RESUMEN
Prevention of infection and propagation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a high priority in the Coronavirus Disease 2019 (COVID-19) pandemic. Here we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin-converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 spike protein, thereby inhibiting viral entry, infectivity and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and, thus, the spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model and, thus, provide a novel avenue to pursue therapy.
Asunto(s)
COVID-19 , Humanos , SARS-CoV-2/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Unión Proteica , Peptidil-Dipeptidasa A/metabolismoRESUMEN
Excitatory/inhibitory (E/I) balance, defined as the balance between excitation and inhibition of synaptic activity in a neuronal network, accounts in part for the normal functioning of the brain, controlling, for example, normal spike rate. In many pathological conditions, this fine balance is perturbed, leading to excessive or diminished excitation relative to inhibition, termed E/I imbalance, reflected in network dysfunction. E/I imbalance has emerged as a contributor to neurological disorders that occur particularly at the extremes of life, including autism spectrum disorder and Alzheimer's disease, pointing to the vulnerability of neuronal networks at these critical life stages. Hence, it is important to develop approaches to rebalance neural networks. In this review, we describe emerging therapies that can normalize the E/I ratio or the underlying abnormality that contributes to the imbalance in electrical activity, thus improving neurological function in these maladies.
Asunto(s)
Trastorno del Espectro Autista , Enfermedades Neurodegenerativas , Encéfalo , Humanos , NeuronasRESUMEN
In the brain, both HIV-1 and methamphetamine (meth) use result in increases in oxidative and nitrosative stress. This redox stress is thought to contribute to the pathogenesis of HIV-associated neurocognitive disorder (HAND) and further worsening cognitive activity in the setting of drug abuse. One consequence of such redox stress is aberrant protein S-nitrosylation, derived from nitric oxide, which may disrupt normal protein activity. Here, we report an improved, mass spectrometry-based technique to assess S-nitrosylated protein in human postmortem brains using selective enrichment of S-nitrosocysteine residues with an organomercury resin. The data show increasing S-nitrosylation of tricarboxylic acid (TCA) enzymes in the setting of HAND and HAND/meth use compared with HIV+ control brains without CNS pathology. The consequence is systematic inhibition of multiple TCA cycle enzymes, resulting in energy collapse that can contribute to the neuronal and synaptic damage observed in HAND and meth use.
Asunto(s)
Ciclo del Ácido Cítrico/efectos de los fármacos , Disfunción Cognitiva/metabolismo , Infecciones por VIH/metabolismo , Metanfetamina/efectos adversos , Procesamiento Proteico-Postraduccional , Trastornos Relacionados con Sustancias/metabolismo , Autopsia , Bancos de Muestras Biológicas , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Encéfalo/patología , Ciclo del Ácido Cítrico/genética , Disfunción Cognitiva/complicaciones , Disfunción Cognitiva/patología , Disfunción Cognitiva/virología , Cisteína/análogos & derivados , Cisteína/metabolismo , Infecciones por VIH/complicaciones , Infecciones por VIH/patología , Infecciones por VIH/virología , VIH-1/crecimiento & desarrollo , VIH-1/patogenicidad , Humanos , Masculino , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Mitocondrias/patología , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/patología , Óxido Nítrico/metabolismo , S-Nitrosotioles/metabolismo , Trastornos Relacionados con Sustancias/complicaciones , Trastornos Relacionados con Sustancias/patología , Trastornos Relacionados con Sustancias/virología , Sinapsis/efectos de los fármacos , Sinapsis/patologíaRESUMEN
Gaining mechanistic insight into interaction between causative factors of complex multifactorial diseases involving photoreceptor damage might aid in devising effective therapies. Oxidative stress is one of the potential unifying mechanisms for interplay between genetic and environmental factors that contribute to photoreceptor pathology. Interestingly, the transcription factor myocyte enhancer factor 2d (MEF2D) is known to be important in photoreceptor survival, as knockout of this transcription factor results in loss of photoreceptors in mice. Here, using a mild light-induced retinal degeneration model, we show that the diminished MEF2D transcriptional activity in Mef2d+/- retina is further reduced under photostimulation-induced oxidative stress. Reactive oxygen species cause an aberrant redox modification on MEF2D, consequently inhibiting transcription of its downstream target, nuclear factor (erythroid-derived 2)-like 2 (NRF2). NRF2 is a master regulator of phase II antiinflammatory and antioxidant gene expression. In the Mef2d heterozygous mouse retina, NRF2 is not up-regulated to a normal degree in the face of light-induced oxidative stress, contributing to accelerated photoreceptor cell death. Furthermore, to combat this injury, we found that activation of the endogenous NRF2 pathway using proelectrophilic drugs rescues photoreceptors from photo-induced oxidative stress and may therefore represent a viable treatment for oxidative stress-induced photoreceptor degeneration, which is thought to contribute to some forms of retinitis pigmentosa and age-related macular degeneration.
