Mitochondrial dysfunction and the metabolism - studies on respiratory chain disorders

Background: Mutations of the mitochondrial assembly factor BCS1L disrupt assembly and function of the respiratory chain complex III (CIII) and thereby result in energy deprivation. In GRACILE syndrome, the BCS1L mutation causes a liver disorder that leads to metabolic disturbances associated with severe growth restriction.Objectives: To clarify the mechanisms and affected metabolic pathways in the CIII deficiency disease progression and perform intervention trials in an experimental disease model. Methods/research questions: A knock-in mouse model, carrying the same missense mutation (Bcs1l c.232A>G) as the GRACILE syndrome patients, is used to study disease mechanisms and pathways involved. Two randomized controlled intervention studies have been performed on homozygous mice and littermate controls: a 4-hour fasting as a metabolic pressure to assess the compensatory capabilities, and a dietary intervention to clarify whether hypoglycemia and survival can be improved by the high-carbohydrate diet (60% dextrose). Further, phenotyping and the disease mechanism in a patient with a novel compound heterozygous BCS1L mutation have been studied and compared to GRACILE patients and mutant mice. In the last study, a metabolic phenotype caused by a single large-scale deletion (SLSD) in mitochondrial (mtDNA), including the genes for structural components of CI and CIII, was analyzed.Methods used in the studies include genetic analysis,WES, WGS, phenotyping, blood and urine chemistry, metabolomics, histopathology, EM, function, and expression analysis. Conventional statistics and linear mixed-effect models (MEMs) were used for the analysis of the metabolic network response to fasting.Results: Fasting mutant animals revealed intact systemic lipid mobilization but disrupted compensatory mechanisms leading to hypoglycemia. The high-carbohydrate diet had an unexpected adverse effect on survival compared to the standard diet. The novel BSC1L mutation had a different phenotype compared to GRACILE syndrome. The patient with the SLSD had a complex metabolic phenotype, which emphasized the importance of diagnostics synergy of clinical awareness, genetic and funtional analysis. Significance: By elucidating metabolic disturbances, the project increases understanding of pathophysiology in CIII deficiency. This has a potential effect on understanding other, more common mitochondrial dysfunctions and raises new queries about this complex system

Fasting reveals largely intact systemic lipid mobilization mechanisms in respiratory chain complex III deficient mice

Mice homozygous for the human GRACILE syndrome mutation (Bcs1l (c.A232G)) display decreased respiratory chain complex III activity, liver dysfunction, hypoglycemia, rapid loss of white adipose tissue and early death. To assess the underlying mechanism of the lipodystrophy in homozygous mice (Bcs1l(p.S)(78G)), these and wild-type control mice were subjected to a short 4-hour fast. The homozygotes had low baseline blood glucose values, but a similar decrease in response to fasting as in wild-type mice, resulting in hypoglycemia in the majority. Despite the already depleted glycogen and increased triacylglycerol content in the mutant livers, the mice responded to fasting by further depletion and increase, respectively. Increased plasma free fatty acids (FAs) upon fasting suggested normal capacity for mobilization of lipids from white adipose tissue into circulation. Strikingly, however, serum glycerol concentration was not increased concomitantly with free FM, suggesting its rapid uptake into the liver and utilization for fuel or gluconeogenesis in the mutants. The mutant hepatocyte mitochondria were capable of responding to fasting by appropriate morphological changes, as analyzed by electron microscopy, and by increasing respiration. Mutants showed increased hepatic gene expression of major metabolic controllers typically associated with fasting response (Ppargc1a, Fgf21, Cd36) already in the fed state, suggesting a chronic starvation-like metabolic condition. Despite this, the mutant mice responded largely normally to fasting by increasing hepatic respiration and switching to FA utilization, indicating that the mechanisms driving these adaptations are not compromised by the CIII dysfunction. Summary statement: Bcs1l mutant mice with severe CIII deficiency, energy deprivation and post-weaning lipolysis respond to fasting similarly to wild-type mice, suggesting largely normal systemic lipid mobilization and utilization mechanisms.Peer reviewe

Effect of High-Carbohydrate Diet on Plasma Metabolome in Mice with Mitochondrial Respiratory Chain Complex III Deficiency

