Défauts intrinsèques de motoneurones spinaux dérivés de cellules souches pluripotentes induites issues d’individus atteints de différentes formes de Sclérose Latérale Amyotrophique

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder characterized by motor neurons death (MNs). Despite several hypothesis trying to explain this selective loss, the exact reasons of MNs degeneration remain unidentified mainly due to the disease heterogeneity. In this respect, the use of human induced pluripotent stem cells (iPSC) are opening up opportunities to model not only familial but also sporadic forms of ALS. In comparison to previously published studies, which focus only on one type of ALS mutation, my thesis had the objective to compare in a same experimental context multiple forms of ALS in order to distinguish similarities and discrepancies inherited by the mutation. Using iPSC obtained from genetic forms of ALS patients (C9ORF72, SOD1, TARDBP) as well as control subjects, we generated pure cultures of human MNs. While ALS MNs were not sensitive to death after few weeks of culture, electrophysiological functional studies revealed a patient-dependent late alteration in MNs excitability. Early defects were also reported, with observations of generic and mutation-specific protein aggregates. Interestingly, some accumulations were localized at the axonal initial segment (AIS) region, which is important for maintaining axonal identity and crucial for action potentials’ initiation. Physical and/or molecular alterations were reported at the AIS in ALS MNs, suggesting that AIS perturbation could be an early event in MN degeneration by disruption of ALS patients’ MNs integrity and functionality.La Sclérose Latérale Amyotrophique (SLA) est une maladie neurodégénérative caractérisée par la mort des motoneurones (MNs). Malgré plusieurs hypothèses pouvant expliquer les mécanismes à l’origine de leur mort sélective, l’hétérogénéité de la SLA rend difficile la compréhension des causes exactes de la dégénérescence. Dans ce contexte, les cellules souches pluripotentes induites humaines (iPSC) permettent l’étude des formes familiales de la maladie comme des formes sporadiques. Contrairement à la majorité des travaux publiés à ce jour qui étudient des iPSC de patients porteurs de mutation dans un seul gène de SLA, mon projet a eu pour objectif de comparer plusieurs formes de SLA dans un même contexte expérimental. A partir d’iPSC de patients présentant différentes formes génétiques de SLA (C9ORF72, SOD1, TARDBP), nous avons obtenu des cultures pures de MNs humains. Alors que nous n’avons pas observé de mort des MNs mutants après plusieurs semaines, des études fonctionnelles d’électrophysiologie ont montré une altération tardive de l’excitabilité des MNs en fonction des patients. De façon plus précoce, nous avons observé la présence d’agrégats protéiques communs ou spécifiques aux différentes formes de SLA, avec certaines accumulations localisées au niveau du segment proximal de l’axone, une région importante pour la maintenance de l’identité axonale et le déclenchement des potentiels d’action. Des altérations physiques ou moléculaires ont été mises en évidence au niveau de ce segment dans les MNs mutants, suggérant qu’une perturbation du segment proximal de l’axone pourrait être un évènement très précoce altérant ainsi l’intégrité et la fonctionnalité des MNs de patients

Comparison of motor neuron intrinsic defects in patients with various forms of Amyotrophic Lateral Sclerosis

