Loss of DJ-1 impairs antioxidant response by altered glutamine and serine metabolism.

The oncogene DJ-1 has been originally identified as a suppressor of PTEN. Further on, loss-of-function mutations have been described as a causative factor in Parkinson's disease (PD). DJ-1 has an important function in cellular antioxidant responses, but its role in central metabolism of neurons is still elusive. We applied stable isotope assisted metabolic profiling to investigate the effect of a functional loss of DJ-1 and show that DJ-1 deficient neuronal cells exhibit decreased glutamine influx and reduced serine biosynthesis. By providing precursors for GSH synthesis, these two metabolic pathways are important contributors to cellular antioxidant response. Down-regulation of these pathways, as a result of loss of DJ-1 leads to an impaired antioxidant response. Furthermore, DJ-1 deficient mouse microglia showed a weak but constitutive pro-inflammatory activation. The combined effects of altered central metabolism and constitutive activation of glia cells raise the susceptibility of dopaminergic neurons towards degeneration in patients harboring mutated DJ-1. Our work reveals metabolic alterations leading to increased cellular instability and identifies potential new intervention points that can further be studied in the light of novel translational medicine approaches.

Transcriptional and metabolic adaptation of human neurons to the mitochondrial toxicant MPP(+).

Assessment of the network of toxicity pathways by Omics technologies and bioinformatic data processing paves the road toward a new toxicology for the twenty-first century. Especially, the upstream network of responses, taking place in toxicant-treated cells before a point of no return is reached, is still little explored. We studied the effects of the model neurotoxicant 1-methyl-4-phenylpyridinium (MPP(+)) by a combined metabolomics (mass spectrometry) and transcriptomics (microarrays and deep sequencing) approach to provide unbiased data on earliest cellular adaptations to stress. Neural precursor cells (LUHMES) were differentiated to homogeneous cultures of fully postmitotic human dopaminergic neurons, and then exposed to the mitochondrial respiratory chain inhibitor MPP(+) (5 µM). At 18-24 h after treatment, intracellular ATP and mitochondrial integrity were still close to control levels, but pronounced transcriptome and metabolome changes were seen. Data on altered glucose flux, depletion of phosphocreatine and oxidative stress (e.g., methionine sulfoxide formation) confirmed the validity of the approach. New findings were related to nuclear paraspeckle depletion, as well as an early activation of branches of the transsulfuration pathway to increase glutathione. Bioinformatic analysis of our data identified the transcription factor ATF-4 as an upstream regulator of early responses. Findings on this signaling pathway and on adaptive increases of glutathione production were confirmed biochemically. Metabolic and transcriptional profiling contributed complementary information on multiple primary and secondary changes that contribute to the cellular response to MPP(+). Thus, combined 'Omics' analysis is a new unbiased approach to unravel earliest metabolic changes, whose balance decides on the final cell fate.

The NOX1/4 inhibitor GKT136901 as selective and direct scavenger of peroxynitrite.

NADPH oxidases (NOX), catalyzing the reduction of molecular oxygen to form the superoxide radical anion (•O2⁻) and hydrogen peroxide (H2O2), are involved in several pathological conditions, such as stroke, diabetes, atherosclerosis, but also in chronic neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, or multiple sclerosis. GKT136901 is a novel NOX-1/4 inhibitor with potential application in the areas of diabetic nephropathy, stroke, or neurodegeneration. In the present study, we investigated additional pharmacological activities of the compound with respect to direct free radical scavenging. GKT136901 did not interact with nitric oxide (•NO), •O2⁻, or hydroxyl radicals (•OH), but it acted as selective scavenger of peroxynitrite (PON) already in the submicromolar concentration range. Alpha synuclein (ASYN) is a protein involved in the pathogenesis of Parkinson's disease and a known target for PON-dependent tyrosine nitration. Submicromolar concentrations of GKT136901 prevented tyrosine nitration and di-tyrosine-dependent dimer formation of ASYN by PON as indicated by Western blot and mass spectrometric analysis. GKT136901 itself was degraded when exposed to PON. In a human neuronal cell model, GKT136901 prevented both the depletion of reduced intracellular glutathione, and the degeneration of neurites when present during PON treatment of the cells. When GKT136901 was applied after PON treatment, no protective effect was observed, thus excluding an impact of GKT136901 on cellular death/survival pathways. In summary, selective scavenging of PON is an additional pharmacological property of the NOX-1/4 inhibitor GKT136901, and this may add to the efficiency of the drug in several disease models.

Hydralazine is a powerful inhibitor of peroxynitrite formation as a possible explanation for its beneficial effects on prognosis in patients with congestive heart failure.

The hemodynamic and anti-ischemic effects of nitroglycerin (GTN) are rapidly blunted as a result of the development of nitrate tolerance. Hydralazine has been shown to prevent tolerance in experimental and clinical studies, all of which may be at least in part secondary to antioxidant properties of this compound. The antioxidant effects of hydralazine were tested in cell free systems, cultured smooth muscle cells, isolated mitochondria, and isolated vessels. Inhibitory effects on the formation of superoxide and/or peroxynitrite formation were tested using lucigenin and L-012 enhanced chemiluminescence as well as DHE-fluorescence. The peroxynitrite scavenging properties were also assessed by inhibition of nitration of phenol. Prevention of impairment of NO downstream signaling and GTN bioactivation was determined by measurement of P-VASP (surrogate parameter for the activity of the cGMP-dependent kinase-I, cGK-I) and mitochondrial aldehyde dehydrogenase (ALDH-2) activity. Hydralazine dose-dependently decreased the chemiluminescence signal induced by peroxynitrite from SIN-1 and by superoxide from HX/XO in a cell free system, by superoxide in smooth muscle cells and mitochondria acutely challenged with GTN. Moreover, hydralazine inhibited the peroxynitrite-mediated nitration of phenols as well as proteins in smooth muscle cells in a dose-dependent fashion. Finally, hydralazine normalized impaired cGK-I activity as well as impaired vascular ALDH-2 activity. Our results indicate that hydralazine is a highly potent radical scavenger. Thus, the combination with isosorbide dinitrate (ISDN) will favorably influence the nitroso-redox balance in the cardiovascular system in patients with congestive heart failure and may explain at least in part the improvement of prognosis in patients with chronic congestive heart failure.