RESUMEN
The accumulation of amyloid-ß (Aß) is a characteristic event in the pathogenesis of Alzheimer's disease (AD). Aquaporin 1 (AQP1) is a membrane water channel protein belonging to the AQP family. AQP1 levels are elevated in the cerebral cortex during the early stages of AD, but the role of AQP1 in AD pathogenesis is unclear. We first determined the expression and distribution of AQP1 in brain tissue samples of AD patients and two AD mouse models (3xTg-AD and 5xFAD). AQP1 accumulation was observed in vulnerable neurons in the cerebral cortex of AD patients, and in neurons affected by the Aß or tau pathology in the 3xTg-AD and 5xFAD mice. AQP1 levels increased in neurons as aging progressed in the AD mouse models. Stress stimuli increased AQP1 in primary cortical neurons. In response to cellular stress, AQP1 appeared to translocate to endocytic compartments of ß- and γ-secretase activities. Ectopic expression of AQP1 in human neuroblastoma cells overexpressing amyloid precussir protein (APP) with the Swedish mutations reduced ß-secretase (BACE1)-mediated cleavage of APP and reduced Aß production without altering the nonamyloidogenic pathway. Conversely, knockdown of AQP1 enhanced BACE1 activity and Aß production. Immunoprecipitation experiments showed that AQP1 decreased the association of BACE1 with APP. Analysis of a human database showed that the amount of Aß decreases as the expression of AQP1 increases. These results suggest that the upregulation of AQP1 is an adaptive response of neurons to stress that reduces Aß production by inhibiting the binding between BACE1 and APP.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/fisiología , Precursor de Proteína beta-Amiloide/fisiología , Amiloide/biosíntesis , Acuaporina 1/fisiología , Enfermedad de Alzheimer/metabolismo , Animales , Acuaporina 1/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones , Neuronas/metabolismoRESUMEN
Transient receptor potential canonical 6 (TRPC6) channels are permeable to Na+ and Ca2+ and are widely expressed in the brain. In this study, the role of TRPC6 was investigated following ischemia/reperfusion (I/R) and oxygen-glucose deprivation (OGD). We found that TRPC6 expression was increased in wild-type (WT) mice cortical neurons following I/R and in primary neurons with OGD, and that deletion of TRPC6 reduced the I/R-induced brain infarct in mice and the OGD- /neurotoxin-induced neuronal death. Using live-cell imaging to examine intracellular Ca2+ levels ([Ca2+] i ), we found that OGD induced a significant higher increase in glutamate-evoked Ca2+ influx compared to untreated control and such an increase was reduced by TRPC6 deletion. Enhancement of TRPC6 expression using AdCMV-TRPC6-GFP infection in WT neurons increased [Ca2+] i in response to glutamate application compared to AdCMV-GFP control. Inhibition of N-methyl-d-aspartic acid receptor (NMDAR) with MK801 decreased TRPC6-dependent increase of [Ca2+] i in TRPC6 infected cells, indicating that such a Ca2+ influx was NMDAR dependent. Furthermore, TRPC6-dependent Ca2+ influx was blunted by blockade of Na+ entry in TRPC6 infected cells. Finally, OGD-enhanced Ca2+ influx was reduced, but not completely blocked, in the presence of voltage-dependent Na+ channel blocker tetrodotoxin (TTX) and dl-α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) blocker CNQX. Altogether, we concluded that I/R-induced brain damage was, in part, due to upregulation of TRPC6 in cortical neurons. We postulate that overexpression of TRPC6 following I/R may induce neuronal death partially through TRPC6-dependent Na+ entry which activated NMDAR, thus leading to a damaging Ca2+ overload. These findings may provide a potential target for future intervention in stroke-induced brain damage.
