A multiomics approach reveals evidence for phenylbutyrate as a potential treatment for combined D,L-2- hydroxyglutaric aciduria.

PURPOSE: To identify therapies for combined D, L-2-hydroxyglutaric aciduria (C-2HGA), a rare genetic disorder caused by recessive variants in the SLC25A1 gene. METHODS: Patients C-2HGA were identified and diagnosed by whole exome sequencing and biochemical genetic testing. Patient derived fibroblasts were then treated with phenylbutyrate and the functional effects assessed by metabolomics and RNA-sequencing. RESULTS: In this study, we demonstrated that C-2HGA patient derived fibroblasts exhibited impaired cellular bioenergetics. Moreover, Fibroblasts form one patient exhibited worsened cellular bioenergetics when supplemented with citrate. We hypothesized that treating patient cells with phenylbutyrate (PB), an FDA approved pharmaceutical drug that conjugates glutamine for renal excretion, would reduce mitochondrial 2-ketoglutarate, thereby leading to improved cellular bioenergetics. Metabolomic and RNA-seq analyses of PB-treated fibroblasts demonstrated a significant decrease in intracellular 2-ketoglutarate, 2-hydroxyglutarate, and in levels of mRNA coding for citrate synthase and isocitrate dehydrogenase. Consistent with the known action of PB, an increased level of phenylacetylglutamine in patient cells was consistent with the drug acting as 2-ketoglutarate sink. CONCLUSION: Our pre-clinical studies suggest that citrate supplementation has the possibility exacerbating energy metabolism in this condition. However, improvement in cellular bioenergetics suggests phenylbutyrate might have interventional utility for this rare disease.

Heptanoic and medium branched-chain fatty acids as anaplerotic treatment for medium chain acyl-CoA dehydrogenase deficiency.

Triheptanoin (triheptanoylglycerol) has shown value as anaplerotic therapy for patients with long chain fatty acid oxidation disorders but is contraindicated in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. In search for anaplerotic therapy for patients with MCAD deficiency, fibroblasts from three patients homozygous for the most common mutation, ACADMG985A/G985A, were treated with fatty acids hypothesized not to require MCAD for their metabolism, including heptanoic (C7; the active component of triheptanoin), 2,6-dimethylheptanoic (dMC7), 6-amino-2,4-dimethylheptanoic (AdMC7), or 4,8-dimethylnonanoic (dMC9) acids. Their effectiveness as anaplerotic fatty acids was assessed in live cells by monitoring changes in cellular oxygen consumption rate (OCR) and mitochondrial protein lysine succinylation, which reflects cellular succinyl-CoA levels, using immunofluorescence (IF) staining. Krebs cycle intermediates were also quantitated in these cells using targeted metabolomics. The four fatty acids induced positive changes in OCR parameters, consistent with their oxidative catalysis and utilization. Increases in cellular IF staining of succinylated lysines were observed, indicating that the fatty acids were effective sources of succinyl-CoA in the absence of media glucose, pyruvate, and lipids. The ability of MCAD deficient cells to metabolize C7 was confirmed by the ability of extracts to enzymatically utilize C7-CoA as substrate but not C8-CoA. To evaluate C7 therapeutic potential in vivo, Acadm-/- mice were treated with triheptanoin for seven days. Dose dependent increase in plasma levels of heptanoyl-, valeryl-, and propionylcarnitine indicated efficient metabolism of the medication. The pattern of the acylcarnitine profile paralleled resolution of liver pathology including reversing hepatic steatosis, increasing hepatic glycogen content, and increasing hepatocyte protein succinylation, all indicating improved energy homeostasis in the treated mice. These results provide the impetus to evaluate triheptanoin and the medium branched chain fatty acids as potential therapeutic agents for patients with MCAD deficiency.

