Antje Anji

Regulation of 5-HT2A receptor mRNA levels and binding sites in rat frontal cortex by the agonist DOI and the antagonist mianserin

In the present study we have characterized the time course of effect of administration of the serotonin(2) (5-HT(2)) receptor antagonist mianserin, or the 5-HT(2) receptor agonist (+/-)-2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI), on 5-HT(2A) receptor binding sites and mRNA levels in rat frontal cortex. Radioligand binding and ribonuclease protection assays were performed with separate hemispheres of frontal cortex from each animal to examine concomitant changes in 5-HT(2A) receptor sites and mRNA levels. The decrease in cortical 5-HT(2A) receptor sites in response to chronic DOI administration was not accompanied by changes in 5-HT(2A) receptor mRNA. A single injection of DOI produced a transient decrease in 5-HT(2A) receptor mRNA levels detected 1 h post-injection. The density of 5-HT(2A) receptor sites, however, was not significantly reduced following a single injection of DOI. The down-regulation of cortical 5-HT(2A) receptor sites in response to a single injection of mianserin was accompanied by reductions in 5-HT(2A) receptor mRNA levels. Following 4 days of mianserin administration, however, we did not observe a change in 5-HT(2A) receptor mRNA levels, although 5-HT(2A) receptor density was decreased. Thus, changes in receptor mRNA may initially contribute to the down-regulation of 5-HT(2A) receptors in response to acute mianserin administration. Sustained changes in 5-HT(2A) receptor mRNA, however, appear not to be involved in maintaining the down-regulation of 5-HT(2A) receptor number with chronic mianserin administration. Mechanisms other than the regulation of receptor mRNA levels appear to underlie the down-regulation of 5-HT(2A) receptor sites in response to chronic administration of the agonist DOI.

A novel RNA binding protein that interacts with NMDA R1 mRNA: regulation by ethanol

Excitatory NMDA receptors are an important target of ethanol. Chronic ethanol exposure, in vivo and in vitro, increases polypeptide levels of NR1 subunit, the key subunit of functional NMDA receptors. In vitro, chronic ethanol treatment increases the half‐life of NR1 mRNA and this observation is dependent on new protein synthesis. The present study was undertaken to locate cis‐acting region(s) within the NR1 3′‐untranslated region (UTR) and identify NR1 3′‐UTR binding trans‐acting proteins expressed in mouse fetal cortical neurons. Utilizing RNA gel shift assays we identified a 156‐nt cis‐acting region that binds to polysomal trans‐acting proteins. This binding was highly specific as inclusion of cyclophilin RNA or tRNA did not interfere with cis–trans interactions. Importantly, the 3′‐UTR binding activity was significantly up‐regulated in the presence of ethanol. UV cross‐link analysis detected three NR1 3′‐UTR binding proteins and their molecular mass calculated by Northwestern analysis was ∼88, 60 and 47 kDa, respectively. Northwestern analysis showed a significant up‐regulation of the 88‐kDa protein after chronic ethanol treatment. The 88‐kDa protein was purified and identified by tandem mass spectrometry as the beta subunit of alpha glucosidase II (GIIβ). That GIIβ is indeed a trans‐acting protein and binds specifically to 3′‐UTR of NR1 mRNA was confirmed by RNA gel mobility supershift assays and immuno RT‐PCR. Western blotting data established a significant increase of GIIβ polypeptide in chronic ethanol‐exposed fetal cortical neurons. We hypothesize that the identified cis‐acting region and the associated RNA‐binding proteins are important regulators of NR1 subunit gene expression.

The role of protein kinase C in the regulation of serotonin‐2A receptor expression

We have investigated in C6 glioma cells the involvement of protein kinase C (PKC) in the regulation of serotonin‐2A receptor (5‐HT2A receptor) expression by agonist treatment. Comparison of the time‐courses of agonist‐induced downregulation of receptor number and mRNA indicate that a decrease in the number of 5‐HT2A receptor binding sites in response to serotonin (5‐HT) treatment is preceded by a decrease in 5‐HT2A receptor mRNA. This decrease in 5‐HT2A receptor mRNA as a result of agonist exposure was not due to a change in the stability or half‐life of the transcript. Pretreatment of cells with the PKC inhibitor bisindolylmaleimide blocked the decrease in 5‐HT2A receptor mRNA levels, and attenuated the down‐regulation of 5‐HT2A receptor binding sites induced by treatment with 5‐HT. Experiments performed with the PKC inhibitors calphostin C and Gö 6976 confirmed that PKC was involved in the regulation of 5‐HT2A receptor mRNA by agonist and implicate the conventional subgroup of PKC isoforms. Western blot analysis, using isoform‐specific anti‐PKC antibodies showed that under our culture conditions C6 glioma cells express the conventional isoforms PKC α, PKC γ, as well as the novel isoforms PKC δ, PKC ε, and the atypical isoforms PKC λ and PKC ι. Upon treatment with 5‐HT for 10 min levels of the conventional isoforms PKC α and PKC γ increased in the nuclear fraction. Taken together, our results implicate PKC α and/or PKC γ in the regulation of 5‐HT2A mRNA receptor and binding sites in response to agonist treatment.