RESUMEN
Delivering neurotherapeutics to target brain-associated diseases is a major challenge. Therefore, we investigated oral delivery of green fluorescence protein (GFP) or myelin basic protein (MBP) fused with the transmucosal carrier cholera toxin B subunit (CTB), expressed in chloroplasts (bioencapsulated within plant cells) to the brain and retinae of triple transgenic Alzheimer's disease (3×TgAD) mice, across the blood-brain barriers (BBB) and blood-retinal barriers (BRB). Human neuroblastoma cells internalized GFP when incubated with CTB-GFP but not with GFP alone. Oral delivery of CTB-MBP in healthy and 3×TgAD mice shows increased MBP levels in different regions of the brain, crossing intact BBB. Thioflavin S-stained amyloid plaque intensity was reduced up to 60% by CTB-MBP incubation with human AD and 3×TgAD mice brain sections ex vivo. Amyloid loads were reduced in vivo by 70% in hippocampus and cortex brain regions of 3×TgAD mice fed with bioencapsulated CTB-MBP, along with reduction in the ratio of insoluble amyloid ß 42 (Aß42) to soluble fractions. CTB-MBP oral delivery reduced Aß42 accumulation in retinae and prevented loss of retinal ganglion cells in 3×TgAD mice. Lyophilization of leaves increased CTB-MBP concentration by 17-fold and stabilized it during long-term storage in capsules, facilitating low-cost oral delivery of therapeutic proteins across the BBB and BRB.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Barrera Hematoencefálica/metabolismo , Barrera Hematorretinal/metabolismo , Cloroplastos/metabolismo , Toxina del Cólera/metabolismo , Proteína Básica de Mielina/metabolismo , Placa Amiloide/tratamiento farmacológico , Administración Oral , Enfermedad de Alzheimer/patología , Animales , Cápsulas , Línea Celular Tumoral , Toxina del Cólera/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína Básica de Mielina/genética , Hojas de la Planta/citología , Placa Amiloide/patología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Intravenous immunoglobulin (IVIg) preparations obtained by fractionating blood plasma, are increasingly being used increasingly as an effective therapeutic agent in treatment of several inflammatory diseases. Its use as a potential therapeutic agent for treatment of stroke and Alzheimer's disease has been proposed, but little is known about the neuroprotective mechanisms of IVIg. In this study, we investigated the effect of IVIg on downstream signaling pathways that are involved in neuronal cell death in experimental models of stroke and Alzheimer's disease. Treatment of cultured neurons with IVIg reduced simulated ischemia- and amyloid ßpeptide (Aß)-induced caspase 3 cleavage, and phosphorylation of the cell death-associated kinases p38MAPK, c-Jun NH2 -terminal kinase and p65, in vitro. Additionally, Aß-induced accumulation of the lipid peroxidation product 4-hydroxynonenal was attenuated in neurons treated with IVIg. IVIg treatment also up-regulated the anti-apoptotic protein, Bcl2 in cortical neurons under ischemia-like conditions and exposure to Aß. Treatment of mice with IVIg reduced neuronal cell loss, apoptosis and infarct size, and improved functional outcome in a model of focal ischemic stroke. Together, these results indicate that IVIg acts directly on neurons to protect them against ischemic stroke and Aß-induced neuronal apoptosis by inhibiting cell death pathways and by elevating levels of the anti-apoptotic protein Bcl2.
Asunto(s)
Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/toxicidad , Isquemia Encefálica/prevención & control , Muerte Celular/efectos de los fármacos , Inmunoglobulinas Intravenosas/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores , Transducción de Señal/efectos de los fármacos , Accidente Cerebrovascular/prevención & control , Péptidos beta-Amiloides/farmacología , Animales , Western Blotting , Isquemia Encefálica/patología , Mapeo Encefálico , Hipoxia de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Glucosa/deficiencia , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Infarto de la Arteria Cerebral Media/patología , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Accidente Cerebrovascular/patología , Resultado del Tratamiento , Regulación hacia ArribaRESUMEN
The cause of elevated level of amyloid ß-peptide (Aß42) in common late-onset sporadic [Alzheimer's disease (AD)] has not been established. Here, we show that the membrane lipid peroxidation product 4-hydroxynonenal (HNE) is associated with amyloid and neurodegenerative pathologies in AD and that it enhances γ-secretase activity and Aß42 production in neurons. The γ-secretase substrate receptor, nicastrin, was found to be modified by HNE in cultured neurons and in brain specimens from patients with AD, in which HNE-nicastrin levels were found to be correlated with increased γ-secretase activity and Aß plaque burden. Furthermore, HNE modification of nicastrin enhanced its binding to the γ-secretase substrate, amyloid precursor protein (APP) C99. In addition, the stimulation of γ-secretase activity and Aß42 production by HNE were blocked by an HNE-scavenging histidine analog in a 3xTgAD mouse model of AD. These findings suggest a specific molecular mechanism by which oxidative stress increases Aß42 production in AD and identify HNE as a novel therapeutic target upstream of the γ-secretase cleavage of APP.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteínas Amiloidogénicas/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Aldehídos/química , Aldehídos/metabolismo , Secretasas de la Proteína Precursora del Amiloide/química , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Encéfalo/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Humanos , Técnicas In Vitro , Peroxidación de Lípido , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Microdominios de Membrana/metabolismo , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Estructura Terciaria de ProteínaRESUMEN
