RESUMEN
Sphingosine-1-phosphate (S1P) is an essential bioactive sphingosine lipid involved in many neurological disorders. Sphingosine kinase 1 (SphK1), a key enzyme for S1P production, is concentrated in presynaptic terminals. However, the role of S1P/SphK1 signaling in exocytosis remains elusive. By detecting catecholamine release from single vesicles in chromaffin cells, we show that a dominant negative SphK1 (SphK1DN ) reduces the number of amperometric spikes and increases the duration of foot, which reflects release through a fusion pore, implying critical roles for S1P in regulating the rate of exocytosis and fusion pore expansion. Similar phenotypes were observed in chromaffin cells obtained from SphK1 knockout mice compared to those from wild-type mice. In addition, extracellular S1P treatment increased the number of amperometric spikes, and this increase, in turn, was inhibited by a selective S1P3 receptor blocker, suggesting extracellular S1P may regulate the rate of exocytosis via activation of S1P3. Furthermore, intracellular S1P application induced a decrease in foot duration of amperometric spikes in control cells, indicating intracellular S1P may regulate fusion pore expansion during exocytosis. Taken together, our study represents the first demonstration that S1P regulates exocytosis through distinct mechanisms: extracellular S1P may modulate the rate of exocytosis via activation of S1P receptors while intracellular S1P may directly control fusion pore expansion during exocytosis. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.
Asunto(s)
Células Cromafines/metabolismo , Exocitosis/fisiología , Lisofosfolípidos/metabolismo , Esfingosina/análogos & derivados , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Esfingosina/metabolismoRESUMEN
The cochlear implant provides auditory cues to profoundly deaf patients by electrically stimulating the primary auditory neurons (ANs) of the cochlea. However, ANs degenerate in deafness; the preservation of a robust AN target population, in combination with advances in cochlear implant technology, may provide improved hearing outcomes for cochlear implant patients. The exogenous delivery of neurotrophins such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 is well known to support AN survival in deafness, and cell-based therapies provide a potential clinically viable option for delivering neurotrophins into the deaf cochlea. This study utilized cells that were genetically modified to express BDNF and encapsulated in alginate microspheres, and investigated AN survival in the deaf guinea pig following (a) cell-based neurotrophin treatment in conjunction with chronic electrical stimulation from a cochlear implant, and (b) long-term cell-based neurotrophin delivery. In comparison to deafened controls, there was significantly greater AN survival following the cell-based neurotrophin treatment, and there were ongoing survival effects for at least six months. In addition, functional benefits were observed following cell-based neurotrophin treatment and chronic electrical stimulation, with a statistically significant decrease in electrically evoked auditory brainstem response thresholds observed during the experimental period. This study demonstrates that cell-based therapies, in conjunction with a cochlear implant, shows potential as a clinically transferable means of providing neurotrophin treatment to support AN survival in deafness. This technology also has the potential to deliver other therapeutic agents, and to be used in conjunction with other biomedical devices for the treatment of a variety of neurodegenerative conditions.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/administración & dosificación , Tratamiento Basado en Trasplante de Células y Tejidos , Sistemas de Liberación de Medicamentos , Fibroblastos/metabolismo , Pérdida Auditiva Sensorineural/terapia , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Supervivencia Celular/efectos de los fármacos , Implantes Cocleares , Estimulación Eléctrica , Femenino , Cobayas , Pérdida Auditiva Sensorineural/cirugía , Masculino , Neuronas/efectos de los fármacos , RatasRESUMEN
