Dissertationen zum Thema „Motorneurone“

1. Electrical characteristics of the cell body of an identified motoneurone, the 'fast' coxal depressor motoneurone (Df), from the cockroach (Periplaneta americana) have been studied under current- and voltage-clamp. 2. In response to low magnitude, relatively long duration depolarising current pulses, Df could generate plateau potentials, regenerative events which often far outlived the duration of the applied depolarisation. 3. Plateau potentials constitute an inherent property of the neurone because they could be evoked in somata that had been surgically isolated from other parts of the neurone (the soma is devoid of synaptic contacts); these experiments also demonstrated that the soma of this neurone can participate in the generation of plateau potentials. 4. Plateau potentials were often surmounted by attenuated action potentials; these correlated 1:1 with axonal impulses recorded extracellularly from the axon of the neuron. 5. Plateau potentials were associated with an increase in membrane conductance. Under voltage- clamp, cells which exhibited plateau potentials possessed a region of negative slope resistance in their current-voltage relationship. 6. Plateau potentials in Df were observed to be calcium-dependent, A series of current- and voltage- damp experiments indicated that the calcium channels involved in plateau potential production differ from those which can mediate calcium-dependent action potentials following pharmacological treatment of this neurone. 7. Plateau potential production in Df was suppressed by the application of GABA (10-4M). Spontaneous plateau potentials could be recorded following application of picrotoxin (10-5M) or pentylenetetrazole (25mM). 8. Recordings taken from two other 'fast' motoneurones, cell 3 (from the cockroach) and FETi (from the locust, Schistocerca gregaria) indicated that the ability to generate plateau potentials may not be restricted to Df. 9. Although freshly dissected, recently impaled neurones responded to relatively brief depolarising current pulses with a series of graded, damped membrane oscillations, the excitability of many preparations increased with time from dissection: many cells became able to generate all-or-none action potentials in response to such pulses (these differed from the attenuated axonal spikes which often surmounted plateau potentials). The appearance of these events did not correlate with consistent changes to the resting potential or input resistance of neurones. 10. Time-dependent action potentials were calcium-dependent and could be recorded from 'intact' cells and isolated neurone somata. These action potentials could also co-exist with plateau potentials; such co-existence provides evidence for different classes of calcium channel in untreated insect neurones.

La sclérose latérale amyotrophique (SLA) et l'amyotrophie spinale (SMA) sont deux maladies du motoneurone (MN) se caractérisant par une dénervation musculaire progressive, pouvant être fatale par insuffisance respiratoire. Dans la SLA, les MN rapides (MNr) sont principalement affectés, tandis que dans la SMA, les MNr et les MN lents (MNl) dégénèrent. Le laboratoire a montré que soumettre des modèles murins adultes de la SLA (B6SJL-Tg(SOD1-G93A)1Gur/J) et de la SMA de type 3 (FVB/NRj-SmnDelta7/Delta7,huSMN2+/+) à un exercice de nage, activant les MNr, induisait une neuroprotection spécifique des MNr dans les deux maladies, tandis que l'entraînement à un exercice de course, activant les MNl, induisait une neuroprotection des MNl, en SMA uniquement. Ces données suggèrent que seules les populations de MN vulnérables et activées par un exercice seraient capables de mettre en place des adaptations permettant leur survie. Afin de tester cette hypothèse, nous avons voulu développer deux approches complémentaires d'isolement des ARNm, l'une axée sur les MNr et l'autre sur les MN activés par l'exercice. La première s'appuie sur l'immunoprécipitation des ARNm des MNr par expression de la PolyA binding protein marquée (PABP-Flag) contrôlée par le système CRE-Lox, dans des souris SLA et SMA exprimant l'enzyme CRE sous le contrôle du promoteur Calcitonin-related-polypeptide alpha (Calca-CRE), marqueur des MNr spinaux. Nous avons développé trois plasmides d'expression de la PABP-Flag dépendante de la CRE, dont deux ont été sélectionnés pour leur efficacité et spécificité d'expression après transfection in vitro d'une lignée cellulaire MNale murine MN1 et encapsidés dans des AAV9. Malheureusement les tests réalisés in vivo de ces deux AAV9-PABP-Flag chez des souris Calca-CRE non mutantes ont révélé une faible efficacité et spécificité d'expression de la PABP-Flag, tant par injection intrathécale qu'intramusculaire et pour des quantités de vecteurs viraux comprises entre 1.5E9 et 3.3E11 Vg. Cette stratégie n'a donc pu être utilisée le cadre de notre étude. La seconde approche repose sur la microdissection laser (MDL) de MNr innervant trois muscles de la patte et activés par les exercices, marqués par le fragment C terminal de la toxine tétanique (TTC), un traceur rétrograde trans-synaptique dépendant de la dépolarisation neuronale. De nouveau, ni l'application d'exercices de nage à différents temps, avant et après injection intramusculaire de la TTC, ni la limitation de l'activité neuromusculaire par immobilisation ne sont parvenus à modifier les populations MNales marquées à la TTC, suggérant que la TTC ne permet pas une sélection spécifique des MNr activés par l'exercice. Nous avons donc décidé de recueillir les ARNm de MNr par utilisation du Fluorogold (FG), un traceur rétrograde pan MN et d'appliquer un filtre d'aire somatique >900µm². Ainsi, nous avons pu mener une analyse transcriptomique croisée sur MNr isolés par MDL de coupes de moelles épinières de souris SLA et SMA adultes non entraînées ou entraînées à la course ou à la nage. Cette analyse a suggéré la mise en place d'adaptations cellulaires spécifiques à la nage participant à la survie des MNr telles qu'une modulation du métabolisme des ARN, de l'homéostasie protéique, de l'excitabilité neuronale et des fonctions synaptiques. Ces adaptations seraient initiées en partie par une modulation fine de la voie des MAP Kinases impliquant des effecteurs propres à chacune des maladies. De manière très intéréssante, notre étude suggère un rôle coordinateur majeur, commun dans les deux maladies, du gène de fusion d'ancrage des protéines kinase A PALM2-AKAP. Ces travaux pionniers permettent une meilleure compréhension des mécanismes de neuroprotection activés par l'exercice dans différents contextes pathologiques, ouvrant la voie pour le développement de nouvelles thérapies potentiellement applicables à de nombreuses maladies neurodégénératives
Amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are two motor neuron (MN) diseases characterized by progressive muscle denervation, which can be fatal due to respiratory failure. In ALS, fast motor neurons (fMNs) are primarily affected, while in SMA, both fMNs and slow motor neurons (sMNs) degenerate. Subjecting adult mouse models of ALS (B6SJL-Tg(SOD1-G93A)1Gur/J) and type 3 SMA (FVB/NRj-SmnDelta7/Delta7, huSMN2+/+) to high-intensity swimming exercise, which activates fMNs, induced specific neuroprotection of fMNs in both diseases, while training to low-intensity running exercise, which activates sMNs, induced neuroprotection of sMNs in SMA only. These data suggest that only vulnerable MN populations activated by exercise are capable of implementing adaptations that enable them to survive. To test this hypothesis, we set out to develop two complementary mRNA isolation approaches, one focusing on fMNs and the other on exercise-activated MNs.The first consist on a CRE recombinase dependant-AAV9-based expression of a tagged Poly-A Binding Protein (PABP) under the control of the Calcitonin related Polypeptide Alpha (Calca), a spinal fMN marker. This adapted ctag-PAPERCLIP technique allows to immunoprecipitate mRNA from fMN in generated heterozygous Calca-CRE ALS and SMA mouse models. To this end, we developed three CRE-dependent PABP-Flag expression plasmids, two plasmids were selected for their expression efficiency and specificity after in vitro transfection of a murine MNal MN1 cell line and encapsidated in AAV9. Unfortunately, after intrathecal or intramuscular injection in non-mutant Calca-CRE mice in quantities ranging from 1,5E9 to 3,3E11 Vg per mouse, these two AAV9-PABP-Flag showed weak PABP-Flag expression efficiency, associated with a non-CRE-dependent leak of expression, therefore non-specific to fMN. Hence, this strategy could not be used in our study. The second approach consist on laser capture microdissection (LCM) of sMNs innervating three hindlimb muscles and activated by exercise labeled by the C-terminal fragment of tetanus toxin (TTC), a depolarization-dependant trans-synaptic retrograde tracer. Once more, neither the application of swimming exercise at different times, before and after intramuscular injection of TTC, nor the limitation of neuromuscular activity by immobilization succeeded in modifying TTC-labeled MNal populations, suggesting that TTC does not allow specific selection of exercise-activated MNr. We therefore decided to collect fMNs mRNA using Fluorogold (FG), a pan MN retrograde tracer, and to apply a somatic area filter >900µm² to the selected MN.This analysis suggested the development of specific cellular adaptations to swimming that contribute to the survival of vulnerable fMNs, such as modulation of RNA metabolism, protein homeostasis, neuronal excitability and synaptic functions. These adaptations would be initiated in part by fine modulation of the MAP Kinase signaling pathway involving effectors specific to each disease. Finally, our study suggests a major coordinating role, common to both diseases, for the PALM2-AKAP protein kinase A anchoring fusion gene. This work provides a better understanding of the neuroprotective mechanisms activated by exercise, a prerequisite for the development of new effective therapies