Chronic intake of nicotine can impair hippocampal plasticity, but the underlying mechanism is poorly understood. Here, we demonstrate that chronic nicotine administration in adult rats inactivates the cyclic AMP-response element binding protein (CREB), a transcription factor that regulates neurogenesis and other plasticity-related processes necessary for learning and memory. Consequently, we showed that impaired CREB signaling is associated with a significant decline in the production of new neurons in the dentate gyrus. Combining retrovirus labeling with gene expression approaches, we found that chronic nicotine administration reduces the number of adult-generated granule neurons by decreasing the survival of newborn cells but not the proliferation of progenitor cells. Additionally, we found that retroviral-mediated expression of a constitutively active CREB in the dentate gyrus rescues survival of newborn cells and reverses the nicotine-induced decline in the number of mature granule neurons. Prolonged nicotine exposure also compromises CREB activation and reduces the viability of progenitor cells in vitro, thereby suggesting that nicotine may exert its adverse effects directly on immature cells in vivo. Taken together, these data demonstrate that inhibition of CREB activation is responsible for the nicotine-induced impairment of hippocampal plasticity.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Giro Dentado/citología , Giro Dentado/efectos de los fármacos , Nicotina/administración & dosificación , Animales , Bromodesoxiuridina/administración & dosificación , Recuento de Células , Muerte Celular , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Giro Dentado/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Neuronas/efectos de los fármacos , Nicotina/efectos adversos , Ratas , Retroviridae/genética , Retroviridae/metabolismo , Células Madre/citología , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Activación Transcripcional , TransfecciónRESUMEN
Endoplasmic reticulum (ER) stress has been implicated as an initiator or contributing factor in neurodegenerative diseases. The mechanisms that lead to ER stress and whereby ER stress contributes to the degenerative cascades remain unclear but their understanding is critical to devising effective therapies. Here we show that knockdown of Herp (Homocysteine-inducible ER stress protein), an ER stress-inducible protein with an ubiquitin-like (UBL) domain, aggravates ER stress-mediated cell death induced by mutant α-synuclein (αSyn) that causes an inherited form of Parkinson's disease (PD). Functionally, Herp plays a role in maintaining ER homeostasis by facilitating proteasome-mediated degradation of ER-resident Ca(2+) release channels. Deletion of the UBL domain or pharmacological inhibition of proteasomes abolishes the Herp-mediated stabilization of ER Ca(2+) homeostasis. Furthermore, knockdown or pharmacological inhibition of ER Ca(2+) release channels ameliorates ER stress, suggesting that impaired homeostatic regulation of Ca(2+) channels promotes a protracted ER stress with the consequent activation of ER stress-associated apoptotic pathways. Interestingly, sustained upregulation of ER stress markers and aberrant accumulation of ER Ca(2+) release channels were detected in transgenic mutant A53T-αSyn mice. Collectively, these data establish a causative link between impaired ER Ca(2+) homeostasis and chronic ER stress in the degenerative cascades induced by mutant αSyn and suggest that Herp is essential for the resolution of ER stress through maintenance of ER Ca(2+) homeostasis. Our findings suggest a therapeutic potential in PD for agents that increase Herp levels or its ER Ca(2+)-stabilizing action.
Asunto(s)
Calcio/metabolismo , Retículo Endoplásmico/fisiología , Proteínas de la Membrana/metabolismo , Estrés Fisiológico , alfa-Sinucleína/metabolismo , Animales , Canales de Calcio/metabolismo , Muerte Celular , Degradación Asociada con el Retículo Endoplásmico , Células HEK293 , Homeostasis , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Proteínas Mutantes/metabolismo , Células PC12 , Interferencia de ARN , Ratas , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , alfa-Sinucleína/genéticaRESUMEN
Acetazolamide (AZA), used in treatment of early or infantile hydrocephalus, is effective in some cases, while its effect on the choroid plexus (CP) remains ill-defined. The drug reversibly inhibits aquaporin-4 (AQP4), the most ubiquitous "water pore" in the brain, and perhaps modulation of AQP1 (located apically on CP cells) by AZA may reduce cerebrospinal fluid (CSF) production. We sought to elucidate the effect of AZA on AQP1 and fluid flow in CP cell cultures.CP tissue culture from 10-day Sprague-Dawley rats and a TRCSF-B cell line were grown on Transwell permeable supports and treated with 100 µM AZA. Fluid assays to assess direction and extent of fluid flow, and AQP1 expression patterns by immunoblot, Immuncytochemistry (ICC), and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) were performed.Immunoblots and ICC analyses showed a decrease in AQP1 protein shortly after AZA treatment (lowest at 12 h), with transient AQP1 reduction mediated by mRNA expression (lowest at 6 h). Transwell fluid assays indicated a fluid shift at 2 h, before significant changes in AQP1 mRNA or protein levels.Timing of AZA effect on AQP1 suggests the drug alters protein transcription, while affecting fluid flow by a concomitant method. It is plausible that other mechanisms account for these phenomena, as the processes may occur independently.
