Pathogenic commonalities between spinal muscular atrophy and amyotrophic lateral sclerosis:Converging roads to therapeutic development

Spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS) are the two most common motoneuron disorders, which share typical pathological hallmarks while remaining genetically distinct. Indeed, SMA is caused by deletions or mutations in the survival motor neuron 1 (SMN1) gene whilst ALS, albeit being mostly sporadic, can also be caused by mutations within genes, including superoxide dismutase 1 (SOD1), Fused in Sarcoma (FUS), TAR DNA-binding protein 43 (TDP-43) and chromosome 9 open reading frame 72 (C9ORF72). However, it has come to light that these two diseases may be more interlinked than previously thought. Indeed, it has recently been found that FUS directly interacts with an Smn-containing complex, mutant SOD1 perturbs Smn localization, Smn depletion aggravates disease progression of ALS mice, overexpression of SMN in ALS mice significantly improves their phenotype and lifespan, and duplications of SMN1 have been linked to sporadic ALS. Beyond genetic interactions, accumulating evidence further suggests that both diseases share common pathological identities such as intrinsic muscle defects, neuroinflammation, immune organ dysfunction, metabolic perturbations, defects in neuron excitability and selective motoneuron vulnerability. Identifying common molecular effectors that mediate shared pathologies in SMA and ALS would allow for the development of therapeutic strategies and targeted gene therapies that could potentially alleviate symptoms and be equally beneficial in both disorders. In the present review, we will examine our current knowledge of pathogenic commonalities between SMA and ALS, and discuss how furthering this understanding can lead to the establishment of novel therapeutic approaches with wide-reaching impact on multiple motoneuron diseases

RNAseq Analyses Identify Tumor Necrosis Factor-Mediated Inflammation as a Major Abnormality in ALS Spinal Cord

ALS is a rapidly progressive, devastating neurodegenerative illness of adults that produces disabling weakness and spasticity arising from death of lower and upper motor neurons. No meaningful therapies exist to slow ALS progression, and molecular insights into pathogenesis and progression are sorely needed. In that context, we used high-depth, next generation RNA sequencing (RNAseq, Illumina) to define gene network abnormalities in RNA samples depleted of rRNA and isolated from cervical spinal cord sections of 7 ALS and 8 CTL samples. We aligned \u3e50 million 2X150 bp paired-end sequences/sample to the hg19 human genome and applied three different algorithms (Cuffdiff2, DEseq2, EdgeR) for identification of differentially expressed genes (DEG’s). Ingenuity Pathways Analysis (IPA) and Weighted Gene Co-expression Network Analysis (WGCNA) identified inflammatory processes as significantly elevated in our ALS samples, with tumor necrosis factor (TNF) found to be a major pathway regulator (IPA) and TNFα-induced protein 2 (TNFAIP2) as a major network “hub” gene (WGCNA). Using the oPOSSUM algorithm, we analyzed transcription factors (TF) controlling expression of the nine DEG/hub genes in the ALS samples and identified TF’s involved in inflammation (NFkB, REL, NFkB1) and macrophage function (NR1H2::RXRA heterodimer). Transient expression in human iPSC-derived motor neurons of TNFAIP2 (also a DEG identified by all three algorithms) reduced cell viability and induced caspase 3/7 activation. Using high-density RNAseq, multiple algorithms for DEG identification, and an unsupervised gene co-expression network approach, we identified significant elevation of inflammatory processes in ALS spinal cord with TNF as a major regulatory molecule. Overexpression of the DEG TNFAIP2 in human motor neurons, the population most vulnerable to die in ALS, increased cell death and caspase 3/7 activation. We propose that therapies targeted to reduce inflammatory TNFα signaling may be helpful in ALS patients

Muscle cells of sporadic amyotrophic lateral sclerosis patients secrete neurotoxic vesicles

