RESUMEN
OBJECTIVES: Booster doses for COVID-19 vaccinations have been shown to amplify the waning immune response after primary vaccination and to enhance protection against emerging variants of concern (VoCs). Here, we aimed to assess the immunogenicity and safety of a booster dose of an inactivated whole-virus COVID-19 vaccine (VLA2001) after primary vaccination with 2 doses of either VLA2001 or ChAdOx1-S (Oxford-Astra Zeneca), including the cross-neutralization capacity against the Delta and Omicron VoCs. METHODS: This interim analysis of an open-label extension of a randomized, controlled phase 3 trial assessed a single booster dose of an inactivated whole-virus COVID-19 vaccine (VLA2001) in healthy or medically stable adults aged 18 years and above, recruited in 21 clinical sites in the UK, who had previously received two doses of either VLA2001 or ChAdOx1-S. Safety outcomes were frequency and severity of solicited injection site and systemic reactions within 7 days after booster vaccination as well as frequency and severity of any unsolicited adverse events (AE) after up to 6 months. Immunogenicity outcomes were the immune response to ancestral SARS-CoV-2 assessed 14 days post booster expressed as geometric mean titres (GMT), GMT fold ratios and seroconversion of specific neutralizing antibodies and S-protein binding IgG antibodies. Immunogenicity against the Delta and Omicron VoCs was assessed as a post-hoc outcome with a pseudovirus neutralization antibody assay. This study is registered with ClinicalTrials.gov, NCT04864561, and is ongoing. RESULTS: A booster dose of VLA2001 was administered to 958 participants, of whom 712 had been primed with VLA2001, and 246 with ChAdOx1-S. Within 7 days following these booster doses, 607 (63.4%) participants reported solicited injection site reactions, and 487 (50.8%) reported solicited systemic reactions. Up to 14 days post booster, 751 (78.4%) participants reported at least one adverse event. The tolerability profile of a booster dose of VLA2001 was similar in VLA2001-primed and ChAdOx1-S-primed participants. In VLA2001-primed participants, the GMT (95% CI) of neutralizing antibodies increased from 32.5 (22.8, 46.3) immediately before to 521.5 (413.0, 658.6) 2 weeks after administration of the booster dose, this corresponds to a geometric mean fold rise (GMFR) of 27.7 (20.0, 38.5). Compared to 2 weeks after the second priming dose, the GMFR was 3.6 (2.8, 4.7). In the ChAdOx1-S primed group, the GMT (95% CI) of neutralizing antibodies increased from 65.8 (43.9, 98.4) immediately before to 188.3 (140.3, 252.8) 2 weeks after administration of the booster dose, a geometric mean fold rise (GMFR) of 3.0 (2.2, 4.0). Compared to 2 weeks after the second priming dose, the GMFR was 1.6 (1.1, 2.2). For S-protein binding IgG antibodies, the pre- versus post-booster GMT fold ratio (95% CI) was 34.6 (25.0, 48.0) in the VLA2001-primed group and 4.0 (3.0, 5.2) in the ChAdOx1-S-primed group. Compared to 2 weeks after the second priming dose, the GMT fold rise of IgG antibodies was 3.8 (3.2, 4.6) in the VLA2001-primed group and 1.2 (0.9, 1.6) in the ChAdOx1-S-primed group. The GMT against Delta (B.1.617.2) and Omicron (BA.4/5) increased from 4.2 to 260, and from 2.7 to 56.7, respectively, when boosting subjects previously primed with VLA2001. Following the boost, 97% of subjects primed with VLA2001 had detectable Delta- and 94% Omicron-neutralizing antibodies. In subjects primed with ChAdOx1-S, the GMT against Delta and Omicron titres increased from 9.1 to 92.5, and from 3.6 to 12.3, respectively. After boosting, 99% of subjects primed with ChAdOx1-S had detectable Delta- and 70% Omicron-neutralizing antibodies. In both VLA2001 and ChAdOx1-S primed subjects, the additional VLA2001 dose boosted T cell responses against SARS-CoV-2 antigens to levels above those observed before the booster dose. CONCLUSION: A booster dose of VLA2001 was safe and well tolerated after primary immunization with VLA2001 and ChAdOx1-S. The tolerability of a booster dose of VLA2001 was similar to the favourable profile observed after the first and second priming doses. Both in a homologous and a heterologous setting, boosting resulted in higher neutralizing antibody titres than after primary immunization and significant increases in cross-neutralization titres against Delta and Omicron were observed after the booster dose. These data support the use of VLA2001 in booster programmes in ChadOx1-S primed groups.