Asunto(s)
Acetazolamida/farmacología , Acuaporina 1/metabolismo , Inhibidores de Anhidrasa Carbónica/farmacología , Plexo Coroideo/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Animales , Animales Recién Nacidos , Permeabilidad Capilar/efectos de los fármacos , Plexo Coroideo/metabolismo , Dextranos , Hidrodinámica , Técnicas de Cultivo de Órganos , ARN Mensajero/metabolismo , Ratas , Rodaminas , Factores de TiempoRESUMEN
BACKGROUND AND PURPOSE: Activation of Notch worsens ischemic brain damage as antisense knockdown or pharmacological inhibition of the Notch pathway reduces the infarct size and improves the functional outcome in a mouse model of stroke. We sought to determine whether Notch activation contributes to postischemic inflammation by directly modulating the microglial innate response. METHODS: The microglial response and the attendant inflammatory reaction were evaluated in Notch1 antisense transgenic (Tg) and in nontransgenic (non-Tg) mice subjected to middle cerebral artery occlusion with or without treatment with a γ-secretase inhibitor (GSI). To investigate the impact of Notch on microglial effector functions, primary mouse microglia and murine BV-2 microglial cell line were exposed to oxygen glucose deprivation or lipopolysaccharide in the presence or absence of GSI. Immunofluorescence labeling, Western blotting, and reverse-transcription polymerase chain reaction were performed to measure microglial activation and production of inflammatory cytokines. The nuclear translocation of nuclear factor-κB in microglia was assessed by immunohistochemistry. The neurotoxic potential of microglia was determined in cocultures. RESULTS: Notch1 antisense mice exhibit significantly lower numbers of activated microglia and reduced proinflammatory cytokine expression in the ipsilateral ischemic cortices compared to non-Tg mice. Microglial activation also was attenuated in Notch1 antisense cultures and in non-Tg cultures treated with GSI. GSI significantly reduced nuclear factor-κB activation and expression of proinflammatory mediators and markedly attenuated the neurotoxic activity of microglia in cocultures. CONCLUSIONS: These findings establish a role for Notch signaling in modulating the microglia innate response and suggest that inhibition of Notch might represent a complementary therapeutic approach to prevent reactive gliosis in stroke and neuroinflammation-related degenerative disorders.
Asunto(s)
Isquemia Encefálica/metabolismo , Núcleo Celular/metabolismo , Gliosis/metabolismo , Microglía/metabolismo , Receptor Notch1/metabolismo , Transporte Activo de Núcleo Celular/genética , Transporte Activo de Núcleo Celular/inmunología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/inmunología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Isquemia Encefálica/genética , Isquemia Encefálica/inmunología , Isquemia Encefálica/patología , Isquemia Encefálica/terapia , Línea Celular , Núcleo Celular/genética , Núcleo Celular/inmunología , Técnicas de Cocultivo , Citocinas/biosíntesis , Citocinas/genética , Citocinas/inmunología , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Gliosis/genética , Gliosis/inmunología , Gliosis/patología , Gliosis/terapia , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Inflamación/terapia , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Ratones , Ratones Transgénicos , Microglía/inmunología , Microglía/patología , FN-kappa B/genética , FN-kappa B/inmunología , FN-kappa B/metabolismo , Oligopéptidos/farmacología , Receptor Notch1/antagonistas & inhibidores , Receptor Notch1/genética , Receptor Notch1/inmunologíaRESUMEN
Notch-1 (Notch) is a cell surface receptor that regulates cell-fate decisions in the developing nervous system, and it may also have roles in synaptic plasticity in the adult brain. Binding of its ligands results in the proteolytic cleavage of Notch by the γ-secretase enzyme complex, thereby causing the release of a Notch intracellular domain (NICD) that translocates to the nucleus, in which it regulates transcription. Here we show that activation of Notch modulates ischemic neuronal cell death in vitro and in vivo. Specifically, our findings from the use of Notch-1 siRNA or the overexpression of NICD indicate that Notch activation contributes to cell death. Using modified NICD, we demonstrate an apoptosis-inducing function of NICD in both the nucleus and the cytosol. NICD transfection-induced cell death was reduced by blockade of calcium signaling, caspase activation, and Janus kinase signaling. Inhibition of the Notch-activating enzyme, γ-secretase, protected against ischemic neuronal cell death by targeting an apoptotic protease, cleaved caspase-3, nuclear factor-κB (NF-κB), and the pro-death BH3-only protein, Bcl-2-interacting mediator of cell death (Bim). Treatment of mice with a γ-secretase inhibitor, compound E, reduced infarct size and improved functional outcome in a model of focal ischemic stroke. Furthermore, γ-secretase inhibition reduced NICD, p-p65, and Bim levels in vivo. These findings suggest that Notch signaling endangers neurons after ischemic stroke by modulating the NF-κB, pro-death protein Bim, and caspase pathways.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Isquemia Encefálica/patología , Muerte Celular/fisiología , FN-kappa B/metabolismo , Neuronas/citología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Receptores Notch/metabolismo , Transducción de Señal , Accidente Cerebrovascular/patología , Animales , Isquemia Encefálica/enzimología , Isquemia Encefálica/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-Dawley , Accidente Cerebrovascular/enzimología , Accidente Cerebrovascular/metabolismoRESUMEN
