Inherited metabolic diseases (IMD) include a broad spectrum of biochemical alterations caused by genetic defects which affect the structure and function of proteins involved in cellular metabolic pathways. More than 700 IMD have been identified that affect the synthesis, metabolism, transport or/and storage of biochemical compounds, and they are considered rare diseases. Our main goal is to identify new modifier genes that could be considered new therapetical targets in these disorders. Emerging evidences have shown: i) that the generation of reactive oxygen species (ROS) contributes to the neurological phenotype of neurodegenerative, chronic-inflammatory and vascular diseases, and cancer; ii) that most of the patients affected by IMD predominantly present neurological symptoms and structural brain abnormalities, and iii) that our previous results have shown an increase in intracellular ROS and apoptosis levels in fibroblasts from patients with defects on cobalamin metabolism and with propionic acidemia.
DETECTION OF INTRACELLULAR ROS AND APOPTOSIS LEVELS IN FIBROBLASTS FROM METHYLMALONIC ACIDEMIA PATIENTS. Intracellular levels of ROS were detected by microscopy (A) and flow cytometry (B) using the H2DCFDA fluorescence probe. The percentage of apoptotic cells was analyzed by flow cytometry using annexin V and caspase 9 fluorescence probes. ROS levels and apoptotic cells were increased in fibroblasts from patients respect to controls. Caspase 9 activation indicates that the intrinsic or mitochondrial pathway is probably the main activated pathway in these patients.
In light of the abovementioned results, our research line at present is mainly focused on the analysis of mitochondrial dysfunction as pathophysiological mechanism and as new therapeutic target using a cellular model (patients´ fibroblasts) and an animal model (knockout PCCA). Specifically, we are analysing in patients´ fibroblasts with defects in cobalamin metabolism and with homocystinuria: i) different parameters related to oxidative stress and apoptosis, ii) mitochondrial morphology, mitochondrial respiration and the opening of mitochondrial permeability transition pore. In addition, the effects of different antioxidants are being evaluated in the cellular model that will be further analysed in the future in the animal model. The results will allow establishing a relationship with the phenotype of these patients, determining its relevance in the pathophysiology of IMD and thus opening the possibility to use antioxidant reagents to prevent cellular damage for a better life quality of the patients.
Gallego-Villar L, Pérez-Cerdá C, Pérez B, Abia D, Ugarte M, Richard E, Desviat LR “Functional characterization of novel genotypes and oxidative stress cellular studies in propionic acidemia”. Journal of Inherited Metabolic Diseases DOI: 10.1007/s10545-012-9545-3 (2012).
Richard E, Desviat LR, Ugarte M, Pérez B. “Oxidative stress and apoptosis in homocystinuria patients with genetic remethylation defects”. Journal of Cellular Biochemistry 114:183-191 (2013).
Jorge-Finnigan A, Gámez A, Pérez B, Ugarte M and Richard E. “Different altered pattern expression of genes related to apoptosis in isolated methylmalonic aciduria cblB type and combined with homocystinuria cblC type”. Biochimica et Biophysica Acta-Molecular Basis of Disease 1802 (11): 959-967 (2010).
Richard E, Jorge-Finnigan A, Garcia-Villoria J, Merinero B, Desviat LR, Gort L, Briones P, Leal F, Pérez-Cerdá C, Ribes A, Ugarte M, Pérez B; MMACHC Working Group. “Genetic and cellular studies of oxidative stress in methylmalonic aciduria (MMA) cobalamin deficiency type C (cblC) with homocystinuria (MMACHC)”. Human Mutation 30 (11): 1558-1566 (2009).