ETHE1 and MOCS1 deficiencies : disruption of mitochondrial bioenergetics, dynamics, redox homeostasis and endoplasmic reticulum-mitochondria crosstalk in patient fibroblasts

Ethylmalonic encephalopathy protein 1 (ETHE1) and molybdenum cofactor (MoCo) defciencies are hereditary disorders that afect the catabolism of sulfur-containing amino acids. ETHE1 defciency is caused by mutations in the ETHE1 gene, while MoCo defciency is due to mutations in one of three genes involved in MoCo biosynthesis (MOCS1, MOCS2 and GPHN). Patients with both disorders exhibit abnormalities of the mitochondrial respiratory chain, among other biochemical fndings. However, the pathophysiology of the defects has not been elucidated. To characterize cellular derangements, mitochondrial bioenergetics, dynamics, endoplasmic reticulum (ER)-mitochondria communication, superoxide production and apoptosis were evaluated in fbroblasts from four patients with ETHE1 defciency and one with MOCS1 defciency. The efect of JP4-039, a promising mitochondrial-targeted antioxidant, was also tested on cells. Our data show that mitochondrial respiration was decreased in all patient cell lines. ATP depletion and increased mitochondrial mass was identifed in the same cells, while variable alterations in mitochondrial fusion and fssion were seen. High superoxide levels were found in all cells and were decreased by treatment with JP4-039, while the respiratory chain activity was increased by this antioxidant in cells in which it was impaired. The content of VDAC1 and IP3R, proteins involved in ER-mitochondria communication, was decreased, while DDIT3, a marker of ER stress, and apoptosis were increased in all cell lines. These data demonstrate that previously unrecognized broad disturbances of cellular function are involved in the pathophysiology of ETHE1 and MOCS1 defciencies, and that reduction of mitochondrial superoxide by JP4-039 is a promising strategy for adjuvant therapy of these disorders

Preclinical Toxicology Studies of Recombinant Human Platelet-Derived Growth Factor-BB Either Alone or in Combination with Beta-Tricalcium Phosphate and Type I Collagen

Human platelet-derived growth factor-BB (hPDGF-BB) is a basic polypeptide growth factor released from platelets at the injury site. It is a multifunctional molecule that regulates DNA synthesis and cell division and induces biological effects that are implicated in tissue repair, atherosclerosis, inflammatory responses, and neoplastic diseases. This paper is an overview of the toxicology data generated from a broad testing platform to determine bone, soft tissue, and systemic responses following administration of rhPDGF-BB. Moreover, the systemic and local toxicity of recombinant human PDGF-BB (rhPDGF-BB) in combination with either beta-tricalcium phosphate (β-TCP) or collagen combined with β-TCP was studied to determine dermal sensitization, irritation, intramuscular tissue responses, pyrogenicity, genotoxicity, and hemolytic properties. All data strongly suggest that rhPDGF-BB either alone or in combination with β-TCP or collagen with β-TCP is biocompatible and has neither systemic nor local toxicity, supporting its safe use in enhancing wound healing in patients

Complex I assembly function and fatty acid oxidation enzyme activity of ACAD9 both contribute to disease severity in ACAD9 deficiency

Acyl-CoA dehydrogenase 9 (ACAD9) is an assembly factor for mitochondrial respiratory chain Complex I (CI), and ACAD9 mutations are recognized as a frequent cause of CI deficiency. ACAD9 also retains enzyme ACAD activity for long-chain fatty acids in vitro, but the biological relevance of this function remains controversial partly because of the tissue specificity of ACAD9 expression: high in liver and neurons and minimal in skin fibroblasts. In this study, we hypothesized that this enzymatic ACAD activity is required for full fatty acid oxidation capacity in cells expressing high levels of ACAD9 and that loss of this function is important in determining phenotype in ACAD9-deficient patients. First, we confirmed that HEK293 cells express ACAD9 abundantly. Then, we showed that ACAD9 knockout in HEK293 cells affected long-chain fatty acid oxidation along with Cl, both of which were rescued by wild type ACAD9. Further, we evaluated whether the loss of ACAD9 enzymatic fatty acid oxidation affects clinical severity in patients with ACAD9 mutations. The effects on ACAD activity of 16 ACAD9 mutations identified in 24 patients were evaluated using a prokaryotic expression system. We showed that there was a significant inverse correlation between residual enzyme ACAD activity and phenotypic severity of ACAD9-deficient patients. These results provide evidence that in cells where it is strongly expressed, ACAD9 plays a physiological role in fatty acid oxidation, which contributes to the severity of the phenotype in ACAD9-deficient patients. Accordingly, treatment of ACAD9 patients should aim at counteracting both CI and fatty acid oxidation dysfunction

Treatment of VLCAD-Deficient Patient Fibroblasts with Peroxisome Proliferator-Activated Receptor δ Agonist Improves Cellular Bioenergetics

