Dissertations / Theses on the topic 'Neuromuscular Junction – drug effects'

A sequential extraction technique was used to rapidly analyse AChE molecular form activity at the mouse neuromuscular junction and also in peripheral parts of muscle fibres. AChE in the synaptic cleft involved in the termination of cholinergic transmission was successfully assessed by the assay method and by an alternative method using a correlation equation which represented the relationship between synaptic AChE and the prolongation of extra-cellular miniature endplate potentials. It was found that inhibition after in vivo Carbamate (CB) dosing could not be maintained during tissue analysis because CB-inhibited enzyme complexes decarbamoylated vary rapidly and could not be prevented even when maintained on ice. The methods employed did not therefore give a measure of inhibition but presented a profile of metabolic responses to continual, low dose CB treatment. Repetitive and continual infusion with low doses of the CBs: pyridostigmine and physostigmine induced a variety of effects on mouse skeletal muscle. Both compounds induced a mild myopathy in the mouse diaphragm during continual infusion which was characterised by endplate deformation without necrosis; such deformation persisted on termination of treatment but had recovered slightly 14 days later. Endplate and non-endplate AChE molecular forms displayed selective responses to CB treatment. During treatment endplate AChE was reduced whereas non-endplate AChE was largely unaffected, and after treatment, endplate AChE recovered, whereas non-endplate AChE was up-regulated. The mechanisms by which these responses become manifest are unclear but may be due to CB-induced effects on nerve-mediated muscle activity, neurotrophic factors or morphological and physiological changes which arise at the neuromuscular junction. As well as inhibiting AChE, CBs also influence the metabolism and regulation of the enzyme and induce persistent endplate deformation.

The effects of organophosphorus compounds which form a rapidly-ageing complex with acetylcholinesterase (AChE) (e.g. pinacolyl S-(2- trimethylaminoethyl)methylphosphonothioate (BOS)) and hence exert a persistent anticholinesterase (anti-ChE) action have been compared with other compounds with a shorter time course of inhibition (e.g. ecothiopate iodide (ECO)). Although the inhibition of AChE produced by BOS lasted longer than that seen with ECO, the time course of the myopathy appeared very similar. BOS also possessed a number of properties which have been seen with other anti-ChEs. BOS and ECO produced significant increases in neuromuscular "jitter" 5 days after injection, not only in the diaphragm but also in the soleus and extensor digitorum longus muscles. Increases in "jitter" produced by ECO could be prevented by pyridostigmine prophylaxis or rapid treatment with pyridine-2- aldoxime methiodide. Some protection from the BOS-induced increases in "jitter" could be gained by repeated treatment with pyridine-2-aldoxime methiodide, an effect which could not be accounted for simply by enzyme reactivation. From experiments performed in Rej 129 mice it was determined that increases in "jitter", although demonstrated in some dystrophic muscles, could not be used as an early diagnostic tool. Because sequalae of inhibition were present some time after intoxication, by which time AChE appeared biochemically normal, experiments were performed to investigate inactivation of physiologically important AChE. The time course of extracellular MEPPs was utilised as an indicator of physiologically important AChE and compared with the AChE activity measured by the technique of Ellman et al. (1961). It was concluded that the degree of persistence of anti-ChE action was unimportant for the induction of myopathy with a time course of 3-24 hours, but had some importance in events of longer duration.

Glutamate is the most abundant excitatory neurotransmitter in the central nervous system. It is implicated in synaptic plasticity including learning and memory and long term potentiation, which are characterized by changes in the number and localization of glutamate receptors (GluRs) in the postsynaptic cell. We have found that proteins important for autophagy, a cellular process that degrades cellular components for reuse, affect the synaptic localization of GluRs at the Drosophila neuromuscular junction. Preliminary data indicated that mutations in atg1 and atg8a lead to a reduction in synaptic GluRs, suggesting that they either indirectly regulate GluR localization or signal independent of autophagy. The present study examined the effects of tissue specific overexpression or knockdown of atg8a to determine that atg8a is important either pre- or postsynaptically for proper GluR localization, and is possibly acting downstream of FOXO signaling. These results also indicate that atg8a may be acting independently of autophagy to determine GluR localization.

TGF-β signaling is particularly well studied for its role in early embryonic patterning and cell proliferation specific to cancer progression. At the larval neuromuscular junction (NMJ) of the model organism Drosophila melanogaster, several ligands from the TGF-β superfamily have been shown to promote synaptic growth and evoked neurotransmitter release. We focused on the retrograde bone morphogenic protein (BMP) signaling pathway and its potential role for the presynaptic expression of WD40, a potential cofactor of the E3 ubiquitin ligase Cullin 4 (Cul4) that is required for evoked neurotransmitter release. The goal of this project was to investigate whether this TGF-β signaling pathway regulates the expression of WD40. In conclusion, our data shows evidence that WD40 might a critical effector of retrograde BMP signaling at Drosophila NMJs.