Thesis (Ph. D.)--University of Oregon, 2004.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 99-113). Also available for download via the World Wide Web; free to University of Oregon users.

The correct polarity of a neuron underlies its ability to integrate precise circuitries in the nervous system. The goal of my thesis was to investigate the pathways that establish and maintain neuron polarity/orientation in vivo. To accomplish this, I used bipolar VC4/5 motor neurons, which innervate the C. elegans egg-laying musculature, as a model system. Vulval proximal VC4/5 neurons extend axons in the left-right (LR) orientation, around the vulva; whereas vulval distal VC1-3,6 neurons extend axons along the anterior-posterior (AP) axis. A previous study showed that vang-1, a core planar cell polarity (PCP) gene, suppresses AP axon growth in VC4/5 neurons. In order to identify new components of this pathway we performed genetic screens for mutants with abnormal VC4/5 polarity/morphology. We isolated and mapped alleles of farnesyl transferase b (fntb-1) and of core PCP genes, prickle- 1 (prkl-1) and dishevelled-1 (dsh-1); all of which display tripolar VC4/5 neurons, similar to vang-1 lof. In prkl-1 and dsh-1 mutants, primary LR and ectopic AP VC4/5 axons are born simultaneously, suggesting an early role in establishing polarity. In addition, prkl-1 and dsh-1 act persistently to maintain neuron morphology/orientation. Genetic analysis of double mutants suggests that prkl-1 interacts with vang-1 in a common PCP pathway to prevent AP axon growth, while dsh-1 also acts in a parallel pathway. Furthermore, prkl-1 functions cell autonomously in neurons, whereas dsh-1 acts both cell autonomously and cell nonautonomously in epithelial cells. Notably, prkl-1 overexpression results in unipolar VC4/5 neurons, in a dose-dependent manner. In contrast, dsh-1 overexpression in VC4/5 neurons results in a lof phenotype, similar to vang-1 lof and overexpression phenotype. Remarkably, prkl-1 overexpression restores normal VC4/5 polarity in dsh-1 and vang-1 mutants, which is suggestive of a downstream role for prkl-1. Both PRKL-1 and DSH-1 are expressed in iii uniformly distributed puncta at the plasma membrane of VC4/5, similar to VANG-1; suggesting that their asymmetric distribution is not critical for neuron polarity. Furthermore, we found that the vulva epithelium induces prkl-1 expression in VC4/5; indicating a functional relationship between the egg-laying organ and neuron morphology. Moreover, a structure-function analysis of PRKL-1 revealed that the conserved PET domain and the Cterminal region are crucial to prevent AP axon growth, whereas the three LIM domains are dispensable for this role. In addition, we showed that dsh-1 also regulates the morphology of AP-oriented PDE neurons. dsh-1 promotes the formation of PDE posterior axons, contrary to its function in VC5 neurons; which indicates a context-dependent role for dsh-1 in neuronal polarity. Altogether, this thesis implicates the PCP signalling pathway in a previously unknown role, in establishing and maintaining neuronal polarity, by controlling AP axon growth in response to organ-derived polarizing cues.

