The Vockley lab research focuses on mitochondrial energy metabolism, branched chain amino acid metabolism, inborn errors of metabolism, and development of novel therapies for inborn errors of metabolism. The acyl-CoA dehydrogenases (ACDs) are a family of mitochondrial enzymes involved in fatty acid and amino acid metabolism which catalyze the transfer of electrons from various acyl-CoA esters to electron transfer flavoprotein. Biochemical and immunological studies have identified 11 distinct members of the ACD enzyme family, each with a narrow substrate specificity. Long, medium and short chain acyl-CoA dehydrogenases (LCAD, MCAD and SCAD) catalyze the first step in the -oxidation cycle with substrate optima of 16, 8 and 4 carbon chains, respectively. Isovaleryl-CoA dehydrogenase (IVD) and 2-methyl branched chain acyl-CoA dehydrogenase (2-meBCAD) catalyze the third step in leucine and isoleucine/valine metabolism, respectively. Each mature enzyme is a homotetramer and each monomer contains a non-covalently bound flavin molecule (FAD) as a prosthetic group. Deficiencies of all these enzymes except 2-meBCAD have been shown to cause disease in humans. One of these conditions (MCAD deficiency) may be the most common inborn error of metabolism in humans. My laboratory focuses on the use of molecular genetic techniques to investigate structure/function relationship in the ACD gene family at the molecular level and relate this information to mutations responsible for clinical deficiencies of these enzymes.
Specific topics of interest in my lab include: 1) analysis of the complex pathway of ACD maturation, focusing on the process of assembly of the enzymes, 2) expression analysis and computer aided molecular modeling of mutant alleles from patients with deficiencies of these enzymes as a source of naturally occurring mutations of functional significance, 3) characterization of structure/function relationships important in the active site, tetramerization, and determination of substrate specificity, 4) novel therapy for deficiencies of these enzymes, 5) functional and structural interactions of the three major pathways of mitochondrial energy pathway, and 6) contribution of energy dysfunction to common disease. The pathway of ACD maturation is studied by metabolic labeling of ACDs in tissue culture cells. Mutant alleles from patients with deficiencies of the ACDs are analyzed via eukaryotic and prokaryotic expression in order to characterize the biochemical and functional nature of the abnormal enzyme proteins produced by these patients' cells. Mouse models and patient cells are used as a platform to design and test novel therapeutics. An additional focus is on understanding the genetic disorder of phenylalanine metabolism PKU and a number of diverse clinical projects. Specific projects include:
Characterization of a Multifunctional Fatty Acid Oxidation Complex. Fatty acid B-oxidation (FAO) and oxidative phosphorylation (OXPHOS) are key pathways involved in cellular energetics. Genetic disorders of FAO and OXPHOS are among the most frequent inborn errors of metabolism. Patients with deficiencies of either FAO or OXPHOS often have clinical or biochemical findings indicative of a disorder of the other pathway. This study examined the physical and functional interactions between these pathways. It provided evidence of a multifunctional FAO complex within mitochondria that is physically associated with OXPHOS supercomplexes and promotes metabolic channeling.
Very-Long-Chain AcylCoA Dehydrogenase (VLCAD) Deficiency. More than 100 cases of VLCAD deficiency have been documented in the literature, with three different disease phenotypes. A severe infant-onset form is characterized by acute metabolic decompensation with hypoketotic hypoglycemia, dicarboxylic aciduria, liver dysfunction, and cardiomyopathy. A second form of the disease presents later in infancy or childhood but has a milder phenotype without cardiac involvement. The third form is of adolescent or adult onset and is dominated by muscle dysfunction often induced by exercise. Not surprisingly, a study found that children with the severe phenotype tended to have null mutations (71% of identified alleles in these patients), whereas patients with the two milder forms of the disease were more likely to have missense mutations (82% and 93% of identified alleles for the milder childhood form and the adult form, respectively). Nevertheless, a few missense mutations were clearly associated with the severe phenotype. Although the data suggest that missense mutations in VLCAD might obviate clinical symptoms due to some degree of residual activity, no correlation was seen between the mutations identified and residual VLCAD activity in fibroblasts. Moreover, the function effects of few of the known VLCAD missense mutations have been directly characterized. Team researchers have previously used prokaryotic expression systems to express, purify, and characterize the biochemical properties of several ACAD enzymes. Several have been crystallized and studied by X-ray diffraction, yielding informative three-dimensional models. The team has used its prokaryotic expression system to study six previously missense mutations described in VLCADdeficient patients (T220M, V243A, R429W, A450P, L462P, and R573W). T220M and V243A are the most frequently reported missense mutations in VLCAD deficient patients. R429W and R573W are among the few missense mutations believed to result in the severe clinical phenotype. A450P and L462P are located in the C-terminal domain unique to VLCAD and ACAD9. Characterization of purified wild-type, A450P, and L462P VLCAD proteins confirmed the long-held assumption that the C-terminus plays a key role in mitochondrial membrane association. The prokaryotic system developed will greatly facilitate investigation of VLCAD structure and function. Funding for this project is included in the above-referenced grant. Based on this progress, the laboratory is developing therapeutic agents for treatment of long-chain fatty acid oxidation disorders. Several potential drugs have already been patented and are moving toward clinical trials.
