RESUMEN
In the immune system, degranulation/exocytosis from lymphocytes is crucial for life through facilitating eradication of infected and malignant cells. Dysfunction of the NK cell exocytosis process has been implicated with devastating immune diseases, such as familial hemophagocytic lymphohistiocytosis, yet the underlying molecular mechanisms of such processes have remained elusive. In particular, although the lytic granule exocytosis from NK cells is strictly Ca2+-dependent, the molecular identity of the Ca2+ sensor has yet to be identified. In this article, we show multiple lines of evidence in which point mutations in aspartic acid residues in both C2 domains of human Munc13-4, whose mutation underlies familial hemophagocytic lymphohistiocytosis type 3, diminished exocytosis with dramatically altered Ca2+ sensitivity in both mouse primary NK cells as well as rat mast cell lines. Furthermore, these mutations within the C2 domains severely impaired NK cell cytotoxicity against malignant cells. Total internal reflection fluorescence microscopy analysis revealed that the mutations strikingly altered Ca2+ dependence of fusion pore opening of each single granule and frequency of fusion events. Our results demonstrate that both C2 domains of Munc13-4 play critical roles in Ca2+-dependent exocytosis and cytotoxicity by regulating single-granule membrane fusion dynamics in immune cells.
Asunto(s)
Células Asesinas Naturales/inmunología , Linfohistiocitosis Hemofagocítica/inmunología , Mastocitos/inmunología , Proteínas de la Membrana/metabolismo , Vesículas Secretoras/metabolismo , Animales , Ácido Aspártico/genética , Señalización del Calcio , Degranulación de la Célula , Células Cultivadas , Citotoxicidad Inmunológica , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Mutación/genética , Dominios Proteicos/genética , RatasRESUMEN
Munc18-1/UNC-18 is believed to prime SNARE-mediated membrane fusion, yet the underlying mechanisms remain enigmatic. Here, we examine how potential gain-of-function mutations of Munc18-1/UNC-18 affect locomotory behavior and synaptic transmission, and how Munc18-1-mediated priming is related to Munc13-1/UNC-13 and Tomosyn/TOM-1, positive and negative SNARE regulators, respectively. We show that a Munc18-1(P335A)/UNC-18(P334A) mutation leads to significantly increased locomotory activity and acetylcholine release in Caenorhabditis elegans, as well as enhanced synaptic neurotransmission in cultured mammalian neurons. Importantly, similar to tom-1 null mutants, unc-18(P334A) mutants partially bypass the requirement of UNC-13. Moreover, unc-18(P334A) and tom-1 null mutations confer a strong synergy in suppressing the phenotypes of unc-13 mutants. Through biochemical experiments, we demonstrate that Munc18-1(P335A) exhibits enhanced activity in SNARE complex formation as well as in binding to the preformed SNARE complex, and partially bypasses the Munc13-1 requirement in liposome fusion assays. Our results indicate that Munc18-1/UNC-18 primes vesicle fusion downstream of Munc13-1/UNC-13 by templating SNARE complex assembly and acts antagonistically with Tomosyn/TOM-1.SIGNIFICANCE STATEMENT At presynaptic sites, SNARE-mediated membrane fusion is tightly regulated by several key proteins including Munc18/UNC-18, Munc13/UNC-13, and Tomosyn/TOM-1. However, how these proteins interact with each other to achieve the precise regulation of neurotransmitter release remains largely unclear. Using Caenorhabditis elegans as an in vivo model, we found that a gain-of-function mutant of UNC-18 increases locomotory activity and synaptic acetylcholine release, that it partially bypasses the requirement of UNC-13 for release, and that this bypass is synergistically augmented by the lack of TOM-1. We also elucidated the biochemical basis for the gain-of-function caused by this mutation. Thus, our study provides novel mechanistic insights into how Munc18/UNC-18 primes synaptic vesicle release and how this protein interacts functionally with Munc13/UNC-13 and Tomosyn/TOM-1.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Proteínas Portadoras/metabolismo , Locomoción/fisiología , Fosfoproteínas/metabolismo , Proteínas SNARE/metabolismo , Transmisión Sináptica/fisiología , Proteínas de Transporte Vesicular/metabolismo , Animales , Proteínas de Caenorhabditis elegans/genética , Proteínas Portadoras/genética , Mutación/genética , Neuronas , Fosfoproteínas/genética , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMEN
Stroke is one of the major causes of mortality and morbidity worldwide, yet novel therapeutic treatments for this condition are lacking. This review focuses on the roles of the transient receptor potential melastatin 2 (TRPM2) ion channels in cellular damage following hypoxia-ischemia and their potential as a future therapeutic target for stroke. Here, we highlight the complex molecular signaling that takes place in neurons, glial cells and the blood-brain barrier following ischemic insult. We also describe the evidence of TRPM2 involvement in these processes, as shown from numerous in vitro and in vivo studies that utilize genetic and pharmacological approaches. This evidence implicates TRPM2 in a broad range of pathways that take place every stage of cerebral ischemic injury, thus making TRPM2 a promising target for drug development for stroke and other neurodegenerative conditions of the central nervous system.