The cell fate determinant Numb exists in four alternatively spliced variants that differ in the length of their PTB (phosphotyrosine-binding domain, either lacking or containing an 11 amino acid insertion) and PRR (proline-rich region, either lacking or containing a 48 amino acid insertion). We previously reported that Numb switches from isoforms containing the PTB insertion to isoforms lacking this insertion in neural cultures subjected to stress induced by trophic factor withdrawal. The switch in Numb isoforms enhances the generation of amyloid-ß peptide (Aß), the principle component of senile plaques in Alzheimer's disease (AD). Here we examine the expression of the Numb isoforms in brains from AD patients and triple transgenic (3xTg) AD mice. We found that levels of the Numb isoforms lacking the PTB insertion are significantly elevated in the parietal cortex but not in the cerebellum of AD patients when compared to control subjects. Levels of Numb isoforms lacking the PTB insertion were also elevated in the cortex but not cerebellum of 12 month-old 3xTg AD mice with Aß deposits compared to younger 3xTg-AD mice and to non-transgenic mice. Exposure of cultured neurons to Aß resulted in an increase in the levels of Numb isoforms lacking the PTB domain, consistent with a role for Aß in the aberrant expression of Numb in vulnerable brain regions of AD patients and mice. Collectively, the data show that altered expression of Numb isoforms in vulnerable neurons occurs during AD pathogenesis and suggest a role for Numb in the disease process.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Regulación de la Expresión Génica/genética , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Isoformas de Proteínas/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/farmacología , Precursor de Proteína beta-Amiloide/genética , Análisis de Varianza , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Corteza Cerebral/citología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Inmunoprecipitación/métodos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Fragmentos de Péptidos/farmacología , Fosfopiruvato Hidratasa/metabolismo , Presenilina-1/genética , Isoformas de Proteínas/genética , Factores de Tiempo , Transfección/métodos , Proteínas de Unión al GTP rab5/metabolismo , Proteínas tau/genéticaRESUMEN
BACKGROUND: Hydrocephalus occurs because of an imbalance of bulk fluid flow in the brain, and aquaporins (AQPs) play pivotal roles in cerebral water movement as essential mediators during edema and fluid accumulation. AQP1 is a water channel found in the choroid plexus (CP), and AQP4 is expressed at the brain-CSF interfaces and astrocytic end feet; excessive fluid accumulation may involve expression of changes in these AQPs during various stages of hydrocephalus. OBJECTIVE: To determine the alterations of CP AQP1 expression in congenital hydrocephalus; detect hydrocephalus-induced AQP1 expression in the cortical parenchyma, ependyma, and pia mater of hydrocephalic animals; and evaluate AQP4 expression in congenital hydrocephalus through progressive stages of the condition. METHODS: We evaluated differential expression of AQPs 1 and 4 in the congenital hydrocephalus Texas rat at postnatal days 5, 10, and 26 in isolated CP and cortex by enzyme-linked immunosorbent assay, Western blot, quantitative reverse transcriptase polymerase chain reaction, and immunohistochemistry. RESULTS: The CP exhibited a 34% decrease in AQP1 expression in young hydrocephalic pups (postnatal days 5 and 10), which became normal (postnatal day 26) just before death. With advancing hydrocephalus, expression of AQPs 1 and 4 increased at the brain-CSF interfaces; AQP1 was localized to the endothelium of cortical capillaries with increased AQP4 expression in surrounding astrocytes end feet. AQP1 expression level was increased in the pia mater, with prominent AQP4 expression in the subpial layers. Subependymal capillaries expressed AQP1 in the endothelium, with increasing AQP4 expression in surrounding astrocytes. Hydrocephalic animals (postnatal day 26) had significant nonendothelial (CD34) AQP1 expression in the septal nucleus of the basal forebrain, an area affected by increased intracranial pressure. CONCLUSION: Biphasic AQP1 expression in the CP with increased AQPs 1 and 4 at the brain-fluid interfaces may indicate compensatory mechanisms to regulate choroidal cerebrospinal fluid secretion and increase parenchymal fluid absorption in the high-pressure hydrocephalic condition.
