Hepatic levels of S-adenosylmethionine regulate the adaptive response to fasting.

There has been an intense focus to uncover the molecular mechanisms by which fasting triggers the adaptive cellular responses in the major organs of the body. Here, we show that in mice, hepatic S-adenosylmethionine (SAMe)-the principal methyl donor-acts as a metabolic sensor of nutrition to fine-tune the catabolic-fasting response by modulating phosphatidylethanolamine N-methyltransferase (PEMT) activity, endoplasmic reticulum-mitochondria contacts, ß-oxidation, and ATP production in the liver, together with FGF21-mediated lipolysis and thermogenesis in adipose tissues. Notably, we show that glucagon induces the expression of the hepatic SAMe-synthesizing enzyme methionine adenosyltransferase α1 (MAT1A), which translocates to mitochondria-associated membranes. This leads to the production of this metabolite at these sites, which acts as a brake to prevent excessive ß-oxidation and mitochondrial ATP synthesis and thereby endoplasmic reticulum stress and liver injury. This work provides important insights into the previously undescribed function of SAMe as a new arm of the metabolic adaptation to fasting.

GABA Receptor Agonists Protect From Excitotoxic Damage Induced by AMPA in Oligodendrocytes.

Oligodendrocytes are the myelin forming cells of the central nervous system, and their vulnerability to excitotoxicity induced by glutamate contributes to the pathogenesis of neurological disorders including brain ischemia and neurodegenerative diseases, such as multiple sclerosis. In addition to glutamate receptors, oligodendrocytes express GABA receptors (GABAR) that are involved in their survival and differentiation. The interactions between glutamate and GABAergic systems are well documented in neurons, under both physiological and pathological conditions, but this potential crosstalk in oligodendrocytes has not been studied in depth. Here, we evaluated the protective effect of GABAR agonists, baclofen (GABAB) and muscimol (GABAA), against AMPA-induced excitotoxicity in cultured rat oligodendrocytes. First, we observed that both baclofen and muscimol reduced cell death and caspase-3 activation after AMPA insult, proving their oligoprotective potential. Interestingly, analysis of the cell-surface expression of calcium-impermeable GluR2 subunits in oligodendrocytes revealed that GABAergic agonists significantly reverted GluR2 internalization induced by AMPA. We determined that baclofen and muscimol also impaired AMPA-induced intracellular calcium increase and subsequent mitochondrial membrane potential alteration, ROS generation, and calpain activation. However, AMPA-triggered activation of Src, Akt, JNK and CREB was not affected by baclofen or muscimol. Overall, our results suggest that GABAR activation initiates alternative molecular mechanisms that attenuate AMPA-mediated apoptotic excitotoxicity in oligodendrocytes by interfering with expression of GluR subunits in membranes and with calcium-dependent intracellular signaling pathways. Together, these findings provide evidence of GABAR agonists as potential oligodendroglial protectants in central nervous system disorders.

Amyloid ß / PKC-dependent alterations in NMDA receptor composition are detected in early stages of Alzheimer´s disease.

Amyloid beta (Aß)-mediated synapse dysfunction is an early event in Alzheimer's disease (AD) pathogenesis and previous studies suggest that NMDA receptor (NMDAR) dysregulation may contribute to these pathological effects. Although Aß peptides impair NMDAR expression and activity, the mechanisms mediating these alterations in the early stages of AD are unclear. Here, we observed that NMDAR subunit NR2B and PSD-95 levels were aberrantly upregulated and correlated with Aß42 load in human postsynaptic fractions of the prefrontal cortex in early stages of AD patients, as well as in the hippocampus of 3xTg-AD mice. Importantly, NR2B and PSD95 dysregulation was revealed by an increased expression of both proteins in Aß-injected mouse hippocampi. In cultured neurons, Aß oligomers increased the NR2B-containing NMDAR density in neuronal membranes and the NMDA-induced intracellular Ca2+ increase, in addition to colocalization in dendrites of NR2B subunit and PSD95. Mechanistically, Aß oligomers required integrin ß1 to promote synaptic location and function of NR2B-containing NMDARs and PSD95 by phosphorylation through classic PKCs. These results provide evidence that Aß oligomers modify the contribution of NR2B to NMDAR composition and function in the early stages of AD through an integrin ß1 and PKC-dependent pathway. These data reveal a novel role of Aß oligomers in synaptic dysfunction that may be relevant to early-stage AD pathogenesis.

