RESUMEN
Whereas many studies have examined the properties of the compromised neocortex in the first several days following ischemia, there is less information regarding the initial 12 h poststroke. In this study we examined live mouse neocortical slices harvested immediately and 12 h after a 30-min middle cerebral artery occlusion (MCAo). We compared nonischemic and ischemic hemispheres with regard to the propensity for tissue swelling and for generating spreading depolarization (SD), as well as evoked synaptic responses and single pyramidal neuron electrophysiological properties. We observed spontaneous SD in 7% of slices on the nonstroked side and 25% in the stroked side following the 30-min MCAo. Spontaneous SD was rare in 12-h recovery slices. The region of the ischemic core and surround in slices was not susceptible to SD induced by oxygen and glucose deprivation. At the neuronal level, neocortical gray matter is surprisingly unaltered in brain slices harvested immediately poststroke. However, by 12 h, the fields of pyramidal and striatal neurons that comprise the infarcted core are electrophysiologically silent because the majority are morphologically devastated. Yet, there remains a subset of diffusely distributed "healthy" pyramidal neurons in the core at 12 h post-MCAo that persist for days poststroke. Their intact electrophysiology and dendritic morphology indicate a surprisingly selective resilience to stroke at the neuronal level. NEW & NOTEWORTHY It is generally accepted that the injured core region of the brain resulting from a focal stroke contains no functioning neurons. Our study shows that some neurons, although surrounded by devastated neighbors, can maintain their structure and electrical activity. This surprising finding raises the possibility of discovering how these neurons are protected to pinpoint new strategies for reducing stroke injury.
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Potenciales Postsinápticos Excitadores , Infarto de la Arteria Cerebral Media/fisiopatología , Neocórtex/fisiopatología , Potenciales de Acción , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Neocórtex/citología , Células Piramidales/fisiologíaRESUMEN
Hippocampus oxidative stress is considered pathogenic in neurodegenerative diseases, such as Alzheimer disease (AD), and in neurodevelopmental disorders, such as Angelman syndrome (AS). Yet clinical benefits of antioxidant treatment for these diseases remain unclear because conventional imaging methods are unable to guide management of therapies in specific hippocampus subfields in vivo that underlie abnormal behavior. Excessive production of paramagnetic free radicals in nonhippocampus brain tissue can be measured in vivo as a greater-than-normal 1/T1 that is quenchable with antioxidant as measured by quench-assisted (Quest) MRI. Here, we further test this approach in phantoms, and we present proof-of-concept data in models of AD-like and AS hippocampus oxidative stress that also exhibit impaired spatial learning and memory. AD-like models showed an abnormal gradient along the CA1 dorsal-ventral axis of excessive free radical production as measured by Quest MRI, and redox-sensitive calcium dysregulation as measured by manganese-enhanced MRI and electrophysiology. In the AS model, abnormally high free radical levels were observed in dorsal and ventral CA1. Quest MRI is a promising in vivo paradigm for bridging brain subfield oxidative stress and behavior in animal models and in human patients to better manage antioxidant therapy in devastating neurodegenerative and neurodevelopmental diseases.-Berkowitz, B. A., Lenning, J., Khetarpal, N., Tran, C., Wu, J. Y., Berri, A. M., Dernay, K., Haacke, E. M., Shafie-Khorassani, F., Podolsky, R. H., Gant, J. C., Maimaiti, S., Thibault, O., Murphy, G. G., Bennett, B. M., Roberts, R. In vivo imaging of prodromal hippocampus CA1 subfield oxidative stress in models of Alzheimer disease and Angelman syndrome.
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Enfermedad de Alzheimer/diagnóstico por imagen , Síndrome de Angelman/diagnóstico por imagen , Región CA1 Hipocampal/patología , Estrés Oxidativo/fisiología , Síntomas Prodrómicos , Aldehído Deshidrogenasa Mitocondrial/genética , Aldehído Deshidrogenasa Mitocondrial/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Síndrome de Angelman/patología , Animales , Antioxidantes , Calcio/metabolismo , Radicales Libres , Imagen por Resonancia Magnética/métodos , Manganeso , Memoria/fisiología , Ratones Noqueados , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Background Nitric oxide (NO) has been heavily implicated in migraine. Nitroglycerin is a prototypic NO-donor, and triggers migraine in humans. However, nitroglycerin also induces oxidative/nitrosative stress and is a source of peroxynitrite - factors previously linked with migraine etiology. Soluble guanylyl cyclase (sGC) is the high affinity NO receptor in the body, and the aim of this study was to identify the precise role of sGC in acute and chronic migraine. Methods We developed a novel brain-bioavailable sGC stimulator (VL-102), and tested its hyperalgesic properties in mice. We also determined the effect of VL-102 on c-fos and calcitonin gene related peptide (CGRP) immunoreactivity within the trigeminovascular complex. In addition, we also tested the known sGC inhibitor, ODQ, within the chronic nitroglycerin migraine model. Results VL-102-evoked acute and chronic mechanical cephalic and hind-paw allodynia in a dose-dependent manner, which was blocked by the migraine medications sumatriptan, propranolol, and topiramate. In addition, VL-102 also increased c-fos and CGRP expressing cells within the trigeminovascular complex. Importantly, ODQ completely inhibited acute and chronic hyperalgesia induced by nitroglycerin. ODQ also blocked hyperalgesia already established by chronic nitroglycerin, implicating this pathway in migraine chronicity. Conclusions These results indicate that nitroglycerin causes migraine-related pain through stimulation of the sGC pathway, and that super-activation of this receptor may be an important component for the maintenance of chronic migraine. This work opens the possibility for negative sGC modulators as novel migraine therapies.