Asunto(s)
Acuaporina 1/biosíntesis , Acuaporina 4/biosíntesis , Hidrocefalia/metabolismo , Animales , Western Blotting , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , Perfilación de la Expresión Génica , Inmunohistoquímica , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Huntington's disease (HD) is an inherited neurodegenerative disorder caused by expanded polyglutamine repeats in the huntingtin (Htt) protein. Mutant Htt may damage and kill striatal neurons by a mechanism involving reduced production of brain-derived neurotrophic factor (BDNF) and increased oxidative and metabolic stress. Because electroconvulsive shock (ECS) can stimulate the production of BDNF and protect neurons against stress, we determined whether ECS treatment would modify the disease process and provide a therapeutic benefit in a mouse model of HD. ECS (50 mA for 0.2 s) or sham treatment was administered once weekly to male N171-82Q Htt mutant mice beginning at 2 months of age. Endpoints measured included motor function, striatal and cortical pathology, and levels of protein chaperones and BDNF. ECS treatment delayed the onset of motor symptoms and body weight loss and extended the survival of HD mice. Striatal neurodegeneration was attenuated and levels of protein chaperones (Hsp70 and Hsp40) and BDNF were elevated in striatal neurons of ECS-treated compared with sham-treated HD mice. Our findings demonstrate that ECS can increase the resistance of neurons to mutant Htt resulting in improved functional outcome and extended survival. The potential of ECS as an intervention in subjects that inherit the mutant Htt gene merits further consideration.
Asunto(s)
Progresión de la Enfermedad , Electrochoque , Enfermedad de Huntington/patología , Enfermedad de Huntington/terapia , Mutación/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Enfermedad de Huntington/genética , Masculino , Ratones , Ratones Transgénicos , Degeneración Nerviosa/genética , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Transducción de Señal , Análisis de SupervivenciaRESUMEN
Wound healing is a complex process involving intrinsic dermal and epidermal cells, and infiltrating macrophages and leukocytes. Excessive oxidative stress and associated inflammatory processes can impair wound healing, and antioxidants have been reported to improve wound healing in animal models and human subjects. Uric acid (UA) is an efficient free radical scavenger, but has a very low solubility and poor tissue penetrability. We recently developed novel UA analogs with increased solubility and excellent free radical-scavenging properties and demonstrated their ability to protect neural cells against oxidative damage. Here we show that the uric acid analog (6, 8 dithio-UA, but not equimolar concentrations of UA or 1, 7 dimethyl-UA) modified the behaviors of cultured vascular endothelial cells, keratinocytes and fibroblasts in ways consistent with enhancement of the wound healing functions of all three cell types. We further show that 6, 8 dithio-UA significantly accelerates the wound healing process when applied topically (once daily) to full-thickness wounds in mice. Levels of Cu/Zn superoxide dismutase were increased in wound tissue from mice treated with 6, 8 dithio-UA compared to vehicle-treated mice, suggesting that the UA analog enhances endogenous cellular antioxidant defenses. These results support an adverse role for oxidative stress in wound healing and tissue repair, and provide a rationale for the development of UA analogs in the treatment of wounds and for modulation of angiogenesis in other pathological conditions.