BACKGROUND: The cause of the motor neuron (MN) death that drives terminal pathology in amyotrophic lateral sclerosis (ALS) remains unknown, and it is thought that the cellular environment of the MN may play a key role in MN survival. Several lines of evidence implicate vesicles in ALS, including that extracellular vesicles may carry toxic elements from astrocytes towards MNs, and that pathological proteins have been identified in circulating extracellular vesicles of sporadic ALS patients. Because MN degeneration at the neuromuscular junction is a feature of ALS, and muscle is a vesicle-secretory tissue, we hypothesized that muscle vesicles may be involved in ALS pathology. METHODS: Sporadic ALS patients were confirmed to be ALS according to El Escorial criteria and were genotyped to test for classic gene mutations associated with ALS, and physical function was assessed using the ALSFRS-R score. Muscle biopsies of either mildly affected deltoids of ALS patients (n = 27) or deltoids of aged-matched healthy subjects (n = 30) were used for extraction of muscle stem cells, to perform immunohistology, or for electron microscopy. Muscle stem cells were characterized by immunostaining, RT-qPCR, and transcriptomic analysis. Secreted muscle vesicles were characterized by proteomic analysis, Western blot, NanoSight, and electron microscopy. The effects of muscle vesicles isolated from the culture medium of ALS and healthy myotubes were tested on healthy human-derived iPSC MNs and on healthy human myotubes, with untreated cells used as controls. RESULTS: An accumulation of multivesicular bodies was observed in muscle biopsies of sporadic ALS patients by immunostaining and electron microscopy. Study of muscle biopsies and biopsy-derived denervation-naïve differentiated muscle stem cells (myotubes) revealed a consistent disease signature in ALS myotubes, including intracellular accumulation of exosome-like vesicles and disruption of RNA-processing. Compared with vesicles from healthy control myotubes, when administered to healthy MNs the vesicles of ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA-processing proteins. The RNA-processing protein FUS and a majority of its binding partners were present in ALS muscle vesicles, and toxicity was dependent on the expression level of FUS in recipient cells. Toxicity to recipient MNs was abolished by anti-CD63 immuno-blocking of vesicle uptake. CONCLUSIONS: ALS muscle vesicles are shown to be toxic to MNs, which establishes the skeletal muscle as a potential source of vesicle-mediated toxicity in ALS

Muscle cells of sporadic amyotrophic lateral sclerosis patients secrete neurotoxic vesicles

Background: The cause of the motor neuron (MN) death that drives terminal pathology in amyotrophic lateral sclerosis (ALS) remains unknown, and it is thought that the cellular environment of the MN may play a key role in MN survival. Several lines of evidence implicate vesicles in ALS, including that extracellular vesicles may carry toxic elements from astrocytes towards MNs, and that pathological proteins have been identified in circulating extracellular vesicles of sporadic ALS patients. Because MN degeneration at the neuromuscular junction is a feature of ALS, and muscle is a vesicle-secretory tissue, we hypothesized that muscle vesicles may be involved in ALS pathology. Methods: Sporadic ALS patients were confirmed to be ALS according to El Escorial criteria and were genotyped to test for classic gene mutations associated with ALS, and physical function was assessed using the ALSFRS-R score. Muscle biopsies of either mildly affected deltoids of ALS patients (n = 27) or deltoids of aged-matched healthy subjects (n = 30) were used for extraction of muscle stem cells, to perform immunohistology, or for electron microscopy. Muscle stem cells were characterized by immunostaining, RT-qPCR, and transcriptomic analysis. Secreted muscle vesicles were characterized by proteomic analysis, Western blot, NanoSight, and electron microscopy. The effects of muscle vesicles isolated from the culture medium of ALS and healthy myotubes were tested on healthy human-derived iPSC MNs and on healthy human myotubes, with untreated cells used as controls. Results: An accumulation of multivesicular bodies was observed in muscle biopsies of sporadic ALS patients by immunostaining and electron microscopy. Study of muscle biopsies and biopsy-derived denervation-naïve differentiated muscle stem cells (myotubes) revealed a consistent disease signature in ALS myotubes, including intracellular accumulation of exosome-like vesicles and disruption of RNA-processing. Compared with vesicles from healthy control myotubes, when administered to healthy MNs the vesicles of ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA-processing proteins. The RNA-processing protein FUS and a majority of its binding partners were present in ALS muscle vesicles, and toxicity was dependent on the expression level of FUS in recipient cells. Toxicity to recipient MNs was abolished by anti-CD63 immuno-blocking of vesicle uptake. Conclusions: ALS muscle vesicles are shown to be toxic to MNs, which establishes the skeletal muscle as a potential source of vesicle-mediated toxicity in ALS

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

Expression of a dominant negative form of Daxx in vivo rescues motoneurons from Fas (CD95)-induced cell death.