Asunto(s)
Barrera Hematoencefálica/fisiopatología , Infarto Cerebral/fisiopatología , Hipoxia/fisiopatología , Neuroglía/fisiología , Neuronas/fisiología , Canales Catiónicos TRPM/fisiología , Animales , Humanos , Canales Catiónicos TRPM/químicaRESUMEN
Understanding how Munc18 proteins govern exocytosis is crucial because mutations of this protein cause severe secretion deficits in neuronal and immune cells. Munc18-2 has indispensable roles in the degranulation of mast cell, partly by binding and chaperoning a subset of syntaxin isoforms. However, the key syntaxin that, crucially, participates in the degranulation whose levels and intracellular localization are regulated by Munc18-2 remains unknown. Here, we demonstrate that double knockdown of Munc18-1 and Munc-2 in mast cells results in greatly reduced degranulation accompanied with strikingly compromised expression levels and localization of syntaxin-3. This phenotype is fully rescued by wild-type Munc18 proteins but not by the K46E, E59K and K46E/E59K mutants of Munc-18 domain 1, each of which exhibits completely abolished binding to 'closed' syntaxin-3. Furthermore, knockdown of syntaxin-3 strongly impairs degranulation. Collectively, our data argue that residues Lys46 and Glu59 of Munc18 proteins are indispensable for mediating the interaction between Munc18 and closed syntaxin-3, which is essential for degranulation by chaperoning syntaxin-3. Our results also indicate that the functional contribution of these residues differs between immune cell degranulation and neuronal secretion.
Asunto(s)
Mastocitos/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Munc18/metabolismo , Unión Proteica/genética , Proteínas Qa-SNARE/metabolismo , Animales , Exocitosis , Humanos , RatasRESUMEN
Glioblastoma, the most common and aggressive type of brain tumors, has devastatingly proliferative and invasive characteristics. The need for finding a novel and specific drug target is urgent as the current approaches have limited therapeutic effects in treating glioblastoma. Xyloketal B is a marine compound obtained from mangrove fungus Xylaria sp. (No. 2508) from the South China Sea, and has displayed antioxidant activity and protective effects on endothelial and neuronal oxidative injuries. In this study, we used a glioblastoma U251 cell line to (1) explore the effects of xyloketal B on cell viability, proliferation, and migration; and (2) investigate the underlying molecular mechanisms and signaling pathways. MTT assay, colony formation, wound healing, western blot, and patch clamp techniques were employed. We found that xyloketal B reduced cell viability, proliferation, and migration of U251 cells. In addition, xyloketal B decreased p-Akt and p-ERK1/2 protein expressions. Furthermore, xyloketal B blocked TRPM7 currents in HEK-293 cells overexpressing TRPM7. These effects were confirmed by using a TRPM7 inhibitor, carvacrol, in a parallel experiment. Our findings indicate that TRPM7-regulated PI3K/Akt and MEK/ERK signaling is involved in anti-proliferation and migration effects of xyloketal B on U251 cells, providing in vitro evidence for the marine compound xyloketal B to be a potential drug for treating glioblastoma.
