Bioenergetics and Protein Metabolism in Sporadic Amyotrophic Lateral Sclerosis

Sporadic Amyotrophic Lateral Sclerosis Clinical Trial

— Fibro-ALS  

Official title:

Bioenergetics and Protein Metabolism in Sporadic Amyotrophic Lateral Sclerosis

Clinical Trial Details — Status: Not yet recruiting

Administrative data

• NCT number: NCT02969759
• Other study ID #: 2016/36
• Status: Not yet recruiting
• Phase: Phase 0
• First received: November 4, 2016
• Last updated: November 22, 2016
• Start date: November 2016
• Est. completion date: November 2019

Study information

• Verified date: November 2016
• Source: University Hospital, Angers
• Contact: n/a
• Is FDA regulated: No
• Health authority: France: Ministry of Health
• Study type: Interventional

Clinical Trial Summary

INTRODUCTION: Amyotrophic Lateral Sclerosis (ALS) is a degenerative disease of the motor neurones of the brain and the spinal cord. The pathophysiological mechanisms of the disease remain unknown. The average age of onset of ALS is about 60 years old, and the mean survival of patients is about 2 years. The disease is responsible for a progressive paralysis leading to death from respiratory failure. The only treatment available is the Riluzole, with a very modest efficiency on the progression of the disease. ALS is the third neurodegenerative disease, affecting 6000 persons in France, 150 000 in the world. Among the protagonists involved in the occurrence of the disease, mitochondrial perturbations and protein accumulations seem to be central elements.

OBJECTIVES: To precise the implication of energy and protein metabolism in the sporadic forms of ALS, to identify potential biomarkers of the disease and to test new therapies.

METHODS: The investigators will study cell growth, bioenergetics, mitochondrial dynamics, free-radicals production, presence of cytoplasmic inclusions, cytoskeleton structure and stress response in primary skin fibroblasts obtained from sporadic ALS patients. The study will be conducted over a period of three years in 3 centres specialized respectively in motor neuron diseases, mitochondrial metabolism and neuronal cytoskeleton.

PROSPECTS: If the investigators achieve to identify differences between ALS fibroblasts and controls, the results will be key elements to reinforce the hypothesis of a systemic disease with an important metabolic participation, to better define ALS pathophysiological mechanisms, to find potential biomarkers and to test new therapies.

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).

Recruitment information / eligibility

• Status: Not yet recruiting
• Enrollment: 30
• Est. completion date: November 2019
• Est. primary completion date: November 2017
• Accepts healthy volunteers: Accepts Healthy Volunteers
• Gender: Both
• Age group: 18 Years to 80 Years

Eligibility

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

Study Design

Allocation: Non-Randomized, Intervention Model: Parallel Assignment, Masking: Open Label, Primary Purpose: Basic Science

Related Conditions & MeSH terms

• Amyotrophic Lateral Sclerosis
• Motor Neuron Disease
• Sclerosis
• Sporadic Amyotrophic Lateral Sclerosis

Intervention

Other:
• Skin Biopsy

Locations

Country	Name	City	State
n/a

Sponsors (1)

Lead Sponsor	Collaborator
University Hospital, Angers

Outcome

Type	Measure	Description	Time frame	Safety issue
Primary	Kinetics of fibroblast growth	The growth rates of the fibroblast lines	24 hours	No
Secondary	Mitochondrial metabolism	the breath parameters of fibroblast will be precisely studied by oxygraphy, a method consisting in measuring the oxygen consumption rates of fibroblasts in different metabolic phases	24 hours	No
Secondary	Protéic metabolism	The presence of intracytoplasmic inclusion of target protein will be sought.	24 hours	No
Secondary	stress in senescence answer	the answer due to the stress of cell lines in hypoxia and nutritionnal lack will be studied.	24 hours	No