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Vacunas contra la COVID-19 , COVID-19 , Adulto , Humanos , Vacunas contra la COVID-19/efectos adversos , COVID-19/prevención & control , SARS-CoV-2 , Anticuerpos Neutralizantes , Inmunoglobulina G , Anticuerpos Antivirales , Inmunogenicidad VacunalRESUMEN
BACKGROUND: The Valneva COVID-19 vaccine (VLA2001; Valneva Austria, Vienna, Austria) is an inactivated whole-virus, adjuvanted SARS-CoV-2 vaccine. We aimed to assess the safety and immunogenicity of primary vaccination with VLA2001 versus the ChAdOx1-S (Oxford-AstraZeneca) adenoviral-vectored vaccine. METHODS: In this immunobridging phase 3 trial (COV-COMPARE), participants aged 18 years and older who were medically stable (as determined by an investigator) were enrolled at 26 sites in the UK. In the double-blind, randomised, controlled arm of the trial, participants aged 30 years and older were randomly assigned (2:1) to receive two doses of VLA2001 (0·5 mL; with 33 antigen units [AU] per dose) or ChAdOx1-S (0·5 mL; with 2·5 × 108 infectious units per dose) on days 1 and 29. In another arm, participants aged 18-29 years received two doses of VLA2001 (same dose) open label on days 1 and 29. The primary immunogenicity outcome was the immune response of a two-dose schedule of VLA2001 on day 43, in adults aged 30 years and older, versus two doses of ChAdOx1-S via superiority of geometric mean titres (GMTs) of neutralising antibodies (GMT ratio of >1 at a two-sided significance level of 5%) and non-inferiority of the seroconversion rate (non-inferiority margin of -10% for the lower limit of the 95% CI for the difference between groups). The primary safety outcome was the frequency and severity of any adverse events in all participants up to day 43. Safety was assessed in all participants who received at least one dose of vaccine. GMTs were assessed in a subset of participants aged 30 years and older who were seronegative at baseline, had at least one evaluable antibody titre measurement after vaccination, and had no confirmed COVID-19 during the study (immunogenicity population); and seroconversion was assessed in the per-protocol population, which comprised the immunogenicity population but excluding any participants with major protocol violations. For each timepoint, only participants with available data were included in the analysis. This study is registered with ClinicalTrials.gov, NCT04864561, and is ongoing. FINDINGS: Between April 28 and June 3, 2021, 4181 individuals were screened and 4017 enrolled, of whom 2975 (74%) were aged 30 years or older and randomly assigned to receive VLA2001 (n=1978) or ChAdOx1-S (n=997), and 1042 (26%) were aged 18-29 years (all received open-label VLA2001). 4012 participants received at least one dose of vaccine (1040 in the open-label VLA2001 group, 1977 in the randomised VLA2001 group, and 995 in the ChAdOx1-S group). The immunogenicity population comprised 492 participants in the randomised VLA2001 group and 498 in the ChAdOx1-S group; three participants in the VLA2001 group were excluded from the per-protocol population. VLA2001 induced higher neutralising GMTs than did ChAdOx1-S (803·5 [95% CI 748·5-862·6] vs 576·6 [543·6-611·7]; GMT ratio 1·39 [95% CI 1·25-1·56]; p<0·0001), and non-inferior seroconversion rates (444 [97·4%] of 456 participants vs 444 [98·9%] of 449; difference -1·5% [95% CI -3·3 to 0·2]. Any adverse event was reported in 963 (92·6%) participants in the open-label VLA2001 group, 1755 (88·8%) in the randomised VLA2001 group, and 976 (98·1%) in the ChAdOx1-S group. Most adverse events reported were mild or moderate in severity. INTERPRETATION: VLA2001 has a favourable tolerability profile and met superiority criteria for neutralising antibodies and non-inferiority criterion for seroconversion rates compared with ChAdOx1-S. The data presented here formed the basis of successful marketing approval for use of VLA2001 in primary vaccination in the EU, the UK, Bahrain, and United Arab Emirates. FUNDING: UK Department of Health and Social Care and Valneva Austria.
