RESUMEN
The energy cost of neuronal activity is mainly sustained by glucose1,2. However, in an apparent paradox, neurons modestly metabolize glucose through glycolysis3-6, a circumstance that can be accounted for by the constant degradation of 6-phosphofructo-2-kinase-fructose-2,6-bisphosphatase-3 (PFKFB3)3,7,8, a key glycolysis-promoting enzyme. To evaluate the in vivo physiological importance of this hypoglycolytic metabolism, here we genetically engineered mice with their neurons transformed into active glycolytic cells through Pfkfb3 expression. In vivo molecular, biochemical and metabolic flux analyses of these neurons revealed an accumulation of anomalous mitochondria, complex I disassembly, bioenergetic deficiency and mitochondrial redox stress. Notably, glycolysis-mediated nicotinamide adenine dinucleotide (NAD+) reduction impaired sirtuin-dependent autophagy. Furthermore, these mice displayed cognitive decline and a metabolic syndrome that was mimicked by confining Pfkfb3 expression to hypothalamic neurons. Neuron-specific genetic ablation of mitochondrial redox stress or brain NAD+ restoration corrected these behavioural alterations. Thus, the weak glycolytic nature of neurons is required to sustain higher-order organismal functions.
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Cognición , Glucólisis , Neuronas , Fosfofructoquinasa-2 , Animales , Neuronas/metabolismo , Ratones , Fosfofructoquinasa-2/metabolismo , Fosfofructoquinasa-2/genética , Cognición/fisiología , Mitocondrias/metabolismo , Metabolismo Energético , NAD/metabolismo , Glucosa/metabolismoRESUMEN
Metabolic bone diseases cover a broad spectrum of disorders that share alterations in bone metabolism that lead to a defective skeleton, which is associated with increasing morbidity, disability, and mortality. There is a close connection between the etiology of metabolic bone diseases and genetic factors, with TP53 being one of the genes associated therewith. The single nucleotide polymorphism (SNP) Arg72Pro of TP53 is a genetic factor associated with several pathologies, including cancer, stroke, and osteoporosis. Here, we aim to analyze the influence of the TP53 Arg72Pro SNP on bone mass in humanized Tp53 Arg72Pro knock-in mice. This work reports on the influence of the TP53 Arg72Pro polymorphism in bone microarchitecture, OPG expression, and apoptosis bone status. The results show that the proline variant of the TP53 Arg72Pro polymorphism (Pro72-p53) is associated with deteriorated bone tissue, lower OPG/RANK ratio, and lower apoptosis in bone tissue. In conclusion, the TP53 Arg72Pro polymorphism modulates bone microarchitecture and may be a genetic biomarker that can be used to identify individuals with an increased risk of suffering metabolic bone alterations.
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Enfermedades Óseas Metabólicas , Proteína p53 Supresora de Tumor , Animales , Ratones , Biomarcadores , Huesos , Estudios de Casos y Controles , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple , Proteína p53 Supresora de Tumor/genética , HumanosRESUMEN
In the era that we seek personalization in material things, it is becoming increasingly clear that the individualized management of medicine and nutrition plays a key role in life expectancy and quality of life, allowing participation to some extent in our welfare and the use of societal resources in a rationale and equitable way. The implementation of precision medicine and nutrition are highly complex challenges which depend on the development of new technologies able to meet important requirements in terms of cost, simplicity, and versatility, and to determine both individually and simultaneously, almost in real time and with the required sensitivity and reliability, molecular markers of different omics levels in biofluids extracted, secreted (either naturally or stimulated), or circulating in the body. Relying on representative and pioneering examples, this review article critically discusses recent advances driving the position of electrochemical bioplatforms as one of the winning horses for the implementation of suitable tools for advanced diagnostics, therapy, and precision nutrition. In addition to a critical overview of the state of the art, including groundbreaking applications and challenges ahead, the article concludes with a personal vision of the imminent roadmap.