Messenger RNA rescues medium-chain acyl-CoA dehydrogenase deficiency in fibroblasts from patients and a murine model.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common inherited disorder of mitochondrial fatty acid ß-oxidation (FAO) in humans. Patients exhibit clinical episodes often associated with fasting. Symptoms include hypoketotic hypoglycemia and Reye-like episodes. With limited treatment options, we explored the use of human MCAD (hMCAD) mRNA in fibroblasts from patients with MCAD deficiency to provide functional MCAD protein and reverse the metabolic block. Transfection of hMCAD mRNA into MCAD- deficient patient cells resulted in an increased MCAD protein that localized to mitochondria, concomitant with increased enzyme activity in cell extracts. The therapeutic hMCAD mRNA-lipid nanoparticle (LNP) formulation was also tested in vivo in Acadm-/- mice. Administration of multiple intravenous doses of the hMCAD mRNA-LNP complex (LNP-MCAD) into Acadm-/- mice produced a significant level of MCAD protein with increased enzyme activity in liver, heart and skeletal muscle homogenates. Treated Acadm-/- mice were more resistant to cold stress and had decreased plasma levels of medium-chain acylcarnitines compared to untreated animals. Furthermore, hepatic steatosis in the liver from treated Acadm-/- mice was reduced compared to untreated ones. Results from this study support the potential therapeutic value of hMCAD mRNA-LNP complex treatment for MCAD deficiency.

A multiomics approach to understanding pathology of Combined D,L-2- Hydroxyglutaric Aciduria and phenylbutyrate as potential treatment.

Combined D, L-2-Hydroxyglutaric Aciduria (D,L-2HGA) is a rare genetic disorder caused by recessive mutations in the SLC25A1 gene that encodes the mitochondrial citrate carrier protein (CIC). SLC25A1 deficiency leads to a secondary increase in mitochondrial 2-ketoglutarate that, in turn, is reduced to neurotoxic 2-hydroxyglutarate. Clinical symptoms of Combined D,L-2HGA include neonatal encephalopathy, respiratory insufficiency and often with death in infancy. No current therapies exist, although replenishing cytosolic stores by citrate supplementation to replenish cytosolic stores has been proposed. In this study, we demonstrated that patient derived fibroblasts exhibited impaired cellular bioenergetics that were worsened with citrate supplementation. We hypothesized treating patient cells with phenylbutyrate, an FDA approved pharmaceutical drug, would reduce mitochondrial 2-ketoglutarate, leading to improved cellular bioenergetics including oxygen consumption and fatty acid oxidation. Metabolomic and RNA-seq analyses demonstrated a significant decrease in intracellular 2-ketoglutarate, 2-hydroxyglutarate, and in levels of mRNA coding for citrate synthase and isocitrate dehydrogenase. Consistent with the known action of phenylbutyrate, detected levels of phenylacetylglutamine was consistent with the drug acting as 2-ketoglutarate sink in patient cells. Our pre-clinical studies suggest citrate supplementation is unlikely to be an effective treatment of the disorder. However, cellular bioenergetics suggests phenylbutyrate may have interventional utility for this rare disease.

Synthetic mRNA rescues very long-chain acyl-CoA dehydrogenase deficiency in patient fibroblasts and a murine model.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is an inborn error of long chain fatty acid ß-oxidation (FAO) with limited treatment options. Patients present with heterogeneous clinical phenotypes affecting predominantly heart, liver, and skeletal muscle. While VLCAD deficiency is a systemic disease, restoration of liver FAO has the potential to improve symptoms more broadly due to increased total body ATP production and reduced accumulation of potentially toxic metabolites. We explored the use of synthetic human VLCAD (hVLCAD) mRNA and lipid nanoparticle encapsulated hVLCAD mRNA (LNP-VLCAD) to generate functional VLCAD enzyme in patient fibroblasts derived from VLCAD deficient patients, mouse embryonic fibroblasts, hepatocytes isolated from VLCAD knockout (Acadvl-/-) mice, and Acadvl-/- mice to reverse the metabolic effects of the deficiency. Transfection of all cell types with hVLCAD mRNA resulted in high level expression of protein that localized to mitochondria with increased enzyme activity. Intravenous administration of LNP-VLCAD to Acadvl-/- mice produced a significant amount of VLCAD protein in liver, which declined over a week. Treated Acadvl-/- mice showed reduced hepatic steatosis, were more resistant to cold stress, and accumulated less toxic metabolites in blood than untreated animals. Results from this study support the potential for hVLCAD mRNA for treatment of VLCAD deficiency.

ITCH deficiency clinical phenotype expansion and mitochondrial dysfunction.