Quercetin alleviates styrene oxide-induced cytotoxicity in cortical neurons in vitro via modulation of oxidative stress and apoptosis.

Styrene 7,8-oxide (SO) is the principal metabolite of styrene, an industrial neurotoxic compound which causes various neurodegenerative disorders. The present study aimed to explore the mechanisms of SO cytotoxicity (0.5 - 4 mM) in primary cortical neurons and to evaluate the neuroprotective potential of quercetin (QUER). Our results showed that exposure to SO decreased viability of cortical neurons in a concentration-dependent manner. In the presence of QUER, cell viability was increased significantly. The neuroprotective effects of QUER were associated with the reduction of intracellular Reactive Oxygen Species (ROS), the decrease in calcium overload and the restoration of mitochondrial membrane depolarization caused by SO. Additionally, to evaluate neuronal death mechanisms triggered by SO, cells were incubated with Ac-DEVD-CHO, Calpeptin and Necrostatin-1, pharmacological inhibitors of caspase-3, calpains and necroptosis respectively. The data showed that the three inhibitors reduced cell death induced by SO and suggested the implication of apoptotic, necrotic and necroptotic pathways. However, western blot analysis showed that QUER attenuated the activation of caspase-3 but did not prevent calpain activity. Taken together, these data indicated that the cytotoxicity of SO was mediated by oxidative stress and apoptosis, necrosis and necroptosis mechanisms, while the neuroprotection provided by QUER against SO depended mainly on its anti-apoptotic activity.

Role of Mitochondrial Dynamics in Microglial Activation and Metabolic Switch.

Microglia act as sensors of injury in the brain, favoring its homeostasis. Their activation and polarization toward a proinflammatory phenotype are associated with injury and disease. These processes are linked to a metabolic reprogramming of the cells, characterized by high rates of glycolysis and suppressed oxidative phosphorylation. This metabolic switch can be reproduced in vitro by microglial stimulation with LPS plus IFN-γ. To understand the mechanisms regulating mitochondrial respiration abolishment, we examined potential alterations in mitochondrial features during this switch using rat primary microglia. Cells did not show any change in mitochondrial membrane potential, suggesting a limited impact in the mitochondrial viability. We provide evidence that reverse operation of F0F1-ATP synthase contributes to mitochondrial membrane potential. In addition, we studied the possible implication of mitochondrial dynamics in the metabolic switch using the mitochondrial division inhibitor-1 (Mdivi-1), which blocks dynamin-related protein 1 (Drp1)-dependent mitochondrial fission. Mdivi-1 significantly reduced the expression of proinflammatory markers in LPS plus IFN-γ-treated microglia. However, this inhibition did not lead to a recovery of the oxidative phosphorylation ablation by LPS plus IFN-γ or to a microglia repolarization. Altogether, these results suggest that Drp1-dependent mitochondrial fission, although potentially involved in microglial activation, does not play an essential role in metabolic reprogramming and repolarization of microglia.

Sephin1 Protects Neurons against Excitotoxicity Independently of the Integrated Stress Response.

