Neuronal Protection by Ha-RAS-GTPase Signaling through Selective Downregulation of Plasmalemmal Voltage-Dependent Anion Channel-1.

The small GTPase RAS acts as a plasma membrane-anchored intracellular neurotrophin counteracting neuronal degeneration in the brain, but the underlying molecular mechanisms are largely unknown. In transgenic mice expressing constitutively activated V12-Ha-RAS selectively in neurons, proteome analysis uncovered a 70% decrease in voltage-dependent anion channel-1 (VDAC-1) in the cortex and hippocampus. We observed a corresponding reduction in the levels of mRNA splicing variant coding for plasma membrane-targeted VDAC-1 (pl-VDAC-1) while mRNA levels for mitochondrial membrane VDAC-1 (mt-VDAC-1) remained constant. In primary cortical neurons derived from V12-Ha-RAS animals, a decrease in pl-VDAC-1 mRNA levels was observed, accompanied by a concomitant reduction in the ferricyanide reductase activity associated with VDAC-1 protein. Application of MEK inhibitor U0126 to transgenic cortical neurons reconstituted pl-VDAC-1 mRNA to reach wild-type levels. Excitotoxic glutamate-induced cell death was strongly attenuated in transgenic V12-Ha-RAS overexpressing cortical cultures. Consistently, a neuroprotective effect could also be achieved in wild-type cortical cultures by the extracellular application of channel-blocking antibody targeting the N-terminus of VDAC-1. These results may encourage novel therapeutic approaches toward blocking pl-VDAC-1 by monoclonal antibody targeting for complementary treatments in transplantation and neurodegenerative disease.

Looking for prosocial genes: ITRAQ analysis of proteins involved in MDMA-induced sociability in mice.

Social behavior plays a fundamental role in life of many animal species, allowing the interaction between individuals and sharing of experiences, needs, and goals across them. In humans, some neuropsychiatric diseases, including anxiety, posttraumatic stress disorder and autism spectrum disorders, are often characterized by impaired sociability. Here we report that N-Methyl-3,4-methylenedioxyamphetamine (MDMA, "Ecstasy") at low dose (3mg/kg) has differential effects on mouse social behavior. In some animals, MDMA promotes sociability without hyperlocomotion, whereas in other mice it elevates locomotor activity without affecting sociability. Both WAY-100635, a selective antagonist of 5-HT1A receptor, and L-368899, a selective oxytocin receptor antagonist, abolish prosocial effects of MDMA. Differential quantitative analysis of brain proteome by isobaric tag for relative and absolute quantification technology (iTRAQ) revealed 21 specific proteins that were highly correlated with sociability, and allowed to distinguish between entactogenic prosocial and hyperlocomotor effects of MDMA on proteome level. Our data suggest particular relevance of neurotransmission mediated by GABA B receptor, as well as proteins involved in energy maintenance for MDMA-induced sociability. Functional association network for differentially expressed proteins in cerebral cortex, hippocampus and amygdala were identified. These results provide new information for understanding the neurobiological substrate of sociability and may help to discover new therapeutic approaches to modulate social behavior in patients suffering from social fear and low sociability.

Differences in the expression of endogenous efflux transporters in MDR1-transfected versus wildtype cell lines affect P-glycoprotein mediated drug transport.

BACKGROUND AND PURPOSE: P-glycoprotein (Pgp) efflux assays are widely used to identify Pgp substrates. The kidney cell lines Madin-Darby canine kidney (MDCK)-II and LLC-PK1, transfected with human MDR1 (ABCB1) are used to provide recombinant models of drug transport. Endogenous transporters in these cells may contribute to the activities of recombinant transporters, so that drug transport in MDR1-transfected cells is often corrected for the transport obtained in parental (wildtype) cells. However, expression of endogenous transporters may vary between transfected and wildtype cells, so that this correction may cause erroneous data. Here, we have measured the expression of endogenous efflux transporters in transfected and wildtype MDCK-II or LLC cells and the consequences for Pgp-mediated drug transport. EXPERIMENTAL APPROACH: Using quantitative real-time RT-PCR, we determined the expression of endogenous Mdr1 mRNA and other efflux transporters in wildtype and MDR1-transfected MDCK-II and LLC cells. Transcellular transport was measured with the test substrate vinblastine. KEY RESULTS: In MDR1-transfected MDCK cells, expression of endogenous (canine) Mdr1 and Mrp2 (Abcc2) mRNA was markedly lower than in wildtype cells, whereas MDR1-transfected LLC cells exhibited comparable Mdr1 but strikingly higher Mrp2 mRNA levels than wildtype cells. As a consequence, transport of vinblastine by human Pgp in efflux experiments was markedly underestimated when transport in MDR1-transfected MDCK cells was corrected for transport obtained in wildtype cells. This problem did not occur in LLC cells. CONCLUSIONS AND IMPLICATIONS: Differences in the expression of endogenous efflux transporters between transfected and wildtype MDCK cells provide a potential bias for in vitro studies on Pgp-mediated drug transport.

Exposure to antiepileptic drugs does not alter the functionality of P-glycoprotein in brain capillary endothelial and kidney cell lines.