Les signaux environnementaux ont une grande influence sur le devenir de certaines populations de motoneurones. J'étudie la population qui exprime le facteur de transcription PEA3, située au niveau brachial, caractérisée et spécifiée par ce facteur, et qui innerve les muscles dorsaux des membres (Livet et al., 2002 ; Vrieseling et al., 2006). Cette population représente un des exemples les mieux compris de l'acquisition d'une identité neuronale par des signaux provenant du muscle cible. Au cours du développement, l'expression de PEA3 se met en place de manière séquentielle. PEA3 est d'abord exprimé dans un premier sous-groupe de neurones localisé en position postérieure dans le domaine (neurones pionniers), puis dans un deuxième sous-groupe de neurones situé en position plus antérieure. Le développement de cette population implique des échanges de signaux entre les neurones pionniers, instruits par le muscle cible, et le deuxième groupe de neurones antérieurs, instruit par les neurones pionniers. Le GDNF, produit par les cellules du futur muscle cible, induit PEA3 dans les neurones pionniers (Haase et al., 2002). Puis le HGF, un autre facteur dérivé du membre, induit les neurones pionniers à sécréter un ‘signal de propagation’, qui agit à distance et induit l'expression de PEA3 dans le deuxième groupe de neurones (neurones recrutés) (Helmbacher et al., 2003). L'objectif initial de ma thèse a été basé sur l'identification de ce signal de propagation. J'ai d'abord utilisé une approche pharmacologique dans un système in vitro de cultures d'explants de moelles épinières d'embryons de souris. En y inhibant la voie EGF, j'ai démontré que le signal de propagation appartient à cette famille de molécules. Les récepteurs de la voie EGF (ErbB1 à ErbB4) sont exprimés chez l'embryon de poulet et de souris dans la moelle épinière brachiale, et spécifiquement dans les motoneurones, au moment où PEA3 est exprimé. Parmi les ligands de la voie EGF, je me suis intéressée aux neurégulines, une famille de glycoprotéines connue pour son implication dans la mise en place du système nerveux. J'ai montré que des isoformes du gène neuréguline1 (nrg1), possédant un domaine immunoglobuline (type I) sont capables d'induire l'expression de pea3 dans la moelle épinière brachiale, et spécifiquement dans les neurones recrutés. J'ai pu démontrer, en utilisant des souris mutantes pour le récepteur à l’HGF (metd/d), que le signal de propagation est vraisemblablement une isoforme NRG1, de type I
Signals derived from the environment have an important influence on development of some motorneurons populations. I study the population that expresses the transcription factor PEA3, localized at the brachial level, characterized and specified by this factor. This population innervates the limb dorsal muscles (Livet et al., 2002 ; Vrieseling et al., 2006). This population represents one of the best understood examples of an acquisition of a neuronal identity induced by signals derived from the target muscle. During development, PEA3 expression is made in two times in motorneurons. Initially, PEA3 is expressed in a first population, localized in the posterior part of the domain (pionneers neurons), then in a second population, localized in a more anterior position. Development of this population implies exchanges of signals between pionneers neurons, instructed by the target muscle, and anterior neurons, instructed by pionneers neurons. GDNF, produced by cells of the future target muscle, induces PEA3 in the pioneers neurons (Haase et al., 2002). Then, HGF, another limb-derived factor, induces pioneers neurons to secrete a propagation signal’, which induces PEA3 expression in the second population of neurons (recruited neurons) (Helmbacher et al., 2003). The initial purpose of my phD was to identify this ‘propagation signal’. First, I used a pharmacological approach in an in vitro assay of mouse embryos spinal cord explants culture. I did inhibition of the EGF pathway in this assay, and I showed that the propagation signal belongs to this family. EGF receptors (ErbB1 à ErbB4) are expressed in chick and mouse embryos, and especially in motorneurons, when PEA3 is expressed. Among the EGF ligands, I studied neuregulins, a family of glycoproteins involved in the nervous system development. I showed that isoforms of neuregulin1 gene (nrg1), which have an immunoglobulin domain (type I) induce pea3 expression in the brachial spinal cord, and especially in the recruited neurons. I observed, by using mice mutants for HGF receptor (metd/d), that the propagation signal is plausibly a typeI NRG1 isoform. Keywords : motorneurons, PEA3, recruitment, ErbB

Stem cell therapy is considered one of the most promising approaches against different neurodegenerative disorders, including Amyotorophic Lateral Sclerosis (ALS). The evidence that the systemic injection of human cord blood mononuclear cells (HuCB-MNC) was able to reduce the clinical outcomes and increase the lifespan in a murine model of fALS1, the SOD1G93A mouse, even if localized far from affected motor neurons, opens the way for new possible candidates and alternative ways of administration. Here the effect of human skeletal muscle-derived stem cell (SkmSCs) was investigated by single administration in lateral ventricles in the most characterized model of spontaneous motor neuron degeneration, the Wobbler (Wr) mouse. Before evaluating clinical progression, we found that SkmSCs (previously labeled with the super paramagnetic contrast agent Endorem™ and/or with the fluorescent nuclear dye Hoeschst 33258): 1) spread along the whole ventricular system as far as the ependymal canal at the spinal cord level; 2) remained for a longer time in the Wr than in the healthy mice, and; 3) did not significantly migrate to the parenchyma. Similar to the SOD1G93A mice treated with HuCB-MNCs, the transplantation of SkmSCs: 1) significantly improved the disease progression of ALS-related Wr motorneuropathology; 2) this effect was not associated with a migration of SkmSCs close to the degenerating motor neurons. Very interestingly, we also found that cell grafting in the Wr brain ventricles significantly increased the gene expression of anti-inflammatory cytokines or chemokines activated in the inflammatory response. These results further confirm the consistency of the hypothesis of the bystander effect of stem cells in motor neurodegenerative disorders by a mechanism of action aimed at reducing the neuroinflammatory response.

