Bioenergetics and Protein Metabolism in Sporadic Amyotrophic Lateral Sclerosis
Mots clés
Abstrait
La description
The investigators will study fibroblasts derived from sporadic ALS patients.
Clinical parameters will be collected such as sex, age, age of onset, clinical presentation, electrophysiological an respiratory parameters, treatments.
Patients and controls derived fibroblasts will be amplified and cultivated from skin biopsies.
Primary fibroblast cell cultures will be derived from skin biopsies obtained from control subjects and from the patients after obtaining written consent. Amplifications will be performed, from passage 1 to 6, allowing the time for cell cryopreservation of 10 samples per cell line. All experiments will be conducted on cells with similar passage numbers, ranging from 6 to 25, to avoid artefacts due to senescence.
Cell growth of each line will be monitored with the IncuCyte ZOOM® system (Essen Biosciences), a microscope gantry that resides in a cell incubator, and a networked external controller hard drive that gathers and processes image data. Cell growth will be automatically calculated at each time point using the relative cell density (ratio of the occupied area to the total area of the acquired regions).
The respiratory parameters of the fibroblasts will be monitored precisely with an high resolution oxygraph (OROBOROS). Enzymatic activity of each complex of the mitochondrial respiratory chain will be measured by spectrometry, and the mitochondrial membrane potential will be studied with fluorescence microscopy (TMRM probe). The investigators will also study mitochondrial dynamics and the architecture of mitochondrial network with fluorescence microscopy and super-resolution microscopy (SRM, Roper). The production of free radicals will be estimated with probes sensitive to oxidative stress. Finally, a metabolomic study (mass spectrometry QTRAP LC/MS/MS 5500 ABSciex) will be performed to characterise precisely the metabolites in the cells and to assess their metabolic profile.
The investigators will search for protein inclusions (intermediate filaments, TDP43, FUS, ubiquitin) with immuno-fluorescence technics. Western Blots will be performed to study the protein metabolism. The architecture of the cytoskeleton will be analysed in SRM and fluorescence microscopy, and the investigators will try to transfect fibroblasts in order to express Neurofilaments and study the consequences on the cytoskeleton and protein metabolism.
The investigators will study the response of the cells to the stress (hypoxia SCI-tive Dual, starvation), and to senescence (high passage numbers).
Rendez-vous
| Dernière vérification: | 10/31/2016 |
| Première soumission: | 11/03/2016 |
| Inscription estimée soumise: | 11/16/2016 |
| Première publication: | 11/20/2016 |
| Dernière mise à jour soumise: | 11/21/2016 |
| Dernière mise à jour publiée: | 11/22/2016 |
| Date de début réelle de l'étude: | 10/31/2016 |
| Date d'achèvement primaire estimée: | 10/31/2017 |
| Date estimée d'achèvement de l'étude: | 10/31/2019 |
Condition ou maladie
Intervention / traitement
Other: Skin Biopsy
Phase
Groupes d'armes
| Bras | Intervention / traitement |
|---|---|
| Other: Patients ALS defined by El Escorial Criteria. | |
| Other: Controls Asymptomatic subjects with normal examination. |
Critère d'éligibilité
| Âges éligibles aux études | 18 Years À 18 Years |
| Sexes éligibles à l'étude | All |
| Accepte les bénévoles en santé | Oui |
| Critères | Inclusion criteria : - a free signed an written consent - aged between 18 and 80 years old - ALS defined by El Escorial Criteria - normal neurological examination Exclusion criteria: - comorbidity or treatment susceptible to impact on metabolism - differential diagnosis suspected, early and late forms (<6 month > 3 years) - ALS not defined by El Escorial Criteria - withdrawal of consent to participate |
Résultat
Mesures des résultats primaires
1. Kinetics of fibroblast growth [24 hours]
Mesures des résultats secondaires
1. Mitochondrial metabolism [24 hours]
2. Protéic metabolism [24 hours]
3. stress in senescence answer [24 hours]
