No new work had been done on this project. Two publications have been published since last report and are listed below.? ? Chronic stress is implicated in the pathogenesis of cardiovascular disease, the metabolic syndrome, neurodegeneration, depression and other psychiatric disorders, and inflammatory diseases. Skeletal myopathy and neuronal cell atrophy are common complications of endogenous and exogenous glucocorticoid excess; yet the underlying mechanisms remain unclear and targeted therapeutic interventions are limited.? ? Mitochondria play a pivotal role in cell homeostasis, housing multiple metabolic pathways that result in energy production in the form of ATP. They also generate most cellular reactive oxygen species (ROS) and regulate programmed cell death, serving as a gatekeeper for apoptosis. Not surprisingly, primary or secondary mitochondrial dysfunction have been associated with the pathogenesis of conditions such as obesity, diabetes, cancer, neurodegeneration, cardiomyopathy, depression and aging, of obvious public health significance. Patients with the aforementioned diseases commonly use wide CAM modalities.? ? To systematically study the contribution of mitochondrial genomics to human physiology and pathophysiology, we built a mitochondria-focused gene database and used it as reference for the development of a customized cDNA microarray (hMitChip), containing 501 mitochondria-related nuclear genes. To validate the ability of this mitochondrial microarray to detect changes in gene expression, we explored the pharmacogenomic effects of dexamethasone (Dex), a synthetic glucocorticoid, on the mitochondrial nuclear transcriptome in human skeletal muscle cells. ? ? Of the 501 mitochondria related genes arrayed in hMitChip, 23 (4.5%) displayed significant transcriptional changes in skeletal myocytes after treatment with Dex. Upregulation of genes involved in amine metabolism (MAOA and SAT) was statistically significant at all time intervals tested, whereas for genes involved in protein processing (AKAP10), oxidative phosphorylation (FDX1, CYBA), heat shock cellular stress response (HSPD1), lipid biosynthesis (FACL2), glycolysis (GCK) and mitochondrial protein synthesis (TSFM) upregulation was stastistically significant only at certain time points. Treatment for 7 days downregulated genes related to lipid metabolism (ACADVL, ACADM), the tricarboxylic acid cycle (DLST), protein processing (MPI, PPIB) and gene transcription (GABPA), whereas it induced genes involved in apoptosis (BNIP3). The CTNNA1 and AK1 genes, involved in cell adhesion and nucleotide metabolism, respectively, were also significantly downregulated after 7 days of exposure to 10-6 M of Dex. Reproducible results were obtained across multiple replicate experiments, and after reverse labeling of the hybridized slides ? ? For selected genes, the transcriptional changes detected with hMitChip were confirmed by QRT-PCR, carried out on RNA from the corresponding microarray experiments, as well as from separate time-course experiments, in which myocytes obtained from two independent healthy male donors were exposed to 10-6 M of Dex for up to 9 days. A significant induction of the MAOA gene was observed at every time point. Fold change increased over time, with a peak induction of 21.212.7 vs. untreated cells at day 5. FDX1 expression differed between Dex-treated and control cells at days 3 and 5, whereas FACL2 expression was different on days 5, 7, and 9, but with no significant increase over time. A trend towards downregulation was observed for ACADVL and ACADM after 7 days of exposure to Dex, consistent with the microarray findings, but failed to reach statistical significance. QRT-PCR results for selected genes, which were not differentially expressed in microarray experiments (i.e., NNT1, TOMM70) and were therefore used as negative controls, were also consistent with the microarray findings.? ? We reported that the catecholamine-metabolizing enzyme MAO-A is a major target for glucocorticoids in human skeletal muscle cells.MAO catalyzes the oxidative deamination of neuroactive, vasoactive (serotonin, dopamine, catecholamines), or dietary (tyramine) amines and xenobiotics, releasing reactive aldehydes and H202. MAO-A activation by glucocorticoids with subsequent excess hydrogen peroxide production could represent a novel pathway for glucocorticoid-induced oxidative tissue damage, and MAO-inhibitors or other allopathic and CAM interventions aimed at different components of that pathway may serve as therapeutic agents.? ? Genetic studies have linked MAO-A and MAO-B to various aspects of human behavior, and environmental interactions modulate the expression of certain MAO-A polymorphisms; however, the hormonal regulation of their expression in humans remains unexplored. We have begun to examine the effects of adrenal, gonadal and neurosteroids, in addition to glucocorticoids, on MAO expression in human fetal astrocytes. We hypothesized that estrogen, progesterone, DHEA and/or other neurosteroids (allopreganolone, THDOC) may elicit effectson on MAO expression opposite to those of glucocorticoids . ? ? It would appear that hMitChip is a reliable and novel tool that may prove useful for systematically studying the contribution of mitochondrial genomics to human health and disease.
| Alesci, S; Manoli, I; Michopoulos, V J et al. (2006) Development of a human mitochondria-focused cDNA microarray (hMitChip) and validation in skeletal muscle cells: implications for pharmaco- and mitogenomics. Pharmacogenomics J 6:333-42 |
| Manoli, Irini; Le, Hanh; Alesci, Salvatore et al. (2005) Monoamine oxidase-A is a major target gene for glucocorticoids in human skeletal muscle cells. FASEB J 19:1359-61 |
| Ilias, Ioannis; Manoli, Irini; Blackman, Marc R et al. (2004) L-Carnitine and acetyl-L-carnitine in the treatment of complications associated with HIV infection and antiretroviral therapy. Mitochondrion 4:163-8 |
