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

Analyse de la composition en acides gras des lipides dans la sclérose latérale amyotrophique : implications dans le processus pathologique

ALS is a fatal neurodegenerative condition characterized by the selective loss of upper and lower motor neurons. The disease is also characterized by alterations of energy homeostasis and lipid metabolism. The objective of this PhD work was, first, to identify changes in fatty acid composition of lipids in blood of ALS patients, as potential diagnosis and prognosis biomarkers of the disease. The second goal was to modulate these changes, by nutritional approaches, in order to better understand the involvement of an altered lipid metabolism in the pathological process. Our results showed that high levels of monounsaturated fatty acid (MUFA), as well as low levels of polyunsaturated fatty acids (PUFA), are associated with a better prognosis. The analysis of the effects of a modified fatty acid composition in both pathological and lesion models of denervation, has confirmed implication of the antagonistic relationship between MUFA and PUFA in the neurodegenerative process. This work suggests a cautious usage of fatty acids in therapeutic approaches targeting ALS.La SLA est une maladie neurodégénérative grave touchant l’adulte qui se caractérise principalement par la perte sélective des neurones moteurs cortico-spinaux et des motoneurones bulbaires et spinaux. La SLA se caractérise également par des altérations de l’homéostasie énergétique et du métabolisme des lipides. L’objectif de ce travail de thèse était, dans un premier temps, d’identifier des changements dans la composition en acides gras, dans le sang, comme potentiels biomarqueurs diagnostiques et pronostiques de la SLA. Dans un second temps, le but était de moduler ces changements, par des approches nutritionnelles, afin de mieux comprendre l’implication d’un métabolisme lipidique altéré dans le processus pathologique de la SLA. Ces travaux ont montré que des taux élevés d’acides gras mono-insaturés (MUFA), ainsi que des taux diminués d’acides gras poly-insaturés (PUFA), sont associés à un meilleur pronostic. L’analyse des effets de la modification de la composition en acides gras, dans des modèles de dénervation pathologique et lésionnelle, a confirmé l’implication de l’antagonisme MUFA/PUFA dans le processus neurodégénératif. Ces travaux font apparaitre des mises en garde quant à l’utilisation des acides gras dans des approches à visée thérapeutique pour la SLA

Analysis of fatty acid composition in lipids of ALS : implications in the pathological process

La SLA est une maladie neurodégénérative grave touchant l’adulte qui se caractérise principalement par la perte sélective des neurones moteurs cortico-spinaux et des motoneurones bulbaires et spinaux. La SLA se caractérise également par des altérations de l’homéostasie énergétique et du métabolisme des lipides. L’objectif de ce travail de thèse était, dans un premier temps, d’identifier des changements dans la composition en acides gras, dans le sang, comme potentiels biomarqueurs diagnostiques et pronostiques de la SLA. Dans un second temps, le but était de moduler ces changements, par des approches nutritionnelles, afin de mieux comprendre l’implication d’un métabolisme lipidique altéré dans le processus pathologique de la SLA. Ces travaux ont montré que des taux élevés d’acides gras mono-insaturés (MUFA), ainsi que des taux diminués d’acides gras poly-insaturés (PUFA), sont associés à un meilleur pronostic. L’analyse des effets de la modification de la composition en acides gras, dans des modèles de dénervation pathologique et lésionnelle, a confirmé l’implication de l’antagonisme MUFA/PUFA dans le processus neurodégénératif. Ces travaux font apparaitre des mises en garde quant à l’utilisation des acides gras dans des approches à visée thérapeutique pour la SLA.ALS is a fatal neurodegenerative condition characterized by the selective loss of upper and lower motor neurons. The disease is also characterized by alterations of energy homeostasis and lipid metabolism. The objective of this PhD work was, first, to identify changes in fatty acid composition of lipids in blood of ALS patients, as potential diagnosis and prognosis biomarkers of the disease. The second goal was to modulate these changes, by nutritional approaches, in order to better understand the involvement of an altered lipid metabolism in the pathological process. Our results showed that high levels of monounsaturated fatty acid (MUFA), as well as low levels of polyunsaturated fatty acids (PUFA), are associated with a better prognosis. The analysis of the effects of a modified fatty acid composition in both pathological and lesion models of denervation, has confirmed implication of the antagonistic relationship between MUFA and PUFA in the neurodegenerative process. This work suggests a cautious usage of fatty acids in therapeutic approaches targeting ALS

Blood Biomarkers for Amyotrophic Lateral Sclerosis: Myth or Reality?