The overall aim of this thesis was to establish strategies to minimize secondary damage to the injured spinal cord. Secondary damage that follows spinal cord injury (SCI) involves inflammatory and excitotoxic pathways. Regulation of these pathways using immunomodulatory and neuroprotective substances potentially protects the injured spinal cord from further damage. We also developed and studied resorbable biomaterials to be used as carriers for potential neuroprotectants to the injured spinal cord. We used transversal spinal cord slice cultures (SCSCs) derived from postnatal mice as a model. SCSCs were maintained on different biomaterials and were studied after treatment with immunomodulatory and/or neurotrophic factors. They were further excitotoxically injured and subsequently treated with interleukin-1 receptor antagonist (IL1RA) or by neural crest stem cell (NCSC)-transplantation. The results show that biocompatible and resorbable hydrogels based on hyaluronic acid (HA) preserved neurons in SCSCs to a much higher extent than a conventional collagen-based biomaterial or standard polyethylene terephthalate (PET) membrane inserts. Glial activation was limited in the cultures maintained on HA-based hydrogel. The anti-inflammatory factor IL1RA protected SCSCs from degenerative mechanisms that occur during in vitro incubation, and IL1RA also protected SCSCs from excitotoxic injury induced by N-Methyl-d-Aspartate (NMDA). IL1RA specifically protected neurons that resided in the ventral horn, while other neuronal populations such as dorsal horn neurons and Renshaw cells did not respond to treatment. Finally, transplantation of NCSCs onto excitotoxically injured SCSCs protected from neuronal loss, apoptosis and glial activation, while NCSCs remained undifferentiated. The results presented in this thesis indicate that carriers based on HA seem to be more suitable than conventional collagen-based biomaterials since they enhance neuronal survival per se. The observed neuroprotection is likely due to biomechanical properties of HA. IL1RA protects SCSCs from spontaneous degeneration and from NMDA-induced injury, suggesting that excitotoxic mechanisms can be modulated through anti-inflammatory pathways. Different neuronal populations are affected by IL1RA to various degrees, suggesting that a combination of different neuroprotectants should be used in treatment strategies after SCI. Finally, NCSCs seem to protect SCSCs from excitotoxic injury through paracrine actions, since they remain undifferentiated and do not migrate into the tissue during in vitro incubation. It seems that combinations of neuroprotectants and carrier substances should be considered rather than one single strategy when designing future treatments for SCI. Incorporation of neuroprotectants such as IL1RA combined with stem cells in injectable biocompatible carriers based on HA is the final goal of our group in the treatment of SCI.

Syfte: Att beskriva närståendes upplevelser av att leva med en person med ALS. Metod: En litteraturöversikt med kvalitativ metod och en induktiv ansats. Resultat: Sjukdomen ALS är ovanlig och de som drabbas av den är i stort behov av omvårdnad. Närstående till en anhörig som har fått diagnosen ALS, upplever att vårdpersonalen har kunskapsbrist angående sjukdomen och vårdandet. De upplever bristande information om sjukdomens förlopp. Egentiden tas ifrån dem, sådant som förr togs förgivet läggs istället åt sidan, då all fokus ligger på den anhöriges omvårdnad. Slutsats: Närstående väljer ofta att vårda den anhöriga i hemmet, trots det snabba sjukdomsförloppet. Vårdandet av den anhöriga leder till känslomässiga påfrestningar hos de närstående, både psykiska och fysiska.
Purpose: To describe next of kin experiences of living with person with ALS. Method: A literature review with qualitative method and an inductive approach. Result: The disease ALS is unusual and those who suffer from it are in great need of nursing. The next of kin those who has been diagnosed with ALS, find that healthcare professionals have a lack of knowledge regarding the disease and care. They experience insufficient information about the course of the disease. The true time is taken away from them, as was previously taking for granted, instead being put aside, as all focus is on the patient's nursing. Conclusion: Next of kin often choose to care for their relatives in spite of the progress of the ilness. The care of the relatives leads to emotional stresses of the related, both mental and physical.

L’atròfia muscular espinal (AME) és una malaltia d’origen genètic que afecta, majoritàriament a la població infantil. La malaltia cursa amb una mort de les motoneurones  i atròfia muscular. El gen implicat és el survival motor neuron (SMN) que està delecionat en un 95% dels casos. El nostre estudi està dividit en dues parts: 1- l’aprofundiment de les alteracions musculars en dos models animals murins transgènics que pateixen les formes més greus d’AME (Tipus 1-2) i 2- estudi dels possibles efectes terapèutics del liti en un d’aquests models d’AME. S’ha trobat alteracions greus en les unions neuromusculars d’animals nounats i prenatals en marcadors relacionats amb l’ancoratge de les vesícules a la membrana presinàptica, organització dels canals de calci presinàptics i altres proteïnes presinàptiques, desorganització i apoptosi de les cèl•lules musculars, apoptosi massiva del timus i alteracions generalitzades en els òrgans limfoides. L’estudi ultraestructural del múscul ens indica que hi ha una mort, per apoptosi, de les cèl•lules satèl•lit, confirmat amb la tècnica de TUNEL. L’augment de les apoptosi, però no es reflexa en un increment, per altra banda esperat, de la densitat dels macròfags. El tractament amb concentracions terapèutiques del liti no millora l’evolució de la malaltia en els ratolins que manifesten l’AME, s’observa una acumulació progressiva dels nivells de liti, provocant toxicitat en l’animal. L’efecte del liti inhibint la GSK3 no es tradueix en el increment d’expressió de SMN, tal com s’ha deduït d’alguns experiments publicats.
La atrofia muscular espinal (AME) es una enfermedad de origen genético que afecta, mayoritariamente a la población infantil. La enfermedad cursa con muerte de las motoneuronas y atrofia muscular. El gen implicado es el “survival motor neuron” (SMN) que está delecionado en un 95% de los casos. Nuestro estudio está dividido en dos partes: 1 - la caracterización de las alteraciones musculares en dos modelos animales murinos transgénicos que sufren las formas más graves de AME (Tipo 1-2) y 2 - estudio de los posibles efectos terapéuticos del litio en uno de estos modelos. Se han encontrado alteraciones pre y postnatales graves en las sinapsis neuromusculares a nivel de marcadores relacionados con el anclaje de las vesículas en la membrana presináptica, en la organización de los canales de calcio presinápticos y en otras proteínas presinápticas, Asimismo se ha hallado desorganización y apoptosis de las células musculares, apoptosis masiva del timo y alteraciones generalizadas en los órganos linfoides. El estudio ultraestructural del músculo nos revela muerte, por apoptosis, de las células satélite, confirmado con la técnica de TUNEL. El aumento de las apoptosis muscular no conlleva un incremento, por otra parte esperado, de la densidad de los macrófagos. El tratamiento con litio no mejora la evolución de la enfermedad en los ratones con AME. Se observa un incremento progresivo de los niveles de litio, provocando toxicidad en el animal. Por otra parte, el efecto del litio inhibiendo la GSK3 no se traduce en un aumento de la expresión de SMN, tal como se ha deducido de algunos experimentos publicados.
The spinal muscular atrophy (SMA) is a pediatric genetic disease. The SMA is a motor neuron disease that affects the motor neurons causing its death and muscle atrophy. The gene involved is the survival motor neuron (SMN) that is mutated in the 95% of the cases. Our study is divided into two parts: 1 – studies of the neuromuscular junction in two transgenic SMA murine models that develop the most severe forms of SMA (type 1-2) and 2 - study of the possible therapeutic effects of lithium on one of these models of SMA. We found severe alterations in the neuromuscular junctions of newborn animals and also in prenatal markers related to the vesicle docking at the presynaptic membrane, lack of organization of presynaptic calcium channels and defects in the expression of other presynaptic proteins. We found also, disruption and apoptosis of muscular cells, massive apoptosis of the thymus and widespread alterations in lymphoid organs. The ultrastructural study of muscle identifies apoptotic satellite cells that was confirmed by the TUNEL technique. The increase in apoptosis is not followed by the expected increase, in the macrophage density. Treatment with therapeutic concentrations of lithium does not improve the course of the disease in SMA mice. There was a progressive accumulation of lithium, causing toxicity in the animal. The effect of lithium inhibiting GSK3 does not determine an increased expression of SMN, as could be deduced from some published experiments.