Asunto(s)
Factor 2 Relacionado con NF-E2/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Degeneración Retiniana/etiología , Abietanos , Animales , Modelos Animales de Enfermedad , Haploinsuficiencia , Luz/efectos adversos , Factores de Transcripción MEF2/genética , Ratones , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Tuberous sclerosis (TSC) is an autosomal dominant disorder caused by heterozygous mutations in the TSC1 or TSC2 gene. TSC is often associated with neurological, cognitive, and behavioral deficits. TSC patients also express co-morbidity with anxiety and mood disorders. The mechanism of pathogenesis in TSC is not entirely clear, but TSC-related neurological symptoms are accompanied by excessive glutamatergic activity and altered synaptic spine structures. To address whether extrasynaptic (e)NMDA-type glutamate receptor (NMDAR) antagonists, as opposed to antagonists that block physiological phasic synaptic activity, can ameliorate the synaptic and behavioral features of this disease, we utilized the Tsc2+/- mouse model of TSC to measure biochemical, electrophysiological, histological, and behavioral parameters in the mice. We found that antagonists that preferentially block tonic activity as found at eNMDARs, particularly the newer drug NitroSynapsin, provide biological and statistically significant improvement in Tsc2+/- phenotypes. Accompanying this improvement was correction of activity in the p38 MAPK-TSC-Rheb-mTORC1-S6K1 pathway. Deficits in hippocampal long-term potentiation (LTP), histological loss of synapses, and behavioral fear conditioning in Tsc2+/- mice were all improved after treatment with NitroSynapsin. Taken together, these results suggest that amelioration of excessive excitation, by limiting aberrant eNMDAR activity, may represent a novel treatment approach for TSC.
Asunto(s)
Antagonistas de Aminoácidos Excitadores/uso terapéutico , Hipocampo/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Esclerosis Tuberosa/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Antagonistas de Aminoácidos Excitadores/farmacología , Hipocampo/metabolismo , Ratones , Ratones Noqueados , Esclerosis Tuberosa/genética , Esclerosis Tuberosa/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismoRESUMEN
Overproduction of nitric oxide (NO) can cause neuronal damage, contributing to the pathogenesis of several neurodegenerative diseases and stroke (i.e., focal cerebral ischemia). NO can mediate neurotoxic effects at least in part via protein S-nitrosylation, a reaction that covalently attaches NO to a cysteine thiol (or thiolate anion) to form an S-nitrosothiol. Recently, the tyrosine phosphatase Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2) and its downstream pathways have emerged as important mediators of cell survival. Here we report that in neurons and brain tissue NO can S-nitrosylate SHP-2 at its active site cysteine, forming S-nitrosylated SHP-2 (SNO-SHP-2). We found that NMDA exposure in vitro and transient focal cerebral ischemia in vivo resulted in increased levels of SNO-SHP-2. S-Nitrosylation of SHP-2 inhibited its phosphatase activity, blocking downstream activation of the neuroprotective physiological ERK1/2 pathway, thus increasing susceptibility to NMDA receptor-mediated excitotoxicity. These findings suggest that formation of SNO-SHP-2 represents a key chemical reaction contributing to excitotoxic damage in stroke and potentially other neurological disorders.