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Vacunas contra la COVID-19 , COVID-19 , Vacunas Virales , Adulto , Humanos , Adenoviridae/genética , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19/prevención & control , Vacunas contra la COVID-19/efectos adversos , Método Doble Ciego , Inmunogenicidad Vacunal , SARS-CoV-2 , Reino UnidoRESUMEN
Progressive neuronal injury following ischaemic stroke is associated with glutamate-induced depolarization, energetic stress and activation of AMP-activated protein kinase (AMPK). We here identify a molecular signature associated with neuronal AMPK activation, as a critical regulator of cellular response to energetic stress following ischaemia. We report a robust induction of microRNA miR-210-3p both in vitro in primary cortical neurons in response to acute AMPK activation and following ischaemic stroke in vivo. Bioinformatics and reverse phase protein array analysis of neuronal protein expression changes in vivo following administration of a miR-210-3p mimic revealed altered expression of phosphatase and tensin homolog (PTEN), 3-phosphoinositide-dependent protein kinase 1 (PDK1), ribosomal protein S6 kinase (p70S6K) and ribosomal protein S6 (RPS6) signalling in response to increasing miR-210-3p. In vivo, we observed a corresponding reduction in p70S6K activity following ischaemic stroke. Utilizing models of glutamate receptor over-activation in primary neurons, we demonstrated that induction of miR-210-3p was accompanied by sustained suppression of p70S6K activity and that this effect was reversed by miR-210-3p inhibition. Collectively, these results provide new molecular insight into the regulation of cell signalling during ischaemic injury, and suggest a novel mechanism whereby AMPK regulates miR-210-3p to control p70S6K activity in ischaemic stroke and excitotoxic injury.
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Proteínas Quinasas Activadas por AMP/metabolismo , Accidente Cerebrovascular Isquémico/patología , MicroARNs/genética , Neuronas/patología , Fosfatidilinositol 3-Quinasas/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Animales , Corteza Cerebral/patología , Biología Computacional , Activación Enzimática , Femenino , Masculino , Ratones Endogámicos C57BL , Fosfohidrolasa PTEN/metabolismo , Reacción en Cadena de la Polimerasa , Cultivo Primario de Células , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/metabolismo , Proteína S6 Ribosómica/metabolismo , Transducción de SeñalRESUMEN
Immunotherapies targeting pathological tau have recently emerged as a promising approach for treatment of neurodegenerative disorders. We have previously showed that the mouse antibody DC8E8 discriminates between healthy and pathological tau, reduces tau pathology in murine tauopathy models and inhibits neuronal internalization of AD tau species in vitro.Here we show, that DC8E8 and antibodies elicited against the first-in-man tau vaccine, AADvac1, which is based on the DC8E8 epitope peptide, both promote uptake of pathological tau by mouse primary microglia. IgG1 and IgG4 isotypes of AX004, the humanized versions of DC8E8, accelerate tau uptake by human primary microglia isolated from post-mortem aged and diseased brains. This promoting activity requires the presence of the Fc-domain of the antibodies.The IgG1 isotype of AX004 showed greater ability to promote tau uptake compared to the IgG4 isotype, while none of the antibody-tau complexes provoked increased pro-inflammatory activity of microglia. Our data suggest that IgG1 has better suitability for therapeutic development.