Les Paraplégies Spastiques Héréditaires sont un groupe de maladies du motoneurone caractérisées par une dégénérescence de l’axe corticospinal menant à la spasticité et une paralysie progressive des membres parfois associés à des troubles cognitifs. Des mutations dans le gène SPG11 codant pour la Spatacsine sont la majeure cause de ces formes complexes de HSP. Pour mieux comprendre les mécanismes responsables de la pathologie liée à SPG11, notre équipe a généré un modèle de souris Knock-out pour ce gène, mimant les déficits cognitifs et moteurs observés chez les patients, corrélés à des altérations histologiques (J.Branchu & al. ; 2017). Etant donné que les troubles moteurs apparaissent avant les premières pertes neuronales, nous avons émis l’hypothèse qu’il existait des dysfonctions neuronales précédant la mort des neurones et mesurables par des techniques d’électrophysiologie. Des enregistrements EEG in vivo du cortex moteur des souris Spg11-/- nous ont permis d’observer l’émergence de décharges pointe-onde (DPO), survenant avant la mort des neurones corticaux NeuN+. Ces signaux sont semblables à ceux observés dans une forme particulière d’épilepsie : l’épilepsie d’absence et suggérent une perturbation de l’excitabilité corticale. Ces signaux ne semblent pas se propager dans le thalamus mais ces DPO répondent de manière dose dépendante à des drogues pro et anti Epilepsie d’Absence. Aucune différence significative n’a été observée dans le nombre d’interneurones GABAergiques, suggérant que l’inhibition corticale médiée par ces derniers n’est pas atteinte. Nous n’avons pas réussi à mettre en évidence de modification de l’expression de gènes liés à l’épilepsie d’absence. Cependant, des expériences de Patch clamp sur des neurones corticaux embryonnaires ont révélé une perturbation de la densité du courant sodique et d’excitabilité dans les neurones Spg11-/-. Des enregistrements ex vivo de l’hippocampe des souris Spg11-/- révèlent une perte de potentialisation à long et à court terme, corrélés avec une perte de mémoire spatiale, suggérant une atteinte d’un ou plusieurs éléments synaptiques. Ces pertes de plasticité hippocampiques ne sont pas observées durant le développement malgré ce qui semble être un défaut de synaptogénèse dans les collatérales de Schaffer. A terme, ces résultats vont étoffer nos connaissances sur les rôles de l’absence de Spatacsine dans la pathogénèse de l’HSP et des maladies du motoneurone et nous fourniront une mesure intéressante et non invasive (EEG) pour l’évaluation de l’efficacité de futur d’essais thérapeutiques
Hereditary Spastic Paraplegia is a group of Motor Neuron Disease characterized by the degeneration of cortico-spinal tract leading to a progressive spasticity and paralysis of lower limbs sometimes associated with cognitive deficits. Mutations in SPG11 gene coding for Spatacsin are a major cause of these complex forms. For a better understanding of SPG11-related HSP mechanisms, our team generated a Knock-Out mouse model (spg11-/-) mimicking the cognitive and motor deficits correlated with histological alterations (J.Branchu & al.; 2017). As motor impairments precede the first neuronal losses, we hypothesized that there may exist some neuronal dysfunctions primary to neuronal death observable with electrophysiological methods. In Vivo EEG recordings of spg11-/- motor cortex highlighted the emergence of spike and wave discharges events (SWD), occurring before the cortical NeuN+ cells loss and suggesting a disturbance of excitability of cortical networks. No propagation to thalamus was found, but these SWLD seems to response in a dose dependent manner to pro and anti-Absence Epilepsy drugs. With our IHC experiments, we didn’t observe any change in GABAergic interneurons number, suggesting no change in cortical inhibition mediated by interneurons. Ex vivo Electrophysiological recordings of adult spg11-/- hippocampi displayed reduced short and long-term potentiation, correlated with a loss of spatial and fear-related memories, suggesting an impairment in synaptic elements. We did not observe those alterations during development although there seem to be a shift from mature to immature dendritic spines. mRNA quantification couldn’t highlight any modification in epilepsy-related gene expression. However, in vitro intracellular recordings of embryonic cortical neurons revealed impairments in sodic current density and excitability in Spg11-/- neurons. Altogether, the results of these experiments will decipher the roles of Spatacsin in the pathogenesis of Motorneurons Diseases and give us a useful and non-invasive read-out for the evaluation of therapeutical assays

