Mahalakshmi Shankaran

Involvement of the serotonin transporter in the formation of hydroxyl radicals induced by 3,4-methylenedioxymethamphetamine

The mechanism of 3,4-methylenedioxymethamphetamine (MDMA)-induced depletion of brain serotonin (5-hydroxytryptamine, 5-HT) has been proposed to involve the generation of reactive oxygen species. In the present study, quantification of the extracellular concentration of 2,3-dihydroxybenzoic acid (2,3-DHBA) from salicylic acid was used as an index of hydroxyl radical generation. Although both MDMA and D-amphetamine markedly increased the extracellular concentration of dopamine in the striatum, only MDMA increased the extracellular concentration of 2,3-DHBA. Treatment with fluoxetine either 1 h prior to or 4 h following the administration of MDMA reduced the MDMA-induced formation of 2,3-DHBA and also attenuated the MDMA-induced depletion of 5-HT in the striatum. These results are supportive of the view that the MDMA-induced generation of hydroxyl radicals and, ultimately, the long-term depletion of 5-HT, is dependent, in part, on the activation of the 5-HT transporter.

A neurotoxic regimen of MDMA suppresses behavioral, thermal and neurochemical responses to subsequent MDMA administration

Abstract Rationale: 3,4-Methylenedioxymethamphetamine (MDMA) produces a long-term depletion of serotonin (5-HT) in the rat brain; this depletion may have some functional consequences. Objective: The aim of the present study was to evaluate the acute effects of MDMA on the extracellular concentrations of dopamine and 5-HT, body temperature and the 5-HT behavioral syndrome in rats 7 days following a neurotoxic regimen of MDMA. Methods: One week after the rats were treated with a neurotoxic regimen of MDMA (10 mg/kg, IP, every 2 h for a total of four injections), the rats were injected with a subsequent injection of MDMA. In vivo microdialysis combined with HPLC was utilized to measure the extracellular concentration of 5-HT and dopamine in the striatum. The increase in body temperature was determined by rectal temperature measurements, and the 5-HT behavioral syndrome was scored using a rating scale following the administration of MDMA.Results: The neurotoxic regimen produced a 45% reduction in brain 5-HT concentrations. The magnitude of the MDMA-induced increase in the extracellular concentration of 5-HT, but not dopamine, in the striatum produced by an acute injection of MDMA (7.5 mg/kg, IP) was reduced in rats treated previously with the neurotoxic regimen of MDMA when compared with that in control animals. In addition, the magnitude of the 5-HT behavioral syndrome, as well as the hyperthermic response, produced by MDMA was markedly diminished in rats that had previously received the neurotoxic regimen of MDMA. Conclusions: It is concluded that the long-term depletion of brain 5-HT produced by MDMA is accompanied by impairments in 5-HT function, as evidenced by the deficits in the neurochemical, thermal and behavioral responses to subsequent MDMA administration.

Ascorbic acid prevents 3,4‐methylenedioxymethamphetamine (MDMA)‐induced hydroxyl radical formation and the behavioral and neurochemical consequences of the depletion of brain 5‐HT

MDMA‐induced 5‐HT neurotoxicity has been proposed to involve oxidative stress due to increased formation of hydroxyl radicals. Recently, MDMA‐induced 5‐HT neurotoxicity has been shown to be accompanied by a suppression of behavioral and neurochemical responses to a subsequent injection of MDMA. The intent of the present study was to examine whether suppression of the MDMA‐induced formation of hydroxyl radicals by an antioxidant, ascorbic acid, attenuates both the MDMA‐induced depletion of 5‐HT and the functional consequences associated with this depletion. Treatment of rats with ascorbic acid suppressed the generation of hydroxyl radicals, as evidenced by the production of 2,3‐dihydroxybenzoic acid from salicylic acid, in the striatum during the administration of a neurotoxic regimen of MDMA. Ascorbic acid also attenuated the MDMA‐induced depletion of striatal 5‐HT content. In rats treated with a neurotoxic regimen of MDMA, the ability of a subsequent injection of MDMA to increase the extracellular concentration of 5‐HT in the striatum, elicit the 5‐HT behavioral syndrome, and produce hyperthermia was markedly reduced compared to the responses in control rats. The concomitant administration of ascorbic acid with the neurotoxic regimen of MDMA prevented the diminished neurochemical and behavioral responses to a subsequent injection of MDMA. Finally, a neurotoxic regimen of MDMA produced significant reductions in the concentrations of vitamin E and ascorbic acid in the striatum and hippocampus. Thus, the MDMA‐induced depletion of brain 5‐HT and the functional consequences thereof appear to involve the induction of oxidative stress resulting from an increased generation of free radicals and diminished antioxidant capacity of the brain. Synapse 40:55–64, 2001.

