary cortical neurons, which was associated to a reduction in GAPDH enzymatic activity thus affecting neuronal glycolytic metabolism [34]. Previously, we have shown that Aβ-induced toxicity is linked with reduced production of pyruvate through glycolysis that can be prevented by several antioxidants [35]. In AD there is also an increase in oxidatively modified glycolytic enzymes, as identified by proteomic studies, including enolase (catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate in glycolysis) which is also up-regulated in MCI, early-onset AD, and AD patients [36]. Interestingly, Aβdependent astrocyte activation leads to a reduction in the rate of glycolysis, which can be reversed by preventing the proteolysis of the hypoxia-inducible factor (HIF)-1alpha [37]. Therefore, HIF-1alpha seems to play a significant role in maintaining the metabolic integrity of the AD brain. Glycolytic ATP generation can be induced by activation of 5' AMP-activated protein kinase (AMPK), a protein that regulates cellular responses to energy changes. This metabolic sensor has been shown to be activated by resveratrol, an enriched bioactive polyphenol in red wine with potential neuroprotective activities. Several epidemiological studies indicate that moderate consumption of red wine is associated with a lower incidence of dementia and AD [38]. It was recently demonstrated that AMPK-deficient mice are Multiple Defects in Energy Metabolism in Alzheimer’s Disease resistant to the metabolic effects of resveratrol [39] and that AMPK signaling activation by resveratrol modulates Aβ metabolism [40]. Resveratrol was also shown to inhibit the AMPK target mTOR (mammalian target of rapamycin) to trigger autophagy and lysosomal degradation of Aβ. Finally, orally administered resveratrol in mice was detected in the brain where it activates AMPK and reduces cerebral Aβ levels and deposition [40]. These data suggest that resveratrol and pharmacological activation of AMPK have therapeutic potential against AD. The pentose phosphate pathway constitutes an alternate pathway for glucose oxidation. Palmer [41] earlier findings showed increased activities of glucose-6-phosphate dehydrogenase and 6-phosphonogluconate dehydrogenase in the temporal cortex of AD subjects, despite unchanged activities of glutathione peroxidase and glutathione reductase. Increased glucose-6-phosphate dehydrogenase immunoreactivity was also observed in pyramidal neurons from hippocampal samples obtained from post-mortem AD patients [42]. Increased oxidative stress markers in AD samples correlated with enhanced activity of the pentose phosphate pathway (e.g. [42]), suggesting that the observed metabolic changes, which were linked to reductive compensation (due to increased levels of NADPH), could play a role in the response against increased brain pro-oxidant activity in AD. Recently, Aβ1-42 was reported to exert its amnestic effects specifically by impairing astrocytic glycolysis; therefore, the astrocytic-specific metabolite acetate, which provides the astrocytes with alternative substrates for oxidative metabolism, could rescue Aβ1-42-induced memory impairment [43]. However, under the same experimental paradigm, neither glucose nor insulin rescued memory inhibition caused by Aβ [43]. In addition, in this paradigm, neither lactate nor the ketone body beta-hydroxybutyrate was able to salvage Aβ-impaired memory [43]. 5. MITOCHONDRIAL DYSFUNCTION 5.1. Deregulation of Tricarboxylic Acid Cycle Based on the hypothesis that reduced energy metabolic fluxes could contribute to cognitive impairment in AD, patients exhibiting cognitive impairment showed changes in neuronal TCA cycle rate (glucose oxidation and metabolization into cerebral glutamate and glutamine) that were correlated with reduced N-acetyl-aspartate/creatine ratio, as determined by magnetic resonance spectroscopy (MRS) [44]. Furthermore, the degree of clinical disability in AD also correlates to the impairment of brain metabolic enzymes of the TCA cycle. Significant decreases were observed in the activities of pyruvate dehydrogenase complex (PDHC), isocitrate dehydrogenase, and the alpha-ketoglutarate dehydrogenase complex (KGDHC), whereas the activities of succinate dehydrogenase (complex II) and malate dehydrogenase were increased in brains from patients with autopsy confirmed AD [45]. Studies of animal and cell culture models of AD suggest that increased levels of oxidative stress (membrane lipid peroxidation, in particular) may disrupt neuronal energy metabolism and ion homeostasis, by impairing the function of membrane ion-motive ATPases and glucose transporters. Current Drug Targets, 2010, Vol. 11, No. 10 1195 Several proteins related with TCA cycle are also oxidatively damaged in advanced AD stages and in MCI (reviewed in [46]). Protein glutathionylation has been proposed as a mechanism underlying oxidative stress-induced inhibition of enzymes of the TCA cycle in AD [47]. Furthermore, neuronal nuclear genes influencing mitochondrial energy metabolism such as genes encoding enzymes of TCA cycle are underexpressed in AD [48, 49] particular in posterior cingulate cortex, in the middle temporal gyrus, in hippocampal CA1 and in entorhinal cortex. The high correlation between the degree of clinical disability in AD and changes in the activity of the mitochondrial PDHC [45] suggests that adequate supplementation of substrates or coenzyme precursors leading to