Asunto(s)
Antineoplásicos/farmacología , Regulación hacia Abajo/efectos de los fármacos , Glioblastoma/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piranos/farmacología , Transducción de Señal/efectos de los fármacos , Canales Catiónicos TRPM/antagonistas & inhibidores , Organismos Acuáticos/química , Organismos Acuáticos/crecimiento & desarrollo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , China , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Océano Pacífico , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Canales Catiónicos TRPM/metabolismo , Humedales , Xylariales/química , Xylariales/crecimiento & desarrolloRESUMEN
Survivin has been widely reported to play a role in diagnosis and prognosis of bladder cancer patients. However, published data on this subject are heterogeneous. Here, we conducted a meta-analysis to obtain a complete evaluation of the association between survivin and recurrence-free survival (RFS), disease-specific survival (DSS), overall survival (OS), and odds ratio (OR) in bladder cancer patients. Published studies on this subject were selected for further assessment by online articles in PubMed, MEDLINE, EMBASE, and OVID databases. Pooled hazard ratios (HR) with 95 % confidence interval (95 % CI) were estimated. Funnel plots were used to evaluate the publication bias. As well, heterogeneity and sensitivity were analyzed. In this meta-analysis, we included 13 studies with the total number of 1,963 patients. Positive survivin expression in bladder cancer was associated with a poor RFS (HR, 1.831; 95 % CI, 1.344-2.49), DSS (HR, 1.721; 95 % CI, 1.477-2.004), or OS (HR, 1.753; 95 % CI, 1.092-2.816) in patients. In addition, a significant association between expression of survivin and age (OR, 0.641; 95 % CI, 0.416-0.987) as well as stage (OR, 0.37; 95 % CI, 0.190-0.750) was revealed. Heterogeneity was observed among the included studies with RFS (x (2) =29.58, p = 0.009, I (2) = 52.7 %), OS (x (2) = 15.67, p = 0.008, I (2) = 68.1 %), and stage (x (2) = 11.97, p = 0.035, I (2) = 58.2 %). There was no publication bias according to Begg's and Egger's tests except for studies with gender. Furthermore, sensitivity analysis obtained the source of heterogeneity and confirmed opposite results of some studies. This study suggests that expression of survivin indicates poor prognosis in older patients and muscle invasive or advanced stage in bladder cancer. Survivin expression could be used in identifying a subgroup of patients with potential to benefit from a targeted therapy against survivin.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas Inhibidoras de la Apoptosis/genética , Pronóstico , Neoplasias de la Vejiga Urinaria/genética , Anciano , Biomarcadores de Tumor , Supervivencia sin Enfermedad , Femenino , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Masculino , Persona de Mediana Edad , Survivin , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Neonatal hypoxic-ischemic encephalopathy causes neurodegeneration and brain injury, leading to sensorimotor dysfunction. Xyloketal B is a novel marine compound isolated from a mangrove fungus Xylaria species (no. 2508) with unique antioxidant effects. In this study, we investigated the effects and mechanism of xyloketal B on oxygen-glucose deprivation-induced neuronal cell death in mouse primary cortical culture and on hypoxic-ischemic brain injury in neonatal mice in vivo. We found that xyloketal B reduced anoxia-induced neuronal cell death in vitro, as well as infarct volume in neonatal hypoxic-ischemic brain injury model in vivo. Furthermore, xyloketal B improved functional behavioral recovery of the animals following hypoxic-ischemic insult. In addition, xyloketal B significantly decreased calcium entry, reduced the number of TUNEL-positive cells, reduced the levels of cleaved caspase-3 and Bax proteins, and increased the level of Bcl-2 protein after the hypoxic-ischemic injury. Our findings indicate that xyloketal B is effective in models of hypoxia-ischemia and thus has potential as a treatment for hypoxic-ischemic brain injury.
