Ensamblaje de los supercomplejos mitocondriales en modelos celulares de salud y enfermedad

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica: Fecha de lectura: 18/07/2014El presente trabajo ha sido financiado por un proyecto de investigación del Instituto de Salud Carlos III (número PI05-0647), concedido a la Dra. Cristina Ugalde Bilbao, así como por un proyecto de la Fundación de Investigación Médica Mutua Madrileña (número 2005- 069), concedido al Dr. Miguel Ángel Martín Casanueva

Mitochondrial respiratory chain dysfunction: implications in neurodegeneration

The definitive version of this article is available at http://www.sciencedirect.com/science/journal/0891584

Mitochondrial complex I plays an essential role in human respirasome assembly

The assembly and function of the mitochondrial respiratory chain (RC) involve the organization of RC enzyme complexes in supercomplexes or respirasomes through an unknown biosynthetic process. This leads to structural interdependences between RC complexes, which are highly relevant from biological and biomedical perspectives, because RC defects lead to severe human disorders. We show that in human cells, respirasome biogenesis involves a complex I assembly intermediate acting as a scaffold for the combined incorporation of complexes III and IV subunits, rather than originating from the association of preassembled individual holoenzymes. The process ends with the incorporation of complex I NADH dehydrogenase catalytic module, which leads to the respirasome activation. While complexes III and IV assemble either as free holoenzymes or by incorporation of free subunits into supercomplexes, the respirasomes constitute the structural units where complex I is assembled and activated, thus explaining the functional significance of the respirasomes for RC function

Impact of the Mitochondrial Genetic Background in Complex III Deficiency

BACKGROUND: In recent years clinical evidence has emphasized the importance of the mtDNA genetic background that hosts a primary pathogenic mutation in the clinical expression of mitochondrial disorders, but little experimental confirmation has been provided. We have analyzed the pathogenic role of a novel homoplasmic mutation (m.15533 A>G) in the cytochrome b (MT-CYB) gene in a patient presenting with lactic acidosis, seizures, mild mental delay, and behaviour abnormalities. METHODOLOGY: Spectrophotometric analyses of the respiratory chain enzyme activities were performed in different tissues, the whole muscle mitochondrial DNA of the patient was sequenced, and the novel mutation was confirmed by PCR-RFLP. Transmitochondrial cybrids were constructed to confirm the pathogenicity of the mutation, and assembly/stability studies were carried out in fibroblasts and cybrids by means of mitochondrial translation inhibition in combination with blue native gel electrophoresis. PRINCIPAL FINDINGS: Biochemical analyses revealed a decrease in respiratory chain complex III activity in patient's skeletal muscle, and a combined enzyme defect of complexes III and IV in fibroblasts. Mutant transmitochondrial cybrids restored normal enzyme activities and steady-state protein levels, the mutation was mildly conserved along evolution, and the proband's mother and maternal aunt, both clinically unaffected, also harboured the homoplasmic mutation. These data suggested a nuclear genetic origin of the disease. However, by forcing the de novo functioning of the OXPHOS system, a severe delay in the biogenesis of the respiratory chain complexes was observed in the mutants, which demonstrated a direct functional effect of the mitochondrial genetic background. CONCLUSIONS: Our results point to possible pitfalls in the detection of pathogenic mitochondrial mutations, and highlight the role of the genetic mtDNA background in the development of mitochondrial disorders

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Residual enzyme activities of mitochondrial respiratory chain complexes in different tissues from the index patient.

<p>Enzyme activities are expressed as</p><p>*cU/U citrate synthase (CS) and</p><p>**nmol.min⁻¹.mg prot⁻¹. CS activity is expressed as mU/mg protein. Abnormal values are indicated in bold. nd, not determined. Complex I, CI; Complex II, CII; Complex III, CIII; Complex IV, CIV.</p

BN-PAGE analysis of mitochondrial respiratory chain complexes in control and mutant fibroblasts and cybrids.

<p>Mitochondrial particles were isolated as described in Methods and 40 µg of protein were analyzed on a 5–15% BN-polyacrylamide gel for the separation of multisubunit complexes. Western-blot analysis was performed using antibodies against the indicated OXPHOS subunits. CI, fully-assembled complex I. CIII₂, complex III dimer. CIV, complex IV. CIII₂+IV indicates the presence of the supercomplex containing complexes III and IV. C1 and C2, control fibroblasts. P, patient's fibroblasts. C, control cybrid. Two independent mutant cybrids are indicated as #1 and #2.</p

Assembly kinetics of respiratory chain complexes in control and mutant cybrids.

<p>Two different control cybrids belonging to haplogroup H, and two independent <i>MT-CYB</i> mutant clones were treated for 6 days with doxycycline (an inhibitor of mitochondrial translation), the medium was replaced by doxycycline-free medium and cells were collected at 0, 6, 15, 24, 48, 72, and 96 hours (indicated as t0–t16). SS indicates the steady-state expression levels of the respiratory chain complexes (A) Example of one control and one mutant clone. 40 µg of crude mitochondrial pellets were analyzed by BN-PAGE in combination with complex I and complex IV-IGA assays. (B) Duplicate gels were blotted and incubated with antibodies against the NDUFA9 complex I subunit, complex III core2 protein, complex IV COX5A subunit and complex II SDHA subunit. (C) The signals from the blots were quantified, expressed as percentage of the untreated cells (SS), normalized with the complex II SDHA subunit and plotted. The restoration curves constitute the mean values ± SD obtained from the two controls and the two independent mutant cybrids. Upper left panel, complex I assembly rates. Upper right panel, complex III assembly rates. Lower left panel, complex IV assembly rates. Lower right panel, supercomplex CIII₂+IV assembly kinetics.</p