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
Graphical abstract
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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2020, Archives of Biochemistry and BiophysicsCitation Excerpt :Results obtained from the knockout mouse model of MMAcidemia revealed alterations of mitochondrial structure and respiratory chain dysfunction with diminished cytochrome c oxidase activity [113,120], besides changes of CAC enzyme activities [121]. Altered respiratory chain complexes activities [122] and disturbance of cellular calcium homeostasis [123] have also been described in tissues from the mouse model of PAcidemia. Patients with β-ketothiolase deficiency are biochemically characterized by large urinary excretion of 2-methyl-3-hydroxybutyric (MHB) and 2-methylacetoacetic (MAA) acids [124,125].
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2020, Biochimica et Biophysica Acta - Molecular Basis of DiseaseCitation Excerpt :In this scenario, considering that heart is a highly energy dependent tissue, mitochondrial dysfunction may be proposed as a contributor factor for the progression of the cardiomyopathy in the affected patients [39]. This hypothesis is supported by previous reports revealing mitochondrial abnormalities in the heart of PAcidemic patients, decreased myocardial levels of coenzyme Q10 [17] and free carnitine [40], as well as reduction of complex I-III [40] and III [41] activities in muscular biopsies of patients, and reduced complex I activity in the heart of the genetic mice model of PAcidemia [18]. Nevertheless, the precise mechanisms of these morphological and biochemical alterations have not yet been elucidated.