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Hiperalgesia/inducido químicamente , Hiperalgesia/enzimología , Trastornos Migrañosos/enzimología , Guanilil Ciclasa Soluble/fisiología , Antagonistas Adrenérgicos beta/administración & dosificación , Antagonistas Adrenérgicos beta/uso terapéutico , Regulación Alostérica , Animales , Anticonvulsivantes/administración & dosificación , Anticonvulsivantes/uso terapéutico , Péptido Relacionado con Gen de Calcitonina/biosíntesis , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Femenino , Hiperalgesia/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Trastornos Migrañosos/inducido químicamente , Trastornos Migrañosos/tratamiento farmacológico , Trastornos Migrañosos/etiología , Terapia Molecular Dirigida , Óxido Nítrico/efectos adversos , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Nitroglicerina/farmacología , Oxadiazoles/administración & dosificación , Oxadiazoles/metabolismo , Oxadiazoles/uso terapéutico , Propranolol/administración & dosificación , Propranolol/uso terapéutico , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Quinoxalinas/administración & dosificación , Quinoxalinas/metabolismo , Quinoxalinas/uso terapéutico , Agonistas del Receptor de Serotonina 5-HT1/administración & dosificación , Agonistas del Receptor de Serotonina 5-HT1/uso terapéutico , Guanilil Ciclasa Soluble/metabolismo , Sumatriptán/administración & dosificación , Sumatriptán/uso terapéutico , Topiramato/administración & dosificación , Topiramato/uso terapéuticoRESUMEN
Tolerance to nitrates such as nitroglycerin (GTN) is associated with oxidative stress, inactivation of aldehyde dehydrogenase 2 (ALDH2), and decreased GTN-induced cGMP accumulation and vasodilation. We hypothesized that GTN-induced inactivation of ALDH2 results in increased 4-hydroxy-2-nonenal (HNE) adduct formation of key proteins involved in GTN bioactivation, and, consequently, an attenuated vasodilator response to GTN (i.e., tolerance). We used an in vivo GTN tolerance model, a cell culture model of nitrate action, and Aldh2(-/-) mice to assess whether GTN exposure resulted in HNE adduct formation, and whether exogenous HNE affected GTN-induced relaxation and cGMP accumulation. Immunoblot analysis indicated a marked increase in HNE adduct formation in GTN-tolerant porcine kidney epithelial cells (PK1) and in aortae from GTN-tolerant rats and untreated Aldh2(-/-) mice. Preincubation of PK1 cells with HNE resulted in a dose-dependent decrease in GTN-induced cGMP accumulation, and pretreatment of isolated rat aorta with HNE resulted in dose-dependent decreases in the vasodilator response to GTN, thus mimicking GTN-tolerance. Pretreatment of aortae from Aldh2(-/-) mice with 10 µM HNE resulted in a desensitized vasodilator response to GTN. In the in vivo rat tolerance model, changes in HNE adduct formation correlated well with the onset of GTN tolerance and tolerance reversal. Furthermore, coadministration of an HNE scavenger during the tolerance induction protocol completely prevented HNE adduct formation and GTN tolerance but did not prevent the inactivation of ALDH2. The data are consistent with a novel mechanism of GTN tolerance suggesting a primary role of HNE adduct formation in the development of GTN tolerance.
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Aldehídos/química , Tolerancia a Medicamentos , Peroxidación de Lípido , Nitratos/farmacología , Aldehído Deshidrogenasa/metabolismo , Animales , Células LLC-PK1 , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias Hepáticas/enzimología , Ratas , Ratas Sprague-Dawley , Porcinos , VasodilataciónRESUMEN
It is generally accepted that nitric oxide (NO) donors, such as sodium nitroprusside (SNP), or phosphodiesterase 5 (PDE5) inhibitors, including sildenafil, each impact human platelet function. Although a strong correlation exists between the actions of NO donors in platelets and their impact on cGMP, agents such as sildenafil act without increasing global intra-platelet cGMP levels. This study was undertaken to identify how PDE5 inhibitors might act without increasing cGMP. Our data identify PDE5 as an integral component of a protein kinase G1beta (PKG1beta)-containing signaling complex, reported previously to coordinate cGMP-mediated inhibition of inositol-1, 4, 5-trisphosphate receptor type 1 (IP(3)R1)-mediated Ca(2+)-release. PKG1beta and PDE5 did not interact in subcellular fractions devoid of IP(3)R1 and were not recruited to IP(3)R1-enriched membranes in response to cGMP-elevating agents. Activation of platelet PKG promoted phosphorylation and activation of the PDE5 fraction tethered to the IP(3)R1-PKG complex, an effect not observed for the nontethered PDE5. Based on these findings, we elaborate a model in which PKG selectively activates PDE5 within a defined microdomain in platelets and propose that this mechanism allows spatial and temporal regulation of cGMP signaling in these cells. Recent reports indicate that sildenafil might prove useful in limiting in-stent thrombosis and the thrombotic events associated with the acute coronary syndromes (ACS), situations poorly regulated with currently available therapeutics. We submit that our findings may define a molecular mechanism by which PDE5 inhibition can differentially impact selected cellular functions of platelets, and perhaps of other cell types.