Amyotrophic lateral sclerosis (ALS) is a fatal condition primarily characterized by the selective loss of upper and lower motor neurons. At present, the diagnosis and monitoring of ALS is based on clinical examination, electrophysiological findings, medical history, and exclusion of confounding disorders. There is therefore an undeniable need for molecular biomarkers that could give reliable information on the onset and progression of ALS in clinical practice and therapeutic trials. From a practical point of view, blood offers a series of advantages, including easy handling and multiple testing at a low cost, that make it an ideal source of biomarkers. In this review, we revisited the findings of many studies that investigated the presence of systemic changes at the molecular and cellular level in patients with ALS. The results of these studies reflect the diversity in the pathological mechanisms contributing to disease (e.g., excitotoxicity, oxidative stress, neuroinflammation, metabolic dysfunction, and neurodegeneration, among others) and provide relatively successful evidence of the usefulness of a wide-ranging panel of molecules as potential biomarkers. More studies, hopefully internationally coordinated, would be needed, however, to translate the application of these biomarkers into benefit for patients

J. Neurosci.

Electrophysiological and neuroanatomical evidence for reciprocal connections with the medial prefrontal cortex (mPFC) and the hippocampus make the reuniens and rhomboid (ReRh) thalamic nuclei a putatively major functional link for regulations of cortico-hippocampal interactions. In a first experiment using a new water escape device for rodents, the double-H maze, we demonstrated in rats that a bilateral muscimol (MSCI) inactivation (0.70 vs 0.26 and 0 nmol) of the mPFC or dorsal hippocampus (dHip) induces major deficits in a strategy shifting/spatial memory retrieval task. By way of comparison, only dHip inactivation impaired recall in a classical spatial memory task in the Morris water maze. In the second experiment, we showed that ReRh inactivation using 0.70 nmol of MSCI, which reduced performance without obliterating memory retrieval in the water maze, produces an as large strategy shifting/memory retrieval deficit as mPFC or dHip inactivation in the double-H maze. Thus, behavioral adaptations to task contingency modifications requiring a shift toward the use of a memory for place might operate in a distributed circuit encompassing the mPFC (as the potential set-shifting structure), the hippocampus (as the spatial memory substrate), and the ventral midline thalamus, and therein the ReRh (as the coordinator of this processing). The results of the current experiments provide a significant extension of our understanding of the involvement of ventral midline thalamic nuclei in cognitive processes: they point to a role of the ReRh in strategy shifting in a memory task requiring cortical and hippocampal functions and further elucidate the functional system underlying behavioral flexibility

Blood Cell Palmitoleate-Palmitate Ratio Is an Independent Prognostic Factor for Amyotrophic Lateral Sclerosis.

Growing evidence supports a link between fatty acid metabolism and amyotrophic lateral sclerosis (ALS). Here we determined the fatty acid composition of blood lipids to identify markers of disease progression and survival. We enrolled 117 patients from two clinical centers and 48 of these were age and gender matched with healthy volunteers. We extracted total lipids from serum and blood cells, and separated fatty acid methyl esters by gas chromatography. We measured circulating biochemical parameters indicative of the metabolic status. Association between fatty acid composition and clinical readouts was studied, including ALS functional rating scale-revised (ALSFRS-R), survival, disease duration, site of onset and body mass index. Palmitoleate (16:1) and oleate (18:1) levels, and stearoyl-CoA desaturase indices (16:1/16:0 and 18:1/18:0) significantly increased in blood cells from ALS patients compared to healthy controls. Palmitoleate levels and 16:1/16:0 ratio in blood cells, but not body mass index or leptin concentrations, negatively correlated with ALSFRS-R decline over a six-month period (p<0.05). Multivariate Cox analysis, with age, body mass index, site of onset and ALSFRS-R as covariables, showed that blood cell 16:1/16:0 ratio was an independent prognostic factor for survival (hazard ratio=0.1 per unit of ratio, 95% confidence interval=0.01-0.57, p=0.009). In patients with high 16:1/16:0 ratio, survival at blood collection was extended by 10 months, as compared to patients with low ratio. The 16:1/16:0 index is an easy-to-handle parameter that predicts survival of ALS patients independently of body mass index. It therefore deserves further validation in larger cohorts for being used to assess disease outcome and effects of disease-modifying drugs

SCD indices are higher in ALS patients than in control subjects.

<p>(A) Palmitoleate to palmitate ratio (16:1/16:0) in serum and blood cells from ALS patients (ALS) and control subjects (CT). (B) Oleate to stearate ratio (18:1/18:0) in serum and blood cells from ALS patients (ALS) and control subjects (CT). *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 (Mann-Withney test, n = 48).</p

Analysis of the survival of ALS patients using multivariate Cox proportional hazards model.

<p>Analysis of the survival of ALS patients using multivariate Cox proportional hazards model.</p