Reticular nucleus-specific changes in alpha3 subunit protein at GABA synapses in genetically epilepsy-prone rats

Differential composition of GABA(A) receptor (GABA(A)R) subunits underlies the variability of fast inhibitory synaptic transmission; alteration of specific GABA(A)R subunits in localized brain regions may contribute to abnormal brain states such as absence epilepsy. We combined immunocytochemistry and high-resolution ImmunoGold electron microscopy to study cellular and subcellular localization of GABA(A)R alpha1, alpha3, and beta2/beta3 subunits in ventral posterior nucleus (VP) and reticular nucleus (RTN) of control rats and WAG/Rij rats, a genetic model of absence epilepsy. In control rats, alpha1 subunits were prominent at inhibitory synapses in VP and much less prominent in RTN; in contrast, the alpha3 subunit was highly evident at inhibitory synapses in RTN. beta2/beta3 subunits were evenly distributed at inhibitory synapses in both VP and RTN. ImmunoGold particles representing all subunits were concentrated at postsynaptic densities with no extrasynaptic localization. Calculated mean number of particles for alpha1 subunit per postsynaptic density in nonepileptic VP was 6.1 +/- 3.7, for alpha3 subunit in RTN it was 6.6 +/- 3.4, and for beta2/beta3 subunits in VP and RTN the mean numbers were 3.7 +/- 1.3 and 3.5 +/- 1.2, respectively. In WAG/Rij rats, there was a specific loss of alpha3 subunit immunoreactivity at inhibitory synapses in RTN, without reduction in alpha3 subunit mRNA or significant change in immunostaining for other markers of RTN cell identity such as GABA or parvalbumin. alpha3 immunostaining in cortex was unchanged. Subtle, localized changes in GABA(A)R expression acting at highly specific points in the interconnected thalamocortical network lie at the heart of idiopathic generalized epilepsy.     

Methionine adenosyltransferase II beta subunit gene expression provides a proliferative advantage in human hepatoma

BACKGROUND & AIMS: Of the 2 genes (MAT1A, MAT2A) encoding methionine adenosyltransferase, the enzyme that synthesizes S-adenosylmethionine, MAT1A, is expressed in liver, whereas MAT2A is expressed in extrahepatic tissues. In liver, MAT2A expression associates with growth, dedifferentiation, and cancer. Here, we identified the beta subunit as a regulator of proliferation in human hepatoma cell lines. The beta subunit has been cloned and shown to lower the K(m) of methionine adenosyltransferase II alpha2 (the MAT2A product) for methionine and to render the enzyme more susceptible to S-adenosylmethionine inhibition. METHODS: Methionine adenosyltransferase II alpha2 and beta subunit expression was analyzed in human and rat liver and hepatoma cell lines and their interaction studied in HuH7 cells. beta Subunit expression was up- and down-regulated in human hepatoma cell lines and the effect on DNA synthesis determined. RESULTS: We found that beta subunit is expressed in rat extrahepatic tissues but not in normal liver. In human liver, beta subunit expression associates with cirrhosis and hepatoma. beta Subunit is expressed in most (HepG2, PLC, and Hep3B) but not all (HuH7) hepatoma cell lines. Transfection of beta subunit reduced S-adenosylmethionine content and stimulated DNA synthesis in HuH7 cells, whereas down-regulation of beta subunit expression diminished DNA synthesis in HepG2. The interaction between methionine adenosyltransferase II alpha2 and beta subunit was demonstrated in HuH7 cells. CONCLUSIONS: Our findings indicate that beta subunit associates with cirrhosis and cancer providing a proliferative advantage in hepatoma cells through its interaction with methionine adenosyltransferase II alpha2 and down-regulation of S-adenosylmethionine levels.     

Role of interferon alpha/beta receptor chain 1 in the structure and transmembrane signaling of the interferon alpha/beta receptor complex.

A previously cloned cDNA encodes one subunit of the human interferon alpha/beta receptor (IFN alpha R), denoted IFN alpha R1. To study the expression and signaling of IFN alpha R1, we used monoclonal antibodies (mAbs) generated against the baculovirus-expressed ectodomain of IFN alpha R1. Immunoprecipitation and immunoblotting of lysates from a variety of human cell lines showed that IFN alpha R1 has an apparent molecular mass of 135 kDa. Binding analysis with 125I-labeled mAb demonstrated high levels of cell surface expression of IFN alpha R1 in human cells and in mouse cells transfected with IFN alpha R1 cDNA, whereas no cross-reactivity was observed in control mouse L929 cells expressing only the endogenous mouse receptor. The subunit was rapidly down-regulated by IFN alpha (80% decrease within 2 hr) and degraded upon internalization. The IFN alpha R1 chain appeared to be constitutively associated with the 115-kDa subunit of the IFN alpha/beta receptor, since the mAbs coprecipitated this protein. IFN alpha/beta treatment induced tyrosine phosphorylation of IFN alpha R1 within 1 min, with kinetics paralleling that of the IFN-activated protein-tyrosine kinases Jak1 and Tyk2. Ligand-induced tyrosine phosphorylation of IFN alpha R1 was blocked by the kinase inhibitors genistein or staurosporine. Although IFN alpha R1 cDNA-transfected mouse cells expressed high levels of this subunit when compared with empty vector-transfected cells the number of binding sites for human IFN alpha (50-75 sites per cell) was not increased. Human IFN alpha induced the expression of a mouse IFN alpha/beta-responsive gene (the 204 gene) in mouse L929 cells transfected with the IFN alpha R1 cDNA, but not in mock-transfected cells. These results suggest that the IFN alpha R1 subunit acts as a species-specific signal transduction component of the IFN alpha/beta receptor complex.     