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Medicina de Precisión , Calidad de Vida , Animales , Caballos , Reproducibilidad de los Resultados , BiomarcadoresRESUMEN
Having direct access to brain vasculature, astrocytes can take up available blood nutrients and metabolize them to fulfil their own energy needs and deliver metabolic intermediates to local synapses1,2. These glial cells should be, therefore, metabolically adaptable to swap different substrates. However, in vitro and in vivo studies consistently show that astrocytes are primarily glycolytic3-7, suggesting glucose is their main metabolic precursor. Notably, transcriptomic data8,9 and in vitro10 studies reveal that mouse astrocytes are capable of mitochondrially oxidizing fatty acids and that they can detoxify excess neuronal-derived fatty acids in disease models11,12. Still, the factual metabolic advantage of fatty acid use by astrocytes and its physiological impact on higher-order cerebral functions remain unknown. Here, we show that knockout of carnitine-palmitoyl transferase-1A (CPT1A)-a key enzyme of mitochondrial fatty acid oxidation-in adult mouse astrocytes causes cognitive impairment. Mechanistically, decreased fatty acid oxidation rewired astrocytic pyruvate metabolism to facilitate electron flux through a super-assembled mitochondrial respiratory chain, resulting in attenuation of reactive oxygen species formation. Thus, astrocytes naturally metabolize fatty acids to preserve the mitochondrial respiratory chain in an energetically inefficient disassembled conformation that secures signalling reactive oxygen species and sustains cognitive performance.
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Astrocitos , Encéfalo , Ratones , Animales , Astrocitos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Encéfalo/metabolismo , Cognición , Ácidos Grasos/metabolismoRESUMEN
Mitochondrial reactive oxygen species (mROS) have been generally considered harmful byproducts wanted to clear when elevated to avoid brain damage. However, the abundance of mROS in astrocytes is very high -about one order of magnitude above that in neurons-, despite they are essential to preserve cell metabolism and animal behavior. Here, we have focused on this apparent ambiguity by discussing (i) the intrinsic mechanisms accounting for the higher production of mROS by the mitochondrial respiratory chain in astrocytes than in neurons, (ii) the specific molecular targets of astrocytic beneficial mROS, and (iii) how decreased astrocytic mROS causes excess neuronal mROS leading to cellular and organismal damage. We hope that this mini-review serves to clarifying the apparent controversy on the beneficial versus deleterious faces of ROS in the brain from molecular to higher-order organismal levels.
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Encéfalo , Mitocondrias , Animales , Especies Reactivas de Oxígeno/metabolismo , Mitocondrias/metabolismo , Encéfalo/metabolismoRESUMEN
Astrocytes show unique anatomical, morphological, and metabolic features to take up substrates from the blood and metabolize them for local delivery to active synapses to sustain neuron function. In the present review, we specifically focus on key molecular aspects of energy and redox metabolism that facilitate this astrocyte-neuronal coupling in a controlled manner. Basal glycolysis is co-ordinated by the anaphase-promoting complex/cyclosome (APC/C)-Cdh1, a ubiquitin ligase that targets the proglycolytic enzyme 6-phosphofructokinase-2,6-bisphosphastate-3 (PFKFB3) for degradation. APC/C-Cdh1 activity is more robust in neurons than in astrocytes, which determine that PFKFB3 abundance and glycolytic rate are weaker in neurons. The low PFKFB3 activity in neurons facilitates glucose-6-phosphate oxidation via the pentose-phosphate pathway, which promotes antioxidant protection. Conversely, the high PFKFB3 activity in astrocytes allows the production and release of glycolytic lactate, which is taken up by neurons that use it as an oxidizable substrate. Importantly, the mitochondrial respiratory chain is tighter assembled in neurons than in astrocytes, thus the bioenergetic efficiency of mitochondria is higher in neurons. Because of this, the production of reactive oxygen species (mROS) by mitochondrial complex I is very low in neurons and very high in astrocytes. Such a naturally occurring high abundance of mROS in astrocytes physiologically determines a specific transcriptional fingerprint that contributes to sustaining cognitive performance. We conclude that the energy and redox metabolism of astrocytes must complementarily match that of neurons to regulate brain function and animal welfare.
