Modulation of an early step in the secretory machinery in hippocampal nerve terminals

In hippocampal neurons, neurotransmitter release can be regulated by protein kinase A (PKA) through a direct action on the secretory machinery. To identify the site of PKA modulation, we have taken advantage of the ability of the neurotoxin Botulinum A to cleave the synaptic protein SNAP-25. Cleavage of this protein decreases the Ca2+ responsiveness of the secretory machinery by partially uncoupling Ca2+-sensing from fusion per se. This is expressed as a shift toward higher Ca2+ levels of the Ca2+ to neurotransmitter release relationship and as a perturbation of synaptic delay under conditions where secretion induced by the Ca2+-independent secretagogue ruthenium red is unimpaired. We find that SNAP-25 cleavage also perturbs PKA-dependent modulation of secretion; facilitation of ruthenium red-evoked neurotransmitter release by the adenylyl cyclase activator forskolin is blocked completely after Botulinum toxin A action. Together with our observation that forskolin modifies the Ca2+ to neurotransmitter release relationship, our results suggest that SNAP-25 acts as a functional linker between Ca2+ detection and fusion and that PKA modulates an early step in the secretory machinery related to calcium sensing to facilitate synaptic transmission.

Disruption of syntaxin-mediated protein interactions blocks neurotransmitter secretion

The membrane protein syntaxin participates in several protein–protein interactions that have been implicated in neurotransmitter release. To probe the physiological importance of these interactions, we microinjected into the squid giant presynaptic terminal botulinum toxin C1, which cleaves syntaxin, and the H3 domain of syntaxin, which mediates binding to other proteins. Both reagents inhibited synaptic transmission yet did not affect the number or distribution of synaptic vesicles at the presynaptic active zone. Recombinant H3 domain inhibited the interactions between syntaxin and SNAP-25 that underlie the formation of stable SNARE complexes in vitro. These data support the notion that syntaxin-mediated SNARE complexes are necessary for docked synaptic vesicles to fuse.

Synaptic sprouting increases the uptake capacities of motoneurons in amyotrophic lateral sclerosis mice

Using adenoviruses encoding reporter genes as retrograde tracers, we assessed the capacity of motoneurons to take up and retrogradely transport adenoviral particles injected into the muscles of transgenic mice expressing the G93A human superoxide dismutase mutation, a model of amyotrophic lateral sclerosis. Surprisingly, transgene expression in the motoneurons was significantly higher in symptomatic mice than in control or presymptomatic mice. Using botulinum toxin to induce nerve sprouting at neuromuscular junctions, we showed that the unexpectedly high level of motoneurons retrograde transduction results, at least in part, from newly acquired uptake properties of the sprouts. These findings demonstrate the remarkable uptake properties of amyotrophic lateral sclerosis motoneurons in response to denervation and the rationale of using intramuscular injections of adenoviruses to overexpress therapeutic proteins in motor neuron diseases.

Bexiga hiperactiva e utilização de toxina botulínica na hiperactividade do detrusor : resultados obtidos pelo Serviço de Urologia do HSM-CHLN