Several major antiepileptic drugs, including carbamazepine, phenytoin and phenobarbital, induce xenobiotic metabolizing enzymes via activation of nuclear receptors, including pregnane X receptor (NR1I2) and constitutive androstane receptor (NR1I3). Via activation of these xenobiotic sensors, antiepileptic drugs may also induce the expression of efflux transporters such as P-glycoprotein (Pgp) in different tissues, including intestine, liver, kidney and brain. Increased expression of Pgp in brain capillary endothelial cells, which form the blood-brain barrier, could limit the penetration of antiepileptic drugs into the brain and therefore decrease their therapeutic efficacy. As a consequence, it is important to know whether antiepileptic drugs alter the expression or functionality of Pgp in endothelial cells. In the present study, we studied the effects of exposure to phenobarbital, phenytoin and carbamazepine on Pgp expression and functionality in the rat brain endothelial cell line GPNT. For comparison with drug effects on endothelial cells, a dog kidney cell line (MDCK II) was used. Furthermore, several known Pgp inducers (dexamethasone, doxorubicin, and rifampicin) were included in the study. Functionality of Pgp was determined by uptake assays, using known Pgp substrates (digoxin and vinblastine) and transport inhibitors (tariquidar, MK571). In GPNT cells, exposure to dexamethasone increased Pgp functionality, while antiepileptic drug exposure at clinically relevant concentrations did not exert any significant induction of Pgp expression or function. Similarly, antiepileptic drug exposure did not affect Pgp in MDCK cells. The lack of antiepileptic drugs to induce Pgp in brain capillary endothelial cells and kidney cells is in contrast to their known effect on Pgp expression in hepatic and intestinal cells, substantiating tissue differences in the regulation of Pgp.

Complex time-dependent alterations in the brain expression of different drug efflux transporter genes after status epilepticus.

PURPOSE: Frequent epileptic seizures or prolonged seizure activity (status epilepticus, SE) is known to increase the brain expression of drug efflux transporter genes and proteins, such as P-glycoprotein (Pgp) and members of the multidrug resistance protein (MRP) family, which might reduce brain levels of antiepileptic drugs and, therefore, be involved in drug resistance. However, the time course of alterations in Pgp or MRPs after seizures or SE is only incompletely known. METHODS: This prompted us to study the time course of alterations in the expression of different efflux transporter genes (Mdr1a, Mdr1b, MRP1, MRP2, MRP5) at various times after a pilocarpine-induced SE in limbic brain regions, using quantitative real-time polymerase chain reaction (RT-PCR) (qPCR). RESULTS: Unexpectedly, between 6 and 24 h after onset of SE, genes encoding Pgp (Mdr1a, Mdr1b), Mrp1, and Mrp5 were downregulated in hippocampus, amygdala, or piriform cortex. This initial decrease in expression was followed by normalization and then increased expression, which became maximal 2 days after SE. One explanation for the initial decrease in transporter expression could be SE-induced acute inflammatory processes, because proinflammatory cytokines are known to suppress the expression of Pgp and other efflux transporters. To directly address this possibility, we quantified the hippocampal mRNA expression of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha, showing a marked SE-induced increase in these cytokines, which paralleled the decreased expression of efflux transporters. DISCUSSION: Taken together, these findings indicate that alterations in expression of drug efflux transporters after prolonged seizure activity are more complex than previously thought.

Novel repressor of the human FMR1 gene - identification of p56 human (GCC)(n)-binding protein as a Krüppel-like transcription factor ZF5.

A series of relatively short (GCC)(n) triplet repeats (n = 3-30) located within regulatory regions of many mammalian genes may be considered as putative cis-acting transcriptional elements (GCC-elements). Fragile X-mental retardation syndrome is caused by an expansion of (GCC)(n) triplet repeats within the 5'-untranslated region of the human fragile X-mental retardation 1 (FMR1) gene. The present study aimed to characterize a novel human (GCC)(n)-binding protein and investigate its possible role in the regulation of the FMR1 gene. A novel human (GCC)(n)-binding protein, p56, was isolated and identified as a Krüppel-like transcription factor, ZF5, by MALDI-TOF analysis. The capacity of ZF5 to specifically interact with (GCC)(n) triplet repeats was confirmed by the electrophoretic mobility shift assay with purified recombinant ZF5 protein. In cotransfection experiments, ZF5 overexpression repressed activity of the GCC-element containing mouse ribosomal protein L32 gene promoter. Moreover, RNA interference assay results showed that endogenous ZF5 acts as a repressor of the human FMR1 gene. Thus, these data identify a new class of ZF5 targets, a subset of genes containing GCC-elements in their regulatory regions, and raise the question of whether transcription factor ZF5 is implicated in the pathogenesis of fragile X syndrome.

Ovarian cancer marker of 11.7 kDa detected by proteomics is a serum amyloid A1.

In this study, to reduce the number of major plasma components, we examined thermostable plasma fractions to search for a biomarker of ovarian cancer. An apparent cancer biomarker of 11.7 kDa was detected in these fractions using ProteinChip SELDI-TOF mass spectrometry system. This peak invariably appeared with another close peak of about 11.5 kDa, suggesting that it is a derivative of a larger mass molecule. Of 27 cancer plasma specimens, 15 (55.6%) demonstrated this peak pair, whereas only 2 of 34 controls specimens (5.8%) were shown to express it with low intensity. Using a method involving cysteine modification by 4-vinylpyridine (4-VP), 2-DE and HPLC, these peaks were identified by mass spectrometry as serum amyloid A1 (11.68 kDa) and its N-terminal arginine-truncated form (11.52 kDa).