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Astrocitos , Metabolismo Energético , Animales , Astrocitos/metabolismo , Glucólisis/fisiología , Oxidación-Reducción , Neuronas/metabolismoRESUMEN
Intracellular Ca2+ concentrations are strictly controlled by plasma membrane transporters, the endoplasmic reticulum, and mitochondria, in which Ca2+ uptake is mediated by the mitochondrial calcium uniporter complex (MCUc), while efflux occurs mainly through the mitochondrial Na+ /Ca2+ exchanger (NCLX). RNAseq database repository searches led us to identify the Nclx transcript as highly enriched in astrocytes when compared with neurons. To assess the role of NCLX in mouse primary culture astrocytes, we inhibited its function both pharmacologically or genetically. This resulted in re-shaping of cytosolic Ca2+ signaling and a metabolic shift that increased glycolytic flux and lactate secretion in a Ca2+ -dependent manner. Interestingly, in vivo genetic deletion of NCLX in hippocampal astrocytes improved cognitive performance in behavioral tasks, whereas hippocampal neuron-specific deletion of NCLX impaired cognitive performance. These results unveil a role for NCLX as a novel modulator of astrocytic glucose metabolism, impacting on cognition.
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Astrocitos , Calcio , Ratones , Animales , Astrocitos/metabolismo , Calcio/metabolismo , Intercambiador de Sodio-Calcio/genética , Mitocondrias/metabolismo , Glucólisis , Cognición , Sodio/metabolismo , Señalización del Calcio/fisiologíaRESUMEN
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline, which is causally related to the accumulation of abnormally folded amyloid-ß (Aß) peptide and hyperphosphorylated tau protein aggregates. The dendritic spine regulator Rho protein kinase 2 (Rock2) accumulates in the brain at the earliest stages of AD and remains increased during disease progression. However, the molecular mechanism that upregulates Rock2 in AD, and its role in the disease progression, are unknown. Here, we found that oligomers of the amyloidogenic fragment 25-35 of the Aß peptide (Aß25-35) trigger Rock2 accumulation and activation in mouse cortical neurons in primary culture and in mouse hippocampus in vivo. Neuronal apoptotic death and memory impairment caused by Aß25-35 administration were rescued by genetic and pharmacological inhibition of Rock2 activity. Mechanistically, Aß25-35 elicited cyclin dependent kinase-5 (Cdk5)-mediated phosphorylation of Cdh1, a cofactor that is essential for the activity of the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) in neurons. Notably, phosphorylated Cdh1 was disassembled from the APC/C complex, causing its inactivation and subsequent Rock2 protein stabilization and activation. Moreover, Aß25-35-induced neuronal apoptosis was prevented by expressing a phosphodefective form of Cdh1, but not by a phosphomimetic Cdh1. Finally, Cdh1 inactivation, using both genetic and pharmacological approaches, enhanced Aß25-35-mediated neuronal death through a mechanism that was prevented by inhibition of Rock2 activity. These results indicate that the Cdk5-Cdh1 signaling pathway accounts for the increased Rock2 activity by amyloidogenic Aß peptides and that this mechanism may contribute to neurodegeneration and memory loss in AD.
RESUMEN
CLN7 neuronal ceroid lipofuscinosis is an inherited lysosomal storage neurodegenerative disease highly prevalent in children. CLN7/MFSD8 gene encodes a lysosomal membrane glycoprotein, but the biochemical processes affected by CLN7-loss of function are unexplored thus preventing development of potential treatments. Here, we found, in the Cln7∆ex2 mouse model of CLN7 disease, that failure in autophagy causes accumulation of structurally and bioenergetically impaired neuronal mitochondria. In vivo genetic approach reveals elevated mitochondrial reactive oxygen species (mROS) in Cln7∆ex2 neurons that mediates glycolytic enzyme PFKFB3 activation and contributes to CLN7 pathogenesis. Mechanistically, mROS sustains a signaling cascade leading to protein stabilization of PFKFB3, normally unstable in healthy neurons. Administration of the highly selective PFKFB3 inhibitor AZ67 in Cln7∆ex2 mouse brain in vivo and in CLN7 patients-derived cells rectifies key disease hallmarks. Thus, aberrant upregulation of the glycolytic enzyme PFKFB3 in neurons may contribute to CLN7 pathogenesis and targeting PFKFB3 could alleviate this and other lysosomal storage diseases.