Transforming growth factor-βs (TGF-βs) are highly expressed in neural development but why the adult nervous system continues to express them is unclear. TGF-β2 is concentrated at mature neuromuscular junctions (NMJs) of mammalian skeletal muscle fibres, and the nerve terminal expresses TβR-II receptors. To test the role of TGF-β2 at mammalian NMJs, I performed four experiments. The first study tested whether TGF-β2 acutely modulates synaptic transmission at mature mammalian NMJs. Second, I asked if chronically reduced TGF-β2 expression disrupts synaptic transmission. Third, I asked if TGF-β2’s effects differ in terminals adapted to different activity patterns in vivo. Lastly, I asked whether TGF-β1, a related peptide to TGF-β2, is distinct in terms of its effects on transmitter release. Using single electrode potential recording, I found TGF-β2 significantly increased the amplitude of spontaneous released single neurotransmitter vesicles (miniature endplate potentials, MEPPs) and nerve stimulation evoked multi-vesicular release (endplate potentials, EPPs). These effects were blocked by L-vesamicol, a vesicular acetylcholine transporter inhibitor, and bafilomycin, a proton pump inhibitor, suggesting the increase in MEPP/EPP amplitude is due to increased vesicle filling presynaptically. These effects were also blocked by the MARK inhibitors, UO126 and PD98059, suggesting TGF-β2 acts via a MARK-dependent pathway. Postsynaptically, two electrode recording showed postsynaptic potential amplitude was enhanced by an increased fibre input resistance, suggesting TGF-β2 also acts postsynaptically. TGF-β2 reduced the number of vesicles released per stimulus (quanta content, QC) but this was blocked by atropine, showing this was indirect through autoreceptor negative feedback. Voltage clamp recording showed TGF-β2 significantly increased the miniature end plate currents (MEPCs), but not the end-plate currents (EPCs), supporting my initial hypothesis that TGF-β2 acts mainly presynaptically to increase vesicle filling. In TGF-β2+/- mice, I found greater MEPP amplitude variability. This supports my previous findings that TGF-β2 modulates vesicle filling. Unexpectedly, there was an excess in larger MEPP sizes (>0.88 mV), perhaps reflecting upregulation of either presynaptic signalling or another synaptic mediator. Two MEPP amplitude populations were induced in TGF-β2-treated TGF-β2+/- mouse NMJs, similar to the bimodal vesicle population in electroplaques. The extensor digitorum longus (EDL, ~95% fast fibres) and soleus (SOL, ~95% slow fibres) were used to investigate whether the TGF-β2-mediated effect differed between fibre types. Overall, TGF-β2 increased the quantal size (MEPP amplitude) in NMJs of both muscles, suggesting this effect is not fibre-type specific and, together with results in mice, that the TGF-β2-mediated increase in vesicle filling is common to all mammalian neuromuscular terminals. With respect to EPP amplitude and QC, the results differed between muscles. In EDL, the EPP amplitude was not significantly changed, whereas it increased in SOL. In EDL, QC was reduced but not in SOL. These difference compared to diaphragm perhaps do reflect muscle fibre-type dependent differences. TGF-β1, at 0.1 ng/ml, significantly reduced quantal size – the opposite of TGF-β2 at any concentration. One explanation would be that a receptor inhibition by TGF-β1 at low concentration interferes with endogenous TGF-β2 binding/receptor activation at the NMJ. However, when the TGF-β1 concentration was raised to 1 ng/ml, like TGF-β2, it significantly increased MEPP amplitude. This suggests that perhaps sufficient binding of TGF-β1 results in the receptor activation of TGF-β2 like signalling. Overall, I conclude that TGF-β2 enhances the size of spontaneous synaptic potentials in all types of muscle fibres, and this is much more rapid (1 hr vs 1 day) than at central neurone synapses in culture. Detailed study in the rat diaphragm shows it increased the evoked EPP amplitude, reduced QC and increased postsynaptic input resistance. Together, TGF-β2 would therefore enhance the postsynaptic depolarisation increasing synaptic strength, and by reducing QC, increase the efficiency of neurotransmission at mammalian NMJs. While unimportant for single stimuli in healthy terminals, by conserving vesicle use, it may help maintain release during stimulus trains, especially during neuromuscular disease.