Autoimmune Disease, Multisystem, with Facial Dysmorphism (ADMFD) is an autosomal recessive disorder due to pathogenic variants in the ITCH gene. It is characterized by failure to thrive, dysmorphic facial features, developmental delay, and systemic autoimmunity that can manifest variably with autoimmune hepatitis, thyroiditis, and enteropathy, among other organ manifestations. It was originally described in 10 consanguineous Old Order Amish patients, and more recently in two patients of White British and Black German ethnicities. While the role of ITCH protein in apoptosis and inflammation has previously been characterized, a defect in cellular bioenergetics has not yet been reported in ITCH deficiency. Here we present a Caucasian female originally evaluated for possible mitochondrial respiratory chain deficiency, who ultimately was found to have two novel variants in ITCH with absence of ITCH protein in patient derived fibroblasts. Clinical studies of patient muscle showed mitochondrial DNA copy number of 57% compared to controls. Functional studies in skin fibroblasts revealed decreased activity of mitochondrial fatty acid oxidation and oxidative phosphorylation, and decreased overall ATP production. Our findings confirm mitochondrial energy dysfunction in a patient with ITCH deficiency offering the opportunity to assess alternative therapeutic options.

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 ß-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.

Mitochondrial dysfunction associated with TANGO2 deficiency.

Transport and Golgi Organization protein 2 Homolog (TANGO2)-related disease is an autosomal recessive disorder caused by mutations in the TANGO2 gene. Symptoms typically manifest in early childhood and include developmental delay, stress-induced episodic rhabdomyolysis, and cardiac arrhythmias, along with severe metabolic crises including hypoglycemia, lactic acidosis, and hyperammonemia. Severity varies among and within families. Previous studies have reported contradictory evidence of mitochondrial dysfunction. Since the clinical symptoms and metabolic abnormalities are suggestive of a broad dysfunction of mitochondrial energy metabolism, we undertook a broad examination of mitochondrial bioenergetics in TANGO2 deficient patients utilizing skin fibroblasts derived from three patients exhibiting TANGO2-related disease. Functional studies revealed that TANGO2 protein was present in mitochondrial extracts of control cells but not patient cells. Superoxide production was increased in patient cells, while oxygen consumption rate, particularly under stress, along with relative ATP levels and ß-oxidation of oleate were reduced. Our findings suggest that mitochondrial function should be assessed and monitored in all patients with TANGO2 mutation as targeted treatment of the energy dysfunction could improve outcome in this condition.

Medium branched chain fatty acids improve the profile of tricarboxylic acid cycle intermediates in mitochondrial fatty acid ß-oxidation deficient cells: A comparative study.

Inherited errors of mitochondrial fatty acid ß-oxidation (FAO) are life threatening, even with optimum care. FAO is the major source of energy for heart and is critical for skeletal muscles especially during physiologic stress. Clinical trials revealed that triheptanoin (commercially known as Dojolvi; C7G), improved heart function and decreased hypoglycemia in long chain FAO disorders, but other symptoms including rhabdomyolysis persisted, suggesting suboptimal tissue distribution/utilization of heptanoic acid (C7) conjugates and/or rapid liver breakdown. In this study, medium branched chain fatty acids were tested as potential anaplerotic treatments in fibroblasts from patients deficient in very long chain acyl-CoA dehydrogenase (VLCAD), long chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD), trifunctional protein (TFP), and carnitine palmitoyltransferase II (CPT II). Cells were cultured to near confluency and treated with C7, 2,6-dimethylheptanoic acid (dMC7), 6-amino-2,4-dimethylheptanoic acid (AdMC7), or 4,8-dimethylnonanoic acid (dMC9) for 72 h and targeted metabolomics performed. The profile of TCA cycle intermediates was improved in cells treated with these branched chain fatty acids compared with C7. Intracellular propionate was higher in AdMC7 treated cells compared with C7 in VLCAD, LCHAD, and TFP deficient cell lines. With AdMC7 treatment, succinate was higher in CPT II and VLCAD deficient cells, compared with C7. Malate and glutamate were consistently higher in AdMC7 treated VLCAD, LCHAD, TFP, and CPT II deficient cells compared with the C7 treatment. The results provide the impetus to further evaluate and consider branched chain fatty acids as viable anaplerotic therapy for fatty acid oxidation disorders and other diseases.

Development and characterization of a mouse model for Acad9 deficiency.

