In vivo evidence of mitochondrial dysfunction and altered redox homeostasis in a genetic mouse model of propionic acidemia: Implications for the pathophysiology of this disorder

https://doi.org/10.1016/j.freeradbiomed.2016.04.007Get rights and content

Highlights

  • We analyze mitochondrial function and redox homeostasis in a propionic acidemia mouse model.

  • Alterations in OXPHOS complexes and/or activities, and mtDNA depletion were present.

  • Increase in superoxide anion production and H2O2 levels, variations in antioxidant defences and lipid oxidative damage were also observed.

  • Mitochondrial dysfunction and redox imbalance probably contribute to the pathophysiology of propionic acidemia.

Abstract

Accumulation of toxic metabolites has been described to inhibit mitochondrial enzymes, thereby inducing oxidative stress in propionic acidemia (PA), an autosomal recessive metabolic disorder caused by the deficiency of mitochondrial propionyl-CoA carboxylase. PA patients exhibit neurological deficits and multiorgan complications including cardiomyopathy. To investigate the role of mitochondrial dysfunction in the development of these alterations we have used a hypomorphic mouse model of PA that mimics the biochemical and clinical hallmarks of the disease. We have studied the tissue-specific bioenergetic signature by Reverse Phase Protein Microarrays and analysed OXPHOS complex activities, mtDNA copy number, oxidative damage, superoxide anion and hydrogen peroxide levels. The results show decreased levels and/or activity of several OXPHOS complexes in different tissues of PA mice. An increase in mitochondrial mass and OXPHOS complexes was observed in brain, possibly reflecting a compensatory mechanism including metabolic reprogramming. mtDNA depletion was present in most tissues analysed. Antioxidant enzymes were also found altered. Lipid peroxidation was present along with an increase in hydrogen peroxide and superoxide anion production. These data support the hypothesis that oxidative damage may contribute to the pathophysiology of PA, opening new avenues in the identification of therapeutic targets and paving the way for in vivo evaluation of compounds targeting mitochondrial biogenesis or reactive oxygen species production.

Introduction

Propionic acidemia (PA) is one of the most common organic acidemias resulting from mutations in either the PCCA or PCCB genes responsible for the two protein subunits of the propionyl-CoA carboxylase enzyme (PCC, E.C.6.4.1.3). PCC catalyzes the conversion of propionyl-CoA to D-methylmalonyl-CoA, which is eventually converted to succinyl-CoA, a substrate in the Krebs cycle. The sources of propionyl-CoA include the catabolism of amino acids valine, isoleucine, threonine and methionine, β-oxidation of odd chain fatty acids and propionate production by gut bacteria [1]. Patients with PA have several metabolic abnormalities including elevated levels of glycine, propionyl-carnitine and methyl-citrate, which are used as biochemical markers for diagnosis. Patients usually present neonatally with hypotonia, lethargy, feeding refusal, failure to thrive, seizures and encephalopathy, reflecting acute metabolic decompensation. Some patients exhibit a milder, late-onset chronic form. Both phenotypes exhibit great morbidity and mortality. The most common complications are cardiomyopathy, pancreatitis, neurological deficits and basal ganglia abnormalities. Some milder forms present in late in childhood with fatal neurological or cardiac symptoms without previous metabolic decompensations [2], [3].

The incidence of PA is estimated to be in the range of 1 in 165,000 to 1 in 300,000 [4] and the disorder is included in the newborn screening programs in most developed countries. Treatment is based on protein restriction, carnitine supplementation and administration of biotin and metronidazole. However, the overall outcome remains unsatisfactory as potentially lethal multiorgan complications persist, even under good metabolic control [5]. PA-related cardiomyopathy may develop in young patients or occur beyond infantile age, progressing rapidly to death [4]. No specific biochemical marker related to cardiomyopathy development has to date been identified [6]. The ultimate mechanism for cardiac alterations in PA remains unclear and is likely multifactorial. Similarly, neurological complications (seizures, basal ganglia abnormalities, extrapyramidal symptoms, brain atrophy and metabolic stroke) are frequent in PA [7], [8] and are often associated with the number of metabolic crises experienced [5] although additional damage resulting from other pathomechanisms may occur.