improvement of the TCA cycle metabolism might be beneficial in AD. Based on brain hypometabolism in AD, ketone bodies have been suggested as a therapeutic alternative brain fuel since they can be converted into acetyl-CoA. Brain ketone bodies supply includes their direct infusion or the administration of a high-fat, low-carbohydrate, low-protein, ketogenic diet. Both strategies have shown efficacy in AD animal models and in AD clinical trials ([50] for review). Exposure to beta-hydroxybutyrate was also shown to protect from Aβ1-42 toxicity in cultured hippocampal neurons [51]. The ability of ketone bodies to protect neurons may be due to their ability to increase mitochondrial efficiency, largely implicating the defects in mitochondrial energy generation in AD pathophysiology (e.g. [51, 50] for review). Aged dogs (used as a model of amyloidosis) receiving medium chain triglycerides (which are rapidly converted to ketone bodies) for a short-term showed improved mitochondrial function, and thus energy metabolism, and decreased levels of APP, as compared to age-matched controls, particularly in the parietal lobe [52]. In addition, the consumption of medium chain triglycerides by human subjects with AD or MCI was followed by an increase in the levels of beta-hydroxybutyrate, increased performance in cognitive tests and greater improvement in paragraph recall, relatively to placebotreated subjects [53]. 5.2. Changes in Oxidative Phosphorylation System Several evidences point towards the involvement of mitochondrial dysfunction in the early alterations that occur in energy metabolism in AD affected brain regions, though the underlying mechanisms are only now beginning to be clarified (reviewed in [9]). A major change associated with AD is the impairment in oxidative phosphorylation (OXPHOS) due to the inhibition of the electron transport chain at complex IV (cytochrome c oxidase, COX). In agreement with brain studies, deficient COX activity has been reported in fibroblasts and platelets from AD patients [54, 55]. Results obtained by Valla and colleagues [56] showed a significant decline in COX activity in platelets from MCI patients, suggesting that mitochondrial dysfunction is present at the earliest symptomatic stages of the disease. Morphological alterations and reduction in mitochondria density have also been described in AD fibroblasts [57]. AD cybrids (cytoplasmic hybrid cells) contain mitochondrial DNA (mtDNA) derived from AD patients and replicate multiple abnormalities found in AD 1196 Current Drug Targets, 2010, Vol. 11, No. 10 brain, including the reduction in COX activity [58]. In addition, altered mitochondria morphology and movement have been reported in AD cybrids [59]. Neuronal genes influencing mitochondrial energy metabolism such as those encoding subunits of the mitochondrial electron transport chain are underexpressed in AD [48, 49]. Furthermore, COX and ATP synthase, two crucial complexes of the OXPHOS system, are oxidized in AD brain, thereby inhibiting enzyme activity and reducing ATP production [60, 61]. In a recent study, deregulation of 24 proteins were observed in vesicular preparations from triple transgenic AD mice (3xTg-AD), and one-third were mitochondrial proteins mainly related to complexes I and IV of the OXPHOS system [62]. Both APP and Aβ have been described to accumulate in mitochondria and to perturb its function (reviewed in [63]). Studies in both human cortical neuronal cells and in a transgenic mouse model for AD revealed that full-length APP progressively accumulates in mitochondria and impairs mitochondrial function inducing a decline in COX activity and ATP levels and disruption of mitochondrial transmembrane potential (ΔΨm) [64]. Mitochondrially localized APP has been shown to be in contact with mitochondrial inner (TIM) and outer (TOM) translocase proteins although the transmembrane-arrested form of APP (ACID domain) prevents the import of its C-terminus into mitochondria. Mitochondrial import pores trapped by APP block the transport of nuclear-encoded proteins to the mitochondria leading to abnormal mitochondrial function (reviewed in [63]). Furthermore, the presence of Aβ was demonstrated in mitochondria from brains of transgenic mice with targeted neuronal overexpression of mutant human APP and AD patients that is associated with diminished enzymatic activity of respiratory chain complexes and a reduction in the rate of oxygen consumption [65]. Other data also demonstrated that Aβ is transported into mitochondria via the TOM machinery [66]. Aβ interacts with the Aβ-binding alcohol dehydrogenase (ABAD) in mitochondria obtained from AD patients and transgenic mice brains [67], and this interaction promotes leakage of ROS, inhibits mitochondria activity and induces cell death [68]. Interaction of mitochondrial Aβ with ABAD contributes to mitochondrial stress by affecting glycolytic, TCA cycle and/or the respiratory chain pathways through the accumulation of deleterious intermediate metabolites (reviewed in [69]). Du and colleagues [70] demonstrated that mitochondrial Aβ also interacts with cyclophilin D (CypD), an integral part of the mitochondrial permeability transition pore (PTP) whose opening leads to cell death. In the same study, the authors have shown that CypD deficiency attenuates mitochondrial dysfunction induced by Aβ, improves learning and memory and synaptic function in an AD mouse model and alleviates Aβ-mediated