Asunto(s)
Isquemia Encefálica/fisiopatología , Óxido Nítrico/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Receptores de N-Metil-D-Aspartato/fisiología , Accidente Cerebrovascular/fisiopatología , Animales , Apoptosis , Inmunohistoquímica , Sistema de Señalización de MAP Quinasas , Ratones , Neuronas/patologíaRESUMEN
Synaptic loss is the cardinal feature linking neuropathology to cognitive decline in Alzheimer's disease (AD). However, the mechanism of synaptic damage remains incompletely understood. Here, using FRET-based glutamate sensor imaging, we show that amyloid-ß peptide (Aß) engages α7 nicotinic acetylcholine receptors to induce release of astrocytic glutamate, which in turn activates extrasynaptic NMDA receptors (eNMDARs) on neurons. In hippocampal autapses, this eNMDAR activity is followed by reduction in evoked and miniature excitatory postsynaptic currents (mEPSCs). Decreased mEPSC frequency may reflect early synaptic injury because of concurrent eNMDAR-mediated NO production, tau phosphorylation, and caspase-3 activation, each of which is implicated in spine loss. In hippocampal slices, oligomeric Aß induces eNMDAR-mediated synaptic depression. In AD-transgenic mice compared with wild type, whole-cell recordings revealed excessive tonic eNMDAR activity accompanied by eNMDAR-sensitive loss of mEPSCs. Importantly, the improved NMDAR antagonist NitroMemantine, which selectively inhibits extrasynaptic over physiological synaptic NMDAR activity, protects synapses from Aß-induced damage both in vitro and in vivo.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Astrocitos/metabolismo , Ácido Glutámico/metabolismo , Inhibición Neural/fisiología , Fragmentos de Péptidos/toxicidad , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/patología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Astrocitos/patología , Técnicas de Cocultivo , Femenino , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Masculino , Ratones , Ratones Transgénicos , Ratas , Receptores Nicotínicos/metabolismo , Sinapsis/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Mutations in the ataxia telangiectasia mutated (ATM) gene, which encodes a kinase critical for the normal DNA damage response, cause the neurodegenerative disorder ataxia-telangiectasia (AT). The substrates of ATM in the brain are poorly understood. Here we demonstrate that ATM phosphorylates and activates the transcription factor myocyte enhancer factor 2D (MEF2D), which plays a critical role in promoting survival of cerebellar granule cells. ATM associates with MEF2D after DNA damage and phosphorylates the transcription factor at four ATM consensus sites. Knockdown of endogenous MEF2D with a short-hairpin RNA (shRNA) increases sensitivity to etoposide-induced DNA damage and neuronal cell death. Interestingly, substitution of endogenous MEF2D with an shRNA-resistant phosphomimetic MEF2D mutant protects cerebellar granule cells from cell death after DNA damage, whereas an shRNA-resistant nonphosphorylatable MEF2D mutant does not. In vivo, cerebella in Mef2d knock-out mice manifest increased susceptibility to DNA damage. Together, our results show that MEF2D is a substrate for phosphorylation by ATM, thus promoting survival in response to DNA damage. Moreover, dysregulation of the ATM-MEF2D pathway may contribute to neurodegeneration in AT.