Asunto(s)
Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/metabolismo , Lipofuscinosis Ceroideas Neuronales/metabolismo , Fosfofructoquinasa-2/metabolismo , Animales , Autofagia , Preescolar , Modelos Animales de Enfermedad , Femenino , Humanos , Enfermedades por Almacenamiento Lisosomal/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/metabolismo , Masculino , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Lipofuscinosis Ceroideas Neuronales/genética , Neuronas/metabolismo , Fosfofructoquinasa-2/genética , Regulación hacia ArribaRESUMEN
Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder and the main cause of dementia in the elderly. The disease has a high impact on individuals and their families and represents a growing public health and socio-economic burden. Despite this, there is no effective treatment options to cure or modify the disease progression, highlighting the need to identify new therapeutic targets. Synapse dysfunction and loss are early pathological features of Alzheimer's disease, correlate with cognitive decline and proceed with neuronal death. In the last years, the E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C) has emerged as a key regulator of synaptic plasticity and neuronal survival. To this end, the ligase binds Cdh1, its main activator in the brain. However, inactivation of the anaphase promoting complex/cyclosome-Cdh1 complex triggers dendrite disruption, synapse loss and neurodegeneration, leading to memory and learning impairment. Interestingly, oligomerized amyloid-ß (Aß) peptide, which is involved in Alzheimer's disease onset and progression, induces Cdh1 phosphorylation leading to anaphase promoting complex/cyclosome-Cdh1 complex disassembly and inactivation. This causes the aberrant accumulation of several anaphase promoting complex/cyclosome-Cdh1 targets in the damaged areas of Alzheimer's disease brains, including Rock2 and Cyclin B1. Here we review the function of anaphase promoting complex/cyclosome-Cdh1 dysregulation in the pathogenesis of Alzheimer's disease, paying particular attention in the neurotoxicity induced by its molecular targets. Understanding the role of anaphase promoting complex/cyclosome-Cdh1-targeted substrates in Alzheimer's disease may be useful in the development of new effective disease-modifying treatments for this neurological disorder.
RESUMEN
One of the most important mechanisms of preconditioning-mediated neuroprotection is the attenuation of cell apoptosis, inducing brain tolerance after a subsequent injurious ischemia. In this context, the antiapoptotic PI3K/AKT signaling pathway plays a key role by regulating cell differentiation and survival. Active AKT is known to increase the expression of murine double minute-2 (MDM2), an E3-ubiquitin ligase that destabilizes p53 to promote the survival of cancer cells. In neurons, we recently showed that the MDM2-p53 interaction is potentiated by pharmacological preconditioning, based on subtoxic stimulation of NMDA glutamate receptor, which prevents ischemia-induced neuronal apoptosis. However, whether this mechanism contributes to the neuronal tolerance during ischemic preconditioning (IPC) is unknown. Here, we show that IPC induced PI3K-mediated phosphorylation of AKT at Ser473, which in turn phosphorylated MDM2 at Ser166. This phosphorylation triggered the nuclear stabilization of MDM2, leading to p53 destabilization, thus preventing neuronal apoptosis upon an ischemic insult. Inhibition of the PI3K/AKT pathway with wortmannin or by AKT silencing induced the accumulation of cytosolic MDM2, abrogating IPC-induced neuroprotection. Thus, IPC enhances the activation of PI3K/AKT signaling pathway and promotes neuronal tolerance by controlling the MDM2-p53 interaction. Our findings provide a new mechanistic pathway involved in IPC-induced neuroprotection via modulation of AKT signaling, suggesting that AKT is a potential therapeutic target against ischemic injury.