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Vacunas contra el Alzheimer/inmunología , Anticuerpos Monoclonales Humanizados/inmunología , Encefalitis/inmunología , Microglía/inmunología , Proteínas tau/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos Monoclonales Humanizados/metabolismo , Transporte Biológico , Células Cultivadas , Encefalitis/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/metabolismo , Adulto Joven , Proteínas tau/metabolismoRESUMEN
Pathologically altered tau protein is a common denominator of neurodegenerative disorders including Alzheimer's disease (AD) and other tauopathies. Therefore, promising immunotherapeutic approaches target and eliminate extracellular pathogenic tau species, which are thought to be responsible for seeding and propagation of tau pathology. Tau isoforms in misfolded states can propagate disease pathology in a template-dependent manner, proposed to be mediated by the release and internalization of extracellular tau. Monoclonal antibody DC8E8, binding four highly homologous and independent epitopes in microtubule-binding domain (MTBD) of diseased tau, inhibits tau-tau interaction, discriminates between healthy and pathologically truncated tau and reduces tau pathology in animal model in vivo. Here, we show that DC8E8 antibody acts via extracellular mechanism and does not influence viability and physiological functions of neurons. Importantly, in vitro functional assays showed that DC8E8 recognises pathogenic tau proteins of different size and origin, and potently blocks their entry into neurons. Next, we examined the mechanisms by which mouse antibody DC8E8 and its humanized version AX004 effectively block the neuronal internalization of extracellular AD tau species. We determined a novel mode of action of a therapeutic candidate antibody, which potently inhibits neuronal internalization of AD tau species by masking of epitopes present in MTBD important for interaction with neuron surface Heparan Sulfate Proteoglycans (HSPGs). We show that interference of tau-heparane sulfate interaction with DC8E8 antibody via steric hindrance represents an efficient and important therapeutic approach halting tau propagation.
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Anticuerpos Monoclonales/metabolismo , Microtúbulos/metabolismo , Neuronas/metabolismo , Proteoglicanos/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/administración & dosificación , Sitios de Unión/fisiología , Encéfalo/efectos de los fármacos , Encéfalo/patología , Sistemas de Liberación de Medicamentos/tendencias , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/genética , Espacio Extracelular/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microtúbulos/efectos de los fármacos , Microtúbulos/genética , Neuronas/efectos de los fármacos , Embarazo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas tau/genéticaRESUMEN
Cell death induced by excessive glutamate receptor overactivation, excitotoxicity, has been implicated in several acute and chronic neurological disorders. While numerous studies have demonstrated the contribution of biochemically and genetically activated cell death pathways in excitotoxic injury, the factors mediating passive, excitotoxic necrosis are less thoroughly investigated. To address this question, we developed a high content screening (HCS) based assay to collect high volumes of quantitative cellular imaging data and elucidated the effects of intrinsic and external factors on excitotoxic necrosis and apoptosis. The analysis workflow consisted of robust nuclei segmentation, tracking and a classification algorithm, which enabled automated analysis of large amounts of data to identify and quantify viable, apoptotic and necrotic neuronal populations. We show that mouse cerebellar granule neurons plated at low or high density underwent significantly increased necrosis compared to neurons seeded at medium density. Increased extracellular Ca2+ sensitized neurons to glutamate-induced excitotoxicity, but surprisingly potentiated cell death mainly through apoptosis. We also demonstrate that inhibition of various cell death signaling pathways (including inhibition of calpain, PARP and AMPK activation) primarily reduced excitotoxic apoptosis. Excitotoxic necrosis instead increased with low extracellular glucose availability. Our study is the first of its kind to establish and implement a HCS based assay to investigate the contribution of external and intrinsic factors to excitotoxic apoptosis and necrosis.