reduction of long term potentiation (LTP). Another study demonstrated that, in the presence of copper, Aβ present in the mitochondria from human leukocytes in culture inhibits COX activity [71]. Furthermore, γ-secretase complex proteins (PS1, APH, PEN-2 and nicastrin) detected in mitochondrial membranes and mitochondria-targeted insulin-degrading enzyme (IDE) isoform, which regulates Aβ levels [72, 73], have been shown to damage the organelle by inducing free radical production and causing oxidative damage (reviewed in [74]). These results are in line with studies conducted in mtDNA-depleted rho0 cells, which Ferreira et al. demonstrated that functional mitochondria are required for Aβ-induced toxicity [75]. Aβ was shown to directly inhibit the activity of COX in isolated non-synaptic rat brain mitochondria [76], suggesting that Aβ can act as a direct mitochondrial toxin. In isolated brain mitochondria, it was shown that Aβ impairs the respiratory chain, uncouples OXPHOS, decreases the energetic levels and exacerbates the susceptibility to PTP opening [7779]. Previous data from our laboratory showed that Aβ inhibits the respiratory chain complexes and reduces ATP levels in PC12 cells [35, 80]. By using human wild-type APP stably transfected neuroblastoma SH-SY5Y cells, a decrease in COX and an increase in complex III activity was recently shown [81]. Moreover, the observed increase in complex III activity, which is probably due to a compensatory response in order to balance the defect of COX, could not prevent the strong impairment of total respiration since the respiratory control ratio (state3/state4) together with ATP production decreased in APP-overexpressing cells [81]. Keil and colleagues [82] have shown that PC12 cells bearing the Swedish double APP mutation (APPsw), exhibit substantial Aβ levels, impaired COX activity and reduced ATP levels that are normalized upon inhibition of intracellular Aβ production. Extracellular treatment of PC12 cells with comparable Aβ concentrations only leads to weak changes, demonstrating the important role of intracellular Aβ. In 3-month-old APP transgenic mice, which exhibit no plaques but already detectable Aβ levels in the brain, significantly decreased ΔΨm under basal conditions and reduced ATP levels were also observed [82]. These results were corroborated by recent studies in mitochondria isolated from adult double Swedish and London mutant APP transgenic mice in which a significant drop of ΔΨm and reduced ATP levels were evident at 3 months of age, when elevated intracellular Aβ but not extracellular Aβ was present [83]. Under these conditions, mitochondrial dysfunction was associated with higher levels of ROS, an altered Bcl-xL/Bax ratio and reduction of COX activity. To further address whether mitochondrial dysfunction precedes the development of AD pathology, Yao and colleagues [84] conducted mitochondrial functional analyses in female 3xTg-AD mice. Decreased mitochondrial respiration was detected as early as 3 months of age and similar results were obtained in embryonic neurons from 3xTg-AD mouse hippocampus. In the same AD animal model, recent findings demonstrated decreased numeric density of succinic dehydrogenase (SDH)-positive mitochondria in hippocampal neurons [85]. Aβ oligomers secreted from APP-expressing cells were shown to cause mitochondrial dysfunction in hippocampal neurons as demonstrated by decreased ΔΨm, COX activity and ATP levels, which were followed by increases in proapoptotic proteins (Bax, Bid and cytochrome c), decreases in anti-apoptotic Bcl-2 and enhanced apoptosis [86]). Previously, Eckert and colleagues [87] demonstrated that both oligomeric and fibrillar, but not disaggregated Aβ decrease ΔΨm in cortical brain cells obtained from P301L tau transgenic mice. Furthermore, the authors found a decrease in state 3 respiration, the respiratory control ratio, and uncoupled respiration when incubating P301L tau mitochondria either with oligomeric or fibrillar preparations of Aβ. It Multiple Defects in Energy Metabolism in Alzheimer’s Disease was also observed that aging specifically increases the sensitivity of mitochondria to oligomeric Aβ damage, suggesting that oligomeric and fibrillar Aβ are both toxic, but exert different degrees of toxicity [87]. A deleterious connection between mitochondria, cholesterol and Aβ was also recently established (reviewed in [88]). APP/PS1 mice exhibit mitochondrial cholesterol loading and glutathione (GSH) depletion [89]. Using mouse models of cholesterol loading, the authors demonstrated that specific mitochondrial cholesterol pool sensitizes neurons to Aβ-induced oxidative stress and release of apoptogenic proteins due to selective mitochondrial GSH depletion induced by cholesterol-mediated perturbation of mitochondrial membrane dynamics. In AD transgenic mice, brain Aβ levels and plaque deposition are increased due to hemizygous deficiency of the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) [90]. In mature APP/PS1 neurons, characterized by increased levels of oxidative damage accompanied by a reduction in ΔΨm, increased vulnerability to Aβ was correlated with low levels of MnSOD [91]. More recently, Massaad and colleagues [92] demonstrated that MnSOD overexpression decreases hippocampal superoxide levels, prevents AD-related learning and memory deficits and reduces Aβ plaques as well as the Aβ1-42/Aβ1-40 ratio in Tg2576 mice. Similar results