Asunto(s)
Isquemia/metabolismo , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Transducción de Señal/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/fisiología , Células HEK293 , Humanos , Precondicionamiento Isquémico/métodos , Ratones , Ratones Endogámicos C57BL , Neuroprotección/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/fisiología , Wortmanina/metabolismoRESUMEN
Cells naturally produce mitochondrial reactive oxygen species (mROS), but the in vivo pathophysiological significance has long remained controversial. Within the brain, astrocyte-derived mROS physiologically regulate behaviour and are produced at one order of magnitude faster than in neurons. However, whether neuronal mROS abundance differentially impacts on behaviour is unknown. To address this, we engineered genetically modified mice to down modulate mROS levels in neurons in vivo. Whilst no alterations in motor coordination were observed by down modulating mROS in neurons under healthy conditions, it prevented the motor discoordination caused by the pro-oxidant neurotoxin, 3-nitropropionic acid (3-NP). In contrast, abrogation of mROS in astrocytes showed no beneficial effect against the 3-NP insult. These data indicate that the impact of modifying mROS production on mouse behaviour critically depends on the specific cell-type where they are generated.
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Astrocitos , Mitocondrias , Animales , Astrocitos/metabolismo , Células Cultivadas , Ratones , Neuronas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Stroke is a major cause of death and long-term disability in the adult. Neuronal apoptosis plays an essential role in the pathophysiology of ischemic brain damage and impaired functional recovery after stroke. The tumor suppressor protein p53 regulates key cellular processes, including cell cycle arrest, DNA repair, senescence, and apoptosis. Under cellular stress conditions, p53 undergoes post-translational modifications, which control protein localization, stability, and proapoptotic activity. After stroke, p53 rapidly accumulates in the ischemic brain, where it activates neuronal apoptosis through both transcriptional-dependent and -independent programs. Over the last years, subcellular localization of p53 has emerged as an important regulator of ischemia-induced neuronal apoptosis. Upon an ischemic insult, p53 rapidly translocates to the mitochondria and interacts with B-cell lymphoma-2 family proteins, which activate the mitochondrial apoptotic program, with higher efficacy than through its activity as a transcription factor. Moreover, the identification of a human single nucleotide polymorphism at codon 72 of the Tp53 gene that controls p53 mitochondrial localization and cell susceptibility to apoptosis supports the important role of the p53 mitochondrial program in neuronal survival and functional recovery after stroke. In this article, we review the relevance of mitochondrial and nuclear localization of p53 on neuronal susceptibility to cerebral ischemia and its impact on functional outcome of stroke patients.
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Mitocondrias/metabolismo , Neuronas/metabolismo , Accidente Cerebrovascular/patología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Muerte Celular , Humanos , Polimorfismo de Nucleótido Simple , Pronóstico , Transporte de Proteínas , Accidente Cerebrovascular/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Failure of neurons to efficiently repair DNA double-strand breaks (DSBs) contributes to cerebral damage after stroke. However, the molecular machinery that regulates DNA repair in this neurological disorder is unknown. Here, we found that DSBs in oxygen/glucose-deprived (OGD) neurons spatiotemporally correlated with the up-regulation of WRAP53 (WD40-encoding p53-antisense RNA), which translocated to the nucleus to activate the DSB repair response. Mechanistically, OGD triggered a burst in reactive oxygen species that induced both DSBs and translocation of WRAP53 to the nucleus to promote DNA repair, a pathway that was confirmed in an in vivo mouse model of stroke. Noticeably, nuclear translocation of WRAP53 occurred faster in OGD neurons expressing the Wrap53 human nonsynonymous single-nucleotide polymorphism (SNP) rs2287499 (c.202C>G). Patients carrying this SNP showed less infarct volume and better functional outcome after stroke. These results indicate that WRAP53 fosters DNA repair and neuronal survival to promote functional recovery after stroke.