Asunto(s)
Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Fármacos Neuroprotectores/uso terapéutico , Piranos/uso terapéutico , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Encéfalo/citología , Encéfalo/efectos de los fármacos , Química Encefálica/efectos de los fármacos , Caspasa 3/análisis , Muerte Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Ratones , Estructura Molecular , Fármacos Neuroprotectores/química , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Piranos/química , Proteína X Asociada a bcl-2/análisisRESUMEN
Transient receptor potential melastatin 7 (TRPM7) is a ubiquitously expressed divalent cation channel that plays a key role in cell functions such as ion homeostasis, cell proliferation, survival, and cytoskeletal dynamics and mediates cells death in hypoxic and ischemic conditions. Previously, TRPM7 was found to play a role in the neurite outgrowth and maturation of primary hippocampal neurons. Either knockdown of TRPM7 with target-specific shRNA or blocking channel conductance by a specific blocker waixenicin A enhanced axonal outgrowth in the primary neuronal culture. In this study, we investigated whether and how TPRM7 is involved in hypoxia-altered neurite outgrowth patterns in E16 hippocampal neuron cultures. We demonstrate that short-term hypoxia activated the MEK/ERK and PI3K/Akt pathways, reduced TRPM7 activity, and enhanced axonal outgrowth of neuronal cultures. On the other hand, long-term hypoxia caused a progressive retraction of axons and dendrites that could be attenuated by the TRPM7-specific inhibitor waixenicin A. Further, we demonstrate that in the presence of astrocytes, axonal retraction in long-term hypoxic conditions was enhanced, and TRPM7 block by waixenicin A prevented this retraction. Our data demonstrate the effect of hypoxia on TRPM7 activity and axonal outgrowth/retraction in cultures with or without astrocytes present.
Asunto(s)
Proteínas Serina-Treonina Quinasas , Canales Catiónicos TRPM , Hipoxia , Proyección Neuronal , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Canales Catiónicos TRPM/metabolismoRESUMEN
Ryanodine receptors (RyR) located on the membrane of the endoplasmic reticulum (ER), are a potent regulator of intracellular calcium levels upon activation. Dysregulated Ca2+ homeostasis is characteristic of hypoxic-ischemic (HI) brain injury and ultimately leads to neurodegeneration. RyRs have thereby been implicated in the Ca2+ imbalance that occurs during and after HI. In this study, we investigated the effects of RyR antagonist, dantrolene, on HI brain injury in neonatal mice. We found that administration of dantrolene (i.p.) on postnatal day 7 mice reduced the infarction volume and morphological damage induced by HI, and improved functional recovery as assessed by neurobehavioral testing. The neuroprotective effect of dantrolene was further demonstrated in neuronal cell culture in vitro, where dantrolene significantly reduced oxygen-glucose deprivation (OGD)-induced cell death. Fura-2 calcium imaging confirmed that dantrolene reduced the intracellular calcium level in cultured cortical neurons in vitro. Finally, Western blot analysis showed that dantrolene treatment reduced cleaved caspase-3 and -9 apoptotic proteins, and elevated pro-survival protein kinase C (PKC) protein levels. Taken together, our results demonstrate that dantrolene exerts neuroprotective effects against neonatal HI brain injury. This suggests that RyRs play a role in mediating the ionic imbalance induced by HI and therefore represent a potential target for drug development.
Asunto(s)
Lesiones Encefálicas , Bloqueadores de los Canales de Calcio , Dantroleno , Hipoxia-Isquemia Encefálica , Fármacos Neuroprotectores , Animales , Animales Recién Nacidos , Lesiones Encefálicas/tratamiento farmacológico , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/uso terapéutico , Dantroleno/uso terapéutico , Homeostasis , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Hipoxia-Isquemia Encefálica/metabolismo , Ratones , Fármacos Neuroprotectores/uso terapéutico , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
Transient receptor potential melastatin 7 (TRPM7), a calcium-permeable, ubiquitously expressed ion channel, is critical for axonal development, and mediates hypoxic and ischemic neuronal cell death in vitro and in vivo. However, the downstream mechanisms underlying the TRPM7-mediated processes in physiology and pathophysiology remain unclear. In this study, we employed a mouse model of hypoxic-ischemic brain cell death which mimics the pathophysiology of hypoxic-ischemic encephalopathy (HIE). HIE is a major public health issue and an important cause of neonatal deaths worldwide; however, the available treatments for HIE remain limited. Its survivors face life-long neurological challenges including mental retardation, cerebral palsy, epilepsy and seizure disorders, motor impairments, and visual and auditory impairments. Through a proteomic analysis, we identified calcium/calmodulin-dependent protein kinase II (CaMKII) and phosphatase calcineurin as potential mediators of cell death downstream from TRPM7 activation. Further analysis revealed that TRPM7 mediates cell death through CaMKII, calmodulin, calcineurin, p38, and cofilin cascade. In vivo, we found a significant reduction of brain injury and improvement of short- and long-term functional outcomes after HI after administration of specific TRPM7 blocker waixenicin A. Our data demonstrate a molecular mechanism of TRPM7-mediated cell death and identifies TRPM7 as a promising therapeutic and drug development target for HIE.