Abstract Collagen XIII is a transmembrane protein consisting of intracellular, transmembrane and extracellular domains. The latter can be cleaved by proteases of the furin family at the plasma membrane and in the trans-Golgi network. Both the transmembrane and shed collagen XIII are expressed at the neuromuscular junctions of mice and humans. Such motor synapse passes the contraction signal from the central nervous system to the muscles and brings about all voluntary movements. Loss of both forms of collagen XIII in mice and loss-of-function mutations in the COL13A1 gene in humans leads to congenital myasthenic syndrome characterized by decreased neuromuscular transmission and muscle weakness. To study the roles of the two collagen XIII forms, a novel mouse line was engineered to harbor only the transmembrane collagen XIII by mutating the furin cleavage site. Transmembrane collagen XIII was discovered to be sufficient to prevent adhesion defects, Schwann cell invagination, the ineffective vesicle accumulation and dispersion of both acetylcholinesterase and acetylcholine receptors, phenotypes seen in the complete lack of collagen XIII. On the other hand, lack of shedding led to acetylcholine receptor fragmentation, aberrantly increased neurotransmission and presynaptic complexity. Remarkably, in vivo and in vitro interaction of collagen XIII and acetylcholinesterase-anchoring ColQ was detected. Furthermore, muscle and neuromuscular junction phenotype in the lack of both forms of collagen XIII closely resembled those in the human patients harboring mutations in the COL13A1 gene and these mice were validated as a good model for studying the human disease. Misexpression of collagen XIII was studied with mice exhibiting transgenic overexpression of the protein. Overexpression of collagen XIII was detected to be mostly extrasynaptic in the muscles of such mice. Exogenous collagen XIII was found at the myotendinous junctions, tenocytes and fibroblast-like cells, in addition to some localization in the near vicinity of the neuromuscular junctions. Collagen XIII expression was found, for the most part, to be normal at the neuromuscular junctions, although some were devoid of collagen XIII. The neuromuscular junction phenotype resembled in many ways the findings made in the lack of collagen XIII. Furthermore, acetylcholine receptor and nerve pattern was discovered to be widened
Tiivistelmä Kollageeni XIII on solukalvoproteiini, jonka rakenne koostuu solunsisäisestä, solukalvon läpäisevästä ja solun ulkoisesta osasta, joka pystytään entsymaattisesti irrottamaan solukalvoilta. Täten se esiintyy kahdessa eri muodossaan; solukalvomuotoisena ja soluvälitilan lihasperäisenä proteiinina hiirten ja ihmisten hermolihasliitoksessa. Tässä motorisessa synapsissa keskushermostosta peräisin oleva lihaksen supistumiskäsky välittyy lihakseen ja aikaan saa tahdonalaiset liikkeet. Molempien kollageeni XIII:n muotojen puute hiirillä ja COL13A1 geenin mutaatiot ihmisillä johtavat synnynnäiseen myasteeniseen oireyhtymään, jossa heikentynyt hermolihasliitoksen toiminta johtaa lihasheikkouteen. Kollageeni XIII:n eri muotojen hermolihasliitosvaikutusten selvittämiseksi luotiin hiirilinja, jossa kollageeni XIII ilmenee geneettisen manipulaation seurauksena ainoastaan solukalvomuodossaan. Tutkimukset osoittivat solukalvomuotoisen kollageeni XIII:n tarvittavan hermon ja lihaksen kiinnittymiseen toisiinsa, hermovälittäjäainerakkuloiden ankkuroimiseen hermopäätteeseen, estämään Schwannin solujen tunkeutuminen synapsirakoon, asetyylikoliiniesteraasin sitomiseen ja asetyylikoliinireseptorien vakaantumiseen. Soluvälitilan kollageeni XIII:n puutos puolestaan johti lihaksen puolen liitoksen pirstaloitumiseen sekä hermopäätteiden liialliseen kasvuun ja aktiivisuuteen. Kollageeni XIII todettiin sitoutuvan asetyylikoliiniesteraasia hermolihasliitokseen ankkuroivan kollageeni Q:n kanssa. Lisäksi molempien kollageeni XIII:n muotojen suhteen poistogeenisten hiirten hermolihas- ja lihaslöydökset todettiin muistuttavan COL13A1 geenin mutaatioista kärsivien ihmisten vastaavia löydöksiä todistaen nämä hiiret hyväksi tautimalliksi tulevaisuuden hoitomuotojen suunnitteluun. Kollageeni XIII:n ylimäärän vaikutusta hermolihasliitokseen ja lihaskudokseen tutkittiin kollageeni XIII:a ylenmäärin ilmentävillä hiirillä. Kollageeni XIII todettiin ilmentyvän ylenmäärin lihaksessa fibroblastinkaltaisissa soluissa, lihasjänneliitoksessa ja hermolihasliitoksen lähettyvillä, mutta ei hermolihasliitoksessa. Osa hermolihasliitoksista näissä hiirissä ilmensi jopa vähemmän kollageeni XIII:a kuin normaalisti. Asetyylikoliinireseptorien ja hermojen valtaama alue todettiin leventyneeksi ja hermolihasliitoslöydökset muistuttivat molempien kollageeni XIII:n muotojen suhteen poistogeenisien hiirten löydöksiä