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Núcleo Celular , Chaperonas Moleculares/metabolismo , Accidente Cerebrovascular , Telomerasa/metabolismo , Animales , Núcleo Celular/metabolismo , Supervivencia Celular/genética , Reparación del ADN , Glucosa/metabolismo , Humanos , Ratones , Neuronas/metabolismo , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Astrocytes take up glucose from the bloodstream to provide energy to the brain, thereby allowing neuronal activity and behavioural responses1-5. By contrast, astrocytes are under neuronal control through specific neurotransmitter receptors5-7. However, whether the activation of astroglial receptors can directly regulate cellular glucose metabolism to eventually modulate behavioural responses is unclear. Here we show that activation of mouse astroglial type-1 cannabinoid receptors associated with mitochondrial membranes (mtCB1) hampers the metabolism of glucose and the production of lactate in the brain, resulting in altered neuronal functions and, in turn, impaired behavioural responses in social interaction assays. Specifically, activation of astroglial mtCB1 receptors reduces the phosphorylation of the mitochondrial complex I subunit NDUFS4, which decreases the stability and activity of complex I. This leads to a reduction in the generation of reactive oxygen species by astrocytes and affects the glycolytic production of lactate through the hypoxia-inducible factor 1 pathway, eventually resulting in neuronal redox stress and impairment of behavioural responses in social interaction assays. Genetic and pharmacological correction of each of these effects abolishes the effect of cannabinoid treatment on the observed behaviour. These findings suggest that mtCB1 receptor signalling can directly regulate astroglial glucose metabolism to fine-tune neuronal activity and behaviour in mice.
Asunto(s)
Astrocitos/metabolismo , Metabolismo Energético , Glucosa/metabolismo , Mitocondrias/metabolismo , Receptor Cannabinoide CB1/metabolismo , Animales , Astrocitos/citología , Astrocitos/efectos de los fármacos , Agonistas de Receptores de Cannabinoides/farmacología , Células Cultivadas , Dronabinol/farmacología , Complejo I de Transporte de Electrón/química , Complejo I de Transporte de Electrón/metabolismo , Metabolismo Energético/efectos de los fármacos , Glucólisis/efectos de los fármacos , Humanos , Factor 1 Inducible por Hipoxia/metabolismo , Ácido Láctico/metabolismo , Masculino , Ratones , Mitocondrias/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Oxidación-Reducción , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Receptor Cannabinoide CB1/agonistas , Conducta SocialRESUMEN
The glycolytic rate in neurons is low in order to allow glucose to be metabolized through the pentose-phosphate pathway (PPP), which regenerates NADPH to preserve the glutathione redox status and survival. This is controlled by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3), the pro-glycolytic enzyme that forms fructose-2,6-bisphosphate, a powerful allosteric activator of 6-phosphofructo-1-kinase. In neurons, PFKFB3 protein is physiologically inactive due to its proteasomal degradation. However, upon an excitotoxic stimuli, PFKFB3 becomes stabilized to activate glycolysis, thus hampering PPP mediated protection of redox status leading to neurodegeneration. Here, we show that selective inhibition of PFKFB3 activity by the small molecule AZ67 prevents the NADPH oxidation, redox stress and apoptotic cell death caused by the activation of glycolysis triggered upon excitotoxic and oxygen-glucose deprivation/reoxygenation models in mouse primary neurons. Furthermore, in vivo administration of AZ67 to mice significantly alleviated the motor discoordination and brain infarct injury in the middle carotid artery occlusion ischemia/reperfusion model. These results show that pharmacological inhibition of PFKFB3 is a suitable neuroprotective therapeutic strategy in excitotoxic-related disorders such as stroke.