Acyl CoA Dehydrogenase 9 (ACAD9) is a member of the family of flavoenzymes that catalyze the dehydrogenation of acyl-CoAs to 2,3 enoyl-CoAs in mitochondrial fatty acid oxidation (FAO). Inborn errors of metabolism of all family members, including ACAD9, have been described in humans, and represent significant causes of morbidity and mortality particularly in children. ACAD9 deficiency leads to a combined defect in fatty acid oxidation and oxidative phosphorylation (OXPHOS) due to a dual role in the pathways. In addition to its function in mitochondrial FAO, ACAD9 has a second function as one of 14 factors responsible for assembly of complex I of the electron transport chain (ETC). Considerable controversy remains over the relative role of these two functions in normal physiology and the disparate clinical findings described in patients with ACAD9 deficiency. To better understand the normal function of ACAD9 and the pathophysiology of its deficiency, several knock out mouse models were developed. Homozygous total body knock out appeared to be lethal as no ACAD9 animals were obtained. Cre-lox technology was then used to generate tissue-specific deletion of the gene. Cardiac-specific ACAD9 deficient animals had severe neonatal cardiomyopathy and died by 17 days of age. They had severe mitochondrial dysfunction in vitro. Muscle-specific mutants were viable but exhibited muscle weakness. Additional studies of heart muscle from the cardiac specific deficient animals were used to examine the evolutionarily conserved signaling Intermediate in toll pathway (ECSIT) protein, a known binding partner of ACAD9 in the electron chain complex I assembly pathway. As expected, ECSIT levels were significantly reduced in the absence of ACAD9 protein, consistent with the demonstrated impairment of the complex I assembly. The various ACAD9 deficient animals should serve as useful models for development of novel therapeutics for this disorder.

Characterization of variants of uncertain significance in isovaleryl-CoA dehydrogenase identified through newborn screening: An approach for faster analysis.

INTRODUCTION: Clinical standard of care for newborn screening (NBS) is acylcarnitine metabolites quantitation by tandem mass spectrometry (MS/MS) from dried blood spots. Follow up sequencing often results in identification of one or more variants of uncertain significance (VUS). Isovaleric acidemia (IVA) is an autosomal recessive inborn error of metabolism caused by deficiency of isovaleryl-CoA dehydrogenase (IVDH) in the Leu catabolism pathway. Many IVD mutations are characterized as VUS complicating IVA clinical diagnoses and treatment. We present a testing platform approach to confirm the functional implication of VUS identified in newborns with IVA applicable to multiple inborn errors of metabolism identified by NBS. METHODS: An IVD null HEK293T cell culture model was generated by using a dual sgRNA CRISPR/Cas9 genome-editing strategy targeting IVD exons 2-3. Clonal cell lines were confirmed by a combination of genomic breakpoint sequencing and droplet digital PCR. The IVD null model had no IVDH antigen signal and 96% reduction in IVDH enzyme activity. The IVD null model was transfected with vectors containing control or variant IVD and functional assays were performed to determine variant pathogenicity. RESULTS: c.149G > C (p.Arg50Pro; precursor numbering), c.986T > C (p.Met329Thr), and c.1010G > A (p.Arg337Gln), c.1179del394 f. mutant proteins had reduced IVDH protein and activity. c.932C > T (p.Ala311Val), c.707C > T (p.Thr236Ile), and c.1232G > A (p.Arg411Gln) had stable IVDH protein, but no enzyme activity. c.521T > G (p.Val174Gly) had normal IVDH protein and activity. IVD variant transfection results confirmed results from IVA fibroblasts containing the same variants. CONCLUSIONS: We have developed an IVD null HEK293T cell line to rapidly allow determination of VUS pathogenicity following identification of novel alleles by clinical sequencing following positive NBS results for suspected IVA. We suggest similar models can be generated via genome-editing for high throughput assessment of VUS function for a multitude of inborn errors of metabolism and can ideally supplement NBS programs.

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.

Clinical, biochemical, mitochondrial, and metabolomic aspects of methylmalonate semialdehyde dehydrogenase deficiency: Report of a fifth case.