Asunto(s)
Plaquetas/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Plaquetas/efectos de los fármacos , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Activación Enzimática , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Nitroprusiato/farmacología , Fosforilación , Piperazinas/farmacología , Agregación Plaquetaria/efectos de los fármacos , Unión Proteica , Purinas/farmacología , Transducción de Señal , Citrato de Sildenafil , Especificidad por Sustrato , Sulfonas/farmacología , Trombina/metabolismoRESUMEN
BACKGROUND: Neuronal damage resulting from increased oxidative stress is important in the development of late onset/age-related Alzheimer's disease (LOAD). We have developed an oxidative stress-related mouse model of LOAD based on gene deletion of aldehyde dehydrogenase 2 (ALDH2), an enzyme important for the detoxification of endogenous aldehydes arising from lipid peroxidation. Compared to wildtype (WT) mice, the knockout (KO) mice exhibit AD-like pathologies and a progressive decline in recognition and spatial memory. This progression presumably has a morphological basis induced by oxidative damage. OBJECTIVE: We performed morphometric analyses in the dorsal hippocampal CA1 region (dCA1) to determine if altered neuronal structure can help account for the progressive cognitive impairment in 3- to 12-month-old KO mice. METHODS: Dendritic morphology was quantitatively analyzed by branched structured analysis and Sholl analysis following Golgi-Cox staining in WT mice (148 neurons) versus KO mice (180 neurons). RESULTS: The morphology and complexity of dCA1 pyramidal neurons were similar at age 3 months in WTs and KOs. However, by 6 months there were significant reductions in apical and basal dendritic length, dendrite complexity, and spine density in KO versus WT mice that were maintained through ages 9 and 12 months. Immunostaining for protein adducts of the lipid peroxidation product 4-hydroxynonenal revealed significant increases in staining in dCA1 (but not ventral CA1) by 3 months, increasing through 12 months. CONCLUSION: This specific and progressive increase in dCA1 oxidative damage preceded detectable synaptic trimming in KO mice, in keeping with studies showing that lesions to dorsal hippocampus primarily impair cognitive memory.
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Envejecimiento/patología , Enfermedad de Alzheimer/patología , Región CA1 Hipocampal/patología , Neuronas/patología , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estrés OxidativoRESUMEN
Prevalence of metabolic disturbances is higher among individuals with neurodevelopmental disorders (NDDs), yet this association has been largely overlooked. Investigation into human disease remains challenging, as a complete pathophysiological understanding relies on accurate modeling and highly controlled variables. Genetically engineered mouse models are widely used to gain insight into the biology of human NDDs, but research focus has been on behavioral and neurophysiological abnormalities. Such models not only allow for evaluating usefulness in reproducing human features, including similarities and discrepancies with rodent phenotypes, but they also represent a unique opportunity to observe and quantify novel anomalies. Here, we present the first characterization and comparison of basal metabolism in three mouse models of NDDs, namely, Down syndrome (DS; Dp(16)Yey/+ mice), 16p11.2 deletion syndrome (16pDel; 16p11.2df/+ mice), and fragile X syndrome [FXS; Fmr1 knock-out (KO) mice] and their wild-type (WT) counterparts. Using the Comprehensive Lab Animal Monitoring System (CLAMS) coupled to EchoMRI, as well as quantification of key plasma metabolites by liquid chromatography mass spectrometry (LC-MS), our in vivo study reveals that each mouse model expresses a unique metabolic signature that is sex-specific, independent of the amount of food consumed and minimally influenced by physical activity. In particular, we identify striking differences in body composition, respiratory exchange ratio (RER), caloric expenditure (CE), and concentrations of circulating plasma metabolites related to mitochondrial function. Providing novel insight into NDD-associated metabolic alterations is an essential prerequisite for future preclinical and clinical interventions.