El sistema neuromuscular és un complex circuit interconnectat en el qual les motoneurones presinàptiques i cèl·lules de Schwann indiquen al múscul esquelètic com créixer, diferenciar-se i funcionar. D’altra banda, el múscul proporciona senyals, incloent les neurotrofines, que regulen la supervivència i les funcions de les motoneurones. En concret, la neurotrofina BDNF regulada per activitat, a l’unir-se al TrkB, pot activar diferents vies incloent les PKCs. Per tant, és important conèixer com opera l’activitat pre- i post-sinàptica en condicions fisiològiques per controlar la funció neuromuscular a través de la regulació del BDNF i PKCs. Per estudiar això, es va estimular el nervi frènic del diafragma de rata amb o sense contracció, per tal de separar els efectes de l’activitat sinàptica i la contracció muscular. A continuació es van realitzar les tècniques ELISA, Western Blot, qRT-PCR, immunofluorescència i electrofisiologia. Els resultats van mostrar que tant l’activitat sinàptica com la contracció muscular regulen la maduració i l’activació de la cPKCβI d’una manera complexa i equilibrada a través de la PDK1 y del BDNF/TrkB. Això determinarà la funcionalitat de la sinapsi ja que els resultats també van mostrar que la cPKCβI potencia l’alliberament d’acetilcolina. No obstant, què ocorre en l’esclerosi lateral amiotròfica (ELA) on l’activitat neuromuscular disminueix? Podria l’exercici físic, incrementant l’activitat neuromuscular, prevenir els símptomes? Per abordar això, es van realitzar protocols d’entrenament basats en la natació i el running i es va analitzar la via del BDNF en el múscul plantaris de ratolins SOD1-G93A mitjançant Western Blot. Els resultats van mostrar que en l’ELA, la senyalització del BDNF està alterada però es pot prevenir aquesta afectació de manera diferent en funció de la natura i/o intensitat de l’exercici físic imposat. En conjunt, aquí es mostra un plantejament mecanicista del rol coordinat entre els components pre- i post-sinàptics per preservar la funció sinàptica.
El sistema neuromuscular es un complejo circuito interconectado en el cual las motoneuronas presinápticas y células de Schwann indican al músculo esquelético cómo crecer, diferenciarse y funcionar. Por otra parte, el músculo proporciona señales, incluyendo las neurotrofinas, que regulan la supervivencia y las funciones de las motoneuronas. Concretamente, la neurotrofina BDNF regulada por actividad, al unirse a TrkB, puede activar diferentes vías incluyendo las PKCs. Por consiguiente, es importante conocer cómo opera la actividad pre- y post-sináptica en condiciones fisiológicas para controlar la función neuromuscular a través de la regulación de BDNF y PKCs. Para realizarlo, se estimuló el nervio frénico del diafragma de rata bloqueando o no la contracción, para separar los efectos de la actividad sináptica y de la contracción muscular. A continuación, se realizaron las técnicas ELISA, Western Blot, qRT-PCR, inmunofluorescencia y electrofisiología. Los resultados mostraron que tanto la actividad sináptica como la contracción muscular regulan la maduración y la activación de la cPKCβI de una manera compleja y balanceada a través de PDK1 y BDNF/TrkB. Esto determinará la funcionalidad de la sinapsis porque los resultados también mostraron que cPKCβI potencia la liberación de acetilcolina. Sin embargo, ¿qué ocurre en la esclerosis lateral amiotrófica (ELA) donde la actividad neuromuscular disminuye? ¿Podría el ejercicio físico, incrementando la actividad neuromuscular, prevenir los síntomas? Para abordar esto, se realizaron protocolos de entrenamiento basados en la natación y el running y se analizó la señalización del BDNF en el músculo plantaris de ratones SOD1-G93A mediante Western Blot. Los resultados mostraron que en ELA, la señalización del BDNF está alterada, pero se puede preservar este deterioro de diferente manera en función de la naturaleza y/o intensidad el ejercicio físico impuesto. En conjunto, aquí se muestra un planteamiento mecanicista del rol coordinado entre los componentes pre- y post-sinápticos para preservar la función sináptica.
The neuromuscular system is a complex and interconnected network in which presynaptic motoneurons and Schwann cells “tell” skeletal muscle to grow, to differentiate and how they should function. Conversely, skeletal muscle provides signals, including neurotrophins, that regulates the survival and function of motoneurons during development, maintenance and/or injury. Neurotrophins as BDNF, are regulated by activity and binding to TrkB triggers different pathways that impact on NMJ function, for example activating presynaptic PKCs. Thus, it is important to address how operates pre- and postsynaptic activities in physiological conditions to balance neuromuscular functionality through regulation of BDNF and PKC signalling. To address that, we stimulated the phrenic nerve of rat diaphragms with or without contraction to differentiate the effects of synaptic activity from that of muscle contraction. Then, we performed ELISA, Western Blot, qRT-PCR, immunofluorescence and electrophysiological techniques. Results showed that both synaptic activity and muscle contraction regulate cPKCβI maturation and activation in a complex and balanced way through PDK1 and BDNF/TrkB signalling. This regulation will determine NMJ functionality since our results also demonstrated that cPKCβI is directly involved in neurotransmission enhancing ACh release. However, what happens in a pathological context such us Amyotrophic lateral sclerosis (ALS) where neuromuscular activity is decreased? Could therapies as physical exercise, increasing activity, prevent the symptoms of ALS? To address that, we performed running and swimming-based training protocols to analyse the BDNF signalling in the plantaris muscle of SOD1-G93A mice by Western Blot. Results showed that in ALS disease where there is a loss of the connection between nerve and muscle, BDNF signalling is impaired but could be prevented in a different way depending on the nature and the intensity of the physical exercise imposed. Altogether, these results provide a mechanistic insight into the coordinated role of pre- and postsynaptic components to accurately preserve NMJ function.