Asunto(s)
Daño del ADN/fisiología , Neuronas/fisiología , Animales , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/deficiencia , Proteínas de la Ataxia Telangiectasia Mutada/fisiología , Supervivencia Celular/fisiología , Células Cultivadas , Cerebelo/citología , Cerebelo/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Células HEK293 , Humanos , Técnicas In Vitro , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/metabolismo , Masculino , Ratones , Ratones Noqueados , Neuronas/citología , Neuronas/metabolismo , Fosforilación , Regiones Promotoras Genéticas/genética , Interferencia de ARN/fisiología , Superóxidos/metabolismoRESUMEN
Oligomerized amyloid-ß (Aß) peptide is thought to contribute to synaptic damage, resulting in dysfunctional neuronal networks in patients with Alzheimer's disease. It has been previously suggested that Aß may be detrimental to neuronal health, at least in part, by triggering oxidative/nitrosative stress. However, the mechanisms underlying this process remain to be elucidated. Here, using rat primary cerebrocortical cultures, we demonstrate that Aß1-42 oligomers trigger a dramatic increase in intracellular nitric oxide (NO) concentration via a process mediated by activation of NMDA-type glutamate receptors (NMDARs). Considering that synaptic NMDARs and extrasynaptic NMDARs (eNMDARs) can have opposite effects on neuronal viability, we explored their respective roles in Aß-induced increases in NO levels. Surprisingly, after pharmacological isolation of eNMDARs, we discovered that eNMDARs are primarily responsible for the increase in neuronal NO triggered by Aß oligomers. Moreover, we found that the eNMDAR-mediated increase in NO can produce S-nitrosylation of Drp1 (dynamin-related protein 1) and Cdk5 (cyclin-dependent kinase 5), targets known to contribute to Aß-induced synaptic damage. These results suggest that pharmacological intervention specifically aimed at eNMDARs may decrease Aß-induced nitrosative stress and thus ameliorate neurotoxic damage to synapses.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Corteza Cerebelosa/citología , Neuronas/efectos de los fármacos , Óxido Nítrico/metabolismo , Fragmentos de Péptidos/farmacología , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Bloqueadores de los Canales de Calcio/farmacología , Células Cultivadas , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Fluoresceínas/metabolismo , Humanos , NG-Nitroarginina Metil Éster/farmacología , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Cyanide is a life-threatening, bioterrorist agent, preventing cellular respiration by inhibiting cytochrome c oxidase, resulting in cardiopulmonary failure, hypoxic brain injury, and death within minutes. However, even after treatment with various antidotes to protect cytochrome oxidase, cyanide intoxication in humans can induce a delayed-onset neurological syndrome that includes symptoms of Parkinsonism. Additional mechanisms are thought to underlie cyanide-induced neuronal damage, including generation of reactive oxygen species. This may account for the fact that antioxidants prevent some aspects of cyanide-induced neuronal damage. Here, as a potential preemptive countermeasure against a bioterrorist attack with cyanide, we tested the CNS protective effect of carnosic acid (CA), a pro-electrophilic compound found in the herb rosemary. CA crosses the blood-brain barrier to up-regulate endogenous antioxidant enzymes via activation of the Nrf2 transcriptional pathway. We demonstrate that CA exerts neuroprotective effects on cyanide-induced brain damage in cultured rodent and human-induced pluripotent stem cell-derived neurons in vitro, and in vivo in various brain areas of a non-Swiss albino mouse model of cyanide poisoning that simulates damage observed in the human brain. Cyanide, a potential bioterrorist agent, can produce a chronic delayed-onset neurological syndrome that includes symptoms of Parkinsonism. Here, cyanide poisoning treated with the proelectrophillic compound carnosic acid, results in reduced neuronal cell death in both in vitro and in vivo models through activation of the Nrf2/ARE transcriptional pathway. Carnosic acid is therefore a potential treatment for the toxic central nervous system (CNS) effects of cyanide poisoning. ARE, antioxidant responsive element; Nrf2 (NFE2L2, Nuclear factor (erythroid-derived 2)-like 2).