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Isquemia Encefálica/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Fosfofructoquinasa-2/genética , Piridinas/farmacología , Pirrolidinas/farmacología , Daño por Reperfusión/prevención & control , Células A549 , Animales , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Corteza Cerebral/irrigación sanguínea , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Fructosadifosfatos/metabolismo , Regulación de la Expresión Génica , Ácido Glutámico/metabolismo , Ácido Glutámico/farmacología , Glucólisis/efectos de los fármacos , Humanos , Masculino , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Vía de Pentosa Fosfato/efectos de los fármacos , Fosfofructoquinasa-1/genética , Fosfofructoquinasa-1/metabolismo , Fosfofructoquinasa-2/antagonistas & inhibidores , Fosfofructoquinasa-2/metabolismo , Cultivo Primario de Células , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis/efectos de los fármacos , Desempeño Psicomotor/efectos de los fármacos , Daño por Reperfusión/genética , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patologíaRESUMEN
The Fizzy-related protein 1 (Fzr1) gene encodes Cdh1 protein, a coactivator of the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C). Previously, we found that genetic ablation of Fzr1 promotes the death of neural progenitor cells leading to neurogenesis impairment and microcephaly in mouse. To ascertain the possible translation of these findings in humans, we searched for mutations in the Fzr1 gene in 390 whole exomes sequenced in trio in individuals showing neurodevelopmental disorders compatible with a genetic origin. We found a novel missense (p.Asp187Gly) Fzr1 gene mutation (c.560A>G) in a heterozygous state in a 4-year-old boy, born from non-consanguineous Spanish parents, who presents with severe antenatal microcephaly, psychomotor retardation, and refractory epilepsy. Cdh1 protein levels in leucocytes isolated from the patient were significantly lower than those found in his parents. Expression of the Asp187Gly mutant form of Cdh1 in human embryonic kidney 293T cells produced less Cdh1 protein and APC/C activity, resulting in altered cell cycle distribution when compared with cells expressing wild-type Cdh1. Furthermore, ectopic expression of the Asp187Gly mutant form of Cdh1 in cortical progenitor cells in primary culture failed to abolish the enlargement of the replicative phase caused by knockout of endogenous Cdh1. These results indicate that the loss of function of APC/C-Cdh1 caused by Cdh1 Asp187Gly mutation is a new cause of prenatal microcephaly, psychomotor retardation, and severe epilepsy. Read the Editorial Highlight for this article on page 8. Cover Image for this issue: doi: 10.1111/jnc.14524.
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Ciclosoma-Complejo Promotor de la Anafase/genética , Antígenos CD/genética , Cadherinas/genética , Epilepsia/genética , Microcefalia/genética , Trastornos Psicomotores/genética , Preescolar , Humanos , Masculino , Mutación MissenseRESUMEN
To satisfy its high energetic demand1, the brain depends on the metabolic cooperation of various cell types2-4. For example, astrocytic-derived lactate sustains memory consolidation5 by serving both as an oxidizable energetic substrate for neurons6 and as a signalling molecule7,8. Astrocytes and neurons also differ in the regulation of glycolytic enzymes9 and in the organization of their mitochondrial respiratory chain10. Unlike neurons, astrocytes rely on glycolysis for energy generation9 and, as a consequence, have a loosely assembled mitochondrial respiratory chain that is associated with a higher generation of mitochondrial reactive oxygen species (ROS)10. However, whether this abundant natural source of mitochondrial ROS in astrocytes fulfils a specific physiological role is unknown. Here we show that astrocytic mitochondrial ROS are physiological regulators of brain metabolism and neuronal function. We generated mice that inducibly overexpress mitochondrial-tagged catalase in astrocytes and show that this overexpression decreases mitochondrial ROS production in these cells during adulthood. Transcriptomic, metabolomic, biochemical, immunohistochemical and behavioural analysis of these mice revealed alterations in brain redox, carbohydrate, lipid and amino acid metabolic pathways associated with altered neuronal function and mouse behaviour. We found that astrocytic mitochondrial ROS regulate glucose utilization via the pentose-phosphate pathway and glutathione metabolism, which modulates the redox status and potentially the survival of neurons. Our data provide further molecular insight into the metabolic cooperation between astrocytes and neurons and demonstrate that mitochondrial ROS are important regulators of organismal physiology in vivo.