International audienceFas-induced death of motoneurons in vitro has been shown to involve two signaling cascades that act together to execute the death program: a Fas-Daxx-ASK-1-p38 kinase-nNOS branch, which controls transcriptional and post-translational events, and the second classical Fas-FADD-caspase-8 branch. To analyze the role of Daxx in the developmental motoneuron cell death, we studied Fas-dependent cell death in motoneurons from transgenic mice that overexpress a dominant-negative form of Daxx. Motoneurons purified from these transgenic mice are resistant to Fas-induced death. This protective effect is specific to Fas because ultraviolet irradiation-triggered death is not affected by the transgene. The Daxx and the FADD pathways work in parallel because only Daxx, but not FADD, is involved in the transcriptional control of neuronal nitric oxide synthase and nitric oxide production. Nevertheless, we do not observe involvement of Daxx in developmental motoneuronal cell death, as the pattern of naturally occurring programmed cell death in vivo is normal in transgenic mice overexpressing the dominant negative form of Daxx, suggesting that Daxx-independent pathways are used during development

Modeling the Morphology and Mechanical Behavior of Shape memory Polyurethanes based on Solid State NMR and Synchrotron SAXS/WAXD

status: publishe

Theme 4 In vivo experimental models

International audienceBackground: In 90% of Amyotrophic Lateral Sclerosis (ALS) cases, the disease is sporadic, the remaining 10% being familial. Many genes have been associated with the disease. The use of next generation sequencing has allowed increasing the number of genes analysed in routine diagnostics. However, this increase raises the issue of genetic variants interpretation within a growing number of ALS-associated-genes. Variant classification is based on a combinatory analysis of multiple factors. Among them, functional analyses provide strong arguments on pathogenicity interpretation.Objectives: We developed a simple animal model, the Zebrafish, for the functional analysis of candidate variants pathogenicity identified by routine genetic testing.Methods: Transient overexpression of different ALS associated genetic variants has been performed by mRNA injection in 1-cell stage zebrafish eggs. Validation of protein overexpression has been done by western blot. Embryos mortality, developmental delay and morphological abnormalities have been assessed within the first two days of development. Cellular phenotype has been investigated by the analysis of axonal length of 2-days old larvae with confocal microscopy. Motor phenotype of 5-days old larvae has been explored by touched-evoked response assay.Results: The model has been validated by the analysis of well-described ALS mutations, SOD1-Gly93Ala and OPTN Glu478Gly. Overexpression of this mutated protein was shown to provoke a shortening of axons and a premature axonal branching, as well as an impairment of motor performances as expected. We did not observe these aberrations in SOD1-WT injected fishes. Two candidate variants observed in ALS-patients have been explored with our model: SOD1 NM_000454.4:c.400_402del, p.Glu134del and OPTN NM_021980.4:c.1475T > G, p. Leu492Arg. Overexpression of both variants induced morphological abnormalities and motor impairment, suggesting a pathogenic involvement of these variants in ALS-patients.Discussion and conclusions: We developed for the first time a simple animal model, the Zebrafish, useful for the functional analysis of variant pathogenicity in order to assist ALS molecular diagnosis. Our model has been used to assess the pathogenicity of SOD1 and OPTN candidate variants, allowing to improve genetic testing interpretation

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

Fetal hemoglobin and hydroxycarbamide modulate both plasma concentration and cellular origin of circulating microparticles in sickle cell anemia children

International audienceMicroparticles are cell membrane-derived microvesicles released during cell apoptosis and activation processes. They have been described as bio-markers in various vascular diseases, including sickle cell anemia, and associated with an increased risk of thrombosis. We investigated the effects of fetal hemoglobin level, a factor known to modulate the clinical expression of sickle cell anemia, and that of hydroxycarbamide treatment which reduces the frequency of vaso-occlusive crises, the canonical clinical manifestation of the disease, on both the plasma concentration and the cellular origin of circulating microparticles. Flow cytometry was used to characterize microparticles in 62 sickle cell anemia children at steady state aged 2 months-16 years; 13 of them were treated with hydroxycarbamide. In untreated children, we observed negative correlations between fetal hemoglobin levels and the absolute plasma concentration of microparticles as well as that of microparticles specifically derived from platelets, erythrocytes, and monocytes. Compared to untreated children, those treated with hydroxyurea showed lower concentrations of total microparticles as a consequence of decreased microparticles shed by platelets and erythrocytes. In conclusion, in our sickle cell patients, neonatal decline of fetal hemoglobin coincided with an increase in circulating microparticles derived from erythrocytes, platelets, and monocytes. Hydroxyurea treatment was associated with a decrease in microparticles derived from erythrocytes and platelets