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Apoptosis , Supervivencia Celular , Cerebelo/citología , Neuronas/citología , Algoritmos , Animales , Calcio/metabolismo , Células Cultivadas , Medios de Cultivo Condicionados , Ratones , Necrosis , Neuronas/efectos de los fármacos , Neuronas/metabolismoRESUMEN
Alzheimer's disease (AD) represents the most common neurodegenerative disorder. Several animal models have been developed in order to test pathophysiological mechanisms of the disease and to predict effects of pharmacological interventions. Here we examine the molecular and behavioral features of R3m/4 transgenic mice expressing human non-mutated truncated tau protein (3R tau, aa151-391) that were previously used for efficacy testing of passive tau vaccine. The mouse model reliably recapitulated crucial histopathological features of human AD, such as pre-tangles, neurofibrillary tangles, and neuropil threads. The pathology was predominantly located in the brain stem. Transgenic mice developed mature sarkosyl insoluble tau complexes consisting of mouse endogenous and human truncated and hyperphosphorylated forms of tau protein. The histopathological and biochemical features were accompanied by significant sensorimotor impairment and reduced lifespan. The sensorimotor impairment was monitored by a highly sensitive, fully-automated tool that allowed us to assess early deficit in gait and locomotion. We suggest that the novel transgenic mouse model can serve as a valuable tool for analysis of the therapeutic efficacy of tau vaccines for AD therapy.
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Encéfalo/metabolismo , Modelos Animales de Enfermedad , Ovillos Neurofibrilares/metabolismo , Tauopatías/metabolismo , Proteínas tau/biosíntesis , Animales , Encéfalo/patología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ovillos Neurofibrilares/patología , Tauopatías/patologíaRESUMEN
Draxin is an important axon guidance cue necessary for the formation of forebrain commissures including the corpus callosum, but the molecular details of draxin signaling are unknown. To unravel how draxin signals are propagated we used murine cortical neurons and genetic and pharmacological approaches. We found that draxin-induced growth cone collapse critically depends on draxin receptors (deleted in colorectal cancer, DCC), inhibition of protein kinase B/Akt, activation of GSK-3ß (glycogen synthase kinase-3ß) and the presence of microtubule-associated protein MAP1B. This study, for the first time elucidates molecular events in draxin repulsion, links draxin and DCC to MAP1B and identifies a novel MAP1B-depenent GSK-3ß pathway essential for chemo-repulsive axon guidance cue signaling.
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Axones/fisiología , Glucógeno Sintasa Quinasa 3/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Células Cultivadas , Receptor DCC , Femenino , Glucógeno Sintasa Quinasa 3/deficiencia , Glucógeno Sintasa Quinasa 3 beta , Masculino , Ratones , Neuronas/fisiología , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Neuronal preconditioning is a phenomenon where a previous exposure to a sub-lethal stress stimulus increases the resistance of neurons towards a second, normally lethal stress stimulus. Activation of the energy stress sensor, AMP-activated protein kinase (AMPK) has been shown to contribute to the protective effects of ischaemic and mitochondrial uncoupling-induced preconditioning in neurons, however, the molecular basis of AMPK-mediated preconditioning has been less well characterized. We investigated the effect of AMPK preconditioning using 5-aminoimidazole-4-carboxamide riboside (AICAR) in a model of NMDA-mediated excitotoxic injury in primary mouse cortical neurons. Activation of AMPK with low concentrations of AICAR (0.1 mM for 2 h) induced a transient increase in AMPK phosphorylation, protecting neurons against NMDA-induced excitotoxicity. Analysing potential targets of AMPK activation, demonstrated a marked increase in mRNA expression and protein levels of the anti-apoptotic BCL-2 family protein myeloid cell leukaemia sequence 1 (MCL-1) in AICAR-preconditioned neurons. Interestingly, over-expression of MCL-1 protected neurons against NMDA-induced excitotoxicity while MCL-1 gene silencing abolished the effect of AICAR preconditioning. Monitored intracellular Ca²âº levels during NMDA excitation revealed that MCL-1 over-expressing neurons exhibited improved bioenergetics and markedly reduced Ca²âº elevations, suggesting a potential mechanism through which MCL-1 confers neuroprotection. This study identifies MCL-1 as a key effector of AMPK-induced preconditioning in neurons.