Mitochondria are the primary site of cellular energy generation and reactive oxygen species (ROS) accumulation. Elevated ROS levels are detrimental to normal cell function and have been linked to the pathogenesis of neurodegenerative disorders such as Down's syndrome (DS) and Alzheimer's disease (AD). RCAN1 is abundantly expressed in the brain and overexpressed in brain of DS and AD patients. Data from nonmammalian species indicates that increased RCAN1 expression results in altered mitochondrial function and that RCAN1 may itself regulate neuronal ROS production. In this study, we have utilized mice overexpressing RCAN1 (RCAN1(ox)) and demonstrate an increased susceptibility of neurons from these mice to oxidative stress. Mitochondria from these mice are more numerous and smaller, indicative of mitochondrial dysfunction, and mitochondrial membrane potential is altered under conditions of oxidative stress. We also generated a PC12 cell line overexpressing RCAN1 (PC12(RCAN1)). Similar to RCAN1(ox) neurons, PC12(RCAN1) cells have an increased susceptibility to oxidative stress and produce more mitochondrial ROS. This study demonstrates that increasing RCAN1 expression alters mitochondrial function and increases the susceptibility of neurons to oxidative stress in mammalian cells. These findings further contribute to our understanding of RCAN1 and its potential role in the pathogenesis of neurodegenerative disorders such as AD and DS.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mitocondrias/metabolismo , Proteínas Musculares/metabolismo , Estrés Oxidativo , Animales , Supervivencia Celular/efectos de los fármacos , Proteínas de Unión al ADN , Femenino , Peróxido de Hidrógeno/toxicidad , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Modelos Biológicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Células PC12 , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Huntingtin-associated protein 1 (HAP1) was initially established as a neuronal binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking and cell signalling. In this study, we establish that HAP1 is important in several steps of exocytosis in adrenal chromaffin cells. Using carbon-fibre amperometry, we measured single vesicle exocytosis in chromaffin cells obtained from HAP1(-/-) and HAP1(+/+) littermate mice. Numbers of Ca(2+)-dependent and Ca(2+)-independent full fusion events in HAP1(-/-) cells are significantly decreased compared with those in HAP1(+/+) cells. We observed no change in the frequency of 'kiss-and-run' fusion events or in Ca(2+) entry. Whereas release per full fusion event is unchanged in HAP1(-/-) cells, early fusion pore duration is prolonged, as indicated by the increased duration of pre-spike foot signals. Kiss-and-run events have a shorter duration, indicating opposing roles for HAP1 in the stabilization of the fusion pore during full fusion and transient fusion, respectively. We use electron microscopy to demonstrate a reduction in the number of vesicles docked at the plasma membrane of HAP1(-/-) cells, where membrane capacitance measurements reveal the readily releasable pool of vesicles to be reduced in size. Our study therefore illustrates that HAP1 regulates exocytosis by influencing the morphological docking of vesicles at the plasma membrane, the ability of vesicles to be released rapidly upon stimulation, and the early stages of fusion pore formation.
Asunto(s)
Médula Suprarrenal/metabolismo , Membrana Celular/metabolismo , Células Cromafines/metabolismo , Exocitosis , Fusión de Membrana , Proteínas del Tejido Nervioso/metabolismo , Vesículas Secretoras/metabolismo , Animales , Calcio/metabolismo , Señalización del Calcio , Catecolaminas/metabolismo , Células Cultivadas , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Vías Secretoras , Factores de TiempoRESUMEN
We have previously shown that Regulator of Calcineurin 1 (RCAN1) regulates multiple stages of vesicle exocytosis. However, the mechanisms by which RCAN1 affects secretory vesicle exocytosis and quantal release kinetics remain unknown. Here, we use carbon fibre amperometry to detect exocytosis from chromaffin cells and identify these underlying mechanisms. We observe reduced exocytosis with repeated stimulations in chromaffin cells over-expressing RCAN1 (RCAN1(ox)), but not in wild-type (WT) cells, indicating a negative effect of RCAN1 on vesicle recycling and endocytosis. Acute exposure to calcineurin inhibitors, cyclosporine A and FK-506, replicates this effect in WT cells but has no additional effect in RCAN1(ox) cells. When we chronically expose WT cells to cyclosporine A and FK-506 we find that catecholamine release per vesicle and pre-spike foot (PSF) signal parameters are decreased, similar to that in RCAN1(ox) cells. Inhibiting calcineurin activity in RCAN1(ox) cells has no additional effect on the amount of catecholamine release per vesicle but further reduces PSF signal parameters. Although electron microscopy studies indicate these changes are not because of altered vesicle number or distribution in RCAN1(ox) cells, the smaller vesicle and dense core size we observe in RCAN1(ox) cells may underlie the reduced quantal release in these cells. Thus, our results indicate that RCAN1 most likely affects vesicle recycling and quantal release kinetics via the inhibition of calcineurin activity.