ACAD9. Mitochondrial B-oxidation of long-chain fatty acyl-CoAs is a primary metabolic pathway for maintenance of energy homeostasis and body temperature. It also recycles carbons from many long-chain fatty acids for lipid synthesis. Little is known about the mechanistic role of the latter in the pathogenesis of symptoms in genetic defects of B-oxidation, and its derangement may, in part, explain features of these disorders, such as neurological dysfunction and acute respiratory distress syndrome, which respond poorly to treatment with alternative energy sources. VLCAD is considered the dominant long-chain acyl-CoA dehydrogenase (LCAD) in energy generation in human muscle and heart cells. In contrast, this study provides evidence that acyl-CoA dehydrogenase 9 (ACAD9) and LCAD more likely function in lipid recycling and synthesis in human brain and lung, respectively, given their unique substrate utilization and tissue distribution pattern. Furthermore, the team has identified a new genetic deficiency of LCAD presenting with congenital surfactant deficiency. This disorder represents the first in an alpha-, beta-oxidation enzyme primarily involved in lipid recycling or synthesis, revealing a new mechanism of pathogenesis in human disease. Funding for this project by the NIH has been renewed, and a supplement through the economic stimulus grant program has been received.
Human ACAD10. In the last half of the 20th century, the incidence of type 2 diabetes mellitus (T2DM), previously unrecognized in the Pima Indians, began to rise. Multiple factors were postulated to be responsible, including environmental factors, such as diet and resultant obesity, along with a number of genetic determinants. ACAD10 was one of 30 genes examined after demonstrating a significant signal for diabetes in a genome-wide association study. To characterize the physiologic role of ACAD10 in intermediary metabolism and its possible link to T2DM, the researchers have characterized an ACAD10 gene trap mouse model. Aging animals become obese on a normal diet and develop insulin-resistant hyperglycemia in response to an intraperitoneal glucose challenge. Tissue and blood acylcarnitine profiles are similar to those previously described for adult humans with T2DM. The findings identify ACAD10 deficiency as a new monogenic cause of T2DM in mice and provide valuable insight into its potential role in the development of T2DM in Pima Indians.
Development of Novel Therapeutics for Disorders of Long Chain Fat Metabolism. Fatty acid -oxidation (FAO) is the major source of energy during times of physiologic stress and is especially critical for heart and skeletal muscle. FAO disorders are a major cause of human disease causing mitochondrial dysfunction and collectively represent the most frequent metabolic disorders in newborns worldwide. Deficiencies of FAO enzymes are often lethal, if diagnosis and treatment are delayed. Symptoms include hypoketotic hypoglycemia, Reye-like syndrome, arrhythmias, cardiomyopathy, and/or rhabdomyolysis. Current treatment protocols are inadequate, and patients are still at risk for cardiomyopathy and muscular symptoms, with significant morbidity and mortality. Therefore, we are developing a series of novel therapies to address the biochemical abnormalities. We present results of our FAOD therapies in development, and discuss prospects of implementation into clinical practice.
Drugs under development can be categorized as follows:
- Chaperones for stabilizing protein and lipid. These include trimetazidine (TMZ) to treat of VLCAD, MCAD, LCHAD, and LCKAT deficiencies, phenylbutyrate to treat MCADD, and a cardiolipin stabilizing peptide to treat LCHAD and TFP deficiencies.
- Mitochondrial-targeted ROS electron scavengers. These include JP4-039 and XJB-5-131. FAODs increase mitochondrial ROS levels that apparently impair a variety of mitochondrial functions including OXPHOS, as well as inducing inflammation. Both drugs reduce ROS levels and improve OXPHOS function in cells from patients with VLCAD, LCHAD, or ACAD9 deficiencies.
- Protein expression enhancers. PPAR agonists enhance the production of defective FAO proteins and their levels of activity. Our preliminary data show that a TMZ/PPAR combination treatment has a superior effect compared to treatment with either alone.
- Anaplerotic agents. We have designed a variety of novel anaplerotic agents that enter the TCA cycle directly and improve energy production in long chain FAODs. These agents would additionally provide benefit for methylmalonic or propionic acidemias. Their role is to alleviate the tertiary deficiency of biochemical intermediates exhausted as a result of the enzymatic block.
Changes in key biochemical markers suggest that damaging biochemical abnormalities in FAODs can be remedied by additional therapies. Our novel drugs with known pharmacodynamics have proven in vitro efficacy, and so provide the impetus to bring them to clinical trials expediently.
A Pig Model of PKU. Phenylalanine hydroxylase (PAH) deficiency, traditionally known as PKU, results in accumulation of phenylalanine (PHE) in the blood of affected individuals and was the original motivation for population based newborn screening. This project is developing a miniature pig model for greater genetic homology and more clinical relevance. The new model system will elucidate biomedical bases, facilitating development of therapeutic approaches, especially for mental retardation and neurological and neuropsychological features. Using bioinformatics and phylogenetic comparison to humans, the researchers initially assembled the entire pig Pah gene encoding a 452 amino acid enzyme and confirmed high expression of PAH in pig liver and kidney. Furthermore, they successfully targeted deletions and inversions of the Pah gene using a CRISPR/Cas9 RNA-guided nuclease approach. Studies over the first eight months of the National PKU Alliance funding period have utilized that in vitro model system and successfully optimized the genome editing reagents and mutation-detection assays for the pig Pah locus. Working with Missouri collaborators, the researchers have shown that the CRISPR gRNAs function in vivo in pig preimplantation embryos, and a pregnancy has been obtained from embryo transfers of genome-edited embryos (~ 35% modified alleles). Affected animals have now been identified, and clinical characterization is proceeding.
Development of a Home PHE Meter. Early identification of PKU and dietary treatment prevent neurological devastation, but neurodevelopmental and psychological problems are regularly diagnosed even in patients who are identified early and treated continuously. The ACMG recently published a clinical treatment guideline that recognizes the difficulty of lifelong compliance to therapeutic regimens. The guideline reports that nearly all adolescents and adults have blood PHE levels that are out of the recommended therapeutic range, leading to diminished executive function and other neurologic and neuropsychiatric problems. To improve PKU patient outcomes, the ACMG guideline says: “Better tools and strategies are required to optimize care for the individual and improve long-term outcomes.” Vockley has recently been awarded two NIH grants to develop and test a home PHE meter for use in treatment of this disease.
Characterizing the Burden of Genetic Disease in Old Order Amish. The Old Order Amish communities (Plain People) of North America have altered health risks that stem from unbalanced population sampling of European founders followed by genetic drift in derivative generations. The population effects have resulted in a high prevalence of specific genetic disorders that vary from the general population and from each other. Several characteristics of those communities facilitate genetic analysis. Most isolates keep excellent historical and genealogical records. Due to their sociologic and/or geographic isolation, there is usually little or no migration into the group, and the members of the group exhibit relatively homogeneous lifestyles. Large nuclear families are frequent, which provides adequate numbers of affected and unaffected siblings within a sibship for blood samples. The primary genetic advantage, however, results from the interaction of two overlapping phenomena: the founder effect and inbreeding. In collaboration with Ghaloul-Gonzalez, Vockley has developed a new program to characterize the genetic variability between the Amish population in Mercer County and Amish populations in other counties by doing whole-exome and mitochondrial DNA sequencing. This will be crucial to determining the phenotype and frequency of other known and unknown genetic disorders in the populations. This project will allow the researchers to characterize the genetic disease load in Old Order Amish of Mercer County and identify disorders that can benefit from early treatment.
Metabolic Imbalance in Treatment-Resistant Depression. Treatment-refractory depression, defined as depression that has no response to three or more maximum-dose treatment trials of adequate duration, is a devastating clinical problem with significant morbidity and mortality that affects at least 15% of adolescents and adults with depression. Its etiology remains unclear. Several metabolic disorders affect neurotransmitter pathways and are associated with psychiatric symptoms, including depression. The team recently published a report of a sentinel patient with severe and unremitting depression and multiple suicide attempts who was unresponsive to pharmacotherapy or electroconvulsive therapy. A neurometabolic evaluation identified a severe deficiency of all metabolites of biopterin. Treatment with sapropterin, a BH4 analog, led to a dramatic remission of his depression and suicidal ideation that has lasted four years and is still maintained. This finding triggered an exploratory case-control trial in which researchers found 24 of 40 additional patients with treatment-refractory depression to have central nervous system neurometabolic abnormalities. Additional therapeutic options were identified for 23 of the 24 patients based on their metabolic findings. Fourteen patients were identified as having cerebral folate deficiency, and subsequent treatment with folinic acid resulted in a sustained improvement of depressive symptoms in 11 of the individuals. The experience has allowed streamlining of the experimental testing protocol in this population. None of the current tools aimed at developing personalized strategies for the treatment of depression (e.g., functional neuroimaging, pharmacogenetics) would have identified the defects or led to effective therapy. The research continues, funded by a generous donor to the UPMC Children's Hospital of Pittsburgh Foundation.
Clinical Research. Vockley continues to coordinate a vigorous program in clinical research for the treatment of inborn errors of metabolism. Expanded detection of inborn errors of mitochondrial fatty acid oxidation via tandem mass spectrometry has placed them among the most frequently identified errors. Vockley formed and leads INFORM, which provides a collaborative framework for clinicians and investigators to exchange information on the disorders and their global effect on metabolism. Two medications for fatty acid oxidation disorders that were developed in the Vockley laboratory have reached phase II and III U.S. Food and Drug Administration trials, including triheptanoin, the first drug in development to treat long-chain fatty acid oxidation disorders. Additional diseases under study include lysosomal storage diseases, disorders of sterol metabolism, disorders of the urea cycle, and abnormalities of bone mineralization.