A la sinapsis de la unió neuromuscular (NMJ), diverses vies de senyalització coordinen les respostes pre-/postsinàptiques i les cèl·lules glials associades. La relació entre aquestes vies modula les vesícules sinàptiques que regulen la neurotransmissió. A més, la PKC fosforila diverses molècules de l'aparell exocitòtic responsable d'aquesta regulació. Munc18-1 i SNAP-25 són substrats de PKC que juguen un paper clau en la maquinària exocitòtica. A més, la PKC està modulada per l'activitat pre-/postsinàptica al múscul esquelètic. No obstant això, encara es desconeix quines isoformes de PKC regulen aquestes molècules clau en la NMJ. cPKCβI i nPKCƐ es troben exclusivament en el terminal nerviós de la NMJ i estan regulades per l'activitat sinàptica. A més, la contracció muscular a través de BDNF/TrkB té un impacte important en aquestes isoformes. Amb tot, l’objectiu d’aquesta tesi és determinar l’expressió, localització i regulació pels activadors de PKC calci i èsters de forbol (PMA) de Munc18-1 i SNAP-25 i la seva fosforilació en el múscul esquelètic. A més, estudiar si aquestes fosforilacions estan afectades per (1) activitat sinàptica i contracció muscular per se; i (2) nPKCƐ, cPKCβI i la senyalització BDNF/TrkB de manera dependent d’activitat neuromuscular. Els principals resultats, obtinguts mitjançant anàlisi Western blot i microscòpia confocal, mostren que Munc18-1 i SNAP-25 s'expressen i fosforilen en condicions basals en el múscul esquelètic, predominantment a la fracció membrana, essent Munc18-1 localitzat al terminal nerviós. La fosforilació de Munc18-1 i SNAP-25 és modulada per calci, PMA, activitat sinàptica i és promoguda per nPKCƐ. D’altra banda, cPKCβI i la senyalització BDNF/TrkB regulen la fosforilació de Munc18-1 però no la de SNAP-25. Finalment, la contracció muscular regula negativament aquestes proteïnes per assolir un estat basal. En conclusió, aquests resultats proporcionen una visió mecànica de com Munc18-1 i SNAP-25 es regulen per aconseguir l'extraordinària precisió i plasticitat de la neurotransmissió.
En la sinapsis de la unión neuromuscular (NMJ), varias vías de señalización coordinan las respuestas pre-/postsinápticas y las células gliales asociadas. La relación entre estas vías modula las vesículas sinápticas que regulan la neurotransmisión. Además, la PKC, modulada por actividad presináptica y postsináptica en el músculo esquelético, fosforila varias moléculas del aparato exocitótico responsable de esta regulación. Munc18-1 y SNAP-25 son sustratos de PKC que juegan un papel clave en la maquinaria exocitótica. Sin embargo, todavía se desconoce qué isoforma de PKC regula estas moléculas clave en la NMJ. cPKCβI y nPKCƐ se encuentran exclusivamente en el terminal nervioso de la NMJ y están reguladas por actividad sináptica. Además, la contracción muscular a través de BDNF/TrkB tiene un impacto importante en estas isoformas. Así mismo, el objetivo de esta tesis es determinar la expresión, localización y la influencia de los activadores de PKC calcio y ésteres de forbol (PMA) de Munc18-1 y SNAP-25 y su fosforilación en el músculo esquelético. Además, estudiar si dichas fosforilaciones están afectadas por (1) actividad sináptica y contracción muscular per se; y (2) nPKCƐ, cPKCβI y la señalización BDNF/TrkB de manera dependiente de actividad neuromuscular. Los principales resultados, obtenidos mediante análisis Western blot y microscopia confocal, muestran que Munc18-1 y SNAP-25 se expresan y fosforilan en condiciones basales en el músculo esquelético, predominantemente en la fracción membrana, localizándose Munc18-1 en el terminal nervioso. La fosforilación de Munc18-1 y SNAP-25 se modula por calcio, PMA, actividad sináptica y es promovida por nPKCƐ. Por otra parte, cPKCβI y la señalización BDNF/TrkB regulan la fosforilación de Munc18-1 pero no de SNAP-25. Finalmente, la contracción muscular regula negativamente estas proteínas hacia un estado basal. En conclusión, estos resultados proporcionan una visión mecánica de cómo Munc18-1 y SNAP-25 se regulan para lograr la extraordinaria precisión y plasticidad de la neurotransmisión.
At the neuromuscular junction (NMJ) synapse, several signaling pathways coordinate pre-, post-synaptic responses and associated glial cells. The relation between these signaling pathways modulates the pool of synaptic vesicles leading to neurotransmission. Moreover, PKC phosphorylates several molecules of synaptic vesicle exocytotic apparatus responsible to the regulation of neurotransmitter release. Munc18-1 and SNAP-25 are two PKC substrates that play a key role in the exocytotic machinery. In addition, PKC is modulated by presynaptic and postsynaptic activity in skeletal muscles. Nevertheless, it is still unknown which PKC regulates these key molecules in the NMJ. cPKCβI and nPKCƐ are exclusively located at the nerve terminal of the NMJ and are regulated by synaptic activity. In addition, muscle contraction through BDNF/TrkB has an important impact on these PKC isoforms. Therefore, this thesis is aimed to determine the expression, location and regulation by the PKC-activators calcium and phorbol esters (PMA) of Munc18-1 and SNAP-25 and their phosphorylated forms in the skeletal muscle. Also, to study whether Munc18-1 and SNAP-25 phosphorylation are affected by (1) synaptic activity and muscle contraction per se; and (2) nPKCƐ, cPKCβI and BDNF/TrkB signaling in a neuromuscular activity-dependent manner. Main results, obtained by Western blot analysis and confocal microscopy, show that Munc18-1 and SNAP-25 are expressed and phosphorylated in basal conditions in the skeletal muscle, predominantly in the membrane fraction, with Munc18-1 being located at the nerve terminal. Munc18-1 and SNAP-25 phosphorylation are modulated by calcium, PMA, synaptic activity and enhanced by nPKCƐ. Otherwise, cPKCβI and BDNF/TrkB signaling pathway regulates Munc18-1 but not SNAP-25 phosphorylation. Finally, muscle contraction downregulates these proteins to reach a basal state. In conclusion, these results provide a mechanistic insight into how Munc18-1 and SNAP-25 phosphorylation is regulated to achieve the extraordinary precision and plasticity of neurotransmission.