Asunto(s)
Calcineurina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Muerte Celular/fisiología , Hipoxia-Isquemia Encefálica/metabolismo , Neuronas/metabolismo , Canales Catiónicos TRPM/metabolismo , Acetatos/farmacología , Animales , Animales Recién Nacidos , Reacción de Prevención/fisiología , Muerte Celular/efectos de los fármacos , Células Cultivadas , Diterpenos/farmacología , Femenino , Células HEK293 , Humanos , Hipoxia-Isquemia Encefálica/patología , Masculino , Ratones , Neuronas/efectos de los fármacos , Neuronas/patologíaRESUMEN
In infants and children, neonatal hypoxic-ischemic (HI) brain injury represents a major cause of chronic neurological morbidity. The transient receptor potential melastatin 2 (TRPM2), a non-selective cation channel that conducts calcium, can mediate neuronal death following HI brain injury. An important endogenous activator of TRPM2 is H2O2, which has previously been reported to be upregulated in the neonatal brain after hypoxic ischemic injury. Here, incorporating both in vitro (H2O2-induced neuronal cell death model) and in vivo (mouse HI brain injury model) approaches, we examined the effects of AG490, which can inhibit the H2O2-induced TRPM2 channel. We found that AG490 elicited neuroprotective effects. We confirmed that AG490 reduced H2O2-induced TRPM2 currents. Specifically, application of AG490 to neurons ameliorated H2O2-induced cell injury in vitro. In addition, AG490 administration reduced brain damage and improved neurobehavioral performance following HI brain injury in vivo. The neuroprotective benefits of AG490 suggest that pharmacological inhibition of H2O2-activated TRPM2 currents can be exploited as a potential therapeutic strategy to treat HI-induced neurological complications.
Asunto(s)
Hipoxia-Isquemia Encefálica/metabolismo , Hipoxia-Isquemia Encefálica/prevención & control , Fármacos Neuroprotectores/uso terapéutico , Canales Catiónicos TRPM/metabolismo , Tirfostinos/uso terapéutico , Animales , Animales Recién Nacidos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Células HEK293 , Humanos , Hipoxia-Isquemia Encefálica/inducido químicamente , Ratones , Fármacos Neuroprotectores/farmacología , Oxidantes/toxicidad , Distribución Aleatoria , Canales Catiónicos TRPM/antagonistas & inhibidores , Tirfostinos/farmacologíaRESUMEN
Williams syndrome (WS) and 7q11.23 duplication syndrome (Dup7q11.23) are neurodevelopmental disorders caused by the deletion and duplication, respectively, of ~ 25 protein-coding genes on chromosome 7q11.23. The general transcription factor 2I (GTF2I, protein TFII-I) is one of these proteins and has been implicated in the neurodevelopmental phenotypes of WS and Dup7q11.23. Here, we investigated the effect of copy number alterations in Gtf2i on neuronal maturation and intracellular calcium entry mechanisms known to be associated with this process. Mice with a single copy of Gtf2i (Gtf2i+/Del) had increased axonal outgrowth and increased TRPC3-mediated calcium entry upon carbachol stimulation. In contrast, mice with 3 copies of Gtf2i (Gtf2i+/Dup) had decreases in axon outgrowth and in TRPC3-mediated calcium entry. The underlying mechanism was that TFII-I did not affect TRPC3 protein expression, while it regulated TRPC3 membrane translocation. Together, our results provide novel functional insight into the cellular mechanisms that underlie neuronal maturation in the context of the 7q11.23 disorders.