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

BDNF-TrkB Signaling in Single-Spine Structural Plasticity

Multiple lines of evidence reveal that activation of the tropomyosin related kinase B (TrkB) receptor is a critical molecular mechanism underlying status epilepticus (SE) induced epilepsy development. However, the cellular consequences of such signaling remain unknown. To this point, localization of SE-induced TrkB activation to CA1 apical dendritic spines provides an anatomic clue pointing to Schaffer collateral-CA1 synaptic plasticity as one potential cellular consequence of TrkB activation. Here, we combine two-photon glutamate uncaging with two photon fluorescence lifetime imaging microscopy (2pFLIM) of fluorescence resonance energy transfer (FRET)-based sensors to specifically investigate the roles of TrkB and its canonical ligand brain derived neurotrophic factor (BDNF) in dendritic spine structural plasticity (sLTP) of CA1 pyramidal neurons in cultured hippocampal slices of rodents. To begin, we demonstrate a critical role for post-synaptic TrkB and post-synaptic BDNF in sLTP. Building on these findings, we develop a novel FRET-based sensor for TrkB activation that can report both BDNF and non-BDNF activation in a specific and reversible manner. Using this sensor, we monitor the spatiotemporal dynamics of TrkB activity during single-spine sLTP. In response to glutamate uncaging, we report a rapid (onset less than 1 minute) and sustained (lasting at least 20 minutes) activation of TrkB in the stimulated spine that depends on N-methyl-D-aspartate receptor (NMDAR)-Ca2+/Calmodulin dependent kinase II (CaMKII) signaling as well as post-synaptically synthesized BDNF. Consistent with these findings, we also demonstrate rapid, glutamate uncaging-evoked, time-locked release of BDNF from single dendritic spines using BDNF fused to superecliptic pHluorin (SEP). Finally, to elucidate the molecular mechanisms by which TrkB activation leads to sLTP, we examined the dependence of Rho GTPase activity - known mediators of sLTP - on BDNF-TrkB signaling. Through the use of previously described FRET-based sensors, we find that the activities of ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) require BDNF-TrkB signaling. Taken together, these findings reveal a spine-autonomous, autocrine signaling mechanism involving NMDAR-CaMKII dependent BDNF release from stimulated dendritic spines leading to TrkB activation and subsequent activation of the downstream molecules Rac1 and Cdc42 in these same spines that proves critical for sLTP. In conclusion, these results highlight structural plasticity as one cellular consequence of CA1 dendritic spine TrkB activation that may potentially contribute to larger, circuit-level changes underlying SE-induced epilepsy.

Tooth Mobility, Periodontal Ligament Space, and the Alveolus During Periodontal Health, Disease, and Disuse

Thesis (Ph.D.)--University of Washington, 2016-07

OnabotulinumtoxinA for hemicrania continua: open label experience in 9 patients

BACKGROUND: Hemicrania continua is a strictly unilateral, continuous headache, typically mild to moderate in severity, with severe exacerbations commonly accompanied by cranial autonomic features and migrainous symptoms. It is exquisitely responsive to Indomethacin. However, some patients cannot tolerate treatment, often due to gastrointestinal side effects. Therapeutic alternatives are limited and controlled evidence lacking. METHODS: We present our experience of nine patients treated with OnabotulinumtoxinA for hemicrania continua. All patients were injected using the PREEMPT (Phase 3 REsearch Evaluating Migraine Prophylaxis Therapy) protocol for migraine. RESULTS: Five of nine patients demonstrated a 50% or more reduction in moderate to severe headache days with OnabotulinumtoxinA with a median reduction in moderate to severe headache days of 80%. Patient estimate of response was 80% or more in five subjects. The median and mean duration of response in the five responders was 11 and 12 weeks (range 6-20 weeks). Improvements were also seen in headache-associated disability CONCLUSIONS: OnabotulinumtoxinA adds a potential option to the limited therapeutic alternatives available in hemicrania continua.

Miotomía extendida en el tratamiento de la acalasia

La acalasia es una enfermedad esofágica poco frecuente que se acompaña de una importante alteración de la calidad de vida de los pacientes. Su etiología no está totalmente aclarada y sus características clínicas principales son la disfagia y la regurgitación. El tratamiento de la acalasia está dirigido al alivio funcional y sintomático mediante la abertura del esfínter esofágico inferior, siendo al momento la miotomía laparoscópica la técnica de elección mientras que las dilataciones neumáticas y la inyección de toxina botulínica deben considerarse como técnicas de recurso en casos seleccionados. Objetivo: Evaluar los resultados de la miotomía extendida más funduplicatura parcial anterior de Dorr como tratamiento de la acalasia por vía laparoscópica, comparándola con nuestra experiencia previa mediante la técnica estándar. Materiales y método: diseño: Estudio prospectivo, descriptivo y longitudinal. Sede: Hospital Latino, Cuenca - Ecuador. Pacientes y método: Desde junio de 1992 hasta diciembre del 2011 se intervinieron 39 pacientes con diagnóstico de acalasia que recibieron tratamiento quirúrgico por medio de cirugía mínimamente invasiva. Se estudió la edad, sintomatología previa, clasificación según Stewart, tiempo de evolución de los síntomas, técnica operatoria realizada, control postoperatorio. Resultados: Se intervinieron 39 paciente, con edad promedio de 66 años, mínima 23 y máxima 81. La sintomatología presentada fue disfagia en el 100%, regurgitación en el 74,4%, pérdida de peso en el 71,8% y odinofagia en el 28.2%. El tiempo de evolución de los síntomas fueron: menor a 2 años 48.7% (n=19), de 2 a 4 años 33.3% (n=13), de 4 a 6 años de 12.8% (n=5), y de 6 a 8 años un 5.1% (n=2). Según Stewart se clasificaron en I 8% (n=3), II 49% (n=19), III 38% (n=15) y IV 5% (n=2).La técnica empleada fue Miotomía + Dorr 57% (n=22), Miotomía extendida + Dorr 20% (n=8), Miotomía sola 18% (n=7), Miotomía + Toupet 5% (n=2). Se ha realizado seguimiento del 75% de pacientes, con resultados excelentes en el 91%, y bueno en el 9%. En los ocho últimos casos se realizó la miotomía extendida más funduplicatura tipo Dorr, brindando resultados excelentes a corto plazo. Conclusión: la miotomía gástrica extendida mejora el resultado de la terapia quirúrgica para la acalasia sin incrementar la tasa de reflujo gastroesofágico anormal cuando se añade una funduplicatura parcial anterior tipo Dorr.