Sephin1 is a derivative of guanabenz that inhibits the dephosphorylation of the eukaryotic initiation factor 2 alpha (eIF2α) and therefore may enhance the integrated stress response (ISR), an adaptive mechanism against different cellular stresses, such as accumulation of misfolded proteins. Unlike guanabenz, Sephin1 provides neuroprotection without adverse effects on the α2-adrenergic system and therefore it is considered a promising pharmacological therapeutic tool. Here, we have studied the effects of Sephin1 on N-methyl-D-aspartic acid (NMDA) receptor signaling which may modulate the ISR and contribute to excitotoxic neuronal loss in several neurodegenerative conditions. Time-course analysis of peIF2α levels after NMDA receptor overactivation showed a delayed dephosphorylation that occurred in the absence of activating transcription factor 4 (ATF4) and therefore independently of the ISR, in contrast to that observed during endoplasmic reticulum (ER) stress induced by tunicamycin and thapsigargin. Similar to guanabenz, Sephin1 completely blocked NMDA-induced neuronal death and was ineffective against AMPA-induced excitotoxicity, whereas it did not protect from experimental ER stress. Interestingly, both guanabenz and Sephin1 partially but significantly reduced NMDA-induced cytosolic Ca2+ increase, leading to a complete inhibition of subsequent calpain activation. We conclude that Sephin1 and guanabenz share common strong anti-excitotoxic properties with therapeutic potential unrelated to the ISR.

Mitochondrial division inhibitor 1 disrupts oligodendrocyte Ca2+ homeostasis and mitochondrial function.

Mitochondrial fission mediated by cytosolic dynamin related protein 1 (Drp1) is essential for mitochondrial quality control but may contribute to apoptosis as well. Blockade of Drp1 with mitochondrial division inhibitor 1 (mdivi-1) provides neuroprotection in several models of neurodegeneration and cerebral ischemia and has emerged as a promising therapeutic drug. In oligodendrocytes, overactivation of AMPA-type ionotropic glutamate receptors (AMPARs) induces intracellular Ca2+ overload and excitotoxic death that contributes to demyelinating diseases. Mitochondria are key to Ca2+ homeostasis, however it is unclear how it is disrupted during oligodendroglial excitotoxicity. In the current study, we have analyzed mitochondrial dynamics during AMPAR activation and the effects of mdivi-1 on excitotoxicity in optic nerve-derived oligodendrocytes. Sublethal AMPAR activation triggered Drp1-dependent mitochondrial fission, whereas toxic AMPAR activation produced Drp1-independent mitochondrial swelling. Accordingly, mdivi-1 efficiently inhibited Drp1-mediated mitochondrial fission and did not prevent oligodendrocyte excitotoxicity. Unexpectedly, mdivi-1 also induced mitochondrial depolarization, ER Ca2+ depletion and modulation of AMPA-induced Ca2+ signaling. These off-target effects of mdivi-1 sensitized oligodendrocytes to excitotoxicity and ER stress and eventually produced oxidative stress and apoptosis. Interestingly, in cultured astrocytes mdivi-1 induced nondetrimental mitochondrial depolarization and oxidative stress that did not cause toxicity or sensitization to apoptotic stimuli. In summary, our results provide evidence of Drp1-mediated mitochondrial fission during activation of ionotropic glutamate receptors in oligodendrocytes, and uncover a deleterious and Drp1-independent effect of mdivi-1 on mitochondrial and ER function in these cells. These off-target effects of mdivi-1 limit its therapeutic potential and should be taken into account in clinical studies.

Inhibition of Casein Kinase 2 Protects Oligodendrocytes From Excitotoxicity by Attenuating JNK/p53 Signaling Cascade.