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Síndrome del Cromosoma X Frágil , Trastornos del Neurodesarrollo , Animales , Metabolismo Basal , Modelos Animales de Enfermedad , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/genética , Humanos , Ratones , Ratones NoqueadosRESUMEN
Hyperplasia of smooth muscle contributes to the thickening of the intestinal wall that is characteristic of inflammation, but the mechanisms of growth control are unknown. Nitric oxide (NO) from enteric neurons expressing neuronal NO synthase (nNOS) might normally inhibit intestinal smooth muscle cell (ISMC) growth, and this was tested in vitro. In ISMC from the circular smooth muscle of the adult rat colon, chemical NO donors inhibited [(3)H]thymidine uptake in response to FCS, reducing this to baseline without toxicity. This effect was inhibited by the guanylyl cyclase inhibitor ODQ and potentiated by the phosphodiesterase-5 inhibitor zaprinast. Inhibition was mimicked by 8-bromo (8-Br)-cGMP, and ELISA measurements showed increased levels of cGMP but not cAMP in response to sodium nitroprusside. However, 8-Br-cAMP and cilostamide also showed inhibitory actions, suggesting an additional role for cAMP. Via a coculture model of ISMC and myenteric neurons, immunocytochemistry and image analysis showed that innervation reduced bromodeoxyuridine uptake by ISMC. Specific blockers of nNOS (7-NI, NAAN) significantly increased [(3)H]thymidine uptake in response to a standard stimulus, showing that nNOS activity normally inhibits ISMC growth. In vivo, nNOS axon number was reduced threefold by day 1 of trinitrobenzene sulfonic acid-induced rat colitis, preceding the hyperplasia of ISMC described earlier in this model. We conclude that NO can inhibit ISMC growth primarily via a cGMP-dependent mechanism. Functional evidence that NO derived from nNOS causes inhibition of ISMC growth in vitro predicts that the loss of nNOS expression in colitis contributes to ISMC hyperplasia in vivo.
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Intestinos/citología , Miocitos del Músculo Liso/efectos de los fármacos , Neuronas/metabolismo , Óxido Nítrico/farmacología , Animales , Células Cultivadas , Técnicas de Cocultivo , Colitis/inducido químicamente , GMP Cíclico/metabolismo , Regulación Enzimológica de la Expresión Génica , Masculino , Plexo Mientérico/citología , Miocitos del Músculo Liso/citología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo I/genética , Óxido Nítrico Sintasa de Tipo I/metabolismo , Ratas , Ratas Sprague-Dawley , Ácido Trinitrobencenosulfónico/toxicidadRESUMEN
The study of late-onset (sporadic) Alzheimer's disease (LOAD) has lacked animal models where impairments develop with aging. Oxidative stress promotes LOAD, so we have developed an oxidative stress-based model of age-related cognitive impairment based on gene deletion of aldehyde dehydrogenase 2 (ALDH2). This enzyme is important for the detoxification of endogenous aldehydes arising from lipid peroxidation. Compared to wildtype (WT) mice, the knockout (KO) mice exhibit a progressive decline in recognition and spatial memory and AD-like pathologies. Here we performed morphometric analyses in the dorsal and ventral hippocampal CA1 regions (dCA1 and vCA1) as well as in overlying primary sensory cortex to determine if altered neuronal structure can help account for the cognitive impairment in 12-month old KO mice. Dendritic morphology was quantitatively analyzed following Golgi-Cox staining using 9 WT mice (108 neurons) and 15 KO mice (180 neurons). Four pyramidal neurons were traced per mouse in each region, followed by branched structured analysis and Sholl analysis. Compared to WT controls, the morphology and complexity of dCA1 pyramidal neurons from KOs showed significant reductions in apical and basal dendritic length, dendrite intersections, ends, and nodes. As well, spine density along dorsal CA1 apical dendrites was significantly lower in KO versus WT. In contrast, pyramidal arborization in the vCA1 and primary sensory cortex were only minimally reduced in KO versus WT mice. These data suggest a region-specific vulnerability to oxidative stress-induced damage and/or a major and specific reduction in synaptic input to the pyramidal neurons of the dorsal hippocampus. This is in keeping with studies showing that lesions to the dorsal hippocampus impair primarily cognitive memory whereas ventral hippocampal lesions cause deficits in stress, emotion, and affect.
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Enfermedad de Alzheimer/patología , Hipocampo/citología , Neocórtex/citología , Células Piramidales/citología , Animales , Región CA1 Hipocampal/citología , Región CA1 Hipocampal/patología , Espinas Dendríticas/patología , Modelos Animales de Enfermedad , Femenino , Hipocampo/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neocórtex/patología , Células Piramidales/patologíaRESUMEN
The Na+/K+-ATPase is a transmembrane ion pump that has a critical homeostatic role within every mammalian cell; however, it is vulnerable to the effects of increased oxidative stress. Understanding how expression of this transporter is influenced by oxidative stress may yield insight into its role in the pathophysiology of neurological and neuropsychiatric diseases. In this study we investigated whether increased oxidative stress could influence Na+/K+-ATPase expression in various brain regions of mice. We utilized two different models of oxidative stress: a behavioural chronic unpredictable stress protocol and the Aldh2-/- mouse model of oxidative stress-based and age-related cognitive impairment. We identified distinct regional baseline mRNA and protein expression patterns of the Na+/K+-ATPase α1 and α3 isoforms within the neocortex, hippocampus, and brainstem of wildtype mice. Consistent with previous studies, there was a higher proportion of α3 expression relative to α1 in the brainstem versus neocortex, but a higher proportion of α1 expression relative to α3 in the neocortex versus the brainstem. The hippocampus had similar expression levels of both α1 and α3. Despite increased staining for oxidative stress in higher brain, no differences in α1 or α3 expression were noted in Aldh2-/- mice versus wildtype, or in mice exposed to a 28-day chronic unpredictable stress protocol. In both models of oxidative stress, gene and protein expression of Na+/K+-ATPase α1 and α3 isoforms within the higher and lower brain was remarkably stable. Thus, Na+/K+-ATPase function previously reported as altered by oxidative stress is not through induced changes in the expression of pump isoforms.