Congenital myasthenic syndromes (CMS) are a rare group of heterogeneous disorders, characterised by compromised neuromuscular transmission and symptoms of fatiguable muscle weakness. CMS is caused by mutations in genes that affect the structure and function of the neuromuscular junction (NMJ). In about 20% of CMS cases, patients have mutations in the gene DOK7; the protein product, DOK7, is crucial for maintaining the dense aggregation of acetylcholine receptor (AChR) clusters at the NMJ. DOK7-CMS patients do not respond to treatment with acetylcholinesterase inhibitors which are the first line treatment for most forms of CMS. Instead, a dramatic response to beta-2 adrenergic receptor (ADRB2) agonists, such as salbutamol, is observed. The aim of this project was to investigate the molecular mechanisms that underlie the beneficial effects of ADRB2 agonists. Firstly, NMJ functioning was modelled in vitro by studying AChR clusters formed on cultured C2C12 mouse myotubes in the presence of WT DOK7. Overexpression of mutant DOK7 led to a significant reduction in the number of AChR clusters, explaining the pathogenic effect of the mutation. Importantly, incubation of myotubes with salbutamol increased the number of AChR clusters and their stability. The results provide the first evidence that ADRB2 agonists directly affect proteins located at the NMJ. However, this disease model suffers from limitations. The rest of the thesis focussed on developing alternative cell culture models to explore the AChR clustering pathway. The first model combined optogenetics and fluorescence lifetime microscopy to study the effects of ADRB2 activation on AChR cluster stability in single live cells. The second used CRISPR/Cas9 genome editing tools to directly introduce Dok7 mutations to the genome of C2C12 cells, thereby overcoming some of the drawbacks associated with DOK7 overexpression. Further manipulations of these novel model systems will be used in the future to examine in more detail the molecular events underlying the pathogenic effects of DOK7 mutations and the mechanisms of ADRB2 agonists.

Thesis (MMed)-- Stellenbosch University, 2013.
ENGLISH ABSTRACT: Introduction: Of the volatile anaesthetic agents, desflurane causes the greatest degree of potentiation of the neuromuscular blocking drugs (NMB). The purpose of this study was to determine whether desflurane prolongs the effects of 3xED95 doses of rocuronium and cisatracurium to the same degree. The two NMB represent potent and less potent classes respectively. Methods: Informed, written consent was obtained from 63 adult patients scheduled for routine surgery. They were randomly allocated to one of four groups to receive either desflurane-sufentanil (end-tidal partial pressure 4.0 kPa) or propofol-sufentanil anaesthesia and either rocuronium (0.9mg/kg) or cisatracurium (0.15mg/kg). All patients received a target-controlled sufentanil infusion (0.5 ng/ml). Neuromuscular blockade was recorded using accelerometry (TOFGUARD ®, Organon) while patients recovered spontaneously to a Train-of-Four ratio of 0.9 (TOFR0.9). Data were analysed using one- and two-way analysis of variance. The main effects were the types of anaesthetic and NMB on indices of recovery. Results: Compared with propofol-sufentanil anaesthesia, mean times to recovery to T125% and TOFR0.9, were prolonged by desflurane-sufentanil (p<0.01). There were no interactions. Mean prolongation of time to TOFR0.9 was 41 min (SD 36) for cisatracurium and 26.6 min (SD 39) for rocuronium. Discussion: Whereas previous studies did not reveal prolongation of the duration of action of rocuronium by desflurane, we demonstrated a statistically significant prolongation of the spontaneous recovery times of both rocuronium and cisatracurium by desflurane. From the data we could not conclude that there was a difference between the two NMB. A power study revealed that in order to detect a difference between times to recovery to TOF0.9, a sample size of 101 subjects per group would be required. Conclusion: Desflurane prolongs the mean time to spontaneous recovery from neuromuscular blockade after 3xED95 doses of both cisatracurium (a potent NMB) and rocuronium (a less potent NMB). There was wide inter-individual variation in times to spontaneous recovery. Any difference in the mean prolongations between the different types of NMB is unlikely to be of clinical importance.
AFRIKAANSE OPSOMMING: Inleiding Van al die vlugtige narkosemiddels veroorsaak desfluraan die grootste mate van potensiasie van die neuromuskulêre blokkeermiddels. Die doel van hierdie studie was om vas te stel of desfluraan wel die effek van driedubbel die ED95 dosis van rokuronium en cisatrakurium tot dieselfde mate sal verleng. Metodiek Geskrewe ingeligte toestemming is verkry van 63 pasiënte wat voorgedoen het vir roetiene chirurgiese prosedures. Pasiënte is lukraak in een van vier groepe ingedeel om of desfluraansufentaniel (eind-gety parsieële druk 4.0 kPa) of propofol-sufentaniel narkose en of rokuronium (0.9 mg/kg) of cisatrakurium (0.15 mg/kg) te ontvang. Alle pasiënte het 'n teiken-beheerde sufentaniel infusie (0.5 ng/ml). Neuromuskulêre blokkade is waargeneem met behulp van aksellerometrie (TOF-GUARD, Organon) terwyl pasiënte spontaan herstel het tot “reeks-van-vier” verhouding (Engels “Train-of-four” ratio) 0.9 (TOFR0.9). Data analise is gedoen met behulp van een- en tweerigting analise van variansie. Resultate Desfluraan-sufentaniel het die gemiddelde hersteltyd tot T125% en TOFR0.9 verleng in vergelyking met propofol-sufentaniel. Geen interaksies is waargeneem nie. Gemiddelde verlenging van TOFR0.9 vir cisatrakurium was 41 minute (standaardafwyking 36) en vir rokuronium 26.6 minute (standaardafwyking 39). Bespreking Vorige studies kon nie vasstel of desfluraan die werkingsduur van rokuronium verleng nie. Ons het in hierdie studie vasgestel dat desfluraan wel 'n statisties beduidende verlenging in die hersteltyd van beide rokuronium en cisatrakurium veroorsaak. Ons kon egter nie 'n verskil tussen die twee neuromuskulêre agente aandui nie. 'n onderskeidingsvermoëstudie het getoon dat ten minste 101 pasiënte per groep benodig sou word om 'n beduidende verskil tussen die hersteltye tot TOFR0.9 te verkry. Gevolgtrekking Desfluraan verleng die gemiddelde hersteltyd tot spontane herstel van neuromuskulêre blokkade na driedubbele ED95 dosisse van beide cisatrakurium en rokuronium. Daar was egter groot interindividuele variasie ten opsigte van spontane hersteltyd. Enige verskille in die gemiddelde verlenging is onwaarskynlik van kliniese belang.