Reduced cortical inhibition in a mouse model of familial childhood absence epilepsy

Mutations in the GABA(A) receptor gamma2 subunit are associated with childhood absence epilepsy and febrile seizures. To understand better the molecular basis of absence epilepsy in man, we developed a mouse model harboring a gamma2 subunit point mutation (R43Q) found in a large Australian family. Mice heterozygous for the mutation demonstrated behavioral arrest associated with 6-to 7-Hz spike-and-wave discharges, which are blocked by ethosuximide, a first-line treatment for absence epilepsy in man. Seizures in the mouse showed an abrupt onset at around age 20 days corresponding to the childhood nature of this disease. Reduced cell surface expression of gamma2(R43Q) was seen in heterozygous mice in the absence of any change in alpha1 subunit surface expression, ruling out a dominant-negative effect. GABA(A)-mediated synaptic currents recorded from cortical pyramidal neurons revealed a small but significant reduction that was not seen in the reticular or ventrobasal thalamic nuclei. We hypothesize that a subtle reduction in cortical inhibition underlies childhood absence epilepsy seen in humans harboring the R43Q mutation.     

Sequence-specific interactions of nuclear factors with conserved sequences of human class II major histocompatibility complex genes

All class II major histocompatibility complex genes contain two highly conserved sequences, termed X and Y, within the promoter regions(s), which may have a role in regulation of expression. To study trans-acting factors that interact with these sequences, sequence-specific DNA binding activity has been examined by the gel electrophoresis retardation assay using the HLA-DQ2 beta gene 5' flanking DNA and nuclear extracts derived from various cell types. Several specific protein-binding activities were found using a 45-base-pair (bp) HinfI/Sau96I (-142 to -98 bp) and a 38-bp Sau96I/Sau96I (-97 to -60 bp) fragment, which include conserved sequence X (-113 to -100 bp) and conserved sequence Y (-80 to -71 bp), respectively. Competition experiments, methylation interference analysis, and DNase I foot-printing demonstrated that distinct proteins in a nuclear extract of Raji cells (a human B lymphoma line) bind to sequence X, to sequence Y, and to DNA 5' of the X sequence (termed sequence W). The factor binding site in the W sequence is also found to be conserved among beta-chain genes and is suggested to be a gamma-interferon control region.     

Ethanol-induced regulation of GABA-A subunit mRNAs in prefrontal fields of cynomolgus monkeys

BACKGROUND: Recent evidence indicates that functional impairment of the orbital and medial fields of the prefrontal cortex may underlie the deficits in executive control of behavior that characterize addictive disorders, including alcohol addiction. Moreover, previous studies have indicated that alcohol alters GABA neurotransmission and one substrate of these effects may be through the reconfiguration of the subunits constituting the GABA(A) receptor complex. Given that GABAergic transmission has an integral role in cortical processing, influencing local and interregional communication, understanding alcohol-induced alterations in GABA(A) receptors in prefrontal fields of the primate brain may provide insight into the functional impairment of these brain regions in the alcohol-addicted state and extend our understanding of the molecular consequences of long-term use in these critical brain regions. METHODS AND RESULTS: To address this problem, the effects of chronic ethanol self-administration in male cynomolgus monkeys on GABA(A) receptor subunit mRNA expression was studied in 3 frontal cortical fields: orbitofrontal cortex (OFC; area 13), anterior cingulate cortex (ACC; area 24), and the dorsolateral prefrontal cortex (DLPFC; area 46). Quantitative polymerase chain reaction revealed significant alterations in GABA(A) subunit mRNA expression in the OFC and DLPFC but not in the ACC. Specifically, expression of the alpha2, alpha4, beta1, beta3, and gamma1 to gamma3 subunit mRNAs was significantly less in the OFC, whereas the expression of beta1, beta2, gamma1, and delta subunit mRNAs was less in the DLPFC of alcohol-treated monkeys. CONCLUSION: These findings suggest that ethanol-induced alterations in GABA(A) function may be due to alterations in GABA(A) subunit mRNA levels and subunit-specific alterations are selective to particular cortical fields.