The role of serotoninergic neurons in amyotrophic lateral sclerosis

La sclérose amyotrophique latérale (SLA) est une maladie neurodégénérative due à la dégénérescence des motoneurones supérieurs dans le cortex moteur et des motoneurones inférieurs situés dans la moelle épinière et le tronc cérébral. La perte des neurones moteurs provoque l’atrophie et la paralysie progressive des muscles. Notre laboratoire a démontré en 2013 que la dégénérescence associée à la SLA n’était pas limitée aux motoneurones mais aussi aux neurones sérotoninergiques chez les patients et les modèles animaux de SLA. L’objectif de ma thèse a été de caractériser le rôle des neurones sérotoninergiques dans la SLA. On a observé une augmentation de l’expression du gène codant pour le récepteur 2B de la sérotonine (5-HT2B) chez les modèles murins de SLA. L’analyse du rôle du récepteur 5-HT2B dans le cas de SLA a montré que ce dernier est un modulateur de la maladie. La perte du récepteur 5-HT2B accélère la progression de la maladie et modifie la régulation de la réponse inflammatoire. En plus, nos travaux ont révélé que la perte des neurones sérotoninergiques est à l’origine du développement de la spasticité, un symptôme douloureux chez les patients SLA. Ces résultats ouvrent la voie à des approches thérapeutiques qui ciblent cette population neuronale affectée lors de la maladie pour traiter la spasticité et la neuroinflammation au cours de la maladie.Amyotrophic lateral sclerosis (SLA) is a neurodegenerative disease characterize by the loss of upper motor neurons in the motor cortex and lower motor neurons in the brainstem and spinal cord. The loss of motor neurons leads to muscle atrophy and progressive paralysis. In 2013 our laboratory identified a new neuronal population affected in ALS. They observed the degeneration of serotoninergic neurons in ALS patients and animal models. For this, the aim of my PhD is to identify the role of serotoninergic neurons in case of ALS. We observed an upregulation of serotonin receptor 5-HT2B in ALS mice models. The investigation of the role of 5-HT2B receptor in case of ALS showed its role as a disease modulator. The loss of 5-HT2B receptor accelerated disease progression and modulated neuroinflammatory response. Moreover, our results showed that the loss of serotoninergic neurons is responsible of the development of spasticity, a painful symptom observed in ALS patients. All these opened the way for therapeutic strategies targeting spasticity and neuroinflammation in case of ALS

Rôle des neurones sérotoninergiques dans la sclérose latérale amyotrophique

Amyotrophic lateral sclerosis (SLA) is a neurodegenerative disease characterize by the loss of upper motor neurons in the motor cortex and lower motor neurons in the brainstem and spinal cord. The loss of motor neurons leads to muscle atrophy and progressive paralysis. In 2013 our laboratory identified a new neuronal population affected in ALS. They observed the degeneration of serotoninergic neurons in ALS patients and animal models. For this, the aim of my PhD is to identify the role of serotoninergic neurons in case of ALS. We observed an upregulation of serotonin receptor 5-HT2B in ALS mice models. The investigation of the role of 5-HT2B receptor in case of ALS showed its role as a disease modulator. The loss of 5-HT2B receptor accelerated disease progression and modulated neuroinflammatory response. Moreover, our results showed that the loss of serotoninergic neurons is responsible of the development of spasticity, a painful symptom observed in ALS patients. All these opened the way for therapeutic strategies targeting spasticity and neuroinflammation in case of ALS.La sclérose amyotrophique latérale (SLA) est une maladie neurodégénérative due à la dégénérescence des motoneurones supérieurs dans le cortex moteur et des motoneurones inférieurs situés dans la moelle épinière et le tronc cérébral. La perte des neurones moteurs provoque l’atrophie et la paralysie progressive des muscles. Notre laboratoire a démontré en 2013 que la dégénérescence associée à la SLA n’était pas limitée aux motoneurones mais aussi aux neurones sérotoninergiques chez les patients et les modèles animaux de SLA. L’objectif de ma thèse a été de caractériser le rôle des neurones sérotoninergiques dans la SLA. On a observé une augmentation de l’expression du gène codant pour le récepteur 2B de la sérotonine (5-HT2B) chez les modèles murins de SLA. L’analyse du rôle du récepteur 5-HT2B dans le cas de SLA a montré que ce dernier est un modulateur de la maladie. La perte du récepteur 5-HT2B accélère la progression de la maladie et modifie la régulation de la réponse inflammatoire. En plus, nos travaux ont révélé que la perte des neurones sérotoninergiques est à l’origine du développement de la spasticité, un symptôme douloureux chez les patients SLA. Ces résultats ouvrent la voie à des approches thérapeutiques qui ciblent cette population neuronale affectée lors de la maladie pour traiter la spasticité et la neuroinflammation au cours de la maladie