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Adaptación Fisiológica/fisiología , Corteza Cerebral/metabolismo , Neuronas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Western Blotting , Corteza Cerebral/efectos de los fármacos , Citometría de Flujo , Hipoglucemiantes/farmacología , Ratones , Microscopía Confocal , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , N-Metilaspartato/toxicidad , Neuronas/efectos de los fármacos , Ribonucleótidos/farmacología , Estrés Fisiológico/fisiologíaRESUMEN
The preconditioning response conferred by a mild uncoupling of the mitochondrial membrane potential (Δψ(m)) has been attributed to altered reactive oxygen species (ROS) production and mitochondrial Ca(2+) uptake within the cells. Here we have explored if altered cellular energetics in response to a mild mitochondrial uncoupling stimulus may also contribute to the protection. The addition of 100 nM FCCP for 30 min to cerebellar granule neurons (CGNs) induced a transient depolarization of the Δψ(m), that was sufficient to significantly reduce CGN vulnerability to the excitotoxic stimulus, glutamate. On investigation, the mild mitochondrial 'uncoupling' stimulus resulted in a significant increase in the plasma membrane levels of the glucose transporter isoform 3, with a hyperpolarisation of Δψ(m) and increased cellular ATP levels also evident following the washout of FCCP. Furthermore, the phosphorylation state of AMP-activated protein kinase (AMPK) (Thr 172) was increased within 5 min of the uncoupling stimulus and elevated up to 1h after washout. Significantly, the physiological changes and protection evident after the mild uncoupling stimulus were lost in CGNs when AMPK activity was inhibited. This study identifies an additional mechanism through which protection is mediated upon mild mitochondrial uncoupling: it implicates increased AMPK signalling and an adaptive shift in energy metabolism as mediators of the preconditioning response associated with FCCP-induced mild mitochondrial uncoupling.
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Proteínas Quinasas Activadas por AMP/metabolismo , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Citoprotección/efectos de los fármacos , Mitocondrias/metabolismo , Neuronas/citología , Neuronas/enzimología , Neurotoxinas/toxicidad , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cerebelo/citología , Metabolismo Energético , Activación Enzimática/efectos de los fármacos , Ácido Glutámico/toxicidad , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Neuronas/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacosRESUMEN
5'-Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a key sensor of cellular energy status. AMPK signaling regulates energy balance at the cellular, organ, and whole-body level. More recently, it has become apparent that AMPK plays also an important role in long-term decisions that determine cell fate, in particular cell cycle progression and apoptosis activation. Here, we describe the diverse mechanisms of AMPK activation and the role of AMPK in the regulation of cellular energy balance. We summarize recent studies implicating AMPK activation in the regulation of neuronal survival and as a key player during ischemic stroke. We also suggest that AMPK activation may have dual functions in the regulation of neuronal survival: AMPK provides a protective effect during transient energy depletion as exemplified in a model of neuronal Ca(2+) overloading, and this effect is partially mediated by the activation of neuronal glucose transporter 3. Prolonged AMPK activation, on the contrary, can lead to neuronal apoptosis via the transcriptional activation of the proapoptotic Bcl-2 family member, bim. Molecular switches that determine the protective versus cell death-inducing effects of AMPK activation are discussed.
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Proteínas Quinasas Activadas por AMP/metabolismo , Supervivencia Celular/fisiología , Neuronas/enzimología , Neuronas/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Animales , Apoptosis/genética , Apoptosis/fisiología , Supervivencia Celular/genética , HumanosRESUMEN
Excitotoxicity after glutamate receptor overactivation induces disturbances in cellular ion gradients, resulting in necrosis or apoptosis. Excitotoxic necrosis is triggered by rapid, irreversible ATP depletion, whereas the ability to recover cellular bioenergetics is suggested to be necessary for the activation of excitotoxic apoptosis. In this study, we demonstrate that even a transient decrease in cellular bioenergetics and an associated activation of adenosine monophosphate-activated protein kinase (AMPK) is necessary for the activation of excitotoxic apoptosis. We show that the Bcl-2 homology domain 3 (BH3)-only protein Bim, a proapoptotic Bcl-2 family member, is activated in multiple excitotoxicity paradigms, mediates excitotoxic apoptosis, and inhibits delayed Ca(2+) deregulation, mitochondrial depolarization, and apoptosis-inducing factor translocation. We demonstrate that bim activation required the activation of AMPK and that prolonged AMPK activation is sufficient to induce bim gene expression and to trigger a bim-dependent cell death. Collectively, our data demonstrate that AMPK activation and the BH3-only protein Bim couple transient energy depletion to stress-induced neuronal apoptosis.