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Piel/lesiones , Ácido Úrico/análogos & derivados , Cicatrización de Heridas/efectos de los fármacos , Animales , Antioxidantes , Células Cultivadas , Depuradores de Radicales Libres , Ratones , Neovascularización Fisiológica , Estrés Oxidativo , Piel/patología , Solubilidad , Compuestos de Sulfhidrilo , Superóxido Dismutasa/efectos de los fármacos , Ácido Úrico/administración & dosificación , Ácido Úrico/farmacología , Ácido Úrico/uso terapéuticoRESUMEN
Glioblastoma multiforme (GBM) is the most frequent and incurable type of brain tumor of adults. Hypoxia has been shown to direct GBM toward a more aggressive and malignant state. Here we show that hypoxia increases Notch1 activation, which in turn induces the expression of transient receptor potential 6 (TRPC6) in primary samples and cell lines derived from GBM. TRPC6 is required for the development of the aggressive phenotype because knockdown of TRPC6 expression inhibits glioma growth, invasion, and angiogenesis. Functionally, TRPC6 causes a sustained elevation of intracellular calcium that is coupled to the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway. Pharmacologic inhibition of the calcineurin-NFAT pathway substantially reduces the development of the malignant GBM phenotypes under hypoxia. Clinically, expression of TRPC6 was elevated in GBM specimens in comparison with normal tissues. Collectively, our studies indicate that TRPC6 is a key mediator of tumor growth of GBM in vitro and in vivo and that TRPC6 may be a promising therapeutic target in the treatment of human GBM.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Invasividad Neoplásica/patología , Receptor Notch1/metabolismo , Canales Catiónicos TRPC/metabolismo , Adulto , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Hipoxia de la Célula/fisiología , Técnica del Anticuerpo Fluorescente , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/patología , Humanos , Inmunohistoquímica , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Invasividad Neoplásica/genética , ARN Interferente Pequeño , Receptor Notch1/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , Canales Catiónicos TRPC/genética , Canal Catiónico TRPC6RESUMEN
The Notch signaling pathway plays an essential role in the regulation of cell specification by controlling differentiation, proliferation, and apoptosis. Numb is an intrinsic regulator of the Notch pathway and exists in four alternative splice variants that differ in the length of their phosphotyrosine-binding domain (PTB) and proline-rich region domains. The physiological relevance of the existence of the Numb splice variants and their exact regulation are still poorly understood. We previously reported that Numb switches from isoforms containing the insertion in PTB to isoforms lacking this insertion in neuronal cells subjected to trophic factor withdrawal (TFW). The functional relevance of the TFW-induced switch in Numb isoforms is not known. Here we provide evidence that the TFW-induced switch in Numb isoforms regulates Notch signaling strength and Notch target gene expression. PC12 cells stably overexpressing Numb isoforms lacking the PTB insertion exhibited higher basal Notch activity and Notch-dependent transcription of the transient receptor potential channel 6 (TRPC6) when compared with those overexpressing Numb isoforms with the PTB insertion. The differential regulation of TRPC6 expression is correlated with perturbed calcium signaling and increased neuronal vulnerability to TFW-induced death. Pharmacological inhibition of the Notch pathway or knockdown of TRPC6 function ameliorates the adverse effects caused by the TFW-induced switch in Numb isoforms. Taken together, our results indicate that Notch and Numb interaction may influence the sensitivity of neuronal cells to injurious stimuli by modulating calcium-dependent apoptotic signaling cascades.
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Proteínas de la Membrana/fisiología , Proteínas del Tejido Nervioso/fisiología , Receptores Notch/metabolismo , Canales Catiónicos TRPC/genética , Animales , Señalización del Calcio , Muerte Celular , Humanos , Neuronas/metabolismo , Células PC12 , Isoformas de Proteínas , Ratas , Transducción de Señal , Estrés Fisiológico , Regulación hacia Arriba/genéticaRESUMEN
beta1 integrin is a cell surface molecule that is critical for endothelial cell adhesion, migration and survival during angiogenesis. In the present study we employed in vivo and in vitro models to elucidate the role of beta1 integrin in vascular remodelling and stroke outcomes. At 24 h after cerebral ischemia and reperfusion (I/R), the ischemic cortex (ipsilateral area) exhibited modest beta1 integrin immunoreactivity and a robust increase was observed at 72 h. Double-label immunohistochemical analysis for beta1 integrin with neuronal (NeuN), microglial (Iba-1), astrocyte (GFAP), progenitor cell (Ng2) and blood vessel (collagen 4) markers showed that beta1 integrin expression only localized to blood vessels. In vitro studies using cultured endothelial cells and a beta1 integrin blocking antibody confirmed that beta1 integrin is required for endothelial cell migration, proliferation and blood vessel formation. In vivo studies in the cerebral I/R model using the beta1 integrin blocking antibody further confirmed that beta1 integrin signaling is involved in vascular formation and recovery following ischemic stroke. Finally, we found that beta1 integrin is critically involved in functional deficits and survival after a stroke. These results suggest that beta1 integrin plays important roles in neurovascular remodelling and functional outcomes following stroke, and that targeting the beta1 integrin signalling may provide a novel strategy for modulating angiogenesis in ischemic stroke and other pathological conditions.
