Open Access

Resveratrol (RSV) is a small compound first identified as an activator of sirtuin 1 (SIRT1), a key factor in mediating the effects of caloric restriction. Since then, RSV received great attention for its widespread beneficial effects on health and in connection to many diseases. RSV improves the metabolism and the mitochondrial function, and more recently it was shown to restore fatty acid β-oxidation (FAO) capacities in patient fibroblasts harboring mutations with residual enzyme activity. Many of RSV's beneficial effects are mediated by the transcriptional coactivator PGC-1α, a direct target of SIRT1 and a master regulator of the mitochondrial fatty acid oxidation. Despite numerous studies RSV's mechanism of action is still not completely elucidated. Our aim was to investigate the effects of RSV on gene regulation on a wide scale, possibly to detect novel genes whose up-regulation by RSV may be of interest with respect to disease treatment. We performed Next Generation Sequencing of RNA on normal fibroblasts treated with RSV. To investigate whether the effects of RSV are mediated through SIRT1 we expanded the analysis to include SIRT1-knockdown fibroblasts. We identified the aspartoacylase (ASPA) gene, mutated in Canavan disease, to be strongly up-regulated by RSV in several cell lines, including Canavan disease fibroblasts. We further link RSV to the up-regulation of other genes involved in myelination including the glial specific transcription factors POU3F1, POU3F2, and myelin basic protein (MBP). We also observe a strong up-regulation by RSV of the riboflavin transporter gene SLC52a1. Mutations in SLC52a1 cause transient multiple acyl-CoA dehydrogenase deficiency (MADD). Our analysis of alternative splicing identified novel metabolically important genes affected by RSV, among which is particularly interesting the α subunit of the stimulatory G protein (Gsα), which regulates the cellular levels of cAMP through adenylyl cyclase. We conclude that in fibroblasts RSV stimulates the PGC-1α and p53 pathways, and up-regulates genes affecting the glucose metabolism, mitochondrial β-oxidation, and mitochondrial biogenesis. We further confirm that RSV might be a relevant treatment in the correction of FAO deficiencies and we suggest that treatment in other metabolic disorders including Canavan disease and MADD might be also beneficial.

Once aberrantly activated, the Wnt/βcatenin pathway may result in uncontrolled proliferation and eventually cancer. Efforts to counter and inhibit this pathway are mainly directed against βcatenin, as it serves a role on the cytoplasm and the nucleus. In addition, speciallygenerated lymphocytes are recruited for the purpose of treating liver cancer. Peripheral blood mononuclear lymphocytes are expanded by the timely addition of interferon γ, interleukin (IL)1β, IL2 and anticluster of differentiation 3 antibody. The resulting cells are called cytokineinduced killer (CIK) cells. The present study utilised these cells and combine them with drugs inhibiting the Wnt pathway in order to examine whether this resulted in an improvement in the killing ability of CIK cells against liver cancer cells. Drugs including ethacrynic acid (EA) and ciclopirox olamine (CPX) were determined to be suitable candidates, as determined by previous studies. Drugs were administered on their own and combined with CIK cells and then a cell viability assay was performed. These results suggest that EAtreated cells demonstrated apoptosis and were significantly affected compared with untreated cells. Unlike EA, CPX killed normal and cancerous cells even at low concentrations. Subsequent to combining EA with CIK cells, the potency of killing was increased and a greater number of cells died, which proves a synergistic action. In summary, EA may be used as an antihepatocellular carcinoma drug, while CPX possesses a high toxicity to cancerous as well as to normal cells. It was proposed that EA should be integrated into present therapeutic methods for cancer.

The human MPV17-related mitochondrial DNA depletion syndrome is an inherited autosomal recessive disease caused by mutations in the inner mitochondrial membrane protein MPV17. Although more than 30 MPV17 gene mutations were shown to be associated with mitochondrial DNA depletion syndrome, the function of MPV17 is still unknown. Mice deficient in Mpv17 show signs of premature aging. In the present study, we used electrophysiological measurements with recombinant MPV17 to reveal that this protein forms a non-selective channel with a pore diameter of 1.8 nm and located the channel's selectivity filter. The channel was weakly cation-selective and showed several subconductance states. Voltage-dependent gating of the channel was regulated by redox conditions and pH and was affected also in mutants mimicking a phosphorylated state. Likewise, the mitochondrial membrane potential (Deltapsim) and the cellular production of reactive oxygen species were higher in embryonic fibroblasts from Mpv17(-/-) mice. However, despite the elevated Deltapsim, the Mpv17-deficient mitochondria showed signs of accelerated fission. Together, these observations uncover the role of MPV17 as a Deltapsim-modulating channel that apparently contributes to mitochondrial homeostasis under different conditions.