were obtained in another transgenic AD mouse model, the Tg19959 mice, in which MnSOD overexpression was shown to improve resistance to Aβ, slow plaque formation and attenuate memory impairment [93]. It was reported that P301L tau transgenic mice have reduced complex I activity and an age-dependent impairment of mitochondrial respiration and ATP synthesis associated with higher levels of ROS [94]. Furthermore, P301L tau transgenic mitochondria displayed increased vulnerability toward Aβ insult, suggesting a synergistic action of tau and Aβ pathology at the level of the mitochondria. The same group demonstrated recently that Aβ and tau synergistically impair the OXPHOS system in 3xTg-AD mice [62]. In this respect, altered protein levels and activity of complex I was shown to be tau-dependent, whereas deregulation of COX was Aβ-dependent [62]. Moreover, caspase-cleaved tau was also shown to induce mitochondrial dysfunction through activation of calcium-dependent calcineurin [95]. The silent information regulator 2 (SIR2, SIRT1 in mammals), a nicotinamide adenine dinucleotide (NAD)+dependent protein deacetylase, has been identified to be a key regulator in the lifespan extending effects of CR in a number of species [96]. Recently, it was found that CR rescues AD neurons decreasing Aβ production through promotion of SIRT1-mediated deacetylase activity and nonamyloidogenic α-secretase [97]. Therefore, SIRT1 activation induced by CR might minimize Aβ accumulation in mitochondria and decrease its toxicity. In this context, SIRT1 overexpression has been found to protect cells against Aβ-induced mitochondrial ROS production and apoptotic death [98]. Calorie-restricted diets were shown to attenuate electron transport chain defects, decrease mitochondrial ROS and damage in neurons from human AD brain [13, 99]. These findings support the view that CR could be a potentially effective, non-pharmacological strategy that Current Drug Targets, 2010, Vol. 11, No. 10 1197 possibly affords protection against the onset and progression of AD. Studies in AD transgenic mice have shown that antioxidants decrease Aβ pathology and ameliorate cognitive deficits [13]. However, contradictory findings were obtained in AD patients using naturally occurring antioxidants, such as vitamins C and E [100, 101]. In the last decade, significant progress has been made in developing antioxidants that target mitochondria, including MitoQ, MitoVitE, MitoPBN, Mitoperoxidase, SS-tetrapeptides, choline esters of GSH and N-acetyl-L-cysteine [102] that are promising candidates for treating AD patients [9]. Latrepirdine (dimebon) is an antihistamine that was studied in Russia as a treatment for AD. In a 6-month trial in patients with mild to moderate AD, dimebon showed significant improvement in all cognitive, behavioral and global outcome measures. A confirmatory phase III trial is now being conducted [103]. The efficacy of dimebon in AD appears to be related to stabilization of mitochondria [104]. 5.3. Altered Mitochondrial Dynamics: Fission and Fusion Mitochondrial shape and structure are maintained by mitochondrial fission and fusion (reviewed in [105]). In a healthy neuron, fission and fusion mechanisms balance equally and mitochondria alter their shape and size to move from cell body to the axons, dendrites, and synapses, and back to the cell body through mitochondrial trafficking. Fission and fusion are controlled by evolutionary conserved, large GTPases which belong to the dynamin family. Mitochondrial fission is controlled and regulated by dynamin-related protein 1 (Drp1), mostly localized in the cytoplasm and also in the mitochondrial outer membrane (MOM), and fission 1 (Fis1), localized to the MOM. On the other hand, mitochondrial fusion is controlled by mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2), localized in the MOM, and dynamin-like guanosine triphosphatase (Opa1), localized in the mitochondrial inner membrane (MIM). Recent findings suggest that abnormal mitochondrial dynamics due to the perturbation of balance between fission and fusion mediates and amplifies mitochondrial dysfunction in AD (reviewed in [74, 106]). Results obtained by Wang and colleagues [107] demonstrated that APP, through Aβ production, causes mitochondrial fragmentation and decreases in Drp1 and Opa1 levels and increases in Fis1 levels. As a consequence, mitochondrial function was impaired as revealed by the elevation of ROS levels, ΔΨm decrease, and reduced ATP production, causing neuronal dysfunction [107]. Drp1 reduction, as a consequence of increased Aβ production, has also been implicated in mitochondrial abnormalities observed in sporadic AD fibroblasts that are rescued by wild-type Drp1 overexpression [108]. More recently, abnormal mitochondrial dynamics was implicated in synaptic abnormalities induced by oligomeric Aβ. In pyramidal neurons of AD brain, it was demonstrated that mitochondria redistribute away from axons, which is associated with a reduction in the levels of Drp1, Opa1, Mfn1, and Mfn2, and an increase in the levels of Fis1 [109]. Manipulation of these mitochondrial fission and fusion proteins in neuronal cells, in order to mimic their changes in AD, caused a similar abnormal mitochondrial distribution pattern. Significantly, exposure to oligomeric amyloid beta- 1198 Current Drug Targets, 2010, Vol. 11, No. 10 Ferreira et al. derived diffusible ligands (ADDLs) caused abnormal mitochondrial