Protein Kinase A deregulation in the medial prefrontal cortex impairs working memory in murine Oligophrenin1 deficiency

Classical and systems genetics have identified wide networks of genes associated with cognitive and neurodevelopmental diseases. In parallel to deciphering the role of each of these genes in neuronal or synaptic function, evaluating the response of neuronal and molecular networks to gene loss-of-function could reveal some pathophysiological mechanisms potentially accessible to non-genetic therapies. Loss of function of the Rho-GAP Oligophrenin-1 is associated with cognitive impairments in both human and mouse. Up-regulation of both PKA and ROCK has been reported in Ophn1-/y mice, but it remains unclear if kinase hyperactivity contributes to the behavioural phenotypes. In this study, we thoroughly characterized a prominent perseveration phenotype displayed by Ophn1 deficient mice using a Y-maze spatial working memory (SWM) test. We report that Ophn1 deficiency in the mouse generated severe cognitive impairments, characterized by both a high occurrence of perseverative behaviours and a lack of deliberation during SWM test. In vivo and in vitro pharmacological experiments suggest that PKA dysregulation in the mPFC underlies cognitive dysfunction in Ophn1 deficient mice, as assessed using a delayed spatial alternation task results. Functionally, mPFC neuronal networks appeared to be affected in a PKA-dependent manner, whereas hippocampal-PFC projections involved in SWM were not affected in Ophn1-/y mice. Thus, we propose that discrete gene mutations in intellectual disability might generate "secondary" pathophysiological mechanisms, which are prone to become pharmacological targets for curative strategies in adult patients.SIGNIFICANCE STATEMENTHere we report that Ophn1 deficiency generates severe impairments in performance at spatial working memory tests, characterized by a high occurrence of perseverative behaviours and a lack of decision making. This cognitive deficit is consecutive to PKA deregulation in the mPFC that prevents Ophn1 KO mice to exploit a correctly acquired rule. Functionally, mPFC neuronal networks appear to be affected in a PKA-dependent manner, whereas behaviourally important hippocampal projections were preserved by the mutation. Thus, we propose that discrete gene mutations in intellectual disability can generate "secondary" pathophysiological mechanisms prone to become pharmacological targets for curative strategies in adults

CA3 hippocampal synaptic plasticity supports ripple physiology during memory consolidation

Consolidation of recent memory depends on hippocampal activities during resting periods that immediately follows the memory encoding. There, Slow Save Sleep phases appear as privileged periods for memory consolidation as hosting the ripple activities, which are fast oscillations generated within the hippocampus whose inactivation leads to memory impairment. If a strong correlation exists between these replays of recent experience and the persistence of behavioural adaptations, the mobilisation, the localization and the importance of synaptic plasticity events in this process is largely unknown. To question this issue, we used cell-surface AMPAR immobilisation to block post-synaptic LTP within the hippocampal region at various steps of the memory process. 1- Our results show that hippocampal synaptic plasticity is engaged during the consolidation but is dispensable during the encoding or recall of a working memory based spatial memory task. 2- Blockade of plasticity during sleep leads to apparent forgetting of the encoded rule. 3- In vivo recordings of ripple activities during resting periods show a strong impact of AMPAR immobilization solely, prominent when a rule has been recently encoded. 4- In situ examination of the interplay between AMPAR mobility, hippocampal plasticity and spontaneous ripple activities pointed that post-synaptic plasticity at CA3-CA3 recurrent synapses support ripple generation. As crucial results were reproduced using another AMPARM blockade strategy, we propose that after rule encoding, post-synaptic AMPAR mobility at CA3 recurrent synapses support the generation of ripples necessary for rule consolidation

VAPB/ALS8 MSP Ligands Regulate Striated Muscle Energy Metabolism Critical for Adult Survival in <i>Caenorhabditis elegans</i>