La Sclérose Latérale Amyotrophique (SLA) est une maladie neurodégénérative caractérisée par une perte sélective des motoneurones et impliquant les effets neurotoxiques des astrocytes. Le but de ce travail est d’explorer les altérations du métabolisme dans les astrocytes induites par des conditions associées à la SLA. Nous avons dans un premier temps mis en place une méthodologie d’analyse spectrométrique (résonance magnétique nucléaire et spectrométries de masse) du métabolome cellulaire. Ensuite, nous avons invalidé les cellules NSC-34 comme modèle in vitro d’étude de l’excitotoxicité induite par le glutamate. Nous avons enfin étudié les altérations métaboliques dans les astrocytes primaires dans des conditions de la SLA et décrit plusieurs dysfonctionnements métaboliques dans ces cellules induits par l’expression de la mutation SOD1G93A, par la présence des motoneurones sauvages et par l’exposition au glutamate. Ce travail met en évidence les relations métaboliques entre la SLA et le métabolisme énergétique cérébral. Nos résultats contribuent à la compréhension des altérations métaboliques des astrocytes dans la SLA et pourraient aider à appréhender de nouvelles cibles thérapeutiques associées aux altérations métaboliques dans la SLA, afin de protéger les motoneurones des perturbations induites par le glutamate
The selective degeneration of motoneuron that characterizes amyotrophic lateral sclerosis (ALS), implicates non-cell-autonomous effects of astrocytes. The aim of this work is to explore the metabolic status of astrocytes exposed to ALS-associated conditions, using metabolomics approach. We first, developed a methodology for the analysis of cellular metabolome using different analytical technologies, and then we evaluated the relevance of differentiated NSC-34 as an in vitro model for glutamate excitotoxicity studies. Finally, we evaluated metabolic alterations in astrocytes in ALS-associated conditions and we described several metabolic dysfunctions in these cells induced by the expression of a SOD1G93A mutation, the presence of wildtype motoneurons and glutamate exposition. These studies highlight major impacts of ALS on the brain energetic metabolism. This work provides novel insight for understanding the metabolic dysfunction of astrocytes in ALS conditions and opens perspective of therapeutics targets though focus on these metabolic ways, in order to protect motoneurons from glutamate injury

Koordinierte Phosphorylierung der C-terminale Domäne von RNA Polymerase II (RNAPII) ist essentiell für eine effiziente Kupplung von naszierender RNA Synthese und co-transkriptionalem RNA Prozessierens. Zirkuläre RNAs (circRNAs) sind eine neue Klasse von RNA Molekülen mit hoher Prävalenz in neuronalen Zelltypen. Die Biogenese von circRNAs ist noch ungeklärt, insbesondere die Frage warum das Intron upstream der circRNA während der Transkription des circRNA Exons zurückbehalten wird um Rück-Spleißen zu ermöglichen. Verschiede Belege suggerieren, dass unzulängliche Rekrutierung des Spleiceosoms zur circRNA Formation führen kann. In dieser Arbeit untersuche ich die Mechanismen die zu Defekten in der Erkennung und des Spleißens des Introns upstream der circRNA führen. Mit diesem Ziel erfasste ich die genomweite Verteilung von chromatinassoziierter RNAPII mit verschiedenen Phosphorylierungen, sowie Spleißfaktoren und Transkriptionsreglern mittels ChIP-seq in neuronaler Differenzierung von murinen embryonalen Stammzellen zu dopaminergen und Motoneuronen. Während der gesamten Differenzierung, aber insbesondere in den differenzieren Neuronen, konnten circRNAs detektiert werden. In meiner Arbeit finde ich, dass circRNAs detektiert werden, wenn Gene hohe Levels an mRNA exprimieren und, dass die Produktion von circRNA mit einer Dysbalance zwischen dem Laden der RNA-Polymerase II auf die DNA und dem Rekruitieren der Splice-Maschinerie zusammen hängt. Um funktionell mit den Pausier-Mechanismen der RNA-Polymerase II zu interferieren, habe ich einen ''promotor-proximal-pausing'' Faktor depletiert. Dabei stellte ich fest, dass diese Depletion genügt, um die circRNA Levels in embryonalen Stamzellen zu erhöhen. Die Ergebnisse die in dieser Arbeit gezeigt werden, beschreiben die Beteiligung des Pausierens der RNA-Polymerase II and der Formierung von circRNAs.
Coordinated phosphorylation of RNA polymerase II (RNAPII) C-terminal domain is essential for efficient coupling of nascent RNA synthesis with co-transcriptional RNA processing events. Circular RNAs (circRNAs) are a novel class of RNAs whose biogenesis remains ill understood, namely why the upstream intron is not spliced before the circRNA-exon is fully transcribed. Indirect evidence suggests that altered spliceosome recruitment can lead to circRNA formation. To investigate the mechanisms that may be involved in deficient recognition and splicing of introns upstream of exons included in circRNAs, I mapped the chromatin occupancy of RNAPII phosphorylated forms, splicing factors, and transcription regulators by ChIP-seq during mouse ESC differentiation to dopaminergic and spinal motor neurons. CircRNAs are detected throughout differentiation, peaking in differentiated neurons, as expected. I found that circRNAs are detected when genes express high levels of mRNA, and that circRNA production is associated with an imbalance between RNAPII loading and recruitment of the splicing machinery. To mechanistically interfere with pausing mechanisms, I depleted an RNAPII promoter-proximal pausing factor, and found that it was sufficient to increase the formation of circRNAs in stem cells. Results shown in this work implicate RNAPII regulation mechanisms in the formation of circRNAs.