distribution in neuronal processes that was correlated with synaptic changes [109]. Drp1 overexpression prevented ADDL-induced synaptic loss, suggesting that abnormal mitochondrial dynamics plays an important role in ADDL-induced synaptic abnormalities [109]. S-nitrosylation of Drp1 was shown to trigger mitochondrial fission, synaptic loss and neuronal damage as a consequence of nitric oxide (NO) production in response to Aβ [110]. Previously, it was shown that mitochondria from cortical neurons undergo fission when neurons are treated with Aβ or NO, in a process prevented by Mfn1 or by dominant-negative Drp1. Conversely, overexpression of Drp1 or Fis1 elicited fission and increased cortical neuronal loss [111]. Overall, the above evidences are consistent with the hypothesis that, in AD neurons, mitochondrial fragmentation and production of defective mitochondria ultimately damage neurons. 6. CALCIUM AND MITOCHONDRIAL DYSFUNCTION 6.1. Mitochondrial Glutamate Receptors Calcium Dyshomeostasis and Abnormal homeostasis of intracellular Ca2+ levels has been observed in aging and AD [112-114]. Apart from generating ATP, mitochondria are positioned within axons, dendrites, and synaptic terminals for Ca2+ buffering [115]. Synaptic compartments, particularly dendritic spines, are regions of neurons that may be exposed to the highest levels of oxidative and metabolic stress and Ca2+ burdens. Ca2+ influx resulting from synaptic N-methyl-D-aspartate receptor (NMDAR) activation induces cAMP response elementbinding (CREB)-dependent gene expression. However, equivalent activation of extrasynaptic NMDARs coupled to a dominant CREB shut-off pathway causing CREB dephosphorylation is less well tolerated, triggering decreased ΔΨm and cell death [116]. Synaptic stimuli evoke Ca2+ entry through both NR2A- and NR2B-containing NMDARs and, in contrast to excitotoxic activation of extrasynaptic NMDARs produce only low-amplitude cytoplasmic Ca2+ spikes and modest, nondamaging mitochondrial Ca2+ accumulation [117]. However, NMDAR signalling can also be due to differences in the composition of the NMDARs as opposed to the location of the receptors. Thus, it has been suggested that excitotoxicity is triggered by the selective activation of NMDARs containing the NR2B subunit [118, 119] irrespective of its location (synaptic or extrasynaptic), whereas NR2A-containing NMDARs promote survival [119]. Due to localized high Ca2+ concentration in microdomains close to mitochondria, Ca2+ is rapidly accumulated within mitochondria (e.g. [120]). In this way, Ca2+ influences energy function by activating mitochondrial matrix dehydrogenases to produce more NADH, which donates more electrons through complex I, driving the synthesis of ATP. Thus, trafficking of mitochondria to locations in neurons where there are large ion fluxes is essential for powering neuronal function. In a recent work Ca2+ influx evoked by glutamate receptor activation caused mitochondria to accumulate at synapses in a Miro1-dependent manner [121]. Studies on AD transgenic mice have provided considerable support for the importance of perturbation of Ca2+ homeostasis in AD [114]. The ability of neurons to regulate the influx, efflux and subcellular compartmentalization of Ca2+ appears to be compromised in AD as the result of agerelated oxidative stress and metabolic impairment, in combination with disease-related accumulation of Aβ. The mitochondrial Ca2+ overload affects ROS production, promotes ionic imbalance and ultimately ATP depletion [112]. Increased intramitochondrial Ca2+ can also induce the opening of the mitochondrial PTP, which is followed by the release of pro-apoptotic factors, particularly cytochrome c and apoptosis-inducing factor (AIF) and the activation of caspases in charge of the “execution” phase of the apoptotic cascade (e.g. [122]). Indeed, the apoptotic process has been shown to be activated locally in synaptic compartments after exposure to Aβ in vulnerable AD neuronal populations [123]. Increased levels of Bcl2 and caspase 3 were also observed in hippocampus from subjects with amnestic MCI [124]. In AD, synapses are the primary sites of Ca2+ deregulation and overactivation of glutamate receptors. These receptors are concentrated on postsynaptic spines of neuronal dendrites and were shown to play an important role in decreased synaptic function observed in aging and early AD ([125-128], reviewed in [129]), thereby altering the ability of neurons to store information. Studies with patients with MCI and AD suggest that synaptic dysfunction and degeneration may occur relatively early in the disease process, and studies in AD mouse models uniformly support this hypothesis [130]. Numerous evidences have demonstrated a direct interaction of Aβ with NMDARs. Aβ has been shown to directly bind to NMDARs, namely by immunoprecipitating with extracellular domains of NR1 subunit, triggering hippocampal neuronal damage through NMDAR-dependent Ca2+ flux [131]. Aβ oligomers were shown to induce inward currents, intracellular Ca2+ increase, mitochondrial Ca2+ overload, oxidative stress, mitochondrial membrane depolarization and apoptotic cell death through a mechanism requiring NMDAR and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) activation in both rat cortical neurons and hippocampal organotypic slices [132]. Moreover, a decrease in the synaptic responses caused by a reduction of the postsynaptic density protein 95 (PSD-95) and the NMDARs was observed in response to Aβ oligomers exposure in rat hippocampal neurons [133]. Accordingly, excitatory synapses containing the NR2B subunit of the NMDAR appear to be principal sites of Aβ oligomers accumulation since the latter colocalize with synaptic markers in AD brains and human cortical neurons, being this effect counteracted by the NMDAR antagonists memantine and ifenprodil [134]. Recently, it was also demonstrated that selective antagonists of NMDARs containing the NR2B subunit prevent Aβ1-42-mediated inhibition of plasticity in the hippocampus in vivo [135]. Moreover, neurons from a genetic mouse model of AD were found to express reduced amounts of surface NMDARs [136] and Aβ1-42 was also found to reduce surface expression of the NR1 subunit of NMDARs, in both cortical and hippocampal neurons [136138]. Sustained Ca2+ influx mediated by NMDARs in hippocampal neurons due to oligomeric Aβ exposure may also involve dynamin1 degradation, as the result of calpain Multiple Defects in Energy Metabolism in Alzheimer’s Disease activation, suggesting that Aβ soluble oligomers cause synaptic dysfunction before the loss of synapses and neurodegeneration [139]. Moreover, memantine improved cognitive function in some AD patients [140] and prevented oxidative stress and Ca2+ influx produced by Aβ oligomers in hippocampal neuronal cultures [131]. In addition to increasing the production of Aβ, amyloidogenic processing of APP can perturb neuronal Ca2+ homeostasis by decreasing the production of a secreted form of APP (sAPPα) [141], and by generating an APP intracellular domain that affects endoplasmic reticulum (ER) Ca2+ release through the regulation of the expression of genes involved in Ca2+ homeostasis [142]. In vitro studies have demonstrated that a reduction in is sufficient to reduce excitotoxic cell death. Uncoupling proteins (UCPs) are mitochondrial inner membrane proteins implicated in the regulation of ΔΨm and cellular energy metabolism [143]. UCPs increase glucose uptake and shift the mode of ATP production from mitochondrial respiration to glycolysis, thereby maintaining cellular ATP levels. This shift in energy metabolism increases the resistance of neurons to oxidative and mitochondrial stress and can reduce excitotoxic-mediated cell death [144, 145]. In AD brains, the expression levels of UCP2, 4, and 5, are significantly reduced [146]. These findings raise the possibility that adopting strategies to improve the actions of UCPs could be a potential novel treatment for AD. ΔΨm 6.2. Mitochondria-Endoplasmic Reticulum Cross-Talk The endoplasmic reticulum (ER) is responsible for the proper folding of newly synthesized proteins destined for secretion, cell surface or intracellular organelles, and Ca2+ sequestration [147]. Accumulation of unfolded or misfolded proteins in the ER lumen triggers an adaptive ER stress response known as the unfolded protein response (UPR). ER stress sensor proteins including inositol requiring transmembrane kinase/endonuclease (IRE1), RNA-dependent protein Kinase (PKR)-like ER membrane-localized kinase (PERK) and activating transcription factor 6 (ATF6), induce signal transduction events that attenuate protein translation, elevate expression of ER chaperones and upregulate proteins needed for ER-associated degradation (ERAD) [148]. Under conditions of severe or prolonged ER stress, these adaptive signalling pathways fail and the ER stress-induced apoptotic pathway is activated (reviewed in [149]). Several findings have linked the ER stress response and UPR to AD. Increased levels of ER stress markers were detected in AD post-mortem brain tissues suggesting that the prolonged activation of the ER stress response is involved in neurodegeneration [150, 151]. In transgenic mice modeling AD, a global molecular profile of hippocampal and cortical gene expression revealed that ER stress-related genes are differentially regulated during the initial and intermediate stages of Aβ deposition [152]. In addition, the ER-resident caspase-12 is strongly up-regulated [153], as well as BiP/Grp78 and CCAAT/enhancer binding protein (C/EBP) homologous protein/DNA damage-inducible gene 153 (CHOP/ GADD153) [154], a pro-apoptotic transcription factor activated under ER stress conditions. Several evidences suggest that APP and the Aβ are intermediaries of ER stress occurring in AD [153, 155-163] and our results Current Drug Targets, 2010, Vol. 11, No. 10 1199 obtained in cultured cortical neurons treated with Aβ also supports this conclusion [164-166]. Perturbation of ER Ca2+ homeostasis, a trigger for the accumulation of unfolded or misfolded proteins and activation of the ER stress response, is also an important step in the beginning or progress of neuronal dysfunction in AD. A markedly decrease of calreticulin immunoreactivity (ER Ca2+ binding protein) was described in AD postmortem brain [160]. Moreover, mutant PS interacts with the InsP3 receptor (InsP3R) Ca2+ release channel, resulting in Ca2+ signaling abnormalities [167,168] that have been suggested to be an early pathogenic event in AD involved in presynaptic dysfunction [169]. Several findings implicate Aβ as a trigger of ER Ca2+ dyshomeostasis. Overexpression of APP was shown to potently enhance cytosolic Ca2+ levels and cell death after ER Ca2+ store depletion and under these conditions CHOP/ GADD153 is significantly upregulated [170]. In cultured cortical neurons, we