Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase.

The attenuation of protein synthesis via the phosphorylation of eIF2α is a major stress response of all eukaryotic cells. The growth-arrest- and DNA-damage-induced transcript 34 (GADD34) bound to the serine/threonine protein phosphatase 1 (PP1) is the necessary eIF2α phosphatase complex that returns mammalian cells to normal protein synthesis following stress. The molecular basis by which GADD34 recruits PP1 and its substrate eIF2α are not fully understood, hindering our understanding of the remarkable selectivity of the GADD34:PP1 phosphatase for eIF2α. Here, we report detailed structural and functional analyses of the GADD34:PP1 holoenzyme and its recruitment of eIF2α. The data highlight independent interactions of PP1 and eIF2α with GADD34, demonstrating that GADD34 functions as a scaffold both in vitro and in cells. This work greatly enhances our molecular understanding of a major cellular eIF2α phosphatase and establishes the foundation for future translational work.

Metabolic profiling of 3-nitropropionic acid early-stage Huntington's disease rat model using gas chromatography time-of-flight mass spectrometry.

3-Nitropropionic acid (3-NP), a potent irreversible inhibitor of mitochondrial complex II enzyme, leads to mitochondrial dysfunction and oxidative stress in Huntington's disease (HD) rat model. In this study, biochemical assays were used to demonstrate the presence of oxidative stress and mitochondrial dysfunction in 3-NP early stage HD rat models. Gas chromatography time-of-flight mass spectrometry (GC/TOFMS) was applied to analyze metabolites in brain and plasma of 3-NP-treated and vehicle-dosed rats. The orthogonal partial least-squares discriminant analysis (OPLS-DA) model generated using brain metabolic profiles robustly differentiated the 3-NP early stage HD rat model from the control. Metabonomic characterization of the 3-NP HD rat model facilitated the detection of biomarkers that define the physiopathological phenotype of early stage HD and elucidated the treatment effect of galantamine. Brain marker metabolites that were identified based on the OPLS-DA model were associated with altered glutathione metabolism, oxidative stress, and impaired energy metabolism. The treatment effect of galantamine in early stage HD could not be concluded mechanistically using the brain metabotype. Our study confirmed that GC/TOFMS is a strategic and complementary platform for the metabonomic characterization of 3-NP induced neurotoxicity in the early stage HD rat model.

Pharmacokinetic and pharmacodynamic properties of cholinesterase inhibitors donepezil, tacrine, and galantamine in aged and young Lister hooded rats.

Physiological alterations that may change pharmacological response accompany aging. Pharmacokinetic/pharmacodynamic properties of cholinesterase inhibitors (ChEIs) used in the treatment of Alzheimer's disease, donepezil, tacrine, and galantamine, were investigated in an aged Lister hooded rat model. Intravenous and oral 6-h blood sampling profiles in old (30 months old) and young (7 months old) rats revealed pharmacokinetic changes similar to those in humans with an approximately 40% increase in C(max) of galantamine and prolonged t(1/2) (1.4-fold) and mean residence time (1.5-fold) of donepezil. Tacrine disposition was maintained with age, and area under the concentration-time curve and clearance in old rats were similar to those in young rats for all drugs tested as was bioavailability. Old rats showed a trend of increased pharmacodynamic sensitivity (<20%) to ChEIs in cholinesterase activity assays, which was attributed to pharmacokinetic effects because a trend of higher blood and brain concentrations was seen in the old rats although brain/blood ratios remained unaffected. Enhanced cholinergic-mediated behaviors such as tremor, hypothermia, salivation, and lacrimation were also observed in the old rats, which could not be accounted for by a similar magnitude of change in pharmacokinetics. A decrease in expression of muscarinic acetylcholine receptor subtype 2 detected in old rat brains was postulated to play a role. Greater age effects in both pharmacokinetics and pharmacodynamics of donepezil and tacrine were seen in previous studies with Fischer 344 rats, indicating a potential risk in overreliance on this rat strain for aging studies.

A sensitive LC/MS/MS bioanalysis assay of orally administered lipoic acid in rat blood and brain tissue.

A sensitive liquid chromatography tandem mass spectrometry (LC/MS/MS) bioanalytical method was developed and validated to analyze lipoic acid (LA) in rat blood and brain samples. Ten mobile phase combinations were investigated during method development. Mobile phase combination of 0.1% acetic acid (pH 4 adjusted with ammonia solution)/acetonitrile was most optimum in terms of sensitivity and peak shape of LA and the internal standard, valproic acid. Sample extraction method was explored using liquid-liquid extraction and protein precipitation methods. Protein precipitation yielded the highest recovery of the analytes from blood and brain ranging from 92 to 115%. The lower limit of quantitation (LLOQ) of LA was 0.1ng/mL (0.485nM) in both blood and brain while on-column lower limit of detection (LLOD) was 0.03pg. The precision (% R.S.D.) ranged from 1.49 to 26.39% and 1.49 to 10.89% for intra- and inter-day assays, respectively. The accuracy ranged from 91.2 to 116.17% for intra-day assay and 102.68 to 114.33% for inter-day assay.