Asunto(s)
Abietanos/farmacología , Lesiones Encefálicas/prevención & control , Cianuros/toxicidad , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Extractos Vegetales/farmacología , Animales , Antioxidantes/farmacología , Bioterrorismo , Encéfalo/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Factor 2 Relacionado con NF-E2/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Nitric oxide (NO) is a gasotransmitter that impacts fundamental aspects of neuronal function in large measure through S-nitrosylation, a redox reaction that occurs on regulatory cysteine thiol groups. For instance, S-nitrosylation regulates enzymatic activity of target proteins via inhibition of active site cysteine residues or via allosteric regulation of protein structure. During normal brain function, protein S-nitrosylation serves as an important cellular mechanism that modulates a diverse array of physiological processes, including transcriptional activity, synaptic plasticity, and neuronal survival. In contrast, emerging evidence suggests that aging and disease-linked environmental risk factors exacerbate nitrosative stress via excessive production of NO. Consequently, aberrant S-nitrosylation occurs and represents a common pathological feature that contributes to the onset and progression of multiple neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's diseases. In the current review, we highlight recent key findings on aberrant protein S-nitrosylation showing that this reaction triggers protein misfolding, mitochondrial dysfunction, transcriptional dysregulation, synaptic damage, and neuronal injury. Specifically, we discuss the pathological consequences of S-nitrosylated parkin, myocyte enhancer factor 2 (MEF2), dynamin-related protein 1 (Drp1), protein disulfide isomerase (PDI), X-linked inhibitor of apoptosis protein (XIAP), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) under neurodegenerative conditions. We also speculate that intervention to prevent these aberrant S-nitrosylation events may produce novel therapeutic agents to combat neurodegenerative diseases.
Asunto(s)
Enfermedades Neurodegenerativas/metabolismo , Proteína S/metabolismo , Animales , HumanosRESUMEN
Intracranial transplantation of neural stem cells (NSCs) delayed disease onset, preserved motor function, reduced pathology and prolonged survival in a mouse model of Sandhoff disease, a lethal gangliosidosis. Although donor-derived neurons were electrophysiologically active within chimeric regions, the small degree of neuronal replacement alone could not account for the improvement. NSCs also increased brain beta-hexosaminidase levels, reduced ganglioside storage and diminished activated microgliosis. Additionally, when oral glycosphingolipid biosynthesis inhibitors (beta-hexosaminidase substrate inhibitors) were combined with NSC transplantation, substantial synergy resulted. Efficacy extended to human NSCs, both to those isolated directly from the central nervous system (CNS) and to those derived secondarily from embryonic stem cells. Appreciating that NSCs exhibit a broad repertoire of potentially therapeutic actions, of which neuronal replacement is but one, may help in formulating rational multimodal strategies for the treatment of neurodegenerative diseases.
Asunto(s)
Encéfalo/citología , Células Madre Embrionarias/citología , Neuronas/citología , Enfermedad de Sandhoff/terapia , Trasplante de Células Madre , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/farmacología , Animales , Humanos , Inmunohistoquímica , Ratones , Ratones Noqueados , Microglía/metabolismo , Técnicas de Placa-Clamp , Enfermedad de Sandhoff/tratamiento farmacológico , beta-N-Acetilhexosaminidasas/antagonistas & inhibidores , beta-N-Acetilhexosaminidasas/genética , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
The activity of Cdk5 and its regulatory subunit p35 is thought to be important in both normal brain function and neurodegenerative disease pathogenesis. Increased Cdk5 activity, via proteolytic cleavage of p35 to a p25 fragment by the calcium-activated protease calpain or by phosphorylation at Cdk5(Tyr15), can contribute to neurotoxicity. Nonetheless, our knowledge of regulation of Cdk5 activity in disease states is still emerging. Here we demonstrate that Cdk5 is activated by S-nitrosylation or reaction of nitric oxide (NO)-related species with the thiol groups of cysteine residues 83 and 157, to form SNO-Cdk5. We then show that S-nitrosylation of Cdk5 contributes to amyloid-ß (Aß) peptide-induced dendritic spine loss. Furthermore, we observed significant levels of SNO-Cdk5 in postmortem Alzheimer's disease (AD) but not in normal human brains. These findings suggest that S-nitrosylation of Cdk5 is an aberrant regulatory mechanism of enzyme activity that may contribute to the pathogenesis of AD.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Quinasa 5 Dependiente de la Ciclina/metabolismo , Espinas Dendríticas/efectos de los fármacos , Espinas Dendríticas/patología , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/patología , Animales , Apoptosis/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Encéfalo/patología , Cisteína/metabolismo , Dinaminas/metabolismo , Activación Enzimática/efectos de los fármacos , Células HEK293 , Humanos , Mutación/genética , N-Metilaspartato/farmacología , Óxido Nítrico/farmacología , Nitrosación/efectos de los fármacos , RatasRESUMEN
After transplantation, individual stem cell-derived neurons can functionally integrate into the host CNS; however, evidence that neurons derived from transplanted human embryonic stem cells (hESCs) can drive endogenous neuronal network activity in CNS tissue is still lacking. Here, using multielectrode array recordings, we report activation of high-frequency oscillations in the ß and γ ranges (10-100 Hz) in the host hippocampal network via targeted optogenetic stimulation of transplanted hESC-derived neurons.