previously demonstrated that Aβ depletes ER Ca2+ stores, promoting Ca2+ release through InsP3R and ryanodine receptor (RyR), thus increasing intracellular Ca2+ levels and compromising cell survival [171]. Additionally, Aβ was described to mediate changes in intracellular Ca2+ homeostasis in neurons through a direct increase of the RyR3 isoform expression and function [172]. Recent studies in AD transgenic mice have shown that enhanced Ca2+ response is associated with increased levels of RyR and alters synaptic transmission and plasticity mechanisms before the onset of histopathology and cognitive deficits [173-175]. Numerous studies indicate that ER stress-induced apoptotic cell death requires a mitochondrial component, highlighting the close cooperation between these two organelles (reviewed in [176]). ER directly communicates with mitochondria through close contacts referred as mitochondriaassociated membranes (MAM) that support Ca2+ transfer from ER to mitochondria. In fact, apoptotic stimuli known to act through Ca2+ release from the ER induce a prolonged increase in the mitochondrial Ca2+ concentration [177-180]. Besides having a role in the mitochondrial-mediated apoptosis, various proteins of the Bcl-2 family, including the antiapoptotic protein Bcl-2, modulate Ca2+ content in both mitochondria and ER [181, 177]. Conversely, overexpression of the pro-apoptotic Bax/Bak proteins favours the transfer of Ca2+ from ER to mitochondria and induces cell death [182-184]. Transmission of a Ca2+ signal from InsP3R to mitochondria was demonstrated to be associated with InsP3-induced opening of PTP and, in turn, cytochrome c release [185]. Similarly, phosphorylation of InsP3R by Akt reduces cellular sensitivity to apoptotic stimuli through a mechanism that involves diminished Ca2+ flux from the ER to the mitochondria [186]. Cytochrome c released from mitochondria can also promote Ca2+ release through the ER InsP3R [187]. Released ER Ca2+ triggers the extrusion of a large amount of cytochrome c from all the mitochondria in the cell, amplifying the death signal [188, 189]. Despite the evidences that demonstrate the involvement of mitochondrial and ER dysfunction in AD pathogenesis, information regarding the role of ER-mitochondria cross-talk in this neurodegenerative disorder is sparse though supporting data is now beginning to emerge. It was recently shown that PS1 and PS2 are highly enriched in a subcompartment of the ER that is associated with mitochondria and that forms a 1200 Current Drug Targets, 2010, Vol. 11, No. 10 physical bridge between the two organelles [190]. Moreover, the association of hyperphosphorylated tau with ER membranes was detected in AD brains and also in the brain of asymptomatic JNPL3 mice that overexpress mutant tau. Interestingly, these mice exhibited more contacts between ER membranes and mitochondria [191], suggesting that accumulation of tau at the surface of ER membranes might contribute to tau-induced neurodegeneration through mitochondria. Results from our laboratory demonstrated that Ca2+ released from ER through InsP3R and RyR receptors in cortical neurons treated with Aβ is responsible for the depolarization of mitochondrial membrane, release of cytochrome c upon translocation of Bax to mitochondria and activation of caspase-9 [164,192], thus implicating the mitochondrial-mediated apoptotic pathway in neurodegeneration Ferreira et al. occurring upon Aβ-induced ER dysfunction. Furthermore, in our studies conducted in rho0 cells it has been shown that the presence of functional mitochondria is required for ER stress-mediated apoptosis triggered by toxic insults such as Aβ [193]. Indeed, we found that ER Ca2+ depletion and increased BiP/Grp78 levels occurring in both Aβ-treated ρ0 and ρ+ cells only proceeds to apoptosis in the parental cell line, as given by the increase in CHOP/GADD153 levels, caspases-9 and -3 activities and DNA fragmentation. Concerning the cooperation between ER and mitochondria during apoptosis, the role of ROS cannot be forgotten (reviewed in [194]. Several studies have demonstrated that ER is sensitive to oxidative stress [195] that may lead to perturbations of the ER Ca2+ homeostasis since many of the proteins involved in the regulation of Ca2+ in the ER are Fig. (1). Pathways involved in metabolic dysfunction in AD. Glucose uptake and utilization is decreased. Several glycolytic enzymes are inhibited reducing the levels of pyruvate. In addition, pyruvate transport to mitochondria and its irreversible conversion to Acetyl-CoA by pyruvate dehydrogenase complex (PDHC) are decreased, as well as the resulting metabolism through the TCA cycle. As a consequence, ATP production is decreased and the availability of reducing equivalents (NADH and FADH2) for the electron transport chain is limited. Aβ (and also APP) accumulates within mitochondria and compromise the activity of mitochondrial electron transport (ETC) chain complexes, leading to ROS production and ATP depletion. Furthermore, pro-apoptotic factors, such as cytochrome c, are released from mitochondria and activate apoptosis-effector caspases, including caspase-3. Aβ also increases cytosolic Ca2+ levels, due to the release of Ca2+ through ER RyR and InsP3R. Due to the close proximity between both organelles, Ca2+ can be transfered from ER to mitochondria through mitochondria-associated membranes (MAM) and can perturb Ca2+ homeostasis in the mitochondria, initiating