Oligodendrocytes are highly vulnerable to glutamate excitotoxicity, a central mechanism involved in tissue damage in Multiple Sclerosis (MS). Sustained activation of AMPA receptors in rat oligodendrocytes induces cytosolic calcium overload, mitochondrial depolarization, increase of reactive oxygen species, and activation of intracelular pathways resulting in apoptotic cell death. Although many signals driven by excitotoxicity have been identified, some of the key players are still under investigation. Casein kinase 2 (CK2) is a serine/threonine kinase, constitutively expressed in all eukaryotic tissues, involved in cell proliferation, malignant transformation and apoptosis. In this study, we identify CK2 as a critical regulator of oligodendrocytic death pathways and elucidate its role as a signal inductor following excitotoxic insults. We provide evidence that CK2 activity is up-regulated in AMPA-treated oligodendrocytes and CK2 inhibition significantly diminished AMPA receptor-induced oligodendroglial death. In addition, we analyzed mitogen-activated protein kinase (MAPK) signaling after excitotoxic insult. We observed that AMPA receptor activation induced a rapid increase in c-Jun N-terminal kinase (JNK) and p38 phosphorylation that was reduced after CK2 inhibition. Moreover, blocking their phosphorylation, we enhanced oligodendrocyte survival after excitotoxic insult. Finally, we observed that the tumor suppressor p53 is activated during AMPA receptor-induced cell death and, interestingly, down-regulated by JNK or CK2 inhibition. Together, these data indicate that the increase in CK2 activity induced by excitotoxic insults regulates MAPKs, triggers p53 activation and mediates subsequent oligodendroglial loss. Therefore, targeting CK2 may be a useful strategy to prevent oligodendrocyte death in MS and other diseases involving central nervous system (CNS) white matter.

Re-examining the potential of targeting ABHD6 in multiple sclerosis: Efficacy of systemic and peripherally restricted inhibitors in experimental autoimmune encephalomyelitis.

α/ß-Hydrolase domain-containing 6 (ABHD6) contributes to the hydrolysis of the major endocannabinoid 2-arachidonoylglycerol (2-AG) in the central nervous system (CNS) and in the periphery. ABHD6 blockade has been proposed as novel strategy to treat multiple sclerosis (MS), based on the observation that the inhibitor WWL70 exerts protective anti-inflammatory effects in experimental autoimmune encephalomyelitis (EAE). According to recent data, WWL70 exhibits off-target anti-inflammatory activity in microglial cells and the potential of ABHD6 as drug target in MS remains controversial. Here we further investigated the role of ABHD6 during autoimmune demyelination by comparing the efficacy of two novel inhibitors with different CNS permeability in vivo. Preventive treatment with the systemically active inhibitor KT182 ameliorated the neurological signs of EAE during the time-course of disease. By contrast, administration of the peripherally restricted compound KT203 was ineffective in attenuating EAE symptomatology. Both inhibitors failed to improve corticospinal tract conduction latency and to attenuate inflammation at EAE recovery phase, despite being equally active at targeting brain ABHD6. Chronic administration of KT182 was associated to a partial loss of brain CB1 receptor coupling ability, suggesting the engagement of CB1 receptor-mediated mechanisms during the EAE disease progression. In cultured neurons, KT182 attenuated NMDA-stimulated excitotoxicity and mitochondrial calcium overload. However, these protective effects were not attributable to ABHD6, as they were not mimicked by the alternative inhibitors KT203, KT195 and WWL70. These results indicate that ABHD6 blockade exerts only modest therapeutic effects against autoimmune demyelination and call into question its utility as novel drug target in MS.

Mangiferin and Morin Attenuate Oxidative Stress, Mitochondrial Dysfunction, and Neurocytotoxicity, Induced by Amyloid Beta Oligomers.

Amyloid beta- (Aß-) mediated ROS overproduction disrupts intraneuronal redox balance and exacerbates mitochondrial dysfunction which leads to neuronal injury. Polyphenols have been investigated as therapeutic agents that promote neuroprotective effects in experimental models of brain injury and neurodegenerative diseases. The aim of this study was to identify the neuroprotective effects of morin and mangiferin against Aß oligomers in cultured cortical neurons and organotypic slices as well as their mechanisms of action. Cell death caused by Aß oligomers in neuronal cultures was decreased in the presence of micromolar concentrations of mangiferin or morin, which in turn attenuated oxidative stress. The neuroprotective effects of antioxidants against Aß were associated with the reduction of Aß-induced calcium load to mitochondria; mitochondrial membrane depolarization; and release of cytochrome c from mitochondria, a key trigger of apoptosis. Additionally, we observed that both polyphenols activated the endogenous enzymatic antioxidant system and restored oxidized protein levels. Finally, Aß induced an impairment of energy homeostasis due to a decreased respiratory capacity that was mitigated by morin and mangiferin. Overall, the beneficial effects of polyphenols in preventing mitochondrial dysfunction and neuronal injury in AD cell models suggest that morin and mangiferin hold promise for the treatment of this neurological disorder.