The accumulation of propionyl-CoA and other organic acids and esters has been shown in vitro to inhibit Krebs cycle enzyme succinyl-CoA synthetase and some respiratory chain complexes. In line with this, oxidative phosphorylation (OXPHOS) defects, mtDNA depletion and ultrastructural mitochondrial abnormalities have been described in patient biopsies, supporting the role of a secondary mitochondrial dysfunction in the disease pathology, notably in organs with a high energy demand such as brain and heart [9], [10], [11], [12].

We have recently described the presence of increased intracellular hydrogen peroxide (H2O2) levels in patients’ fibroblasts, correlating with the activation of the JNK and p38 signalling pathways [13]. Different antioxidants could significantly reduce H2O2 content, arguing in favour of further assessment of antioxidant strategies in PA as adjuvant therapy [14].

Mouse models of PA have been generated and used initially to study the possible benefits of gene therapy. A knockout mouse model for the Pcca gene [15] proved to be quite a stringent model to test for gene therapy as it dies within 1–2 days after birth, making systemic treatment challenging. Subsequently, a hypomorphic mouse model was generated on the Pcca KO background [16]. These mice carry a human transgene with the mutation p.A138T, identified in human patients and retaining 9% residual activity. The Pcca hypomorphic mice survive into adulthood, while accurately recapitulating biochemical and clinical biomarkers similar to those in patients with PA. Brain natriuretic peptide, a biomarker for cardiomyopathy and cardiac dysfunction, was found elevated in heart of the PA mice, correlating with a significant increase in heart size [16].

In this work, we have investigated mitochondrial function, superoxide anion and H2O2 levels and oxidative damage in relevant tissues of the hypomorphic mice, to gain insight into the pathophysiological processes contributing to the multiorgan complications observed in PA. We present compelling evidence of the presence of mitochondrial dysfunction and alterations in redox homeostasis in PA, identifying parameters related to these cellular functions that qualify as biomarkers of pathology and offering a basis for in vivo testing of novel adjuvant therapies in this disorder.

Section snippets

Mice handling

All mice used, wild-type and hypomorphic Pcca-/-(A138T) [16], were adult males or females (3–10 months old) in an FVB background. Mice were maintained on standard chow. Animal experiments were carried out in a pathogen-free environment at the Animal Facility of Centro de Biología Molecular Severo Ochoa, in accordance with the Spanish Law on Animal Protection. All animal studies were approved by the Institutional Animal Experimentation Ethical Committee (Universidad Autónoma de Madrid, reference

Mitochondrial function

The bioenergetic phenotype in relevant tissues of the hypomorphic PA mouse model was analysed using Reverse Phase Protein Microarrays (RPPMA) and by activity measurement of respiratory chain complexes. RPPMA has been validated as a technique to identify and quantify biomarkers of energy metabolism in human pathology, to phenotype tissue energy metabolism in mouse models of rare diseases and to monitor response to therapy [18], [19], [20], [31]. In this work, we applied RPPMA to analyze tissue

Discussion

PA has been linked to increased ROS levels and secondary mitochondrial dysfunction [10], [13], [35]. In this work, we have investigated for the first time these parameters in the hypomorphic PA mouse model, which will serve as basis for future investigations in therapeutic approaches for PA.

Several disturbances in mitochondrial function and redox homeostasis were observed in PA mice tissues, including increased superoxide anion production and in vivo mitochondrial H2O2 levels, mtDNA depletion,

Conclusions

Currently, the available therapies for PA only treat the symptoms but do not prevent completely the progression of neurological deficits and cardiac complications. Several biochemical parameters were shown to be altered in PA mice such as bioenergetic signature, lipid peroxidation, hydrogen peroxide and superoxide anion levels, and antioxidant defences, which may be considered biomarkers of PA pathology. The present study provides in vivo evidence obtained in the PA mouse model suggesting that

Author disclosure statement

No competing financial interests exist.

Acknowledgements

The technical assistance of Isabel Manso and Laura Rufian is gratefully acknowledged. The authors thank Esmeralda Alonso and Aitor Delmiro for their collaboration, and Drs. Pedro Ruiz-Sala and María Sánchez-Aragó for helpful discussions. This work was supported by grants SAF2013-43005-R from Ministerio de Economía y Competitividad, MITOLAB S2010/BMD-2402 from Comunidad de Madrid and ISCIII PI12_01683 from Ministerio de Economía y Competitividad (Fondo Europeo de Desarrollo Regional-FEDER).

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    These authors contributed equally to the work.

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