the mitochondrial-mediated cell death pathway. Translocation of Bax, release of cytochrome c and activation of caspase-9 and -3 can occur upon ER Ca2+ release. Aβinduced ER Ca2+ release is also responsible for the accumulation of ROS, which can enhance the impairment of mitochondrial function. In addition to ER, another source of mitochondrial Ca2+ dyshomeostasis are the NMDARs. Overactivation of this subtype of glutamate receptors induces a significant rise in the levels of cytosolic Ca2+ that is taken up by mitochondria through the Ca2+ uniporter. Current Drug Targets, 2010, Vol. 11, No. 10 Multiple Defects in Energy Metabolism in Alzheimer’s Disease changed by oxidants [196]. Furthermore, CHOP/GADD153 can lead to the transfer of electrons to molecular oxygen, forming ROS [197]. In addition, Ca2+ that results from the depletion of ER Ca2+ stores can be taken up by juxtaposed mitochondria as described before, inducing ROS formation [198]. Recently, we demonstrated that the release of Ca2+ from ER in cortical neurons treated with Aβ is responsible for the depletion of the antioxidant GSH and accumulation of ROS that are involved in loss of ΔΨm [192], thus highlighting ROS as potential mediators of ER-mitochondria cross-talk in AD. 1201 (project reference PTDC/SAU-NEU/71675/2006) and the pharmaceutical company Lundbeck. ABBREVIATIONS 3xTg-AD = Triple transgenic mice model of AD ABAD = Aβ-binding alcohol dehydrogenase AD = Alzheimer’s disease ADDL = Amyloid beta-derived diffusible ligand AGE = Advanced glycation endproducts 7. CONCLUSIONS AIF = Apoptosis-inducing factor Several lines of evidence suggest that region specific declines in the cerebral glucose metabolism are an early and progressive feature of AD, occurring before pathology and symptoms manifest. In vivo imaging demonstrated that reduced cerebral glucose utilization in AD patients is more severe than in normal aging and correlates with symptom severity. Moreover, biochemical analysis performed in postmortem brain tissue and peripheral samples showed that several enzymes involved in extramitochondrial and mitochondrial metabolic pathways, namely glycolysis, the pentose phosphate pathway, the TCA cycle and oxidative phosphorylation, are affected either as a result of activity inhibition or transcription downregulation. In recent years, the cellular and molecular mechanisms underlying changes in metabolic pathways occurring in AD brain have begun to be clarified. Some of these mechanisms are depicted in Fig. (1). Numerous evidences point to mitochondrial dysfunction occurring upon inhibition of mitochondrial respiratory chain, in particular of complex IV (COX), as a prominent feature driving AD pathogenesis. Recent studies in AD transgenic mice and in vitro cellular models have implicated impaired mitochondrial oxidative phosphorylation in ROS generation, ATP depletion and activation of apoptotic cell death pathways. Moreover, changes in mitochondrial dynamics as a result of fission/fusion unbalance have been associated with loss of mitochondrial function. APP and Aβ have been demonstrated to accumulate in mitochondria and act as direct mitochondrial toxins. In addition, several triggers for mitochondrial dysfunction have been identified which can operate through perturbation of mitochondrial Ca2+ homeostasis. Overactivation of NMDARs and ER-mitochondria cross-talk may cause mitochondrial Ca2+ overload and subsequent neuronal death. Due to multiple targets of oligomeric Aβ converging on mitochondrial dysfunction (being mitochondria itself a target) and neurodegeneration, the development of effective treatments or preventive strategies for AD is urging. Considering that an increasing body of evidence implicates hypometabolism, resulting from alterations in glucose metabolism and mitochondrial enzymes, as a crucial player during the pathogenesis and progression of AD, combined treatment acting on multiple metabolic pathways and selective NMDAR subunits may constitute a way for intervention in the disease process. AMPAR = α-Amino-3-hydroxy-5-methyl-4isoxazolepropionic acid receptor ApoE = Apolipoprotein E APP = Amyloid beta precursor protein APPsw = Swedish mutation in APP ATF6 = Activating transcription factor 6 Aβ = Amyloid-beta peptide BDNF = Brain-derived neurotrophic factor CHOP = CCAAT/enhancer binding protein (C/EBP) homologous protein CMRglc = Cerebral metabolic rate for glucose COX = Cytochrome c oxidase CREB = cAMP response element-binding CSF = Cerebrospinal fluid CypD = Cyclophilin D Drp1 = Dynamin-related protein 1 ER = Endoplasmic reticulum ERAD = ER-associated degradation FAD = Familial AD FDG-PET = [18F]-Fluorodeoxyglucose positron emission tomography fMRI = Functional magnetic resonance imaging Gadd153 = DNA damage-inducible gene 153 GAPDH = Glyceraldehyde-3-phosphate dehydrogenase GLUT = Glucose transporter GSH = Reduced glutathione HIF = Hypoxia-inducible factor IDE = Insulin-degrading enzyme iNOS = Inducible nitric oxide synthase InsP3 = Inositol 1,4,5-trisphosphate InsP3R = Inositol 1,4,5-trisphosphate receptor IRE1 = Inositol requiring transmembrane kinase/endonuclease KGDHC = Alpha-ketoglutarate dehydrogenase complex LTP = Long term potentiation ACKNOWLEDGEMENTS The authors acknowledge financial support from Fundação para a Ciência e a Tecnologia (FCT), Portugal 1202 Current Drug Targets, 2010, Vol. 11, No. 10 Ferreira et al. 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