Asunto(s)
Neuronas/metabolismo , Canales Catiónicos TRPC/metabolismo , Factores de Transcripción TFII/metabolismo , Animales , Axones/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Aberraciones Cromosómicas , Modelos Animales de Enfermedad , Ratones , Neuritas/metabolismo , Fenotipo , Factores de TiempoRESUMEN
Glioblastoma (GBM), the most common and aggressive brain tumor in the central nervous system, remains a lethal diagnosis with a median survival of < 15 months. Aberrant expression of the TRPM7 channel has been linked to GBM functions. In this study, using the human GBM cell line U87, we evaluated the TRPM7 activator naltriben on GBM viability, migration, and invasiveness. First, using the whole-cell patch-clamp technique, we showed that naltriben enhanced the endogenous TRPM7-like current in U87 cells. In addition, with Fura-2 Ca2+ imaging, we observed robust Ca2+ influx following naltriben application. Naltriben significantly enhanced U87 cell migration and invasion (assessed with scratch wound assays, Matrigel invasion experiments, and MMP-2 protein expression), but not viability and proliferation (evaluated with MTT assays). Using Western immunoblots, we also detected the protein levels of p-Akt/t-Akt, and p-ERK1|2/t-ERK1|2. We found that naltriben enhanced the MAPK/ERK signaling pathway, but not the PI3k/Akt pathway. Therefore, potentiated TRPM7 activity contributes to the devastating migratory and invasive characteristics of GBM.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Naltrexona/análogos & derivados , Proteínas Serina-Treonina Quinasas/metabolismo , Canales Catiónicos TRPM/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Calcio/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Immunoblotting , Metaloproteinasa 2 de la Matriz/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Naltrexona/farmacología , Invasividad Neoplásica , Técnicas de Placa-Clamp , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
AIMS: Glycogen synthase kinase 3ß (GSK-3ß) is activated following hypoxic-ischemic (HI) brain injury. TDZD-8 is a specific GSK-3ß inhibitor. Currently, the impact of inhibiting GSK-3ß in neonatal HI injury is unknown. We aimed to investigate the effect of TDZD-8 following neonatal HI brain injury. METHODS: Unilateral common carotid artery ligation followed by hypoxia was used to induce HI injury in postnatal day 7 mouse pups pretreated with TDZD-8 or vehicle. The infarct volume, whole-brain imaging, Nissl staining, and behavioral tests were used to evaluate the protective effect of TDZD-8 on the neonatal brain and assess functional recovery after injury. Western blot was used to evaluate protein levels of phosphorylated protein kinase B (Akt), GSK-3ß, and cleaved caspase-3. Protein levels of cleaved caspase-3, neuronal marker, and glial fibrillary acidic protein were detected through immunohistochemistry. RESULTS: Pretreatment with TDZD-8 significantly reduced brain damage and improved neurobehavioral outcomes following HI injury. TDZD-8 reversed the reduction of phosphorylated Akt and GSK-3ß, and the activation of caspase-3 induced by hypoxia-ischemia. In addition, TDZD-8 suppressed apoptotic cell death and reduced reactive astrogliosis. CONCLUSION: TDZD-8 has the therapeutic potential for hypoxic-ischemic brain injury in neonates. The neuroprotective effect of TDZD-8 appears to be mediated through its antiapoptotic activity and by reducing astrogliosis.
Asunto(s)
Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Tiadiazoles/farmacología , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Astrocitos/efectos de los fármacos , Astrocitos/enzimología , Astrocitos/patología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Caspasa 3/metabolismo , Proteínas de Unión al ADN , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Proteína Ácida Fibrilar de la Glía/metabolismo , Gliosis/tratamiento farmacológico , Gliosis/metabolismo , Gliosis/patología , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Hipoxia-Isquemia Encefálica/metabolismo , Hipoxia-Isquemia Encefálica/patología , Ratones , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Distribución AleatoriaRESUMEN
Transient receptor potential melastatin 2 (TRPM2), a calcium-permeable non-selective cation channel, is reported to mediate brain damage following ischemic insults in adult mice. However, the role of TRPM2 channels in neonatal hypoxic-ischemic brain injury remains unknown. We hypothesize that TRPM2+/- and TRPM2-/- neonatal mice have reduced hypoxic-ischemic brain injury. To study the effect of TRPM2 on neonatal brain damage, we used 2,3,5-triphenyltetrazolium chloride (TTC) staining to assess the infarct volume and whole brain imaging to assess morphological changes in the brain. In addition, we also evaluated neurobehavioral outcomes for sensorimotor function 7days following hypoxic-ischemic brain injury. We report that the infarct volumes were significantly smaller and behavioral outcomes were improved in both TRPM2+/- and TRPM2-/- mice compared to that of wildtype mice. Next, we found that TRPM2-null mice showed reduced dephosphorylation of GSK-3ß following hypoxic ischemic injury unlike sham mice. TRPM2+/- and TRPM2-/- mice also had reduced activation of astrocytes and microglia in ipsilateral hemispheres, compared to wildtype mice. These findings suggest that TRPM2 channels play an essential role in mediating hypoxic-ischemic brain injury in neonatal mice. Genetically eliminating TRPM2 channels can provide neuroprotection against hypoxic-ischemic brain injury and this effect is elicited in part through regulation of GSK-3ß.