Effect of 3,4-methylenedioxymethamphetamine (MDMA) on hippocampal dopamine and serotonin

The 3,4-methylenedioxymethamphetamine (MDMA)-induced increase in the extracellular concentration of dopamine and the long-term depletion of 5-HT were studied in the hippocampus of the rat brain. MDMA produced a dose-dependent increase in the extracellular concentration of dopamine in the hippocampus, as well as in the striatum. The MDMA-induced increase in the extracellular concentration of dopamine in the hippocampus, but not in the striatum, was suppressed in rats treated with the norepinephrine uptake inhibitor, desipramine, and in rats in which noradrenergic neurons in the hippocampus were lesioned with DSP4 (N-(2- chloroethyl)-N-ethyl-2-bromo benzylamine). However, the long-term depletion of 5-HT in the hippocampus produced by MDMA was unaltered in desipramine-treated rats. These results are supportive of the view that the MDMA-induced increase in the extracellular concentration of dopamine in the hippocampus is the result of an enhanced release of dopamine from noradrenergic neurons. In addition, the MDMA-induced depletion of 5-HT in the hippocampus appears not to involve dopamine-initiated processes, because suppression of MDMA-induced dopamine release did not attenuate the long-term depletion of 5-HT in the hippocampus.

Mazindol Attenuates the 3,4-Methylenedioxymethamphetamine-Induced Formation of Hydroxyl Radicals and Long-Term Depletion of Serotonin in the Striatum

Abstract: The formation of hydroxyl radicals following the systemic administration of 3,4‐methylenedioxymethamphetamine (MDMA) was studied in the striatum of the rat by quantifying the stable adducts of salicylic acid and D‐phenylalanine, namely, 2,3‐dihydroxybenzoic acid (2,3‐DHBA) and p‐tyrosine, respectively. The repeated administration of MDMA produced a sustained increase in the extracellular concentration of 2,3‐DHBA and p‐tyrosine, as well as dopamine. The MDMA‐induced increase in the extracellular concentration of both dopamine and 2,3‐DHBA was suppressed in rats treated with mazindol, a dopamine uptake inhibitor. Mazindol also attenuated the long‐term depletion of serotonin (5‐HT) in the striatum produced by MDMA without altering the acute hyperthermic response to MDMA. These results are supportive of the view that MDMA produces a dopamine‐dependent increase in the formation of hydroxyl radicals in the striatum that may contribute to the mechanism whereby MDMA produces a long‐term depletion of brain 5‐HT content.

Greater muscle protein synthesis and mitochondrial biogenesis in males compared with females during sprint interval training

Improved endurance exercise performance in adult humans after sprint interval training (SIT) has been attributed to mitochondrial biogenesis. However, muscle protein synthesis (MPS) and mitochondrial biogenesis during SIT have not been measured, nor have sex‐specific differences. We hypothesized that males and females would have similar rates of MPS, mitochondrial biogenesis, and synthesis of individual proteins during SIT. Deuterium oxide (D2O) was orally administered to 21 adults [11 male, 10 female; mean age, 23±1 yr; body mass index (BMI), 22.8±0.6 kg/m2; mean± se] for 4 wk, to measure protein synthesis rates while completing 9 sessions of 4–8 bouts of 30 s duration on a cycle ergometer separated by 4 min of active recovery. Samples of the vastus lateralis were taken before and 48 h after SIT. SIT increased maximum oxygen uptake (VO2max, males 43.4±2.1–44.0±2.3; females 39.5±0.9–42.5±1.3 ml/kg/min; P=0.002). MPS was greater in the males than in the females in the mixed (~150%; P < 0.001), cytosolic (~135%; P=0.038), and mitochondrial (~135%; P=0.056) fractions. The corresponding ontological clusters of individual proteins were significantly greater in the males than in the females (all P<0.00001). For the first time, we document greater MPS and mitochondrial biogenesis during SIT in males than in females and describe the synthetic response of individual proteins in humans during exercise training.—Scalzo, R. L., Peltonen, G. L., Binns, S. E., Shankaran, M., Giordano, G. R., Hartley, D. A., Klochak, A. L., Lonac, M. C., Paris, H. L. R., Szallar, S. E., Wood, L. M., Peelor, F. F., III, Holmes, W. E., Hellerstein, M. K., Bell, C., Hamilton, K. L., Miller, B. F. Greater muscle protein synthesis and mitochondrial biogenesis in males than in females during sprint interval training. FASEB J. 28, 2705–2714 (2014). www.fasebj.org

The Effect of Long Term Calorie Restriction on in Vivo Hepatic Proteostatis: A Novel Combination of Dynamic and Quantitative Proteomics