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Vasos Sanguíneos/metabolismo , Regulación de la Expresión Génica/fisiología , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/fisiopatología , Interferón beta/metabolismo , Neovascularización Patológica/metabolismo , Animales , Anticuerpos/farmacología , Anticuerpos/uso terapéutico , Antígenos/metabolismo , Encéfalo/patología , Proteínas de Unión al Calcio/metabolismo , Línea Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Proliferación Celular/efectos de los fármacos , Proliferación Celular/ética , Colágeno/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Proteína Ácida Fibrilar de la Glía/metabolismo , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/patología , Interferón beta/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos , Neovascularización Patológica/tratamiento farmacológico , Fosfopiruvato Hidratasa/metabolismo , Proteoglicanos/metabolismo , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismo , Estadísticas no ParamétricasRESUMEN
Mitochondrial dysfunction and reactive oxygen species (ROS) production are at the heart of the aging process and are thought to underpin age-related diseases. Mitochondria are not only the primary energy-generating system but also the dominant cellular source of metabolically derived ROS. Recent studies unravel the existence of mechanisms that serve to modulate the balance between energy metabolism and ROS production. Among these is the regulation of proton conductance across the inner mitochondrial membrane that affects the efficiency of respiration and heat production. The field of mitochondrial respiration research has provided important insight into the role of altered energy balance in obesity and diabetes. The notion that respiration and oxidative capacity are mechanistically linked is making significant headway into the field of aging and age-related diseases. Here we review the regulation of cellular energy and ROS balance in biological systems and survey some of the recent relevant studies that suggest that respiratory adaptation and thermodynamics are important in aging and age-related diseases.
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Adaptación Fisiológica , Envejecimiento/metabolismo , Regulación de la Temperatura Corporal/fisiología , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , Animales , Restricción Calórica , Respiración de la Célula , Diabetes Mellitus Tipo 2/metabolismo , Metabolismo Energético , Humanos , Canales Iónicos/metabolismo , Ratones , Proteínas Mitocondriales/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Obesidad/metabolismo , Protones , Ratas , Especies Reactivas de Oxígeno/metabolismo , Proteína Desacopladora 1RESUMEN
Increased bioenergetics demand can stimulate compensatory increases in glucose metabolism. We previously reported that neural cells expressing the brain uncoupling protein UCP4 exhibit enhanced dependency on glucose for support of cellular bioenergetics and survival. The switch from oxidative toward glycolytic metabolism reduces the production of toxic reactive oxygen species (ROS) and increases cellular resistance to toxicity induced by 3-nitropropionic acid, a mitochondrial complex II inhibitor that compromises cellular bioenergetics. In this study we elucidate the underlying mechanism whereby expression of UCP4 promotes bioenergetics adaptation and cell survival. We found that activation of extracellular signal-regulated kinases (ERKs) is necessary and sufficient for the increased dependency on glucose utilization. Pharmacological inhibition of ERKs not only abrogated bioenergetics adaptation but also reduced the activation of cAMP-responsive element-binding (CREB) protein suggesting that CREB protein signaling contributes in part to UCP4-dependent cell death rescue from 3-nitropropionic acid-induced toxicity. We also demonstrated that activation of ERKs by growth factors ameliorated the bioenergetics compromise and reduced cellular toxicity induced by 3-nitropropionic acid. Collectively, our results support the involvement of ERKs in UCP4 dependent bioenergetics adaptation and cell survival.
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Metabolismo Energético/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Neuronas/efectos de los fármacos , Neurotoxinas/toxicidad , Nitrocompuestos/toxicidad , Propionatos/toxicidad , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Proteína de Unión a CREB/metabolismo , Muerte Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Corteza Cerebral/citología , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Embrión de Mamíferos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Glucosa/metabolismo , Humanos , Proteínas de Transporte de Membrana/genética , Proteínas Desacopladoras Mitocondriales , Factor de Crecimiento Nervioso/farmacología , Células PC12 , Ratas , Estadísticas no Paramétricas , Factores de TiempoRESUMEN
The endoplasmic reticulum (ER) is a key organelle regulating intracellular Ca(2+) homeostasis. Oxidants and mitochondria-derived free radicals can target ER-based Ca(2+) regulatory proteins and cause uncontrolled Ca(2+) release that may contribute to protracted ER stress and apoptosis. Several ER stress proteins have been suggested to counteract the deregulation of ER Ca(2+) homeostasis and ER stress. Here we showed that knockdown of Herp, an ubiquitin-like domain containing ER stress protein, renders PC12 and MN9D cells vulnerable to 1-methyl-4-phenylpyridinium-induced cytotoxic cell death by a mechanism involving up-regulation of CHOP expression and ER Ca(2+) depletion. Conversely, Herp overexpression confers protection by blocking 1-methyl-4-phenylpyridinium-induced CHOP up-regulation, ER Ca(2+) store depletion, and mitochondrial Ca(2+) accumulation in a manner dependent on a functional ubiquitin-proteasomal protein degradation pathway. Deletion of the ubiquitin-like domain of Herp or treatment with a proteasomal inhibitor abolished the central function of Herp in ER Ca(2+) homeostasis. Thus, elucidating the underlying molecular mechanism(s) whereby Herp counteracts Ca(2+) disturbances will provide insights into the molecular cascade of cell death in dopaminergic neurons and may uncover novel therapeutic strategies to prevent and ameliorate Parkinson disease progression.