Spinal muscular atrophy (SMA) and Charcot-Marie-Tooth disease type 2D (CMT2D)/distal SMA type V (dSMAV) are two incurable neuromuscular disorders that predominantly manifest during childhood and adolescence. Both conditions are caused by mutations in widely and constitutively expressed genes that encode proteins with essential housekeeping functions, yet display specific lower motor neuron pathology. SMA results from recessive inactivating mutations in the survival motor neuron 1 (SMN1) gene, while CMT2D/dSMAV manifests due to dominant point mutations in the glycyl-tRNA synthetase (GlyRS) gene, GARS. Using a number of different model systems, ranging from Caenorhabditis elegans to the mouse, this thesis aimed to identify potential novel therapeutic compounds for SMA, and to increase our understanding of the mechanisms underlying both diseases. I characterised a novel C. elegans allele, which possesses a point mutation in the worm SMN1 orthologue, smn-1, and showed its potential for large-scale screening by highlighting 4-aminopyridine in a screen for compounds able to improve the mutant motility defect. Previously, the gene encoding three isoforms of chondrolectin (Chodl) was shown to be alternatively spliced in the spinal cord of SMA mice before disease onset. I performed functional analyses of the three isoforms in neuronal cells with experimentally reduced Smn levels, and determined that the dysregulation of Chodl likely reflects a combination of compensatory mechanism and contributor to pathology, rather than mis-splicing. Finally, working with two Gars mutant mice and a new Drosophila model, I have implicated semaphorin-plexin pathways and axonal guidance in the GlyRS toxic gain-of-function disease mechanism of CMT2D/dSMAV.

Levetiracetam (LEV) is an antiepileptic drug (AED) that has been shown to mainly enhance synaptic depression and modulate certain voltage and ligand-gated channels, after it gains entry into neurons through endocytosis. Since synaptic terminals and distal axons are the first compartments exposed to LEV, we utilized a crayfish motor axon preparation to investigate whether LEV modulates axonal excitability. Two electrode current clamp from the inhibitor axon of the crayfish opener showed that LEV reduced action potential amplitude (APamp) and enhanced synaptic depression, although these events did not occur at the same time, the latter occurred later than the reduction in APamp. Further examinations of these effects and comparison of antidromic and orthodromic conducting action potentials in LEV suggests that this drug preferentially reduces excitability of the proximal axon despite the expectation that it enters the axon at terminals and reaches distal branches first. Results presented here demonstrate that LEV modulates axonal excitability, which may also contribute to its antiepileptic effects.

碩士
國立中山大學
生物科學系研究所
98
The transient receptor potential (TRP) channel superfamily is a non-selective Ca2+-permeable cation channels involved in sensory physiology. Here we show that 2-aminoethoxydiphenyl borate (2-APB), a compound commonly used as TRP channel inhibitor, dose-dependently induce a significant facilitation on the frequency of spontaneous neurotransmitter release at developing Xenopus neuromuscular junction through, surprisingly, TRP channel activation. Bath application of universal TRP channel inhibitors either SKF96365, flufenamic acid or RuR cease the 2-APB-induced synaptic facilitation. Exclusion of Ca2+ from culture medium or bath application of the pharmacological Ca2+ channel inhibitor cadmium, membrane-permeable Ca2+ chelator BAPTA-AM, effectively hampered the facilitation of neurotransmitter release induced by 2-APB, suggesting Ca2+ influx is requisite for 2-APB-induced synaptic facilitation. Blockade of the voltage-dependent Ca2+ channel with either nifedipine, verapamil or ω-CTX failed to abolish the SSC facilitating effect of 2-APB. Electrophysiological recording of 2-APB induced single channel currents by using cell-attached patch-clamp technique reveals 2-APB evoked a robust single channel activity recorded at different pipette voltages. Furthermore, the 2-APB-evoked single-channel events are significantly abolished in the presence of SKF96365. Either pretreatment of the cultures with inhibitor of phospholipase C (U73122) or tyrosine kinase (Genistein) abolishes 2-APB induced potentiation of synaptic transmission. The structure of PMA is analogous to diacylglycerol (DAG), which abolishes 2-APB induced synaptic facilitation. 2-APB no longer elicited any changes in SSC frequency when serum is eliminated from culture medium. Overall, results from our current study provide evidences that 2-APB induces the opening of TRP channels and Ca2+ influx which resulting in facilitation of spontaneous neurotransmitter release at developing Xenopus neuromuscular synapse. Serum may activate tyrosine kinase to turn on PI3K and phospholipase C. Then phospholipase C cleavage PIP2 to IP3 and diacylglycerol, and diacylglycerol induced TRP channel opening. 2-APB potentiates and sensitizes the TRP channel, increasing Ca2+ inffux. Elevated [Ca2+]i resulted in enhancement of neurotransmitter release from presynaptic nerve terminal.