Calorie restriction (CR) promotes longevity. A prevalent mechanistic hypothesis explaining this effect suggests that protein degradation, including mitochondrial autophagy, is increased with CR, removing damaged proteins and improving cellular fitness. At steady state, increased catabolism must be balanced by increasing mitochondrial biogenesis and protein synthesis, resulting in faster protein replacement rates. To test this hypothesis, we measured replacement kinetics and relative concentrations of hundreds of proteins in vivo in long-term CR and ad libitum-fed mice using metabolic 2H2O-labeling combined with the Stable Isotope Labeling in Mammals protocol and LC-MS/MS analysis of mass isotopomer abundances in tryptic peptides. CR reduced absolute synthesis and breakdown rates of almost all measured hepatic proteins and prolonged the half-lives of most (∼80%), particularly mitochondrial proteins (but not ribosomal subunits). Proteins with related functions exhibited coordinated changes in relative concentration and replacement rates. In silico expression pathway interrogation allowed the testing of potential regulators of altered network dynamics (e.g. peroxisome proliferator-activated receptor gamma coactivator 1-alpha). In summary, our combination of dynamic and quantitative proteomics suggests that long-term CR reduces mitochondrial biogenesis and mitophagy. Our findings contradict the theory that CR increases mitochondrial protein turnover and provide compelling evidence that cellular fitness is accompanied by reduced global protein synthetic burden.

Discovery of Novel Hippocampal Neurogenic Agents by Using an in Vivo Stable Isotope Labeling Technique

Neurogenesis occurs in discrete regions of adult mammalian brain, including the subgranular zone of the hippocampus. Hippocampal neurogenesis is enhanced by different classes of antidepressants, but screening for neurogenic actions of novel antidepressants has been inefficient because of limitations of 5-bromo-2′-deoxyuridine labeling techniques. We describe an efficient in vivo method for measuring hippocampal neurogenesis involving incorporation of the stable isotope, 2H, into genomic DNA during labeling with 2H2O (heavy water). Male rodents received 8 to 10% 2H2O in drinking water; DNA was isolated from hippocampal progenitor cells or neurons. Label incorporation into progenitor cells of Swiss-Webster mice revealed subpopulation kinetics: 16% divided with t1/2 of 2.7 weeks; the remainder did not divide over 1 year. Progenitor cell proliferation rates in mice were strain-dependent. Chronic antidepressant treatment for 3 weeks, with 2H2O administered during the final week, increased progenitor cell proliferation across all the strains tested. Fluoxetine treatment increased 2H incorporation into DNA of gradient-enriched neurons or flow-sorted neuronal nuclei 4 weeks after 2H2O labeling, representing the survival and differentiation of newly divided cells into neurons. By screening 11 approved drugs for effects on progenitor cell proliferation, we detected previously unrecognized, dose-dependent enhancement of hippocampal progenitor cell proliferation by two statins and the anticonvulsant topiramate. We also confirmed stimulatory activity of other anticonvulsants and showed inhibition of progenitor cell proliferation by isotretinoin and prednisolone. In conclusion, stable isotope labeling is an efficient, high-throughput in vivo method for measuring hippocampal progenitor cell proliferation that can be used to screen for novel neurogenic drugs.

Circulating protein synthesis rates reveal skeletal muscle proteome dynamics

Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders.

Differential In Vivo Effects on Target Pathways of a Novel Arylpyrazole Glucocorticoid Receptor Modulator Compared with Prednisolone

Glucocorticoids are widely prescribed to treat autoimmune and inflammatory diseases. Although they are extremely potent, their utility in clinical practice is limited by a variety of adverse side effects. Development of compounds that retain the potent immunomodulating and anti-inflammatory properties of classic glucocorticoids while exhibiting reduced adverse actions is therefore a priority. Using heavy water labeling and mass spectrometry to measure fluxes through multiple glucocorticoid-responsive, disease-relevant target pathways in vivo in mice, we compared the effects of a classic glucocorticoid receptor (GR) ligand, prednisolone, with those of a novel arylpyrazole-based compound, L5 {[1-(4-fluorophenyl)-4a-methyl-5,6,7,8-tetrahydro-4H-benzo[f]indazol-5-yl]-[4-(trifluoromethyl)phenyl]methanol}. We show for the first time that L5 exhibits clearly selective actions on disease-relevant pathways compared with prednisolone. Prednisolone reduced bone collagen synthesis, skin collagen synthesis, muscle protein synthesis, and splenic lymphocyte counts, proliferation, and cell death, whereas L5 had none of those actions. In contrast, L5 was a more rapid and potent inhibitor of hippocampal neurogenesis than prednisolone, and L5 and prednisolone induced insulin resistance equally. Administration of prednisolone or L5 increased expression comparably for one GR-regulated gene involved in protein degradation in skeletal muscle (Murf1) and one GR-regulated gluconeogenic gene in liver (PEPCK). In summary, L5 dissociates the pleiotropic effects of the GR ligand prednisolone in intact animals in ways that neither gene expression nor cell-based models were able to fully capture or predict. Because multiple actions can be measured concurrently in a single animal, this method is a powerful systems approach for characterizing and differentiating the effects of ligands that bind nuclear receptors.