Background: Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is an autosomal recessive disease that prevents the body from utilizing long-chain fatty acids for energy, most needed during stress and fasting. Symptoms can appear from infancy through childhood and adolescence or early adulthood, and include hypoglycemia, recurrent rhabdomyolysis, myopathy, hepatopathy, and cardiomyopathy. REN001 is a peroxisome-proliferator-activated receptor delta (PPARδ) agonist that modulates the expression of the genes coding for fatty acid β-oxidation enzymes and proteins involved in oxidative phosphorylation. Here, we assessed the effect of REN001 on VLCAD-deficient patient fibroblasts. Methods: VLCAD-deficient patient and control fibroblasts were treated with REN001. Cells were harvested for gene expression analysis, protein content, VLCAD enzyme activity, cellular bioenergetics, and ATP production. Results: VLCAD-deficient cell lines responded differently to REN001 based on genotype. All cells had statistically significant increases in ACADVL gene expression. Small increases in VLCAD protein and enzyme activity were observed and were cell-line- and dose-dependent. Even with these small increases, cellular bioenergetics improved in all cell lines in the presence of REN001, as demonstrated by the oxygen consumption rate and ATP production. VLCAD-deficient cell lines containing missense mutations responded better to REN001 treatment than one containing a duplication mutation in ACADVL. Discussion: Treating VLCAD-deficient fibroblasts with the REN001 PPARδ agonist results in an increase in VLCAD protein and enzyme activity, and a decrease in cellular stress. These results establish REN001 as a potential therapy for VLCADD as enhanced expression may provide a therapeutic increase in total VLCAD activity, but suggest the need for mutation-specific treatment augmented by other treatment measures

ACAD10 protein expression and Neurobehavioral assessment of Acad10-deficient mice.

Acyl-CoA dehydrogenase 10 (Acad10)-deficient mice develop impaired glucose tolerance, peripheral insulin resistance, and abnormal weight gain. In addition, they exhibit biochemical features of deficiencies of fatty acid oxidation, such as accumulation of metabolites consistent with abnormal mitochondrial energy metabolism and fasting induced rhabdomyolysis. ACAD10 has significant expression in mouse brain, unlike other acyl-CoA dehydrogenases (ACADs) involved in fatty acid oxidation. The presence of ACAD10 in human tissues was determined using immunohistochemical staining. To characterize the effect of ACAD10 deficiency on the brain, micro-MRI and neurobehavioral evaluations were performed. Acad10-deficient mouse behavior was examined using open field testing and DigiGait analysis for changes in general activity as well as indices of gait, respectively. ACAD10 protein was shown to colocalize to mitochondria and peroxisomes in lung, muscle, kidney, and pancreas human tissue. Acad10-deficient mice demonstrated subtle behavioral abnormalities, which included reduced activity and increased time in the arena perimeter in the open field test. Mutant animals exhibited brake and propulsion metrics similar to those of control animals, which indicates normal balance, stability of gait, and the absence of significant motor impairment. The lack of evidence for motor impairment combined with avoidance of the center of an open field arena and reduced vertical and horizontal exploration are consistent with a phenotype characterized by elevated anxiety. These results implicate ACAD10 function in normal mouse behavior, which suggests a novel role for ACAD10 in brain metabolism

Complex I assembly function and fatty acid oxidation enzyme activity of ACAD9 both contribute to disease severity in ACAD9 deficiency

Acyl-CoA dehydrogenase 9 (ACAD9) is an assembly factor for mitochondrial respiratory chain Complex I (CI), and ACAD9 mutations are recognized as a frequent cause of CI deficiency. ACAD9 also retains enzyme ACAD activity for long-chain fatty acids in vitro, but the biological relevance of this function remains controversial partly because of the tissue specificity of ACAD9 expression: high in liver and neurons and minimal in skin fibroblasts. In this study, we hypothesized that this enzymatic ACAD activity is required for full fatty acid oxidation capacity in cells expressing high levels of ACAD9 and that loss of this function is important in determining phenotype in ACAD9-deficient patients. First, we confirmed that HEK293 cells express ACAD9 abundantly. Then, we showed that ACAD9 knockout in HEK293 cells affected long-chain fatty acid oxidation along with Cl, both of which were rescued by wild type ACAD9. Further, we evaluated whether the loss of ACAD9 enzymatic fatty acid oxidation affects clinical severity in patients with ACAD9 mutations. The effects on ACAD activity of 16 ACAD9 mutations identified in 24 patients were evaluated using a prokaryotic expression system. We showed that there was a significant inverse correlation between residual enzyme ACAD activity and phenotypic severity of ACAD9-deficient patients. These results provide evidence that in cells where it is strongly expressed, ACAD9 plays a physiological role in fatty acid oxidation, which contributes to the severity of the phenotype in ACAD9-deficient patients. Accordingly, treatment of ACAD9 patients should aim at counteracting both CI and fatty acid oxidation dysfunctions