Asunto(s)
Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Hipoxia-Isquemia Encefálica/metabolismo , Canales Catiónicos TRPM/metabolismo , Análisis de Varianza , Animales , Animales Recién Nacidos , Reacción de Prevención/fisiología , Citocinas/genética , Citocinas/metabolismo , Embrión de Mamíferos , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Hipoxia-Isquemia Encefálica/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Proteína Oncogénica v-akt/genética , Proteína Oncogénica v-akt/metabolismo , ARN Mensajero/metabolismo , Reflejo/genética , Transducción de Señal/genética , Canales Catiónicos TRPM/genéticaRESUMEN
Transient receptor potential melastatin 7 (TRPM7) is a calcium-permeable divalent cation channel and mediates neuronal cell death under ischemic stresses. In this study, we investigated the contribution of TRPM7 to neuronal development in mouse primary hippocampal neurons. We demonstrated that TRPM7 channels are highly expressed in the tips of the growth cone. Either knockdown of TRPM7 with target-specific shRNA or blocking channel conductance by a specific blocker waixenicin A enhanced axonal outgrowth in culture. Blocking TRPM7 activity by waixenicin A reduced calcium influx and accelerated the polarization of the hippocampal neurons as characterized by the development of distinct axons and dendrites. Furthermore, TRPM7 coprecipitated and colocalized with F-actin and α-actinin-1 at the growth cone. We conclude that calcium influx through TRPM7 inhibits axonal outgrowth and maturation by regulating the F-actin and α-actinin-1 protein complex. Inhibition of TRPM7 channel promotes axonal outgrowth, suggesting its therapeutic potential in neurodegenerative disorders.
Asunto(s)
Axones/fisiología , Hipocampo/crecimiento & desarrollo , Neurogénesis/fisiología , Neuronas/fisiología , Canales Catiónicos TRPM/fisiología , Acetatos/farmacología , Animales , Axones/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Células Cultivadas , Diterpenos/farmacología , Relación Dosis-Respuesta a Droga , Hipocampo/citología , Hipocampo/efectos de los fármacos , Ratones , Neurogénesis/efectos de los fármacos , Neuronas/efectos de los fármacos , Canales Catiónicos TRPM/antagonistas & inhibidoresRESUMEN
The postsynaptic density-95 inhibitor NA-1 uncouples NMDA glutamate receptors from downstream neurotoxic signaling pathways without affecting normal glutamate receptor function. NA-1 attenuates NMDA receptor-mediated neuronal cell death after stroke in multiple models and species. However, its efficacy in providing neuroprotection in models of neonatal hypoxic-ischemic brain injury has not yet been tested. In this study, a modified version of the Rice-Vannucci method for the induction of neonatal hypoxic-ischemic brain injury was performed on postnatal day 7 mouse pups. Animals received a single dose of NA-1 intraperitoneally either before or after right common carotid artery occlusion. All experiments were performed in a blinded manner. Infarct volumes were measured 1 and 7 days after the injury, while behavioral tests were conducted 1, 3, and 7 days after injury. Administration of NA-1 before right common carotid artery occlusion or immediately after ischemia significantly reduced infarct volume and improved neurobehavioral outcomes 1, 3, and 7 days post-injury. The neuroprotection and improvement in neurobehavioral outcomes conferred by NA-1 in this mouse neonatal hypoxic-ischemic injury model imply that NA-1 will be effective in reducing neonatal stroke damage and thus could potentially serve as a therapeutic drug for prevention or treatment of neonatal stroke.