Methylmalonate semialdehyde dehydrogenase deficiency (MMSDD; MIM 614105) is a rare autosomal recessive defect of valine and pyrimidine catabolism. Four prior MMSDD cases are published. We present a fifth case, along with functional and metabolomic analysis. The patient, born to non-consanguineous parents of East African origin, was admitted at two weeks of age for failure to thrive. She was nondysmorphic, had a normal brain MRI, and showed mild hypotonia. Gastroesophageal reflux occurred with feeding. Urine organic acid assessment identified excess 3-hydroxyisobutyrate and 3-hydroxypropionate, while urine amino acid analysis identified elevated concentrations of ß-aminoisobutyrate and ß-alanine. Plasma amino acids showed an elevated concentration of ß-aminoisobutyrate with undetectable ß-alanine. ALDH6A1 gene sequencing identified a homozygous variant of uncertain significance, c.1261C > T (p.Pro421Ser). Management with valine restriction led to reduced concentration of abnormal analytes in blood and urine, improved growth, and reduced gastroesophageal reflux. Western blotting of patient fibroblast extracts demonstrated a large reduction of methylmalonate semialdehyde dehydrogenase (MMSD) protein. Patient cells displayed compromised mitochondrial function with increased superoxide production, reduced oxygen consumption, and reduced ATP production. Metabolomic profiles from patient fibroblasts demonstrated over-representation of fatty acids and fatty acylcarnitines, presumably due to methylmalonate semialdehyde shunting to ß-alanine and subsequently to malonyl-CoA with ensuing increase of fatty acid synthesis. Previously reported cases of MMSDD have shown variable clinical presentation. Our case continues the trend as clinical phenotypes diverge from prior cases. Recognition of mitochondrial dysfunction and novel metabolites in this patient provide the opportunity to assess future patients for secondary changes that may influence clinical outcome.

Reticular Dysgenesis and Mitochondriopathy Induced by Adenylate Kinase 2 Deficiency with Atypical Presentation.

Reticular dysgenesis is an autosomal recessive form of severe combined immunodeficiency (SCID) that usually manifests in newborns. It is a unique example of an immune deficiency that is linked to dysfunctional mitochondrial energy metabolism and caused by adenylate kinase 2 (AK2) deficiency. It is characterized by an early differentiation arrest in the myeloid lineage, impaired lymphoid maturation, and sensorineural hearing loss. In this study, a novel AK2 homozygous mutation, c.622 T > C [p.Ser208Pro], was identified in an Old Order Amish patient through whole exome sequencing. Functional studies showed that the patient's cells have no detectable AK2 protein, as well as low oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and proton production rate (PPR). An increased production of reactive oxygen species, mitochondrial membrane permeability, and mitochondrial mass, and decreased ATP production, were also observed. The results confirm the pathogenicity of the AK2 mutation and demonstrate that reticular dysgenesis should be considered in Amish individuals presenting with immune deficiency. We also describe other pathophysiological aspects of AK2 deficiency not previously reported.

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) deficiencies are hereditary disorders that affect the catabolism of sulfur-containing amino acids. ETHE1 deficiency is caused by mutations in the ETHE1 gene, while MoCo deficiency 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 findings. 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 fibroblasts from four patients with ETHE1 deficiency and one with MOCS1 deficiency. The effect 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 identified in the same cells, while variable alterations in mitochondrial fusion and fission 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 deficiencies, and that reduction of mitochondrial superoxide by JP4-039 is a promising strategy for adjuvant therapy of these disorders.

Mitochondrial energetics is impaired in very long-chain acyl-CoA dehydrogenase deficiency and can be rescued by treatment with mitochondria-targeted electron scavengers.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is the most common defect of mitochondrial long-chain fatty acid ß-oxidation. Patients present with heterogeneous clinical phenotypes affecting heart, liver and skeletal muscle predominantly. The full pathophysiology of the disease is unclear and patient response to current therapeutic regimens is incomplete. To identify additional cellular alterations and explore more effective therapies, mitochondrial bioenergetics and redox homeostasis were assessed in VLCAD-deficient fibroblasts, and several protective compounds were evaluated. The results revealed cellular and tissue changes, including decreased respiratory chain (RC) function, increased reactive oxygen species (ROS) production and altered mitochondrial function and signaling pathways in a variety of VLCAD-deficient fibroblasts. The mitochondrially enriched electron and free radical scavengers JP4-039 and XJB-5-131 improved RC function and decreased ROS production significantly, suggesting that they are viable candidate compounds to further develop to treat VLCAD-deficient patients.

Evaluation of mitochondrial bioenergetics, dynamics, endoplasmic reticulum-mitochondria crosstalk, and reactive oxygen species in fibroblasts from patients with complex I deficiency.

Mitochondrial complex I (CI) deficiency is the most frequent cause of oxidative phosphorylation (OXPHOS) disorders in humans. In order to benchmark the effects of CI deficiency on mitochondrial bioenergetics and dynamics, respiratory chain (RC) and endoplasmic reticulum (ER)-mitochondria communication, and superoxide production, fibroblasts from patients with mutations in the ND6, NDUFV1 or ACAD9 genes were analyzed. Fatty acid metabolism, basal and maximal respiration, mitochondrial membrane potential, and ATP levels were decreased. Changes in proteins involved in mitochondrial dynamics were detected in various combinations in each cell line, while variable changes in RC components were observed. ACAD9 deficient cells exhibited an increase in RC complex subunits and DDIT3, an ER stress marker. The level of proteins involved in ER-mitochondria communication was decreased in ND6 and ACAD9 deficient cells. |ΔΨ| and cell viability were further decreased in all cell lines. These findings suggest that disruption of mitochondrial bioenergetics and dynamics, ER-mitochondria crosstalk, and increased superoxide contribute to the pathophysiology in patients with ACAD9 deficiency. Furthermore, treatment of ACAD9 deficient cells with JP4-039, a novel mitochondria-targeted reactive oxygen species, electron and radical scavenger, decreased superoxide level and increased basal and maximal respiratory rate, identifying a potential therapeutic intervention opportunity in CI deficiency.

Investigating the link of ACAD10 deficiency to type 2 diabetes mellitus.

The Native American Pima population has the highest incidence of insulin resistance (IR) and type 2 diabetes mellitus (T2DM) of any reported population, but the pathophysiologic mechanism is unknown. Genetic studies in Pima Indians have linked acyl-CoA dehydrogenase 10 (ACAD10) gene polymorphisms, among others, to this predisposition. The gene codes for a protein with a C-terminus region that is structurally similar to members of a family of flavoenzymes-the acyl-CoA dehydrogenases (ACADs)-that catalyze α,ß-dehydrogenation reactions, including the first step in mitochondrial FAO (FAO), and intermediary reactions in amino acids catabolism. Dysregulation of FAO and an increase in plasma acylcarnitines are recognized as important in the pathophysiology of IR and T2DM. To investigate the deficiency of ACAD10 as a monogenic risk factor for T2DM in human, an Acad-deficient mouse was generated and characterized. The deficient mice exhibit an abnormal glucose tolerance test and elevated insulin levels. Blood acylcarnitine analysis shows an increase in long-chain species in the older mice. Nonspecific variable pattern of elevated short-terminal branch-chain acylcarnitines in a variety of tissues was also observed. Acad10 mice accumulate excess abdominal adipose tissue, develop an early inflammatory liver process, exhibit fasting rhabdomyolysis, and have abnormal skeletal muscle mitochondria. Our results identify Acad10 as a genetic determinant of T2DM in mice and provide a model to further investigate genetic determinants for insulin resistance in humans.

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.

Long-chain acyl-CoA dehydrogenase deficiency as a cause of pulmonary surfactant dysfunction.

Long-chain acyl-CoA dehydrogenase (LCAD) is a mitochondrial fatty acid oxidation enzyme whose expression in humans is low or absent in organs known to utilize fatty acids for energy such as heart, muscle, and liver. This study demonstrates localization of LCAD to human alveolar type II pneumocytes, which synthesize and secrete pulmonary surfactant. The physiological role of LCAD and the fatty acid oxidation pathway in lung was subsequently studied using LCAD knock-out mice. Lung fatty acid oxidation was reduced in LCAD(-/-) mice. LCAD(-/-) mice demonstrated reduced pulmonary compliance, but histological examination of lung tissue revealed no obvious signs of inflammation or pathology. The changes in lung mechanics were found to be due to pulmonary surfactant dysfunction. Large aggregate surfactant isolated from LCAD(-/-) mouse lavage fluid had significantly reduced phospholipid content as well as alterations in the acyl chain composition of phosphatidylcholine and phosphatidylglycerol. LCAD(-/-) surfactant demonstrated functional abnormalities when subjected to dynamic compression-expansion cycling on a constrained drop surfactometer. Serum albumin, which has been shown to degrade and inactivate pulmonary surfactant, was significantly increased in LCAD(-/-) lavage fluid, suggesting increased epithelial permeability. Finally, we identified two cases of sudden unexplained infant death where no lung LCAD antigen was detectable. Both infants were homozygous for an amino acid changing polymorphism (K333Q). These findings for the first time identify the fatty acid oxidation pathway and LCAD in particular as factors contributing to the pathophysiology of pulmonary disease.