Auswahl der wissenschaftlichen Literatur 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 &gt;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<br>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 &gt;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.<br>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.