Vector analysis of porosity evidences bone loss at the epiphysis in the BTX rat model of disuse osteoporosis

International audience

Paciente bruxómano e as suas opções terapêuticas

O bruxismo caracteriza-se por um ato involuntário de apertar e/ou ranger os dentes sendo este hábito um fator de risco que pode comprometer a função do sistema estomatognático com várias sequelas associadas (dor dentária e muscular, desgaste dentário, etc.). Esta pesquisa bibliográfica pretende rever as possibilidades terapêuticas que se podem apresentar como soluções no controlo desta parafunção. A bibliografia ainda é inconclusiva sobre qual o melhor tratamento a seguir sugerindo uma abordagem multidisciplinar, tendo em conta a etiologia e a sintomatologia para a definição de um plano de tratamento mais completo e adequado. Têm surgido cada vez mais alternativas terapêuticas e cada uma delas, ao longo do tempo, tem desenvolvido novas metodologias mais eficazes no tratamento do bruxismo.

Efecto de los dispositivos de emisión de microondas, radiofrecuencia no ablativa y ultrasonido microfocalizado en el tratamiento de la hiperhidrosis primaria revisión sistemática de la literatura

Antecedentes y objetivos: La hiperhidrosis primaria afecta el 2,8% de la población de Estados Unidos. Condición que impacta el desarrollo social de los individuos afectados, ocasionando fobia social. Existen opciones disponibles para el tratamiento de la hiperhidrosis incluyendo medicamentos tópico, sistémico, inyectable y quirúrgico. El objetivo de ésta revisión sistemática de la literatura es determinar la efectividad y seguridad de los dispositivos de emisión de microondas, radiofrecuencia no ablativa y sistema de ultrasonido microfocalizado para el tratamiento de la hiperhidrosis primaria. Materiales y métodos: Se realizó una revisión sistemática de la literatura de artículos obtenidos de bases de datos: Medline, Cochrane, Embase, Ovid y Scielo. Se incluyeron ensayos clínicos aleatorizados controlados, ensayos cuasiexperimentales desde el 2011; donde evaluaran el uso de estos dispositivos en el manejo de hiperhidrosis primaria. Resultados: Se seleccionaron 21 artículos en total. Se encontró que con los tres dispositivos se logra una reducción significativa a puntajes entre 1 y 2 de la escala de Severidad de la Hiperhidrosis; en 3 estudios se encontró mejoría en la calidad de vida; los eventos adversos fueron transitorios, siendo más frecuentes con el dispositivo de emisión de microondas. Conclusión: Primera revisión sistemática de la literatura sobre el efecto de estos tres dispositivos en el manejo de hiperhidrosis. Se espera aportar a la literatura existente una recomendación acerca de la efectividad y seguridad de estos dispositivos para que sea aplicado en los pacientes con diagnóstico de hiperhidrosis primaria.