Asunto(s)
Homólogo 4 de la Proteína Discs Large/antagonistas & inhibidores , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Fármacos Neuroprotectores/uso terapéutico , Péptidos/uso terapéutico , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Caspasa 3/metabolismo , Supervivencia Celular/efectos de los fármacos , Homólogo 4 de la Proteína Discs Large/metabolismo , Hipoxia-Isquemia Encefálica/patología , Ratones , Neuroprotección/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Tamaño de los Órganos/efectos de los fármacos , Péptidos/farmacología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Sulfonylureas are ATP-sensitive potassium (KATP) channel blockers commonly used in the treatment of type 2 diabetes mellitus (T2DM). Activation of KATP channels plays a neuroprotective role in ischemia; thus, whether sulfonylureas affect the outcomes of stroke in patients with T2DM needs to be further studied. In our study, streptozotocin (STZ)-induced diabetic mice subjected to transient middle cerebral artery occlusion (MCAO) showed larger areas of brain damage and poorer behavioral outcomes. Blocking the KATP channel by tolbutamide increased neuronal injury induced by oxygen-glucose deprivation (OGD) in vitro and permanent MCAO (pMCAO) in vivo. Activating the KATP channel by diazoxide reduced the effects of both the OGD and pMCAO. Western blot analysis in STZ mouse brains indicated an early increase in protein levels of N-methyl-d-aspartate receptor 2B and postsynaptic density protein-95, followed by a decrease in phosphorylation of glycogen synthase kinase 3ß. Our systematic meta-analysis indicated that patients with T2DM treated with sulfonylureas had a higher odds ratio for stroke morbidity than those who received comparator drugs. Taken together, these results suggest that sulfonylurea treatment in patients with T2DM may inhibit the neuroprotective effects of KATP channels and increase the risk of stroke.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Canales KATP/metabolismo , Compuestos de Sulfonilurea/efectos adversos , Compuestos de Sulfonilurea/uso terapéutico , Animales , Western Blotting , Células Cultivadas , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Homólogo 4 de la Proteína Discs Large , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Guanilato-Quinasas/metabolismo , Inmunohistoquímica , Canales KATP/antagonistas & inhibidores , Masculino , Proteínas de la Membrana/metabolismo , Metaanálisis como Asunto , Ratones , Ratones Endogámicos C57BL , Fosforilación/efectos de los fármacos , Ensayos Clínicos Controlados Aleatorios como Asunto , Receptores de N-Metil-D-Aspartato/metabolismo , Factores de Riesgo , Accidente Cerebrovascular/metabolismo , Tolbutamida/farmacologíaRESUMEN
Glioblastomas are progressive brain tumors with devastating proliferative and invasive characteristics. Ion channels are the second largest target class for drug development. In this study, we investigated the effects of the TRPM7 inhibitor carvacrol on the viability, resistance to apoptosis, migration, and invasiveness of the human U87 glioblastoma cell line.The expression levels of TRPM7 mRNA and protein in U87 cells were detected by RT-PCR, western blotting and immunofluorescence. TRPM7 currents were recorded using whole-cell patch-clamp techniques. An MTT assay was used to assess cell viability and proliferation. Wound healing and transwell experiments were used to evaluate cell migration and invasion. Protein levels of p-Akt/t-Akt, p-ERK1/2/t-ERK1/2, cleaved caspase-3, MMP-2 and phosphorylated cofilin were also detected.TRPM7 mRNA and protein expression in U87 cells is higher than in normal human astrocytes. Whole-cell patch-clamp recording showed that carvacrol blocks recombinant TRPM7 current in HEK293 cells and endogenous TRPM7-like current in U87 cells. Carvacrol treatment reduced the viability, migration and invasion of U87 cells. Carvacrol also decreased MMP-2 protein expression and promoted the phosphorylation of cofilin. Furthermore, carvacrol inhibited the Ras/MEK/MAPK and PI3K/Akt signaling pathways.Therefore, carvacrol may have therapeutic potential for the treatment of glioblastomas through its inhibition of TRPM7 channels.