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Adenilato Quinasa/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Adenilato Quinasa/genética , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Muerte Celular , Células Cultivadas , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Microscopía Confocal , Neuronas/metabolismo , Proteínas Proto-Oncogénicas/genética , RatasRESUMEN
Ischemic and excitotoxic events within the brain result in rapid and often unfavorable depletions in neuronal energy levels. Here, we investigated the signaling pathways activated in response to the energetic stress created by transient glutamate excitation in cerebellar granule neurons. We characterized a glucose dependent hyperpolarization of the mitochondrial membrane potential (Delta psi(m)) in the majority of neurons after transient glutamate excitation. Expression levels of the primary neuronal glucose transporters (GLUTs) isoforms 1, 3, 4, and 8 were found to be unaltered within a 24 h period after excitation. However, a significant increase only in GLUT3 surface expression was identified 30 min after excitation, with this high surface expression remaining significantly above control levels in many neurons for up to 4 h. Glutamate excitation induced a rapid alteration in the AMP:ATP ratio that was associated with the activation of the AMP-activated protein kinase (AMPK). Interestingly, pharmacological activation of AMPK with AICAR (5-aminoimidazole-4-carboxamide riboside) alone also increased GLUT3 surface expression, with a hyperpolarization of Delta psi(m) evident in many neurons. Notably, inhibition of the CaMKK (calmodulin-dependent protein kinase kinase) had little affect on GLUT translocation, whereas the inhibition or knockdown of AMPK (compound C, siRNA) activity prevented GLUT3 translocation to the cell surface after glutamate excitation. Furthermore, gene silencing of GLUT3 eradicated the increase in Delta psi(m) associated with transient glutamate excitation and potently sensitized neurons to excitotoxicity. In summary, our data suggest that the activation of AMPK and its regulation of cell surface GLUT3 expression is critical in mediating neuronal tolerance to excitotoxicity.
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Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Aminoácidos Excitadores/toxicidad , Regulación de la Expresión Génica/fisiología , Transportador de Glucosa de Tipo 3/biosíntesis , Ácido Glutámico/toxicidad , Neuronas/efectos de los fármacos , Aminoimidazol Carboxamida/análogos & derivados , Bisbenzimidazol , Western Blotting , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/fisiología , Membrana Celular/fisiología , Cerebelo/citología , Cerebelo/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica/genética , Glucosa/fisiología , Transportador de Glucosa de Tipo 3/genética , Humanos , Microscopía Confocal , Mitocondrias/fisiología , Mitocondrias/ultraestructura , ARN Interferente Pequeño , Receptores de Superficie Celular/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleótidos/fisiologíaRESUMEN
Changes in the electrochemical gradients across biological membranes are excellent indicators of pathophysiological processes, drug action, or drug toxicity. Our previous studies have utilized the potentiometric probe tetramethylrhodamine methyl ester (TMRM) to characterize changes in mitochondrial function by monitoring alterations in the mitochondrial membrane potential (Deltapsi(m)) over time during glutamate excitotoxicity. However, fluorescently charged dyes such as TMRM respond to changes in both Deltapsi(m) and the plasma membrane (Deltapsi(p)) potentials making whole cell fluorescence data difficult to interpret. Here we have implemented a mathematical model that exploits the Nernstian behaviour of TMRM and uses automated Newton based root-finding fitting (TOXI-SIM) to model changes in TMRM fluorescence from multiple cells simultaneously, providing output on changes in Deltapsi(m) and Deltapsi(p) over time. Based on Ca(2+) responses, TOXI-SIM allows for an accurate modelling of TMRM traces for different injury paradigms (necrosis, apoptosis, tolerance). TOXI-SIM is provided as a user friendly public web service for trace analysis, with an additional online data base provided for the storage and retrieval of experimental traces (http://systemsbiology.rcsi.ie/tmrm/index.html).