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ATPasa Intercambiadora de Sodio-Potasio , Sodio , Animales , Encéfalo/metabolismo , Ratones , Estrés Oxidativo , Isoformas de Proteínas/metabolismo , Sodio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
BACKGROUND: Dysregulated signaling in neurons and astrocytes participates in pathophysiological alterations seen in the Alzheimer's disease brain, including increases in amyloid-ß, hyperphosphorylated tau, inflammation, calcium dysregulation, and oxidative stress. These are often noted prior to the development of behavioral, cognitive, and non-cognitive deficits. However, the extent to which these pathological changes function together or independently is unclear. OBJECTIVE: Little is known about the temporal relationship between calcium dysregulation and oxidative stress, as some reports suggest that dysregulated calcium promotes increased formation of reactive oxygen species, while others support the opposite. Prior work has quantified several key outcome measures associated with oxidative stress in aldehyde dehydrogenase 2 knockout (Aldh2-/-) mice, a non-transgenic model of sporadic Alzheimer's disease. METHODS: Here, we tested the hypothesis that early oxidative stress can promote calcium dysregulation across aging by measuring calcium-dependent processes using electrophysiological and imaging methods and focusing on the afterhyperpolarization (AHP), synaptic activation, somatic calcium, and long-term potentiation in the Aldh2-/- mouse. RESULTS: Our results show a significant age-related decrease in the AHP along with an increase in the slow AHP amplitude in Aldh2-/- animals. Measures of synaptic excitability were unaltered, although significant reductions in long-term potentiation maintenance were noted in the Aldh2-/- animals compared to wild-type. CONCLUSION: With so few changes in calcium and calcium-dependent processes in an animal model that shows significant increases in HNE adducts, Aß, p-tau, and activated caspases across age, the current findings do not support a direct link between neuronal calcium dysregulation and uncontrolled oxidative stress.
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Aldehído Deshidrogenasa Mitocondrial/deficiencia , Enfermedad de Alzheimer/metabolismo , Calcio/metabolismo , Modelos Animales de Enfermedad , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Factores de Edad , Aldehído Deshidrogenasa Mitocondrial/genética , Enfermedad de Alzheimer/genética , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Imagen Molecular/métodos , Neuronas/química , Técnicas de Cultivo de ÓrganosRESUMEN
PURPOSE: Hypoxia contributes to drug resistance in solid cancers, and studies have revealed that low concentrations of nitric oxide (NO) mimetics attenuate hypoxia-induced drug resistance in tumor cells in vitro. Classic NO signaling involves activation of soluble guanylyl cyclase, generation of cyclic GMP (cGMP), and activation of cGMP-dependent protein kinase. Here, we determined whether chemosensitization by NO mimetics requires cGMP-dependent signaling and whether low concentrations of NO mimetics can chemosensitize tumors in vivo. EXPERIMENTAL DESIGN: Survival of human prostate and breast cancer cells was assessed by clonogenic assays following exposure to chemotherapeutic agents. The effect of NO mimetics on tumor chemosensitivity in vivo was determined using a mouse xenograft model of human prostate cancer. Drug efflux in vitro was assessed by measuring intracellular doxorubicin-associated fluorescence. RESULTS: Low concentrations of the NO mimetics glyceryl trinitrate (GTN) and isosorbide dinitrate attenuated hypoxia-induced resistance to doxorubicin and paclitaxel. Similar to hypoxia-induced drug resistance, inhibition of various components of the NO signaling pathway increased resistance to doxorubicin, whereas activation of the pathway with 8-bromo-cGMP attenuated hypoxia-induced resistance. Drug efflux was unaffected by hypoxia and inhibitors of drug efflux did not significantly attenuate hypoxia-induced chemoresistance. Compared with mice treated with doxorubicin alone, tumor growth was decreased in mice treated with doxorubicin and a transdermal GTN patch. The presence of GTN and GTN metabolites in plasma samples was confirmed by gas chromatography. CONCLUSION: Tumor hypoxia induces resistance to anticancer drugs by interfering with endogenous NO signaling and reactivation of NO signaling represents a novel approach to enhance chemotherapy.