La Sclérose Latérale Amyotrophique (SLA) est une maladie neurodégénérative caractérisée par la mort des motoneurones (MNs). Malgré plusieurs hypothèses pouvant expliquer les mécanismes à l’origine de leur mort sélective, l’hétérogénéité de la SLA rend difficile la compréhension des causes exactes de la dégénérescence. Dans ce contexte, les cellules souches pluripotentes induites humaines (iPSC) permettent l’étude des formes familiales de la maladie comme des formes sporadiques. Contrairement à la majorité des travaux publiés à ce jour qui étudient des iPSC de patients porteurs de mutation dans un seul gène de SLA, mon projet a eu pour objectif de comparer plusieurs formes de SLA dans un même contexte expérimental. A partir d’iPSC de patients présentant différentes formes génétiques de SLA (C9ORF72, SOD1, TARDBP), nous avons obtenu des cultures pures de MNs humains. Alors que nous n’avons pas observé de mort des MNs mutants après plusieurs semaines, des études fonctionnelles d’électrophysiologie ont montré une altération tardive de l’excitabilité des MNs en fonction des patients. De façon plus précoce, nous avons observé la présence d’agrégats protéiques communs ou spécifiques aux différentes formes de SLA, avec certaines accumulations localisées au niveau du segment proximal de l’axone, une région importante pour la maintenance de l’identité axonale et le déclenchement des potentiels d’action. Des altérations physiques ou moléculaires ont été mises en évidence au niveau de ce segment dans les MNs mutants, suggérant qu’une perturbation du segment proximal de l’axone pourrait être un évènement très précoce altérant ainsi l’intégrité et la fonctionnalité des MNs de patients.Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder characterized by motor neurons death (MNs). Despite several hypothesis trying to explain this selective loss, the exact reasons of MNs degeneration remain unidentified mainly due to the disease heterogeneity. In this respect, the use of human induced pluripotent stem cells (iPSC) are opening up opportunities to model not only familial but also sporadic forms of ALS. In comparison to previously published studies, which focus only on one type of ALS mutation, my thesis had the objective to compare in a same experimental context multiple forms of ALS in order to distinguish similarities and discrepancies inherited by the mutation. Using iPSC obtained from genetic forms of ALS patients (C9ORF72, SOD1, TARDBP) as well as control subjects, we generated pure cultures of human MNs. While ALS MNs were not sensitive to death after few weeks of culture, electrophysiological functional studies revealed a patient-dependent late alteration in MNs excitability. Early defects were also reported, with observations of generic and mutation-specific protein aggregates. Interestingly, some accumulations were localized at the axonal initial segment (AIS) region, which is important for maintaining axonal identity and crucial for action potentials’ initiation. Physical and/or molecular alterations were reported at the AIS in ALS MNs, suggesting that AIS perturbation could be an early event in MN degeneration by disruption of ALS patients’ MNs integrity and functionality

Structure-based optimization of a PDZ binding motif within a viral peptide stimulates neurite outgrowth

International audienceProtection of neuronal homeostasis is a major goal in the management of neurodegenerative diseases. Microtubule-associated Ser/Thre kinase 2 (MAST2), inhibits neurite outgrowth, and its inhibition therefore represents a potential therapeutic strategy. We previously reported that a viral protein (G-protein from rabies virus) capable of interfering with protein-protein interactions between the PDZ domain of MAST2 and the C-terminal moieties of its cellular partners counteracts MAST2-mediated suppression of neurite outgrowth. Here, we designed peptides derived from the native viral protein to increase the affinity of these peptides for the MAST2 PDZ domain. Our strategy involved modifying the length and flexibility of the non-interacting sequence linking the two subsites anchoring the peptide to the PDZ domain. Three peptides, Neurovita1 (NV1), NV2, and NV3, were selected, and we found that they all had increased affinities for the MAST2 PDZ domain, with Kd values decreasing from 1300 to 60 nM, while target selectivity was maintained. A parallel biological assay evaluating neurite extension and branching in cell cultures revealed that the NV peptides gradually improved neural activity, with the efficacies of these peptides for stimulating neurite outgrowth mirroring their affinities for MAST2-PDZ. We also show that NVs can be delivered into the cytoplasm of neurons as a gene or peptide. In summary, our findings indicate that virus-derived peptides targeted to MAST2-PDZ stimulate neurite outgrowth in several neuron types, opening up promising avenues for potentially using NVs in the management of neurodegenerative diseases

HDAC6 inhibition reverses axonal transport defects in motor neurons derived from FUS-ALS patients

International audienceAmyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disorder due to selective loss of motor neurons (MNs). Mutations in the fused in sarcoma (FUS) gene can cause both juvenile and late onset ALS. We generated and characterized induced pluripotent stem cells (iPSCs) from ALS patients with different FUS mutations, as well as from healthy controls. Patient-derived MNs show typical cytoplasmic FUS pathology, hypoexcitability, as well as progressive axonal transport defects. Axonal transport defects are rescued by CRISPR/Cas9-mediated genetic correction of the FUS mutation in patient-derived iPSCs. Moreover, these defects are reproduced by expressing mutant FUS in human embryonic stem cells (hESCs), whereas knockdown of endogenous FUS has no effect, confirming that these pathological changes are mutant FUS dependent. Pharmacological inhibition as well as genetic silencing of histone deacetylase 6 (HDAC6) increase α-tubulin acetylation, endo-plasmic reticulum (ER)–mitochondrial overlay, and restore the axonal transport defects in patient-derived MNs

HDAC6 inhibition reverses axonal transport defects in motor neurons derived from FUS-ALS patients

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disorder due to selective loss of motor neurons (MNs). Mutations in the fused in sarcoma (FUS) gene can cause both juvenile and late onset ALS. We generated and characterized induced pluripotent stem cells (iPSCs) from ALS patients with different FUS mutations, as well as from healthy controls. Patient-derived MNs show typical cytoplasmic FUS pathology, hypoexcitability, as well as progressive axonal transport defects. Axonal transport defects are rescued by CRISPR/Cas9-mediated genetic correction of the FUS mutation in patient-derived iPSCs. Moreover, these defects are reproduced by expressing mutant FUS in human embryonic stem cells (hESCs), whereas knockdown of endogenous FUS has no effect, confirming that these pathological changes are mutant FUS dependent. Pharmacological inhibition as well as genetic silencing of histone deacetylase 6 (HDAC6) increase α-tubulin acetylation, endoplasmic reticulum (ER)-mitochondrial overlay, and restore the axonal transport defects in patient-derived MNs.Amyotrophic lateral sclerosis (ALS) leads to selective loss of motor neurons. Using motor neurons derived from induced pluripotent stem cells from patients with ALS and FUS mutations, the authors demonstrate that axonal transport deficits that are observed in these cells can be rescued by HDAC6 inhibition.status: publishe

Characteristics and outcomes of an international cohort of 600 000 hospitalized patients with COVID-19

Background: We describe demographic features, treatments and clinical outcomes in the International Severe Acute Respiratory and emerging Infection Consortium (ISARIC) COVID-19 cohort, one of the world's largest international, standardized data sets concerning hospitalized patients. Methods: The data set analysed includes COVID-19 patients hospitalized between January 2020 and January 2022 in 52 countries. We investigated how symptoms on admission, co-morbidities, risk factors and treatments varied by age, sex and other characteristics. We used Cox regression models to investigate associations between demographics, symptoms, co-morbidities and other factors with risk of death, admission to an intensive care unit (ICU) and invasive mechanical ventilation (IMV). Results: Data were available for 689 572 patients with laboratory-confirmed (91.1%) or clinically diagnosed (8.9%) SARS-CoV-2 infection from 52 countries. Age [adjusted hazard ratio per 10 years 1.49 (95% CI 1.48, 1.49)] and male sex [1.23 (1.21, 1.24)] were associated with a higher risk of death. Rates of admission to an ICU and use of IMV increased with age up to age 60 years then dropped. Symptoms, co-morbidities and treatments varied by age and had varied associations with clinical outcomes. The case-fatality ratio varied by country partly due to differences in the clinical characteristics of recruited patients and was on average 21.5%. Conclusions: Age was the strongest determinant of risk of death, with a ∼30-fold difference between the oldest and youngest groups; each of the co-morbidities included was associated with up to an almost 2-fold increase in risk. Smoking and obesity were also associated with a higher risk of death. The size of our international database and the standardized data collection method make this study a comprehensive international description of COVID-19 clinical features. Our findings may inform strategies that involve prioritization of patients hospitalized with COVID-19 who have a higher risk of death

The value of open-source clinical science in pandemic response: lessons from ISARIC

International audienc