Gene transfer vectors based on the adeno-associated virus (AAV) are used for various experimental and clinical therapeutic approaches. In the present study, we demonstrate the utility of rAAV as a tumoricidal agent in human colorectal cancer. We constructed an rAAV vector that expresses tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) and used it to transduce human colorectal cancer cells. TRAIL belongs to the TNF superfamily of cytokines that are involved in various immune responses and apoptotic processes. It has been shown to induce cell death specifically in cancer cells.

Recently, gamma-glutamyl transpeptidase, which initiates cleavage of extracellular glutathione, has been shown to promote oxidative damage to cells. Here we examined a murine disease model of glomerulosclerosis, involving loss of the Mpv17 gene coding for a peroxisomal protein. In Mpv17-/- cells, enzyme activity and mRNA expression (examined by quantitative RT-PCR) of membrane-bound gamma-glutamyl transpeptidase were increased, while plasma glutathione peroxidase and superoxide dismutase levels were lowered. Superoxide anion production in these cells was increased as documented by electron spin resonance spectroscopy. In the presence of Mn(III)tetrakis(4-benzoic acid)porphyrin, the activities of gamma-glutamyl transpeptidase and plasma glutathione peroxidase were unchanged, suggesting a relationship between enzyme expression and the amount of reactive oxygen species. Inhibition of gamma-glutamyl transpeptidase by acivicin reverted the lowered plasma glutathione peroxidase and superoxide dismutase activities, indicating reciprocal control of gene expression for these enzymes.

The mutant Mpv17 mouse is a transgenic strain that fails to express a protein that is normally expressed in the kidney and that is associated with peroxisomes. The present studies provide a quantitative examination of renal function and structure in this strain compared to its control CFW strain. By 52 wk of age, the mutant strain developed proteinuria (urinary protein to creatinine ratio: 25 +/- 14 versus 3 +/- 1, mutant versus control), albuminuria (urinary albumin to creatinine ratio: 23 +/- 15 versus 0.1 +/- 0.1, mutant versus control), and hypoalbuminemia (2.1 +/- 0.4 versus 2.5 +/- 0.2 G/dl, mutant versus control), but without arterial hypertension or major reduction in filtration (serum creatinine 0.14 +/- 0.04 versus 0.18 +/- 0.12 mg/dl, mutant versus control). The Mpv17 glomeruli were enlarged (0.98 +/- 0.12 versus 0.52 +/- 0.02 micrometer(3) x 10(6), mutant versus control). Glomerular sclerosis became widespread (95 +/- 3 versus 23 +/- 32%, mutant versus control) and was preceded by mesangiolysis and microaneurysms. Tubulointerstitial disease was conspicuous by its absence. The intrarenal vasculature was normal in the mutant mice. Electron microscopy demonstrated focal foot process fusion and mesangiolysis. Thus, this mutant strain of mouse develops proteinuria and a distinct glomerulopathy including mesangiolysis but little interstitial injury all due to the loss of expression of a single gene.

We have generated transgenic mice carrying the URR of the human papillomavirus type 11 ligated in front of the Escherichia coli beta-galactosidase coding region sequence. Using X-Gal staining to demonstrate beta-galactosidase production, we observed a hair-specific transcription of the reporter gene. This transcription was limited to the epithelial cells of the hair bulge region. The transgene was developmentally regulated, as no LacZ staining was demonstrated during embryogenesis and specific staining was first observed after birth. Surprisingly, dexamethasone and ultraviolet B, but not phorbol myristate acetate or progesterone treatment of the animals resulted in an increase in number and intensity of hair follicles expressing the reporter gene.

Focal segmental glomerulosclerosis is a steroid-resistant glomerular disease characterized by foot process flattening and heavy proteinuria. A similar disease was found to occur spontaneously in mice in which the Mpv17 gene was inactivated by retroviral insertion (Mpv17-/- mice). Here evidence is provided that glomerular damage in this murine model is due to overproduction of oxygen radicals and accumulation of lipid peroxidation adducts that were found in isolated glomeruli of Mpv17-/- mice. The development of glomerular disease in Mpv17-/- mice was inhibited by scavengers of oxygen radicals (dithiomethylurea) and lipid peroxidation (probucol), but not by steroid treatment. Although the glomerular polyanion was greatly reduced in proteinuric Mpv17-/- mice, it was preserved by antioxidative therapy. These results indicate that the glomerular disease in Mpv17-/- mice qualifies as a model of steroid-resistant focal segmental glomerulosclerosis and that experimental therapies with scavengers of oxygen radicals and lipid peroxidation efficiently ameliorate glomerular damage.

The transgenic mouse strain Mpv17 develops severe morphological degeneration of the inner ear and nephrotic syndrome at a young age (Meyer zum Gottesberge et al., 1996; Weiher et al., 1990). The audiograms (1-32 kHz) of Mpv17-negative mice were determined from auditory brain stem responses in young (2 months) and old (7 months) animals. Audiograms of age-matched wild-type mice with the same genetic background, but wild-type at the Mpv17 locus, were also determined. Furthermore, young Mpv17-negative mice that carried a human Mpv17 homologue gene were studied. NMRI mice served as a reference for normal hearing. Mpv17-negative mice suffer from severe sensorineural hearing loss as early as 2 months after birth. In the old Mpv17-negative mice no responses could be elicited at all. The 2 month old wild-type mice had normal audiograms, at 7 months only high threshold responses were seen. The poor audiograms of the Mpv17-negative mice are assumed to be the functional correlate of the morphological degeneration of the cochlea described earlier (Meyer zum Gottesberge et al., 1996). The finding that 2 out of 4 Mpv17-negative mice with the human Mpv17 gene had normal audiograms, shows that the gene inactivation can be functionally compensated by the human Mpv17 gene product.

The Mpv17 mouse strain is a recessive transgenic mouse mutant that develops glomerulosclerosis and nephrotic syndrome at a young age. The phenotype results from a loss of function of a gene coding for a hydrophobic peroxisomal protein of 176 amino acids of 20 kDa following its destruction by retroviral integration. To investigate a potential effect of the missing Mpv17 function on the inner ear light and electron microscopic investigations were performed on the inner ears of Mpv17 mice and controls. These revealed degeneration of the stria vascularis and spiral ligament, loss of cochlear neurons and degeneration of the organ of Corti. The alterations observed here were similar to those described for Alport's syndrome, an inherited disorder characterized by progressive nephritis and neurosensory deafness. These findings indicate that although the molecular cause is different, the Mpv17 mouse model may share pathological mechanisms involved in patients with Alport's syndrome. At present the Mpv17 mouse appears to be a suitable animal model for this disease and may help to further elucidate the relationship between the kidney and the inner ear.

The germ line insertion of a defective retrovirus into the Mpv17 gene of mice is associated with a recessive phenotype. Mice homozygous for the integration develop glomerulosclerosis at a young age. The phenotype resembles human glomerulosclerosis in its physiological parameters as well as in histology. A human homologue of the Mpv17 gene has been identified, isolated and analyzed. We here show that this gene, which has a role in the production of reactive oxygen species, can rescue the phenotype of Mpv17 deficient mice when introduced by transgenesis. This provides formal proof for the hypothesis that the phenotype is caused by the loss of function of the Mpv17 gene. It also provides evidence for the functional conservation of the Mpv17 gene in mammals and points to a potential role of this gene in human kidney disease.

The mutant mouse strain Mpv17 carries a retroviral insert in its genome which inactivates the Mpv17 gene. At a young age these mice develop glomerulosclerosis and nephrotic syndrome which resembles human disease. We show here that the Mpv17 gene product is highly conserved and encodes a peroxisomal protein. Loss of the Mpv17 protein does not impair peroxisome biogenesis but instead leads to a reduced ability to produce reactive oxygen species (ROS). In turn, overproduction of the Mpv17 gene in transfected cells results in dramatically enhanced levels of intracellular ROS indicating a direct involvement of Mpv17 in ROS production. These data reveal a role for the Mpv17 protein in peroxisomal reactive oxygen metabolism and establish a novel link between peroxisomal ROS production and glomerulosclerosis.