Energy-Aware RFID Anti-Collision Protocol.

The growing interest in mobile devices is transforming wireless identification technologies. Mobile and battery-powered Radio Frequency Identification (RFID) readers, such as hand readers and smart phones, are are becoming increasingly attractive. These RFID readers require energy-efficient anti-collision protocols to minimize the tag collisions and to expand the reader's battery life. Furthermore, there is an increasing interest in RFID sensor networks with a growing number of RFID sensor tags. Thus, RFID application developers must be mindful of tag anti-collision protocols. Energy-efficient protocols involve a low reader energy consumption per tag. This work presents a thorough study of the reader energy consumption per tag and analyzes the main factor that affects this metric: the frame size update strategy. Using the conclusion of this analysis, the anti-collision protocol Energy-Aware Slotted Aloha (EASA) is presented to decrease the energy consumption per tag. The frame size update strategy of EASA is configured to minimize the energy consumption per tag. As a result, EASA presents an energy-aware frame. The performance of the proposed protocol is evaluated and compared with several state of the art Aloha-based anti-collision protocols based on the current RFID standard. Simulation results show that EASA, with an average of 15 mJ consumed per tag identified, achieves a 6% average improvement in the energy consumption per tag in relation to the strategies of the comparison.

Mitochondrial Division Inhibitor 1 (mdivi-1) Protects Neurons against Excitotoxicity through the Modulation of Mitochondrial Function and Intracellular Ca2+ Signaling.

Excessive dynamin related protein 1 (Drp1)-triggered mitochondrial fission contributes to apoptosis under pathological conditions and therefore it has emerged as a promising therapeutic target. Mitochondrial division inhibitor 1 (mdivi-1) inhibits Drp1-dependent mitochondrial fission and is neuroprotective in several models of brain ischemia and neurodegeneration. However, mdivi-1 also modulates mitochondrial function and oxidative stress independently of Drp1, and consequently the mechanisms through which it protects against neuronal injury are more complex than previously foreseen. In this study, we have analyzed the effects of mdivi-1 on mitochondrial dynamics, Ca2+ signaling, mitochondrial bioenergetics and cell viability during neuronal excitotoxicity in vitro. Time-lapse fluorescence microscopy revealed that mdivi-1 blocked NMDA-induced mitochondrial fission but not that triggered by sustained AMPA receptor activation, showing that mdivi-1 inhibits excitotoxic mitochondrial fragmentation in a source specific manner. Similarly, mdivi-1 strongly reduced NMDA-triggered necrotic-like neuronal death and, to a lesser extent, AMPA-induced toxicity. Interestingly, neuroprotection provided by mdivi-1 against NMDA, but not AMPA, correlated with a reduction in cytosolic Ca2+ ([Ca2+]cyt) overload and calpain activation indicating additional cytoprotective mechanisms. Indeed, mdivi-1 depolarized mitochondrial membrane and depleted ER Ca2+ content, leading to attenuation of mitochondrial [Ca2+] increase and enhancement of the integrated stress response (ISR) during NMDA receptor activation. Finally, lentiviral knockdown of Drp1 did not rescue NMDA-induced mitochondrial fission and toxicity, indicating that neuroprotective activity of mdivi-1 is Drp1-independent. Together, these results suggest that mdivi-1 induces a Drp1-independent protective phenotype that prevents predominantly NMDA receptor-mediated excitotoxicity through the modulation of mitochondrial function and intracellular Ca2+ signaling.

Blockade and knock-out of CALHM1 channels attenuate ischemic brain damage.