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Antineoplásicos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Experimentales/metabolismo , Óxido Nítrico/metabolismo , Transducción de Señal/fisiología , Animales , Neoplasias de la Mama/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/fisiología , Línea Celular Tumoral , GMP Cíclico/metabolismo , Femenino , Humanos , Dinitrato de Isosorbide/metabolismo , Dinitrato de Isosorbide/farmacología , Masculino , Ratones , Neoplasias Experimentales/tratamiento farmacológico , Donantes de Óxido Nítrico/sangre , Donantes de Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Nitroglicerina/sangre , Nitroglicerina/metabolismo , Nitroglicerina/farmacología , Neoplasias de la Próstata/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
OBJECTIVE: Patients with chronic heart failure (CHF) often require higher doses of nitroglycerin (glyceryl trinitrate, GTN) than patients with normal cardiac function to achieve a given haemodynamic goal. Two pathways leading to biotransformation of GTN have been characterized; a high-affinity pathway operative in nanomolar concentration ranges yielding predominantly 1,2-glyceryl dinitrate (1,2-GDN), and a low-affinity pathway operative at higher, micromolar concentrations of GTN associated with a greater proportion of 1,3-GDN formation. We tested the hypothesis that, at a given GTN-induced blood pressure reduction, the CHF group would present with: (i) higher concentrations of GTN; and (ii) decreased ratios of 1,2-GDN/GTN and 1,2-GDN/1,3-GDN compared with healthy subjects (HS). METHODS: Twelve patients with CHF (left ventricular ejection fraction 20 +/- 5%, NYHA III) and nine HS were investigated during a right cardiac catheterization. GTN was titrated intravenously until mean arterial blood pressure (MAP) was reduced by 15%. RESULTS: At arterial GTN concentrations of 27.2 [10.0-57.8] nmol l(-1) in CHF and 2.8 [2.5-3.5] nmol l(-1) in HS [median (quartile range), P<0.05 between groups], MAP and mean capillary wedge pressures were reduced similarly in both groups (approx. 15% and 65%, respectively, P = NS between groups). The ratios of 1,2-GDN/GTN and 1,2-GDN/1,3-GDN were lower in CHF (0.86 [0.28-1.58] and 5.8 [5.6-6.3]) compared with HS [1.91 (1.54-2.23) and 7.6 (7.2-10.2), P<0.05], with a negative correlation between the 1,2-GDN/1,3-GDN ratio and the arterial GTN concentrations in the CHF patients (R = -0.8, P<0.05). CONCLUSION: Patients with CHF have attenuated GTN responsiveness and decreased relative formation of 1,2-GDN in comparison with HS, indicating an altered biotransformation of GTN.
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Insuficiencia Cardíaca/metabolismo , Nitroglicerina/farmacocinética , Vasodilatadores/farmacocinética , Adulto , Biotransformación , Enfermedad Crónica , Insuficiencia Cardíaca/tratamiento farmacológico , Humanos , Masculino , Persona de Mediana Edad , Nitroglicerina/análogos & derivados , Nitroglicerina/química , Nitroglicerina/uso terapéutico , Vasodilatadores/química , Vasodilatadores/uso terapéuticoRESUMEN
Many conditions adversely affecting learning, memory, and cognition are associated with reductions in forebrain acetylcholine (ACh), most notably aging and Alzheimer's disease. In the current study, we demonstrate that bilateral depletion of neocortical and hippocampal ACh in rats produces deficits in a spatial learning task and in a recently described, delayed visual matching-to-sample task. Oral administration of the novel nitrate, GT1061 (4-methyl-5-(2-nitroxyethyl) thiazole HCl), and the acetylcholinesterase inhibitor, donepezil, reversed the cognitive deficits in both memory tasks in a dose-dependent manner. GT1061 was superior in the delayed matching-to-sample task. GT1061 was absorbed rapidly after oral administration, crossed the blood brain barrier, and achieved brain concentrations that were slightly higher than those found in plasma. The activity of GT1061 was NO mimetic: soluble guanylyl cyclase (sGC) was activated, but selectivity was observed for sGC in the hippocampus relative to the vasculature; and hippocampal levels of phosphorylated ERK1/2, which is a postulated intermediary in the formation of long-term memory, were increased. The beneficial effect on visual and spatial memory task performance supports the concept that stimulating the NO/sGC/cGMP signal transduction system can provide new, effective treatments for cognitive disorders. This approach may be superior to that of current drugs that attempt only to salvage the residual function of damaged cholinergic neurons.
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Acetilcolina/deficiencia , Trastornos del Conocimiento/tratamiento farmacológico , Nitratos/uso terapéutico , Nootrópicos/uso terapéutico , Prosencéfalo/patología , Análisis de Varianza , Animales , Conducta Animal/efectos de los fármacos , Conducta de Elección/efectos de la radiación , Modelos Animales de Enfermedad , Donepezilo , Relación Dosis-Respuesta a Droga , Femenino , Indanos/farmacología , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Nitratos/sangre , Nitratos/farmacocinética , Nootrópicos/sangre , Nootrópicos/farmacocinética , Fosforilación/efectos de los fármacos , Piperidinas/farmacología , Prosencéfalo/metabolismo , Unión Proteica/efectos de los fármacos , Pruebas Psicológicas , Ratas , Ratas Long-Evans , Ratas Sprague-Dawley , Tiempo de Reacción/efectos de los fármacosRESUMEN
Oxidative damage resulting from increased lipid peroxidation (LPO) is considered an important factor in the development of late onset/age-related Alzheimer's disease (AD). Deuterium-reinforced polyunsaturated fatty acids (D-PUFAs) are more resistant to the reactive oxygen species-initiated chain reaction of LPO than regular hydrogenated (H-) PUFAs. We investigated the effect of D-PUFA treatment on LPO and cognitive performance in aldehyde dehydrogenase 2 (Aldh2) null mice, an established model of oxidative stress-related cognitive impairment that exhibits AD-like pathologies. Mice were fed a Western-type diet containing either D- or H-PUFAs for 18 weeks. D-PUFA treatment markedly decreased cortex and hippocampus F2 -isoprostanes by approximately 55% and prostaglandin F2α by 20-25% as compared to H-PUFA treatment. D-PUFAs consistently improved performance in cognitive/memory tests, essentially resetting performance of the D-PUFA-fed Aldh2-/- mice to that of wild-type mice fed a typical laboratory diet. D-PUFAs therefore represent a promising new strategy to broadly reduce rates of LPO, and combat cognitive decline in AD.