Metabolic profiling of rat brain and cognitive behavioral tasks: potential complementary strategies in preclinical cognition enhancement research.

In this study, the correlation between the metabolic profiles of rats undergoing cognition enhancement drug therapy and their associated cognitive behavioral outcomes were investigated. Male Lister Hooded rats were administered either with donepezil, galantamine, or vehicle and subjected to Atlantis watermaze training and novel object recognition tests. An UPLC/MS/MS method was developed to profile 21 neurologically related metabolites in the rat brains. Pharmacologically induced behavioral changes were compared subsequently with the metabolic fluctuations of neurologically related metabolites from multiple neurotransmitter pathways using multivariate and univariate statistical analyses. Significant improvements in cognitive behavioral outcomes were demonstrated in the rats administered with donepezil and galantamine using both AWM training (P < 0.05) and NOR (P < 0.05) tests as compared to those dosed with the vehicle. This corroborated with the significant elevation of eight prominent biomarkers after the cognitive enhancement therapy. An orthogonal partial least-squares discriminant analysis model generated using only the 8 metabolites identified as discriminating the drug-dosed rats from the vehicle-dosed rats gave a Q(2) = 0.566, receiver operator characteristic (ROC) AUC = 1.000, using 7-fold cross validation. Our study suggests that metabolic profiling of rat brain is a potential complementary strategy to the cognitive behavioral tasks for characterizing neurobiological responses to cognition enhancement drug testing.

Investigation of the drug-drug interaction between alpha-lipoic acid and valproate via mitochondrial beta-oxidation.

PURPOSE: To investigate the potential drug-drug interaction (DDI) between lipoic acid (LA) and valproate (VA) via the mitochondrial beta-oxidation pathway in rats. METHODS: In vitro mitochondrial assays were performed to compare the biotransformation of VA to valproyl-CoA (VA-CoA), in the absence and presence of LA. In vitro microsomal and protein binding assays were performed to elucidate their potential DDI at the microsomal metabolism and distribution levels. A pharmacokinetic study was conducted in Lister Hooded rats to ascertain the in vivo DDI between LA and VA. RESULTS: LA was shown to decrease significantly (p < 0.05) the in vitro formation of VA-CoA in a concentration-dependent manner. Our in vitro assay results confirmed that there was minimal interaction between LA and VA in microsomal metabolism and protein binding. Based on the pharmacokinetic data, the absolute bioavailability of VA was determined to be 1.3 in the presence of LA. CONCLUSIONS: Our study demonstrated for the first time that there is a potential DDI between LA and VA at the mitochondrial beta-oxidation level. While further clinical study is essential, our preliminary finding suggested that medical practitioners need to be prudent when managing epileptic patients who are co-administered with both VA and LA.

Mitochondrial protection by the JNK inhibitor leflunomide rescues mice from acetaminophen-induced liver injury.

UNLABELLED: Acetaminophen (APAP) is a widely used analgesic and antipyretic drug that is safe at therapeutic doses but which can precipitate liver injury at high doses. We have previously found that the antirheumatic drug leflunomide is a potent inhibitor of APAP toxicity in cultured human hepatocytes, protecting them from mitochondria-mediated cell death by inhibiting the mitochondrial permeability transition. The purpose of this study was to explore whether leflunomide protects against APAP hepatotoxicity in vivo and to define the molecular pathways of cytoprotection. Male C57BL/6 mice were treated with a hepatotoxic dose of APAP (750 mg/kg, ip) followed by a single injection of leflunomide (30 mg/kg, ip). Leflunomide (4 hours after APAP dose) afforded significant protection from liver necrosis as assessed by serum ALT activity and histopathology after 8 and 24 hours. The mechanism of protection by leflunomide was not through inhibition of cytochrome P450 (CYP)-catalyzed APAP bioactivation or an apparent suppression of the innate immune system. Instead, leflunomide inhibited APAP-induced activation (phosphorylation) of c-jun NH2-terminal protein kinase (JNK), thus preventing downstream Bcl-2 and Bcl-XL inactivation and protecting from mitochondrial permeabilization and cytochrome c release. Furthermore, leflunomide inhibited the APAP-mediated increased expression of inducible nitric oxide synthase and prevented the formation of peroxynitrite, as judged from the absence of hepatic nitrotyrosine adducts. Even when given 8 hours after APAP dose, leflunomide still protected from massive liver necrosis. CONCLUSION: Leflunomide afforded protection against APAP-induced hepatotoxicity in mice through inhibition of JNK-mediated activation of mitochondrial permeabilization.