Overactivation of purinergic receptors during cerebral ischemia results in a massive release of neurotransmitters, including adenosine triphosphate (ATP), to the extracellular space which leads to cell death. Some hypothetical pathways of ATP release are large ion channels, such as calcium homeostasis modulator 1 (CALHM1), a membrane ion channel that can permeate ATP. Since this transmitter contributes to postischemic brain damage, we hypothesized that CALHM1 activation may be a relevant target to attenuate stroke injury. Here, we analyzed the contribution of CALHM1 to postanoxic depolarization after ischemia in cultured neurons and in cortical slices. We observed that the onset of postanoxic currents in neurons in those preparations was delayed after its blockade with ruthenium red or silencing of Calhm1 gene by short hairpin RNA, as well as in slices from CALHM1 knockout mice. Subsequently, we used transient middle cerebral artery occlusion and found that ruthenium red, a blocker of CALHM1, or the lack of CALHM1, substantially attenuated the motor symptoms and reduced significantly the infarct volume. These results show that CALHM1 channels mediate postanoxic depolarization in neurons and brain damage after ischemia. Therefore, targeting CALHM1 may have a high therapeutic potential for treating brain damage after ischemia.

Measuring baseline Ca(2+) levels in subcellular compartments using genetically engineered fluorescent indicators.

Intracellular Ca(2+) signaling is involved in a series of physiological and pathological processes. In particular, an intimate crosstalk between bioenergetic metabolism and Ca(2+) homeostasis has been shown to determine cell fate in resting conditions as well as in response to stress. The endoplasmic reticulum and mitochondria represent key hubs of cellular metabolism and Ca(2+) signaling. However, it has been challenging to specifically detect highly localized Ca(2+) fluxes such as those bridging these two organelles. To circumvent this issue, various recombinant Ca(2+) indicators that can be targeted to specific subcellular compartments have been developed over the past two decades. While the use of these probes for measuring agonist-induced Ca(2+) signals in various organelles has been extensively described, the assessment of basal Ca(2+) concentrations within specific organelles is often disregarded, in spite of the fact that this parameter is vital for several metabolic functions, including the enzymatic activity of mitochondrial dehydrogenases of the Krebs cycle and protein folding in the endoplasmic reticulum. Here, we provide an overview on genetically engineered, organelle-targeted fluorescent Ca(2+) probes and outline their evolution. Moreover, we describe recently developed protocols to quantify baseline Ca(2+) concentrations in specific subcellular compartments. Among several applications, this method is suitable for assessing how changes in basal Ca(2+) levels affect the metabolic profile of cancer cells.

Endoplasmic reticulum Ca(2+) release through ryanodine and IP(3) receptors contributes to neuronal excitotoxicity.

Overactivation of ionotropic glutamate receptors induces a Ca(2+) overload into the cytoplasm that leads neurons to excitotoxic death, a process that has been linked to several neurodegenerative disorders. While the role of mitochondria and its involvement in excitotoxicity have been widely studied, the contribution of endoplasmic reticulum (ER), another crucial intracellular store in maintaining Ca(2+) homeostasis, is not fully understood. In this study, we analyzed the contribution of ER-Ca(2+) release through ryanodine (RyR) and IP(3) (IP(3)R) receptors to a neuronal in vitro model of excitotoxicity. NMDA induced a dose-dependent neuronal death, which was significantly decreased by ER-Ca(2+) release inhibitors in cortical neurons as well as in organotypic slices. Furthermore, ryanodine and 2APB, RyR and IP(3)R inhibitors respectively, attenuated NMDA-triggered intracellular Ca(2+) increase and oxidative stress, whereas 2APB reduced mitochondrial membrane depolarization and caspase-3 cleavage. Consistent with ER-Ca(2+) homeostasis disruption, we observed that NMDA-induced ER stress, characterized here by eIF2alpha phosphorylation and over-expression of GRP chaperones which were regulated by ER-Ca(2+) release inhibitors. These results demonstrate that Ca(2+) release from ER contributes to neuronal death by both promoting mitochondrial dysfunction and inducing specific stress and apoptosis pathways during excitotoxicity.