Asunto(s)
Enfermedad de Alzheimer/prevención & control , Cognición/efectos de los fármacos , Deuterio/química , Ácidos Grasos Insaturados/farmacología , Aldehído Deshidrogenasa Mitocondrial/genética , Aldehído Deshidrogenasa Mitocondrial/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/fisiopatología , Animales , Cognición/fisiología , Modelos Animales de Enfermedad , F2-Isoprostanos/metabolismo , Ácidos Grasos Insaturados/administración & dosificación , Ácidos Grasos Insaturados/química , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/fisiopatología , Humanos , Hidrogenación , Peroxidación de Lípido/efectos de los fármacos , Memoria/efectos de los fármacos , Memoria/fisiología , Ratones NoqueadosRESUMEN
NO is an important messenger molecule in the brain, playing an important role in learning and memory, in particular via the ERK/CREB signaling pathway. NO is also a neuroprotective agent; multiple mechanisms having been demonstrated that can contribute to cell survival as levels of antioxidants and trophic factors are reduced with aging. Small molecules that mimic the biological activity of NO, including NO donors, may thus ameliorate cognition and provide neuroprotection. Several lines of evidence have linked the neurodegeneration and dementia characteristic of Alzheimer's disease with the action of beta-amyloid protein at the alpha7-nicotinic acetylcholine receptor. The interplay of Abeta with alpha7-nicotinic ACh receptors operating via the ERK signaling cascade links the amyloid cascade and the cholinergic hypothesis in pathways that impact synaptic plasticity and memory. This interplay also provides linkages to disruption of NO/cGMP signaling in AD, and in addition, recent direct evidence has been found demonstrating that Abeta downregulates the NO/cGMP/CREB pathway. Activation of soluble guanylyl cyclase elevating cGMP in the brain represents the central element of a therapeutic approach to the treatment of AD and other neurodegenerative diseases, furthermore, evidence suggests that NO may display cGMP-independent activity and may operate via multiple biochemical signaling pathways to ensure the survival of neurons subjected to stress. GT 1061 is an NO chimera, an NO mimetic compound that contains an ancillary, synergistic pharmacophore, currently in clinical trials for Alzheimer's. NO chimeras and hybrid nitrates hold promise as therapeutics for AD with multiple sites of action.
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Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Nitratos/uso terapéutico , Óxido Nítrico/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/efectos de los fármacos , Animales , Materiales Biomiméticos/uso terapéutico , Humanos , Nitratos/metabolismo , Óxido Nítrico/análogos & derivados , Óxido Nítrico/uso terapéutico , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
cAMP-response element-binding protein (CREB) plays a central role in various aspects of central nervous system (CNS) function, ranging from the developmental stages to neuronal plasticity and survival in adult brain. Activation of CREB plays a crucial role in learning and memory and is at the convergence of multiple intracellular signaling cascades including CAMKII and MAPK. This review focuses on the important functions of nitric oxide (NO) in activating CREB via the NO receptor, soluble guanylyl cyclase (sGC), and production of the second messenger, cGMP. The involvement of the NO/cGMP signaling pathway in synaptic plasticity suggests several avenues for therapeutic intervention, and targeting early synaptic degeneration could be an attractive approach for the development of novel disease-modifying approaches to treat cognition and memory dysfunction in neurodegenerative diseases.