Asunto(s)
Membrana Celular/fisiología , Cerebelo/citología , Potencial de la Membrana Mitocondrial/fisiología , Potenciales de la Membrana/fisiología , Modelos Estadísticos , Neuronas/ultraestructura , Animales , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Células Cultivadas , Maleato de Dizocilpina/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Glutamatos/farmacología , Potenciales de la Membrana/efectos de los fármacos , Necrosis/metabolismo , Necrosis/patología , Neuronas/fisiología , Ratas , Rodaminas/metabolismo , Factores de TiempoRESUMEN
A failure of mitochondrial bioenergetics has been shown to be closely associated with the onset of apoptotic and necrotic neuronal injury. Here, we developed an automated computational model that interprets the single-cell fluorescence for tetramethylrhodamine methyl ester (TMRM) as a consequence of changes in either delta psi(m) or delta psi(p), thus allowing for the characterization of responses for populations of single cells and subsequent statistical analysis. Necrotic injury triggered by prolonged glutamate excitation resulted in a rapid monophasic or biphasic loss of delta psi(m) that was closely associated with a loss of delta psi(p) and a rapid decrease in neuronal NADPH and ATP levels. Delayed apoptotic injury, induced by transient glutamate excitation, resulted in a small, reversible decrease in TMRM fluorescence, followed by a sustained hyperpolarization of delta psi(m) as confirmed using the delta psi(p)-sensitive anionic probe DiBAC2(3). This hyperpolarization of delta psi(m) was closely associated with a significant increase in neuronal glucose uptake, NADPH availability, and ATP levels. Statistical analysis of the changes in delta psi(m) or delta psi(p) at a single-cell level revealed two major correlations; those neurons displaying a more pronounced depolarization of delta psi(p) during the initial phase of glutamate excitation entered apoptosis more rapidly, and neurons that displayed a more pronounced hyperpolarization of delta psi(m) after glutamate excitation survived longer. Indeed, those neurons that were tolerant to transient glutamate excitation (18%) showed the most significant increases in delta psi(m). Our results indicate that a hyperpolarization of delta psi(m) is associated with increased glucose uptake, NADPH availability, and survival responses during excitotoxic injury.
Asunto(s)
Apoptosis/fisiología , Membrana Celular/metabolismo , Cerebelo/fisiología , Ácido Glutámico/metabolismo , Potenciales de la Membrana/fisiología , Membranas Mitocondriales/fisiología , Animales , Apoptosis/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Células Cultivadas , Cerebelo/citología , Cerebelo/efectos de los fármacos , Tolerancia a Medicamentos/fisiología , Femenino , Ácido Glutámico/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Necrosis/inducido químicamente , Necrosis/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Ratas WistarRESUMEN
The capacity of yeast cells to produce reactive oxygen species (ROS), both as a response to manipulation of mitochondrial functions and to growth conditions, was estimated and compared with the viability of the cells. The chronological ageing of yeast cells (growth to late-stationary phase) was accompanied by increased ROS accumulation and a significantly higher loss of viability in the mutants with impaired mitochondrial functions than in the parental strain. Under these conditions, the ectopic expression of mammalian Bcl-x(L), which is an anti-apoptotic protein, allowed cells to survive longer in stationary phase. The protective effect of Bcl-x(L) was more prominent in respiratory-competent cells that contained defects in mitochondrial ADP/ATP translocation, suggesting a model for Bcl-x(L) regulation of chronological ageing at the mitochondria. Yeast can also be triggered into apoptosis-like cell death, at conditions leading to the depletion of the intramitochondrial ATP pool, as a consequence of the parallel inhibition of mitochondrial respiration and ADP/ATP translocation. If respiratory-deficient (rho(0)) cells were used, no correlation between the numbers of ROS-producing cells and the viability loss in the population was observed, indicating that ROS production may be an accompanying event. The protective effect of Bcl-x(L) against death of these cells suggests a mitochondrial mechanism which is different from the antioxidant activity of Bcl-x(L).