碩士
高雄醫學大學
醫學研究所碩士班
93
Childhood spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder, characterized by degeneration of the anterior horn cells of the spinal cord and by symmetrical proximal muscle weakness and atrophy. Three forms of SMA have been recognized, based on varying clinical severity. The survival motor neuron (SMN) gene is present in humans in a telomeric copy, SMN1, and several centromeric copies, SMN2. Homozygous mutation of SMN1 is associated with SMA. We produced mouse lines deficient for mouse Smn and transgenic mouse lines that expressed human SMN2. In contrast, transgenic mice harboring SMN2 in the Smn-/- background showed pathological changes in the spinal cord and skeletal muscles similar to those of SMA patients. The variable phenotypes of Smn-/-SMN2 mice reflect those seen in SMA patients, providing a mouse model for this disease. To investigate the role of apoptosis in SMA. We sacrife the mice before dying on ice and collect the spinal cords from type I SMA mice. The amount of apoptosis was assessed by H&E stain, double labeling with TUNEL (TdT-mediated dUTP-biotin nick end labeling ) and Nisslstainings, and electron microscopy. The morphological changes in H&E staining revealed that chromatin condensation and fragmentation in the motorneuron of type I SMA mice spinal cord. The percentage of apoptosis was higher in SMA mice than in age-matched control mice and wild type mice. In contrast, the motorneuron numbers of spinal cord in thoracic and lumbar of type I IV SMA mice were less than two control groups. We hope this novel finding can provide a new insight to the understanding of the pathogenesis of SMA and the investigation of new therapies for this devastating illness of childhood.

The remodeling of the central nervous system (CNS) during metamorphosis in Drosophila melanogaster is a prime model system in which to study the genetic control of the sexual dimorphisms in the abdominal ganglion of the CNS. I have been using a P[tau-lacZ] enhancer trap line, 4.078, to label a segmentally repeated subset of abdominal motorneurons in order to assess the function of the sex determination hierarchy in controlling sex-specific development of the adult nervous system. In both the male and female larva there are 8 sets of these labeled abdominal motorneurons but only six sets in males and five sets in females survive in the adult. When this P[tau-lacZ] reporter construct is placed into a doublesex (dsx) mutant background, all 8 sets of these labeled abdominal motorneurons survive in both male and female adults. These results strongly suggest that dsx plays a role in the sex-specific survival of larval neurons that have functions in the adult. During the construction of mutant strains containing the sex determining genes transformer (tra) and transformer-2 (tra2), a genetic interactor was discovered in the P[tau-lacZ] 4.078 line. Female flies heterozygous for either tra or tra-2 alleles and the P[tau-lacZ] 4.078 developed with masculinized external and internal sex-specific structures. The external sex-specific structures, such as the genitalia, and ventral muscles are dependent on dsx gene function and a dorsal sex-specific muscle is dependent on fruitless (fru) gene function. From standard genetic crosses, I have characterized and demonstrated that the genetic interaction is linked to the P-element insertion site, which maps to the 85-87 region on the right arm of the third chromosome. By genetic analysis, this new genetic interactor appears to interfere with the tra and tra2 regulated female specific functions of both dsx and fru, potentially by reducing the female-specific splicing of the primary transcripts of the genes dsx and fru. To test the possibility that this newly described genetic interactor was allelic to a known gene, B52, that maps to the same region of the chromosome and alters dsx splicing, complementation tests were conducted which showed that the P[tau-lacZ] is not allelic B52. Additional phenotypes were observed in the crosses that first detected the interaction, suggesting that this newly described locus may affect other gene functions as well. Among the phenotypes observed were XX intersexes, male-female gynandromorphs (XX//XO mosaics), and non-disjunction events evident as XO males and XXY females. This new locus may represent a new member of the family of genes that influence regulated splicing events.
Graduation date: 1999

M. Tech. Chiro.
The purpose of this study was to determine the effect that C5 and C6 spinal manipulative therapy had on alpha motorneuron excitability of the musculocutaneous nerve in the biceps brachii muscle. Readings were recorded using Electromyography (EMG). Eighty asymptomatic subjects participated in this study. The subjects selected had to be between 18 and 35 years of age, having experienced no cervical pain, discomfort or pathology. The subjects had to present with a C5 or C6 joint motion restriction based on motion and static palpation. Patients were selected based on their response to advertisements posted throughout the Technikon Witwatersrand. Forty of the subjects were placed in the test group and received C5 or C6 joint manipulation once off. The other forty subjects were placed in the control group. EMG readings were taken before, at the time of, and after the spinal manipulative therapy, to ascertain whether the adjustment influenced the strength of the biceps brachii contraction and the reflexive action of the muscle. Pressure algometry was used on the biceps brachii as a subjective test on each patient. A repeated measure student’s t-test was performed, including a Normality and Equal Variance Test for control and trial groups. This study showed that spinal manipulative therapy momentarily increases alpha motorneuron excitability in the biceps brachii muscle in 71.4% of the patients with C5-C6 joint motion restrictions.
Dr. C. Yelverton Dr. C. J. Hay