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Enfermedad de Alzheimer/patología , Sistema Nervioso Central/metabolismo , GMP Cíclico/metabolismo , Enfermedades Neurodegenerativas/patología , Óxido Nítrico/metabolismo , Enfermedad de Alzheimer/metabolismo , Proteína de Unión a CREB/metabolismo , Humanos , Enfermedades Neurodegenerativas/metabolismo , Plasticidad Neuronal/fisiología , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/metabolismo , Transducción de Señal , Guanilil Ciclasa Soluble/antagonistas & inhibidores , Guanilil Ciclasa Soluble/metabolismo , Sinapsis/metabolismoRESUMEN
BACKGROUND: Clinical failures singularly targeting amyloid-ß pathology indicate a critical need for alternative Alzheimer's disease (AD) therapeutic strategies. The mixed pathology reported in a large population of AD patients demands a multifunctional drug approach. Since activation of cAMP response element binding protein (CREB) plays a crucial role in synaptic strengthening and memory formation, we retooled a clinical drug with known neuroprotective and anti-inflammatory activity to activate CREB, and validated this novel multifunctional drug, NMZ, in 4 different mouse models of AD. RESULTS: NMZ was tested in three mouse models of familial AD and one model of sporadic AD. In 3 × Tg hippocampal slices, NMZ restored LTP. In vivo, memory was improved with NMZ in all animal models with robust cognitive deficits. NMZ treatment lowered neurotoxic forms of Aß in both APP/PS1 and 3 × Tg transgenic mice while also restoring neuronal plasticity biomarkers in the 3 × Tg mice. In EFAD mice, incorporation of the major genetic AD risk factor, hAPOE4, did not mute the beneficial drug effects. In a novel sporadic mouse model that manifests AD-like pathology caused by accelerated oxidative stress in the absence of any familial AD mutation, oral administration of NMZ attenuated hallmark AD pathology and restored biomarkers of synaptic and neuronal function. CONCLUSIONS: The multifunctional approach, embodied by NMZ, was successful in mouse models of AD incorporating Aß pathology (APP/PS1), tau pathology (3xTg), and APOE4, the major human genetic risk factor for AD (EFAD). The efficacy observed in a novel model of sporadic AD (Aldh2 (-/-) ) demonstrates that the therapeutic approach is not limited to rare, familial AD genetic mutations. The multifunctional drug, NMZ, was not designed directly to target Aß and tau pathology; however, the attenuation of this hallmark pathology suggests the approach to be a highly promising, disease-modifying strategy for AD and mixed pathology dementia.
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Enfermedad de Alzheimer/terapia , Precursor de Proteína beta-Amiloide/metabolismo , Trastornos del Conocimiento/metabolismo , Memoria/fisiología , Plasticidad Neuronal/fisiología , Proteínas tau/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Ratones Noqueados , Ratones TransgénicosRESUMEN
Nitric oxide is multifunctional messenger molecule in the brain, playing important roles including in learning and memory and in regulating the expression of trophic factors that may be reduced with aging. Small molecules that mimic the biological activity of NO, NO mimetics, will bypass cholinergic receptor activation and are anticipated to provide multiple pathways of treating and circumventing dementia in Alzheimer's disease. Activation of soluble guanylyl cyclase and cGMP formation in the brain represents one element of effective neuroprotective pathways mediated by NO. Substantial evidence suggests that NO mimetics may display cGMP-dependent and cGMP-independent activity and may operate via multiple biochemical signaling pathways, both to ensure the survival of neurons subjected to stress and also to provide cognition-enabling pathways to circumvent dementia. GT 1061 is an NO mimetic compound currently in clinical trials for Alzheimer's. A survey of current research indicates that NO mimetics will provide a combined neuroprotective and cognition-enabling approach to anti-neurodegenerative therapy.
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Enfermedad de Alzheimer/tratamiento farmacológico , Materiales Biomiméticos/uso terapéutico , Óxido Nítrico/uso terapéutico , Enfermedad de Alzheimer/metabolismo , Animales , Materiales Biomiméticos/metabolismo , Materiales Biomiméticos/farmacología , Humanos , Aprendizaje/efectos de los fármacos , Aprendizaje/fisiología , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacologíaRESUMEN
BACKGROUND: The study of late-onset/age-related Alzheimer's disease (AD)(sporadic AD, 95% of AD cases) has been hampered by a paucity of animal models. Oxidative stress is considered a causative factor in late onset/age-related AD, and aldehyde dehydrogenase 2 (ALDH2) is important for the catabolism of toxic aldehydes associated with oxidative stress. One such toxic aldehyde, the lipid peroxidation product 4-hydroxynonenal (HNE), accumulates in AD brain and is associated with AD pathology. Given this linkage, we hypothesized that in mice lacking ALDH2, there would be increases in HNE and the appearance of AD-like pathological changes. RESULTS: Changes in relevant AD markers in Aldh2 (-/-) mice and their wildtype littermates were assessed over a 1 year period. Marked increases in HNE adducts arise in hippocampi from Aldh2 (-/-) mice, as well as age-related increases in amyloid-beta, p-tau, and activated caspases. Also observed were age-related decreases in pGSK3ß, PSD95, synaptophysin, CREB and pCREB. Age-related memory deficits in the novel object recognition and Y maze tasks begin at 3.5-4 months and are maximal at 6.5-7 months. There was decreased performance in the Morris Water Maze task in 6 month old Aldh2 (-/-) mice. These mice exhibited endothelial dysfunction, increased amyloid-beta in cerebral microvessels, decreases in carbachol-induced pCREB and pERK formation in hippocampal slices, and brain atrophy. These AD-associated pathological changes are rarely observed as a constellation in current AD animal models. CONCLUSIONS: We believe that this new model of age-related cognitive impairment will provide new insight into the pathogenesis and molecular/cellular mechanisms driving neurodegenerative diseases of aging such as AD, and will prove useful for assessing the efficacy of therapeutic agents for improving memory and for slowing, preventing, or reversing AD progression.