Asunto(s)
Células Madre Embrionarias/trasplante , Hipocampo/fisiología , Células-Madre Neurales/trasplante , Neuronas/trasplante , Potenciales de Acción/fisiología , Animales , Células Madre Embrionarias/citología , Femenino , Hipocampo/citología , Humanos , Masculino , Células-Madre Neurales/citología , Neurogénesis/fisiología , Neuronas/citología , Optogenética , Ratas , Ratas Sprague-DawleyRESUMEN
Prevention of infection and propagation of SARS-CoV-2 is of high priority in the COVID-19 pandemic. Here, we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 Spike protein, thereby inhibiting viral entry, infectivity, and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and thus spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E-protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model, and thus provide a novel avenue for therapy.
RESUMEN
Activation of the Keap1/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway and consequent induction of phase 2 antioxidant enzymes is known to afford neuroprotection. Here, we present a series of novel electrophilic compounds that protect neurons via this pathway. Natural products, such as carnosic acid (CA), are present in high amounts in the herbs rosemary and sage as ortho-dihydroquinones, and have attracted particular attention because they are converted by oxidative stress to their active form (ortho-quinone species) that stimulate the Keap1/Nrf2 transcriptional pathway. Once activated, this pathway leads to the production of a series of antioxidant phase 2 enzymes. Thus, such dihydroquinones function as redox-activated 'pro-electrophiles'. Here, we explored the concept that related para-dihydroquinones represent even more effective bioactive pro-electrophiles for the induction of phase 2 enzymes without producing toxic side effects. We synthesized several novel para-hydroquinone-type pro-electrophilic compounds (designated D1 and D2) to analyze their protective mechanism. DNA microarray, PCR, and western blot analyses showed that compound D1 induced expression of heat-shock proteins (HSPs), including HSP70, HSP27, and DnaJ, in addition to phase 2 enzymes such as hemeoxygenase-1 (HO-1), NADP(H) quinine-oxidoreductase1, and the Na(+)-independent cystine/glutamate exchanger (xCT). Treatment with D1 resulted in activation of Nrf2 and heat-shock transcription factor-1 (HSF-1) transcriptional elements, thus inducing phase 2 enzymes and HSPs, respectively. In this manner, D1 protected neuronal cells from both oxidative and endoplasmic reticulum (ER)-related stress. Additionally, D1 suppressed induction of 78 kDa glucose-regulated protein (GRP78), an ER chaperone protein, and inhibited hyperoxidation of peroxiredoxin 2 (PRX2), a molecule that is in its reduced state can protect from oxidative stress. These results suggest that D1 is a novel pro-electrophilic compound that activates both the Nrf2 and HSF-1 pathways, and may thus offer protection from oxidative and ER stress.