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Astrocitos/metabolismo , Conducta Animal , Encéfalo/metabolismo , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Células Cultivadas , Metabolismo Energético , Glucosa/metabolismo , Glucólisis/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oxidación-Reducción , Vía de Pentosa Fosfato/fisiologíaRESUMEN
Cerebral preconditioning (PC) confers endogenous brain protection after stroke. Ischemic stroke patients with a prior transient ischemic attack (TIA) may potentially be in a preconditioned state. Although PC has been associated with the activation of pro-survival signals, the mechanism by which preconditioning confers neuroprotection is not yet fully clarified. Recently, we have described that PC-mediated neuroprotection against ischemic insult is promoted by p53 destabilization, which is mediated by its main regulator MDM2. Moreover, we have previously described that the human Tp53 Arg72Pro single nucleotide polymorphism (SNP) controls susceptibility to ischemia-induced neuronal apoptosis and governs the functional outcome of patients after stroke. Here, we studied the contribution of the human Tp53 Arg72Pro SNP on PC-induced neuroprotection after ischemia. Our results showed that cortical neurons expressing the Pro72-p53 variant exhibited higher PC-mediated neuroprotection as compared with Arg72-p53 neurons. PC prevented ischemia-induced nuclear and cytosolic p53 stabilization in Pro72-p53 neurons. However, PC failed to prevent mitochondrial p53 stabilization, which occurs in Arg72-p53 neurons after ischemia. Furthermore, PC promoted neuroprotection against ischemia by controlling the p53/active caspase-3 pathway in Pro72-p53, but not in Arg72-p53 neurons. Finally, we found that good prognosis associated to TIA within 1 month prior to ischemic stroke was restricted to patients harboring the Pro72 allele. Our findings demonstrate that the Tp53 Arg72Pro SNP controls PC-promoted neuroprotection against a subsequent ischemic insult by modulating mitochondrial p53 stabilization and then modulates TIA-induced ischemic tolerance.
Asunto(s)
Isquemia Encefálica/genética , Hipoxia de la Célula/genética , Precondicionamiento Isquémico/métodos , Neuronas/patología , Polimorfismo de Nucleótido Simple/genética , Proteína p53 Supresora de Tumor/genética , Anciano , Anciano de 80 o más Años , Animales , Apoptosis/genética , Arginina/genética , Isquemia Encefálica/prevención & control , Caspasa 3/metabolismo , Células Cultivadas , Corteza Cerebral/citología , Estudios de Cohortes , Complejo IV de Transporte de Electrones/metabolismo , Embrión de Mamíferos , Agonistas de Aminoácidos Excitadores/farmacología , Femenino , Glucosa/deficiencia , Humanos , Masculino , Potenciales de la Membrana/genética , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Persona de Mediana Edad , N-Metilaspartato/farmacología , Prolina/genética , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/patologíaRESUMEN
Neurodegeneration in selective brain areas underlies the pathology of Alzheimer's disease (AD). Although oligomeric amyloid-ß (Aß) plays a central role in the AD pathogenesis, the mechanism of neuronal loss in response to Aß remains elusive. The p53 tumor suppressor protein, a key regulator of cell apoptosis, has been described to accumulate in affected brain areas from AD patients. However, whether p53 plays any role in AD pathogenesis remains unknown. To address this issue, here we investigated the involvement of p53 on Aß-induced neuronal apoptosis. We found that exposure of neurons to oligomers of the amyloidogenic fragment 25-35 of the Aß peptide (Aß25-35) promoted p53 protein phosphorylation and stabilization, leading to mitochondrial dysfunction and neuronal apoptosis. To address the underlying mechanism, we focused on cyclin dependent kinase-5 (Cdk5), a known p53-phosphorylating kinase. The results revealed that Aß25-35 treatment activated Cdk5, and that inhibiting Cdk5 activity prevented p53 protein stabilization. Furthermore, Aß25-35-mediated mitochondrial dysfunction and neuronal apoptosis were prevented by both genetic and pharmacological inhibition of either p53 or Cdk5 activities. This effect was mimicked with the full-length peptide Aß1-42. To confirm the mechanism in vivo, Aß25-35 was stereotaxically injected in the cerebral right ventricle of mice, a treatment that caused p53 protein accumulation, dendrite disruption and neuronal death. Furthermore, these effects were prevented in p53 knockout mice or by pharmacologically inhibiting p53. Thus, Aß25-35 triggers Cdk5 activation to induce p53 phosphorylation and stabilization, which leads to neuronal damage. Inhibition of the Cdk5-p53 pathway may therefore represent a novel therapeutic strategy against Aß-induced neurodegeneration.