<div><p>Mutations in VAPB/ALS8 are associated with amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), two motor neuron diseases that often include alterations in energy metabolism. We have shown that <i>C. elegans</i> and Drosophila neurons secrete a cleavage product of VAPB, the N-terminal major sperm protein domain (vMSP). Secreted vMSPs signal through Roundabout and Lar-like receptors expressed on striated muscle. The muscle signaling pathway localizes mitochondria to myofilaments, alters their fission/fusion balance, and promotes energy production. Here, we show that neuronal loss of the <i>C. elegans</i> VAPB homolog triggers metabolic alterations that appear to compensate for muscle mitochondrial dysfunction. When vMSP levels drop, cytoskeletal or mitochondrial abnormalities in muscle induce elevated DAF-16, the Forkhead Box O (FoxO) homolog, transcription factor activity. DAF-16 promotes muscle triacylglycerol accumulation, increases ATP levels in adults, and extends lifespan, despite reduced muscle mitochondria electron transport chain activity. Finally, <i>Vapb</i> knock-out mice exhibit abnormal muscular triacylglycerol levels and FoxO target gene transcriptional responses to fasting and refeeding. Our data indicate that impaired vMSP signaling to striated muscle alters FoxO activity, which affects energy metabolism. Abnormalities in energy metabolism of ALS patients may thus constitute a compensatory mechanism counterbalancing skeletal muscle mitochondrial dysfunction.</p></div

DAF-16 localization and activity in wild-type and mutant worms.

<p>(A) Transgenic strains expressing DAF-16::GFP under its endogenous promoter. Transgenic controls raised at 20°C are similar to those raised at 20°C then shifted to 35°C for 30 minutes (see panel B for quantification). Close up images of boxed areas are shown. Anterior is to the left in all panels. Low magnification bar, 50 µm; high magnification bar, 25 µm. (B) Quantification of DAF-16::GFP localization in control (n = 157) and <i>vpr-1(tm1411)</i> mutants (n = 49). (−), incubation under normal growth condition; (+), incubation at 35°C for 30 minutes. (C) Magnified images showing transgenic lines expressing GFP under the <i>sod-3</i> promoter. <i>arx-2</i> encodes Arp2. Arrows indicate vulva muscle region. Anterior is to the left in all panels. Bar, 50 µm.</p

Fat levels in body wall muscle of wild-type and <i>vpr-1</i> mutant worms.

<p>(A) DIC images of muscle in live adult hermaphrodites. Arrowheads indicate lipid-like droplets. Bar, 5 µm. (B) Transmission electron micrographs of body wall muscle cytoplasm in wild-type and <i>vpr-1(tm1411)</i> mutant hermaphrodites. Light blue color demarcates muscle boundary. L, Lipid-like droplet. Bar, 0.5 µm. (C) Fluorescent images of muscle in live adult hermaphrodites fed Bodipy-FAs. Close-up images of boxed areas are shown below. Arrowheads indicate examples of Bodipy-FA-stained droplets. Anterior is to the left in all panels. Bars, 50 µm. (D) High magnification images of muscle showing Bodipy-FA-stained fluorescent droplets and droplets observed by DIC microscopy. Bar, 5 µm. (E) Comparison of total ion chromatograms of wild-type and <i>vpr-1(tm1411)</i> mutant adults extracts for 18∶0 TAG (Neutral Loss 284) and phosphatidylethanolamine (Neutral Loss 141).</p

Effect of tissue-specific <i>vpr-1</i> expression on fat levels.

<p>(A) DIC images of muscle in live wild-type and <i>vpr-1(tm1411)</i> mutant hermaphrodites expressing wild-type VPR-1 or VPR-1(P56S) under indicated tissue-specific promoters. Arrowheads indicate lipid-like droplets. Bar, 5 µm. (B) Sudan Black B staining images of <i>vpr-1</i> mutants expressing <i>vpr-1</i> under the <i>unc-119</i> pan-neuronal promoter. Arrows indicate muscle fat droplets. Anterior is to the left in all panels. Wild-type controls (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003738#pgen.1003738.s003" target="_blank">Figure S3</a>) are similar to transgenic <i>vpr-1(tm1411)</i> mutants expressing <i>unc119p::vpr-1</i>. Low magnification bars, 50 µm; high magnification bars, 25 µm.</p

Effect of Arp2/3 inactivation on muscle fat levels.

<p>DIC and fluorescent images of muscle in live 3-day-old hermaphrodite worms fed Bodipy-FAs. <i>arx-2</i> encodes the Arp2 component of the Arp2/3 complex. Arrowheads indicate Bodipy-FA-stained fat droplets. Bar, 5 µm.</p