Asunto(s)
Antioxidantes/metabolismo , Proteínas de Unión al ADN/fisiología , Factor 2 Relacionado con NF-E2/fisiología , Fármacos Neuroprotectores/farmacología , Quinonas/farmacología , Epitelio Pigmentado de la Retina/enzimología , Transducción de Señal/fisiología , Factores de Transcripción/fisiología , Antioxidantes/síntesis química , Antioxidantes/fisiología , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Chaperón BiP del Retículo Endoplásmico , Factores de Transcripción del Choque Térmico , Humanos , Factor 2 Relacionado con NF-E2/metabolismo , Fármacos Neuroprotectores/síntesis química , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Quinonas/síntesis química , Epitelio Pigmentado de la Retina/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismoRESUMEN
Emerging evidence suggests that myocyte enhancer factor 2 (MEF2) transcription factors act as effectors of neurogenesis in the brain, with MEF2C the predominant isoform in developing cerebrocortex. Here, we show that conditional knockout of Mef2c in nestin-expressing neural stem/progenitor cells (NSCs) impaired neuronal differentiation in vivo, resulting in aberrant compaction and smaller somal size. NSC proliferation and survival were not affected. Conditional null mice surviving to adulthood manifested more immature electrophysiological network properties and severe behavioral deficits reminiscent of Rett syndrome, an autism-related disorder. Our data support a crucial role for MEF2C in programming early neuronal differentiation and proper distribution within the layers of the neocortex.
Asunto(s)
Diferenciación Celular , Factores Reguladores Miogénicos/metabolismo , Neuronas/citología , Células Madre/citología , Factores de Transcripción/metabolismo , Animales , Animales Recién Nacidos , Conducta , Cognición , Electrofisiología , Desarrollo Embrionario , Factores de Transcripción MEF2 , Ratones , Ratones Noqueados , Mitosis , Neocórtex/embriología , Neocórtex/patología , Neuronas/patología , FenotipoRESUMEN
Cell-based therapies require a reliable source of cells that can be easily grown, undergo directed differentiation, and remain viable after transplantation. Here, we generated stably transformed murine ES (embryonic stem) cells that express a constitutively active form of myocyte enhancer factor 2C (MEF2CA). MEF2C has been implicated as a calcium-dependent transcription factor that enhances survival and affects synapse formation of neurons as well as differentiation of cardiomyocytes. We now report that expression of MEF2CA, both in vitro and in vivo, under regulation of the nestin enhancer effectively produces "neuronal" progenitor cells that differentiate into a virtually pure population of neurons. Histological, electrophysiological, and behavioral analyses demonstrate that MEF2C-directed neuronal progenitor cells transplanted into a mouse model of cerebral ischemia can successfully differentiate into functioning neurons and ameliorate stroke-induced behavioral deficits.
Asunto(s)
Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/trasplante , Factores Reguladores Miogénicos/genética , Trasplante de Células Madre/métodos , Células Madre/metabolismo , Factores de Transcripción/genética , Animales , Apoptosis/genética , Isquemia Encefálica/terapia , Trasplante de Tejido Encefálico/métodos , Diferenciación Celular/fisiología , Línea Celular Transformada , Proliferación Celular , Supervivencia Celular/genética , Células Cultivadas , Células Madre Embrionarias/citología , Regulación de la Expresión Génica/genética , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Factores de Transcripción MEF2 , Masculino , Ratones , Ratones Endogámicos C57BL , Degeneración Nerviosa/etiología , Degeneración Nerviosa/fisiopatología , Degeneración Nerviosa/terapia , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Nestina , Neuronas/citología , Neuronas/metabolismo , Técnicas de Cultivo de Órganos , Células Madre/citologíaRESUMEN
Damage to neonatal and adult tissues always incites an influx of inflammatory neutrophils and macrophages. Besides clearing the wound of invading microbes, these cells are believed to be crucial coordinators of the repair process, acting both as professional phagocytes to clear wound debris and as a major source of wound growth factor signals. Here we report wound healing studies in the PU.1 null mouse, which is genetically incapable of raising the standard inflammatory response because it lacks macrophages and functioning neutrophils. Contrary to dogma, we show that these "macrophageless" mice are able to repair skin wounds with similar time course to wild-type siblings, and that repair appears scar-free as in the embryo, which also heals wounds without raising an inflammatory response. The growth factor and cytokine profile at the wound site is changed, cell death is reduced, and dying cells are instead engulfed by stand-in phagocytic fibroblasts. We also show that hyperinnervation of the wound site, previously believed to be a consequence of inflammation, is present in the PU.1 null wound, too.