The nervous system is made up of specialized cells which receive and respond to environmental stimuli. Intercellular communication in the nervous system is achieved predominantly through chemical synaptic transmission. Within the chemical synapse, the actin cytoskeleton plays a major role in regulating synaptic activities, although the extent and clarity in our understanding of these processes is still limited. Using the genetically pliable model, Drosophila melanogaster, this thesis begins to unravel contributions of actin binding proteins to synaptic development and physiology at the larval neuromuscular junction (NMJ). Two actin binding proteins, Moesin and Nonmuscle Myosin II (NMMII) were selected for study based on previous studies implicating them in synaptic development. Combining genetics, fluorescent imaging and electrophysiological recordings this thesis unveils previously unidentified functions for Moesin and NMMII in morphology and physiology of the Drosophila NMJ. Moesin was found to help restrain synaptic growth but did not affect synaptic physiology. By correlating morphological and electrophysiological measurements in Moesin mutants, it was determined that physiology and morphology can be independently regulated at the NMJ. NMMII was used to investigate a role for actin binding proteins in physiology at the Drosophila NMJ. By using the fluorescent imaging technique, FRAP, this becomes the first research to implicate NMMII in unstimulated synaptic vesicle mobility. FRAP indicated that vesicle mobility was highly dependent on the expression level of NMMII. Electrophysiological analysis of NMMII indicated distinct mechanisms for spontaneous and evoked vesicle release. NMMII expression exhibited a positive correlation with basal synaptic transmission and was important in mobilizing vesicles for synaptic potentiation. In addition, NMMII was found to be involved in a high frequency dependent low frequency depression. This work begins to identify how vesicles traverse within boutons and suggests differential mechanisms of synaptic release, both of which are partially dependent of NMMII expression. Studying Moesin and NMMII have revealed a complex interplay between the actin cytoskeleton and synaptic function and together this research furthers our understanding of how the actin cytoskeleton regulates synaptic activity.

博士
國立中興大學
生命科學系所
101
Botulinum toxin type A (BoNT-A), a kind of botulinum toxin, is used for spasticity block recently. The mechanism of this drug is to block the secretion of pre-synaptic acetylcholine, thus makes muscle weakness. Botulinum toxin injection is usually performed in cerebral palsy to reduce spasticity. Treadmill training for cerebral palsy is a well-documented program. Although botulinum toxin injection and treadmill training have usually been used in combination, the effects of treadmill training in muscles after intramuscular botulinum toxin injection are not well established. We showed that after Botulinum toxin type A injection, treadmill training improved the sciatic functional index, the maximal contraction force of the gastrocnemius muscle, and the percentage of activated muscle fibers which was demonstrated by differences in amplitude and area of compound muscle action potential. Upregulation of GAP-43, IGF-1, Myo-D, Myf-5, myogenin, AChR subunits α and β were also found following treadmill running which may have contributed to enhanced recovery of gastrocnemius strength. In clinical practice, when considering the therapeutic strategies of combining these two treatments, clinicians should take this potential counteraction effect into consideration. Muscle atrophy is a common symptom associated with reduced skeletal muscle activity following bone fracture, trauma, ligament tear, inflammatory disease and nerve injury. To date, there are no pharmacological strategies for preventing or treating muscle atrophy. Myostatin is a potent inhibitor of skeletal muscle growth. Myostatin propeptide (MPro) is showed to improve muscle growth. However, the underlying mechanism of muscle atrophy attenuation effects of myostatin propeptide in atrophied muscles is not well established. We constructed both wild type MPro (MSPP) and mutant MPro construct (MSPPD75A) with resistance to proteinase. Using the C2C12 in vitro cell model and BoNT-induced muscle atrophy model, we evaluated the effects of myostatin propeptide gene therapy. We also observed changes in gene activation associated with neuromuscular junction, muscle and nerve. We showed that through MSPPD75A gene delivery, gastrocnemius muscle atrophy caused by botulinum toxin was attenuated. Through the delivery of either MSPP or MSPPD75A, the expressions of downstream proteins in ubiquitine-proteosome pathway Smad3 and MuRF1 were decreased, and the expressions of the muscle regulatory factors, IGF-1, GAP43, and acetylcholine receptors were increased. The data suggested that gene therapy may be a promising treatment for muscle atrophy. And the results could be used as basic knowledge for clinical rehabilitation, as a basis for developing treatment strategy of gene therapy.

The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes. The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations.