AAV-mediated expression of wild-type and ALS-linked mutant VAPB selectively triggers death of motoneurons through a Ca2+-dependent ER-associated pathway

A dominant mutation in the gene coding for the vesicle-associated membrane protein-associated protein B (VAPB) was associated with amyotrophic lateral sclerosis, a fatal paralytic disorder characterized by the selective loss of motoneurons in the brain and spinal cord. Adeno-associated viral vectors that we show to transduce up to 90% of motoneurons in vitro were used to model VAPB-associated neurodegenerative process. We observed that Adeno-associated viral-mediated over-expression of both wild-type and mutated form of human VAPB selectively induces death of primary motoneurons, albeit with different kinetics. We provide evidence that ER stress and impaired homeostatic regulation of calcium (Ca2+) are implicated in the death process. Finally, we found that completion of the motoneuron death program triggered by the over-expression of wild-type and mutant VAPB implicates calpains, caspase 12 and 3. Our viral-based in vitro model, which recapitulates the selective vulnerability of motoneurons to the presence of mutant VAPB and also to VAPB gene dosage effect, identifies aberrant Ca2+ signals and ER-derived death pathways as important events in the motoneuron degenerative process

Necdin Protects Embryonic Motoneurons from Programmed Cell Death

NECDIN belongs to the type II Melanoma Associated Antigen Gene Expression gene family and is located in the Prader-Willi Syndrome (PWS) critical region. Necdin-deficient mice develop symptoms of PWS, including a sensory and motor deficit. However, the mechanisms underlying the motor deficit remain elusive. Here, we show that the genetic ablation of Necdin, whose expression is restricted to post-mitotic neurons in the spinal cord during development, leads to a loss of 31% of specified motoneurons. The increased neuronal loss occurs during the period of naturally-occurring cell death and is not confined to specific pools of motoneurons. To better understand the role of Necdin during the period of programmed cell death of motoneurons we used embryonic spinal cord explants and primary motoneuron cultures from Necdin-deficient mice. Interestingly, while Necdin-deficient motoneurons present the same survival response to neurotrophic factors, we demonstrate that deletion of Necdin leads to an increased susceptibility of motoneurons to neurotrophic factor deprivation. We show that by neutralizing TNFα this increased susceptibility of Necdin-deficient motoneurons to trophic factor deprivation can be reduced to the normal level. We propose that Necdin is implicated through the TNF-receptor 1 pathway in the developmental death of motoneurons

Neurodegenerative mechanisms associated with the inflammatory process in amyotrophic lateral sclerosis

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative incurable, qui touche les motoneurones de la moelle épinière et du cerveau. Elle se manifeste par une faiblesse musculaire qui évolue rapidement vers une paralysie générale, entrainant la mort du patient. Les principes moléculaires conduisant à la dégénérescence sélective des motoneurones demeurent encore mal connus, entravant le développement de nouvelles thérapies. Mon travail de thèse a permis l'identification d'une nouvelle voie de mort spécifique aux motoneurones, qui dépend du récepteur LT-βR et de son ligand LIGHT. De plus, cette voie de mort peut être déclenchée par une cytokine pro-inflammatoire, qui est l'interféron gamma (IFNγ). Nous avons pu montrer des signes d'activation de cette voie de mort chez des souris modèles de la SLA ainsi que dans les tissus de patients atteints de la maladie. En effet, on observe au cours de la maladie une augmentation des niveaux d'IFNγ dans les astrocytes et les motoneurones. Une approche génétique a par la suite permis de démontrer l'implication fonctionnelle de cette voie de mort dans le processus pathologique.Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease affecting primarily motoneurons in the brain and spinal cord. Symptoms of the disease include general muscle weakness, rapidly evolving in an overall paralysis, leading to the death of the patient. The precise mechanisms responsible for the selective vulnerability of motoneurons remain largely unknown, impeding therefore the development of effective therapies. My thesis work led to the discovery of a novel motoneuron selective death pathway dependent on the activation of LT-βR by LIGHT. This death pathway might also be triggered by the pro-inflammatory cytokine interferon gamma (IFNγ). Interestingly, we have documented signs of activation of this pathway in ALS mice and sporadic ALS patients, with IFNγ being upregulated in astrocytes and motoneurons. Furthermore, a genetic approach has provided evidence of the functional involvement of this death pathway in the pathogenic process

Mécanismes de neurodégénérescence associés au processus inflammatoire dans la sclérose latérale amyotrophique

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative incurable, qui touche les motoneurones de la moelle épinière et du cerveau. Elle se manifeste par une faiblesse musculaire qui évolue rapidement vers une paralysie générale, entrainant la mort du patient. Les principes moléculaires conduisant à la dégénérescence sélective des motoneurones demeurent encore mal connus, entravant le développement de nouvelles thérapies. Mon travail de thèse a permis l'identification d'une nouvelle voie de mort spécifique aux motoneurones, qui dépend du récepteur LT-βR et de son ligand LIGHT. De plus, cette voie de mort peut être déclenchée par une cytokine pro-inflammatoire, qui est l'interféron gamma (IFNγ). Nous avons pu montrer des signes d'activation de cette voie de mort chez des souris modèles de la SLA ainsi que dans les tissus de patients atteints de la maladie. En effet, on observe au cours de la maladie une augmentation des niveaux d'IFNγ dans les astrocytes et les motoneurones. Une approche génétique a par la suite permis de démontrer l'implication fonctionnelle de cette voie de mort dans le processus pathologique.Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease affecting primarily motoneurons in the brain and spinal cord. Symptoms of the disease include general muscle weakness, rapidly evolving in an overall paralysis, leading to the death of the patient. The precise mechanisms responsible for the selective vulnerability of motoneurons remain largely unknown, impeding therefore the development of effective therapies. My thesis work led to the discovery of a novel motoneuron selective death pathway dependent on the activation of LT-βR by LIGHT. This death pathway might also be triggered by the pro-inflammatory cytokine interferon gamma (IFNγ). Interestingly, we have documented signs of activation of this pathway in ALS mice and sporadic ALS patients, with IFNγ being upregulated in astrocytes and motoneurons. Furthermore, a genetic approach has provided evidence of the functional involvement of this death pathway in the pathogenic process.AIX-MARSEILLE2-Bib.electronique (130559901) / SudocSudocFranceF

Bicistronic aav vector for rna interference in als

The present invention relates to a bicistronic expression vector for silencing a gene specifically in astrocytes and neurons, comprising two expression cassettes comprising a first and a second silencer sequence, respectively, wherein the expression of said first silencer sequence within astrocytes is regulated by an astrocyte-specific promoter and the expression of said second silencer sequence within neurons is regulated by a neuron-specific promoter. In a preferred embodiment, said first and second silencer sequences are SOD1 silencer sequences. Pharmaceutical composition comprising said bicistronic vector and the use of the same in the treatment of motoneuron diseases are further described

Cerebrospinal fluid-targeted delivery of neutralizing anti-IFN gamma antibody delays motor decline in an ALS mouse model

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder characterized by the selective and gradual loss of motoneurons in the brain and spinal cord. A persistent inflammation, typified by the activation of astrocytes and microglia, accompanies the progressive degeneration of motoneurons. Interferon gamma (IFN), a potent proinflammatory cytokine that is aberrantly present in the spinal cord of ALS mice and patients, has been proposed to contribute to motoneuron death by eliciting the activation of the lymphotoxin- receptor (LT-R) through its ligand LIGHT. However, the implication of IFN in the pathogenic process remains elusive. Here, we show that an antagonistic anti-IFN antibody efficiently rescues motoneurons from IFN-induced death. When transiently delivered in the cerebrospinal fluid through a subcutaneously implanted osmotic minipump, the neutralizing anti-IFN antibody significantly retarded motor function decline in a mouse model of ALS. However, this transient infusion of anti-IFN antibody did not increase the life expectancy of ALS mice. Our results suggest that IFN contributes to ALS pathogenesis and represents a potential therapeutic target for ALS. (C) 2013 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins

Somatic and axonal LIGHT signaling elicit degenerative and regenerative responses in motoneurons, respectively

A receptor-ligand interaction can evoke a broad range of biological activities in different cell types depending on receptor identity and cell type-specific post-receptor signaling intermediates. Here, we show that the TNF family member LIGHT, known to act as a death-triggering factor in motoneurons through LT-beta R, can also promote axon outgrowth and branching in motoneurons through the same receptor. LIGHT-induced axonal elongation and branching require ERK and caspase-9 pathways. This distinct response involves a compartment-specific activation of LIGHT signals, with somatic activation-inducing death, while axonal stimulation promotes axon elongation and branching in motoneurons. Following peripheral nerve damage, LIGHT increases at the lesion site through expression by invading B lymphocytes, and genetic deletion of Light significantly delays functional recovery. We propose that a central and peripheral activation of the LIGHT pathway elicits different functional responses in motoneurons

Dysregulated miRNA biogenesis downstream of cellular stress and ALS-causing mutations: a new mechanism for ALS

Interest in RNA dysfunction in amyotrophic lateral sclerosis (ALS) recently aroused upon discovering causative mutations in RNA-binding protein genes. Here, we show that extensive down-regulation of miRNA levels is a common molecular denominator for multiple forms of human ALS. We further demonstrate that pathogenic ALS-causing mutations are sufficient to inhibit miRNA biogenesis at the Dicing step. Abnormalities of the stress response are involved in the pathogenesis of neurodegeneration, including ALS. Accordingly, we describe a novel mechanism for modulating microRNA biogenesis under stress, involving stress granule formation and re-organization of DICER and AGO2 protein interactions with their partners. In line with this observation, enhancing DICER activity by a small molecule, enoxacin, is beneficial for neuromuscular function in two independent ALS mouse models. Characterizing miRNA biogenesis downstream of the stress response ties seemingly disparate pathways in neurodegeneration and further suggests that DICER and miRNAs affect neuronal integrity and are possible therapeutic targets