La maladie d’Alzheimer est une maladie neurodégénérative déterminée par deux caractéristiques : les plaques extracellulaires composées d’amyloïde-β et l’accumulation intracellulaire de tau hyperphosphorylée, appelée enchevêtrements neurofibrillaires. Malgré le nombre important d’études, la nature de la toxicité des espèces tau hyperphosphorylée et hypophosphorylée reste mal connue. Notre projet de recherche vise à caractériser quel état de phosphorylation de la tau contribue le plus à la toxicité neuronale ainsi que d’identifier les mécanismes sous-jacents. Pour répondre à ces objectifs, nous avons généré des modèles transgéniques de C. elegans exprimant soit une tau hyperphosphorylée humaine (12 glutamates pour mimer l’hyperphosphorylation de la tau trouvée chez des patients Alzheimer), une tau sauvage, ou une tau hypophosphorylée (12 alanines pour mimer l’hypophosphorylation), dans les motoneurones GABAergiques. Ensuite, pour caractériser nos modèles, nous avons mesuré leur comportement principalement avec des tests de locomotion en utilisant le logiciel WormLab. Nos résultats ont montré que la tau phosphorylée est l’espèce la plus toxique car la souche hyperP a montré une perturbation du système locomoteur se traduisant par une neurodégénérescence ainsi que des problèmes développementaux (longueur des vers). Puis nous avons testé certains médicaments utilisés dans des modèles de tauopathies, afin d’identifier des voies biologiques impliquées dans la toxicité de la tau hyperphosphorylée. Pour conclure, nos modèles vont être des outils utiles pour identifier des modificateurs génétiques et pharmacologiques dans la toxicité de la tau.
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by two hallmarks: extracellular plaques composed of amyloid-β (Aβ) deposits and intraneuronal accumulation of hyper and abnormal phosphorylated tau, also called neurofibrillary tangles (NFT). Despite many decades of research, the nature hypophosphorylated or hyperphosphorylated Tau toxicity remains ill understood. Our research project aims to characterize which state of Tau phosphorylation contributes to neuronal toxicity and identify the underlying mechanisms. To assess these objectives, we generated transgenic C. elegans models expressing either a human hyperphosphorylated tau (incorporation of 12 glutamate residues to mimic Tau hyperphosphorylation found in AD’s patients) human wild type Tau, or a human hypophosphorylated tau (incorporation of 12 alanine residues to mimic Tau hypophosphorylation) in the GABAergic motoneurons. Then, to characterize our models, we measured their behavior mainly with locomotion’s test using WormLab software. Our results showed that hyperphosphorylation of tau is the most toxic species for our models because hyperP strain showed an impair in the locomotor system translating into neurodegeneration, as well as developmental problems such as worm length. Then we tested some drugs used in taupathies C. elegans models to see if we could identify some biological pathways implicated in the toxicity. To conclude, our models may be a useful tool to identify genetic and pharmacological modifiers of tau toxicity.

碩士
國立臺灣大學
藥理學研究所
88
Part I： 1.The assembly of fibronectin matrix is a key event in regulating cell adhesion, migration, and differentiation. The signal transduction pathway that regulates fibronectin assembly is still unclear. To elucidate the regulatory role of protein kinase in the formation of fibronectin matrix, we quantitatively examined the fibronectin assembly by fibroblast in Xenopus cell cultures using fluorescent immunocytochemistry. 2.Soluble form of bovine fibronectin was bath-applied to the cultures. Fibroblast changed the soluble form of fibronectin into immobilized form time-dependently. 3.Treatment with RGDS, Rhodostomin, genistein, cytochalasin D, H-7, Ro-31-8220 and forskolin inhibited the assembly of immobilized fibronectin. 4.When the fibronectin assembly was shortened to 1 hr, co-incubation with TPA significantly enhanced the formation of immobilized fibronectin. 5.The clustering of integrin and integrin were enhanced and inhibited by TPA and forskolin, respectively. 6.After one day’s incubation with soluble fibronectin to form immobilized fibronectin network, treatment with genistein, cytochalasin D, H-7 or forskolin for 30 min degraded the fibronectin networks, indicating that fibronectin fibrillogenesis is dynamic and modulated by protein kinases. 7.Aprotinin, leupeptin and matrix metalloproteinase inhibitor 1 (2μM) selectively antagonized the fibronectin matrix degradative action of forskolin, but not that caused by genistein, cytochalasin D and H-7. On the other hand, the higher concentration of matrix metalloproteinase inhibitor 1 (150μM) antagonized the fibronectin matrix degradative action caused by all these drugs. 8.These results suggest that the formation of fibronectin matrix beneath the fibronblast is dynamic. Cytoskeleton organization, tyrosine kinase and serine-threonine kinases are all involved in the regulation of fibronectin fibrillogenesis. Protein kinase C potentiates and protein kinase A inhibits the assembly of fibronectin matrix. Part II： 1.Numerous processes including secretion, gene expression and proliferation are controlled by hormones which act through the second messenger cAMP. Although cAMP was the first discovered second messenger of hormone action, much remains to be learned about its mode of action and the regulation of signaling pathways which utilize this second messenger. 2.Extracellular matrix (ECM) proteins such as fibronectin, laminin and collagen have been implicated in a wide variety of cellular properties which include cell adhesion, migration, differentiation and proliferation. The previous data of our laboratory showed that fibronectin and laminin markedly increased spontaneous ACh release via increasing PKA or PKC activity. The aim of the present study is to further analyze the modulation of protein kinase A activity in synaptic transmission by matrix proteins at developing motoneurons. 3.The cultures of spinal neurons and myotomal cells were prepared from 1-day-old Xenopus embryos. Spontaneous synaptic currents (SSCs) were recorded from innervated myocytes of natural synapses by whole-cell voltage-clamped recordings (Vh=-60~-65 mV). 4.Bath application of an adenylate cyclase activator forskolin (20 μM) resulted in significant enhancement of the SSC frequency in cultures grown on fibronectin substratum but not on laminin- or collagen-coated glass. 5.Bath application of a PKA activator DBcAMP (1mM) resulted in significant enhancement of the SSC frequency in cultures grown on fibronectin substratum but not on laminin- or collagen-coated glass. 6.Application of 3μM CGRP did not significantly affect the spontaneous ACh release in cultures grown on fibronectin substratum. 7.Bath application of a non-selective β agonist isoproterenol (10μM) resulted in significant enhancement of the SSC frequency in cultures grown on fibronectin substratum but not on laminin- or collagen-coated glass. 8.Bath application of a selective β2 agonist albuterol (10μM) resulted in significant enhancement of the SSC frequency in cultures grown on fibronectin substratum but not on laminin- or collagen-coated glass. 9.When cells were cultured on non-coated glasses, application of isoproterenol (10μM) or soluble fibronectin (6μg/ml) alone did not significantly affect the spontaneous ACh release. However, sequential application of soluble fibronectin and isoproterenol markedly increased the SSC frequency. 10.Bath application of BDNF (30 ng/ml), CNTF (150 ng/ml), GDNF (30 ng/ml) or NT-3 (50 ng/ml) was bath applied to 4 hr‘s cultures grown on fibronectin-coated glasses. After one day’s incubation, BDNF inhibited but CNTF, GDNF and NT-3 potentiated SSC enhancement in response to addition to DBcAMP. 11.In summary, these results suggest that fibronectin matrix protein potentiates the activation of PKA and may play an important role in regulating synaptic transmission at developing motoneuron.