Asunto(s)
Calcineurina/metabolismo , Calcineurina/farmacocinética , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas Musculares/fisiología , Vesículas Secretoras/metabolismo , Animales , Inhibidores de la Calcineurina , Proteínas de Unión al Calcio , Células Cultivadas , Células Cromafines/citología , Células Cromafines/metabolismo , Células Cromafines/fisiología , Endocitosis/fisiología , Femenino , Cinética , Masculino , Ratones , Ratones Mutantes , Teoría Cuántica , Vesículas Secretoras/fisiologíaRESUMEN
RCAN1 is a chromosome 21 gene that controls secretion in endocrine cells, regulates mitochondrial function, and is sensitive to oxidative stress. Regulator of calcineurin 1 (RCAN1) is also an endogenous inhibitor of the protein phosphatase calcineurin, the inhibition of which leads to hypoinsulinemia and diabetes in humans and mice. However, the presence or the role of RCAN1 in insulin-secreting ß-cells and its potential role in the pathogenesis of diabetes is unknown. Hence, the aim of this study is to investigate the presence of RCAN1 in ß-cells and identify its role in ß-cell function. RCAN1 is expressed in mouse islets and in the cytosol of pancreatic ß-cells. We find RCAN1 is a glucose-responsive gene with a 1.5-fold increase in expression observed in pancreatic islets in response to chronic hyperglycemia. The overexpression of the human RCAN1.1 isoform in mice under the regulation of its endogenous promoter causes diabetes, age-associated hyperglycemia, reduced glucose tolerance, hypoinsulinemia, loss of ß-cells, reduced ß-cell insulin secretion, aberrant mitochondrial reactive oxygen species production, and the down-regulation of key ß-cell genes. Our data therefore identifies a novel molecular link between the overexpression of RCAN1 and ß-cell dysfunction. The glucose-responsive nature of RCAN1 provides a potential mechanism of action associated with the ß-cell dysfunction observed in diabetes.
Asunto(s)
Diabetes Mellitus/metabolismo , Intolerancia a la Glucosa/metabolismo , Hiperglucemia/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Musculares/metabolismo , Animales , Proteínas de Unión al Calcio , Diabetes Mellitus/genética , Diabetes Mellitus/patología , Intolerancia a la Glucosa/genética , Intolerancia a la Glucosa/patología , Hiperglucemia/genética , Hiperglucemia/patología , Secreción de Insulina , Células Secretoras de Insulina/patología , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Musculares/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
How fusion pore formation during exocytosis affects the subsequent release of vesicle contents remains incompletely understood. It is unclear if the amount released per vesicle is dependent upon the nature of the developing fusion pore and whether full fusion and transient kiss and run exocytosis are regulated by similar mechanisms. We hypothesise that if consistent relationships exist between these aspects of exocytosis then they will remain constant across any age. Using amperometry in mouse chromaffin cells we measured catecholamine efflux during single exocytotic events at P0, 1 month and 6 months. At all ages we observed full fusion (amperometric spike only), full fusion preceded by fusion pore flickering (pre-spike foot (PSF) signal followed by a spike) and pure "kiss and run" exocytosis (represented by stand alone foot (SAF) signals). We observe age-associated increases in the size of all 3 modes of fusion but these increases occur at different ages. The release probability of PSF signals or full spikes alone doesn't alter across any age in comparison with an age-dependent increase in the incidence of "kiss and run" type events. However, the most striking changes we observe are age-associated changes in the relationship between vesicle size and the membrane bending energy required for exocytosis. Our data illustrates that vesicle size does not regulate release probability, as has been suggested, that membrane elasticity or flexural rigidity change with age and that the mechanisms controlling full fusion may differ from those controlling "kiss and run" fusion.