Mitochondrial disorders cause energy failure and metabolic derangements. Metabolome profiling in patients and animal models may identify affected metabolic pathways and reveal new biomarkers of disease progression. Using liver metabolomics we have shown a starvation-like condition in a knock-in (Bcs1l(c.232A>G)) mouse model of GRACILE syndrome, a neonatal lethal respiratory chain complex III dysfunction with hepatopathy. Here, we hypothesized that a high-carbohydrate diet (HCD, 60% dextrose) will alleviate the hypoglycemia and promote survival of the sick mice. However, when fed HCD the homozygotes had shorter survival (mean +/- SD, 29 +/- 2.5 days, n = 21) than those on standard diet (33 +/- 3.8 days, n = 30), and no improvement in hypoglycemia or liver glycogen depletion. We investigated the plasma metabolome of the HCD- and control diet-fed mice and found that several amino acids and urea cycle intermediates were increased, and arginine, carnitines, succinate, and purine catabolites decreased in the homozygotes. Despite reduced survival the increase in aromatic amino acids, an indicator of liver mitochondrial dysfunction, was normalized on HCD. Quantitative enrichment analysis revealed that glycine, serine and threonine metabolism, phenylalanine and tyrosine metabolism, and urea cycle were also partly normalized on HCD. This dietary intervention revealed an unexpected adverse effect of high-glucose diet in complex III deficiency, and suggests that plasma metabolomics is a valuable tool in evaluation of therapies in mitochondrial disorders.Peer reviewe

Respiratory chain complex III deficiency due to mutated BCS1L : a novel phenotype with encephalomyopathy, partially phenocopied in a Bcs1l mutant mouse model

Background: Mitochondrial diseases due to defective respiratory chain complex III (CIII) are relatively uncommon. The assembly of the eleven-subunit CIII is completed by the insertion of the Rieske iron-sulfur protein, a process for which BCS1L protein is indispensable. Mutations in the BCS1L gene constitute the most common diagnosed cause of CIII deficiency, and the phenotypic spectrum arising from mutations in this gene is wide. Results: A case of CIII deficiency was investigated in depth to assess respiratory chain function and assembly, and brain, skeletal muscle and liver histology. Exome sequencing was performed to search for the causative mutation(s). The patient's platelets and muscle mitochondria showed respiration defects and defective assembly of CIII was detected in fibroblast mitochondria. The patient was compound heterozygous for two novel mutations in BCS1L, c.306A > T and c.399delA. In the cerebral cortex a specific pattern of astrogliosis and widespread loss of microglia was observed. Further analysis showed loss of Kupffer cells in the liver. These changes were not found in infants suffering from GRACILE syndrome, the most severe BCS1L-related disorder causing early postnatal mortality, but were partially corroborated in a knock-in mouse model of BCS1L deficiency. Conclusions: We describe two novel compound heterozygous mutations in BCS1L causing CIII deficiency. The pathogenicity of one of the mutations was unexpected and points to the importance of combining next generation sequencing with a biochemical approach when investigating these patients. We further show novel manifestations in brain, skeletal muscle and liver, including abnormality in specialized resident macrophages (microglia and Kupffer cells). These novel phenotypes forward our understanding of CIII deficiencies caused by BCS1L mutations.Peer reviewe

The phenotype of polycythemia due to Croatian homozygous VHL (571C\u3eG:H191D) mutation is different from that of Chuvash polycythemia (VHL 598C\u3eT:R200W)

Mutations of VHL (a negative regulator of hypoxia-inducible factors) have position-dependent distinct cancer phenotypes. Only two known inherited homozygous VHL mutations exist and they cause polycythemia: Chuvash R200W and Croatian H191D. We report a second polycythemic Croatian H191D homozygote distantly related to the first propositus. Three generations of both families were genotyped for analysis of shared ancestry. Biochemical and molecular tests were performed to better define their phenotypes, with an emphasis on a comparison with Chuvash polycythemia. The VHL H191D mutation did not segregate in the family defined by the known common ancestors of the two subjects, suggesting a high prevalence in Croatians, but haplotype analysis indicated an undocumented common ancestor ~six generations ago as the founder of this mutation. We show that erythropoietin levels in homozygous VHL H191D individuals are higher than in VHL R200W patients of similar ages, and their native erythroid progenitors, unlike Chuvash R200W, are not hypersensitive to erythropoietin. This observation contrasts with a report suggesting that polycythemia in VHL R200W and H191D homozygotes is due to the loss of JAK2 regulation from VHL R200W and H191D binding to SOCS1. In conclusion, our studies further define the hematologic phenotype of VHL H191D and provide additional evidence for phenotypic heterogeneity associated with the positional effects of VHL mutations. © 2013 Ferrata Storti Foundation