Genotoxicity investigation of a cyanobacterial toxin, cylindrospermopsin

Cylindrospermopsin (CYN), a potent cyanobacterial hepatotoxin produced by Cylindrospermopsis raciborskii and other cyanobacteria, is regularly found in water supplies in many parts of the world, and has been associated with the intoxication of humans and livestock. In this study, Balb/c mice were injected via the intraperitoneal (IP) route with a single dose of 0.2 mg/kg CYN. Animals were sacrificed at 6, 12, 24, 48 and 72 It. DNA was isolated from the mouse livers, and examined for strand breakage by alkaline gel electrophoresis (pH 12). Significant DNA strand breakage was observed in the mouse liver exposed to CYN, suggesting that induction of DNA strand breakage is probably one of the key mechanisms for CYN genotoxicity. (C) 2002 Elsevier Science Ltd. All rights reserved.

Phoneutria nigriventer spider toxin Tx2-6 causes priapism and death: A histopathological investigation in mice

Phoneutria nigriventer spider bite causes priapism, an effect attributed to the peptide toxins Tx2-5 and Tx2-6 and involving nitric oxide. Tx2-6 (MW = 5287) is known to delay the inactivation of Sodium channels in the same fashion as many other venom toxins. In the present study we evaluated the i.p. dose that induces priapism and the other symptoms in mice. Animals killed by the toxin or crude venom (0.85 mg/kg) were autopsied and a pathological study of brain, lung, kidney, liver and heart was undertaken using standard techniques. The same protocol was employed with animals injected with crude venom. Results showed that priapism is the first sign of intoxication, followed by piloerection, abundant salivation and tremors. An i.p. injection of about 0.3 mu g/kg induced only priapism with minimal side-effects. The most remarkable histological finding was a general vascular congestion in all organs studied. Penis showed no necrosis or damage. Lungs showed vascular congestion and alveolar hemorrhage. Heart showed also sub-endothelial hemorrhage. Brain showed only a mild edema and vascular congestion. Results obtained with crude venom closely resemble those of purified toxin. We conclude that Tx2-6 have profound effects on the vascular bed especially in lungs and heart, which may be the cause of death. Interestingly brain tissue was less affected and the observed edema may be attributed to respiratory impairment. To the best of our knowledge this is the first histopathological investigation on this toxin and venom suggesting a possible cause of death. (C) 2012 Elsevier Ltd. All rights reserved.

A study of the effect of ionizing radiation on resistance, germination, and toxin synthesis of Clostridium botulinum spores, types A, B, and E

At head of title: The University of Michigan, College of Engineering, Dept. of Chemical and Metallurgical Engineering. Final report.

Genotoxicity investigation of chlorinated degradation products of a cyanobacterial toxin, cylindrospermopsin

Cylindrospermopsin (CYN), a potent cyanobacterial hepatotoxin produced by Cylindrospermopsis raciborskii and other cyanobacteria, is regularly found in water supplies in many parts of the world and has been associated with the intoxication of humans and livestock.Water treatment via chlorination can degrade the toxin effectively but result in the production of several byproducts. In this study, male and female Balb/c mice were injected via the intraperitoneal (IP) route with a single dose of 10 mg/kg 5-chlorouracil and 10 mg/kg 5-chloro-6-hydroxymethyluracil; these two compounds are the predicted chlorinated degradation products of CYN.DNA was isolated from the mouse livers and examined for strand breakage by alkaline gel electrophoresis (pH 12). The median molecular length (MML) of the DNA distributed in the gel was determined by estimating the midpoint of the DNA size distribution by densitometry. The toxicity of 5-chlorouracil (as measured by DNA strand breakage) was significantly influenced by time from dosing. There was no significant difference in MML between mice dosed with 5-chloro-6-hydroxymethyluracil and the controls. In another experiment, mice were dosed with 0, 0.1, 1, 10 and 100 mg/kg body weight 5-chlorouracil and 0, 0.1, 1, 10 and 20 mg/kg 5-chloro-6-hydroxymethyluracil via IP injection. The heart, liver, kidney, lung and spleen were removed, fixed and examined under electron microscopy. Liver was the main target organ. The EM results revealed marked distortion on the nuclear membrane of liver cells in mice dosed with 1.0 mg/kg 5-chlorouracil or 10 mg/kg 5-chloro-6-hydroxymethyluracil, or higher.