Asunto(s)
Envejecimiento/fisiología , Células Cromafines/fisiología , Exocitosis/fisiología , Fusión de Membrana/fisiología , Animales , Calcio/metabolismo , Catecolaminas/metabolismo , Membrana Celular/metabolismo , Membrana Celular/fisiología , Células Cultivadas , Células Cromafines/metabolismo , Técnicas Electroquímicas/métodos , Cinética , Ratones , Ratones Endogámicos C57BL , Factores de Tiempo , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/fisiologíaRESUMEN
Genes located on chromosome 21, over-expressed in Down syndrome (DS) and Alzheimer's disease (AD) and which regulate vesicle trafficking, are strong candidates for involvement in AD neuropathology. Regulator of calcineurin activity 1 (RCAN1) is one such gene. We have generated mutant mice in which RCAN1 is either over-expressed (RCAN1(ox)) or ablated (Rcan1-/-) and examined whether exocytosis from chromaffin cells, a classic cellular model of neuronal exocytosis, is altered using carbon fibre amperometry. We find that Rcan1 regulates the number of vesicles undergoing exocytosis and the speed at which the vesicle fusion pore opens and closes. Cells from both Rcan1-/- and RCAN1(ox) mice display reduced levels of exocytosis. Changes in single-vesicle fusion kinetics are also evident resulting in the less catecholamine released per vesicle with increasing Rcan1 expression. Acute calcineurin inhibition did not replicate the effect of RCAN1 overexpression. These changes are not due to alterations in Ca2+ entry or the readily releasable vesicle pool size. Thus, we illustrate a novel regulator of vesicle exocytosis, Rcan1, which influences both exocytotic rate and vesicle fusion kinetics. If Rcan1 functions similarly in neurons then overexpression of this protein, as occurs in DS and AD brains, will reduce both the number of synaptic vesicles undergoing exocytosis and the amount of neurotransmitter released per fusion event. This has direct implications for the pathogenesis of these diseases as sufficient levels of neurotransmission are required for synaptic maintenance and the prevention of neurodegeneration and vesicle trafficking defects are the earliest hallmark of AD neuropathology.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Síndrome de Down/metabolismo , Exocitosis , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fusión de Membrana , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Animales , Calcineurina/metabolismo , Calcio/metabolismo , Células Cromafines/fisiología , Vesículas Citoplasmáticas/metabolismo , Proteínas de Unión al ADN , Humanos , Cinética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones TransgénicosRESUMEN
Arboviruses of the families Togaviridae and Flaviviridae are widely distributed and are important causative agents of viral encephalitis, a severe and often fatal disease. The only internationally available vaccine against these diseases is expensive and laborious to manufacture and difficult to administer. Therefore, new vaccines are required against these pathogens. This study investigates the use of a DNA-prime, orally delivered protein boost vaccination strategy against viral encephalitis. This vaccination strategy was immunogenic and provided partial protection against viral encephalitis in a murine model, demonstrating the possible applicability of this vaccination strategy for the management of endemic encephalitis.
Asunto(s)
Infecciones por Alphavirus/prevención & control , Encefalitis Viral/prevención & control , Virus Sindbis/inmunología , Vacunas/farmacología , Administración Oral , Infecciones por Alphavirus/inmunología , Animales , ADN Viral/inmunología , ADN Viral/farmacología , Modelos Animales de Enfermedad , Encefalitis Viral/inmunología , Femenino , Inmunización Secundaria , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Virus Sindbis/genéticaRESUMEN
Japanese encephalitis (JE) is a severe disease that is widespread throughout Asia and is spreading beyond its traditional boundaries. Three vaccines are currently in use against JE but only one is available internationally, a mouse-brain-derived inactivated vaccine first used in the 1930s. Although this vaccine has been effective in reducing the incidence of JE, it is relatively expensive and has been linked to severe allergic and neurological reactions. Cell-culture-derived inactivated and attenuated vaccines have been developed but are only used in the People's Republic of China. Other vaccines currently in various stages of development are DNA vaccines, a chimeric yellow fever-JE viral vaccine, virus-like particle vaccines and poxvirus-based vaccines. Poxvirus-based vaccines and the chimeric yellow fever-JE vaccine have been tested in Phase I clinical trials. These new vaccines have the potential to significantly reduce the impact of JE in Asia, particularly if used in an oral vaccine delivery strategy.