A cis-acting region in the N-methyl-D-aspartate R1 3′-untranslated region interacts with the novel RNA-binding proteins beta subunit of alpha glucosidase II and annexin A2: effect of chronic ethanol exposure in vivo

A cis‐acting region, Δ4, located in the 3′‐untranslated region of N‐methyl‐d‐aspartate R1(NR1) mRNA interacts with several trans‐acting proteins present in polysomes purified from fetal cortical neurons. Chronic ethanol exposure of fetal cortical neurons increases Δ4 RNA–protein interactions. This increased interaction is due to an increase in one of the Δ4‐binding trans‐acting proteins identified as beta subunit of alpha glucosidase II (GIIβ). In this study, we examined whether ethanol‐mediated regulation of NR1 mRNA in vivo is similar to that in vitro and whether Δ4–trans interactions are important for ethanol‐mediated NR1 mRNA stability. Our data show that polysomal proteins from adult mouse cerebral cortex (CC) formed a complex with Δ4 RNA, suggesting the presence of NR1 mRNA‐binding trans‐acting proteins in CC polysomes. The intensity of the Δ4 RNA–protein complex was increased with polysomes from chronic ethanol‐exposed CC. The Δ4 RNA–protein complex harbored GIIβ and a second trans‐acting protein identified as annexin A2 (AnxA2). Ethanol‐sensitive GIIβ was upregulated by 70% in ethanol‐exposed CC. Heparin, a known binding partner of AnxA2, inhibited Δ4 RNA–protein complex formation. Transient transfection studies using chimeric constructs with and without the Δ4 region revealed that cis–trans interactions are important for ethanol‐mediated stability of NR1 mRNA. Furthermore, our data highlight, for the first time, the presence of a binding site on the 3′‐untranslated region of NR1 mRNA for AnxA2 and demonstrate the regulation of NR1 mRNA by AnxA2, GIIβ and a third NR1 mRNA‐binding protein, which is yet to be identified.

The Molecular Effects of Alcohol

Abstract: Chronic ethanol treatment (50 mM, five days) induces stabilization of NR1 receptor subunit mRNA in cultured fetal cortical neurons. In this paper, we investigate the mechanism(s) by which ethanol mediates its effects on NR1 mRNA. Specifically, we have determined if cellular localization of NR1 mRNA in cortical neurons and/or de novo protein synthesis is essential for ethanol‐mediated stabilization of NR1 mRNA. Subcellular fractionation studies show that all detectable NR1 mRNA is associated with rough endoplasmic reticulum, indicating that subcellular distribution of NR1 mRNA in fetal cortical neurons does not play a role in ethanol‐mediated NR1 mRNA stabilization. However, inhibition of protein synthesis by cycloheximide abolished the mRNA stabilizing effect of ethanol on NR1 mRNA, thus suggesting de novo protein synthesis is crucial for the action of ethanol on NR1 mRNA stabilization.

An Old Story with a New Twist: Do NMDAR1 mRNA Binding Proteins Regulate Expression of the NMDAR1 Receptor in the Presence of Alcohol?

Abstract: NMDA receptors not only play a pivotal role in normal physiological processes in the central nervous system (CNS), but have been identified as an important target of ethanol. Chronic exposure to ethanol induces a number of adaptive processes in the CNS, including an upregulation of NMDA receptor number and function. The increase in NMDA receptor number in response to chronic ethanol exposure both in vivo and in vitro is accompanied by an increase in NMDAR1 and NMDAR2B polypeptide levels. It is widely believed that these adaptive changes play an important role in the development of alcohol dependence and withdrawal syndrome. At the molecular level, chronic ethanol exposure of fetal cortical neurons selectively increases expression of NMDAR1 splice variants lacking exon 5 and exon 22. Chronic ethanol exposure of fetal cortical neurons also increases NMDAR1 mRNA half‐life in these neurons. However, when new protein synthesis is inhibited, the half‐life of NR1 mRNA in these neurons returns to control values, strongly suggesting that ethanol induces the synthesis of protein(s) that may regulate the decay of NR1 mRNA. In recent years, it has become apparent that regulation of mRNA stability is an important aspect of regulation of gene expression. Changes in mRNA stability can be accomplished by interaction between cis‐acting sequences in the 3′ untranslated region (3′UTR) of mRNAs and trans‐acting proteins expressed in cells. Such interactions may protect RNAs from degradation by ribonucleases, thereby increasing the half‐life of mRNAs.

Effect of Ethanol on Lipid‐Mediated Transfection of Primary Cortical Neurons

Abstract: Successful introduction of nucleic acids into mammalian neurons has revolutionized the analyses of gene regulation and cellular function. Various methods, including viral infection, have been developed to introduce plasmid DNA into primary neuronal cultures. However, transfection of primary cultures of neurons using the calcium phosphate precipitation method and electroporation have been comparatively inefficient. In this paper, we describe a method to successfully transfect cultured fetal cortical neurons using a cationic lipid reagent, lipofectamine. Cells were cultured in the absence and presence of 50 mM ethanol. To monitor transfection of neurons, we employed three mammalian expression vectors containing Renilla luciferase and/or firefly luciferase, or the beta‐galactosidase reporter gene. Fetal cortical neurons were isolated and cultured in the absence or presence of 50 mM ethanol, for two days. On day 3, neurons were washed, fed with serum‐free medium, and transfected with the DNA‐lipofectamine complex. After two hours, cells were washed, fed complete medium lacking or containing 50 mM ethanol and cultured for two additional days with a change of medium after 24 h. Cultures were terminated 48 h after transfection. Cells were either stained for beta‐galactosidase activity using X‐gal or lysed to prepare cell extracts to assay for luciferase activity using a luminometer. When neurons were cotransfected, Renilla luciferase was used as an internal control to normalize the expression of the firefly luciferase reporter gene. Analysis of results showed that expression of the reporter gene, firefly luciferase, was approximately 2.5 times greater in ethanol treated neuronal cultures than for neurons cultured in the absence of ethanol. An increased number of neurons expressing beta‐galactosidase was also observed in ethanol‐treated neurons. These data suggest that perhaps ethanol treatment of fetal cortical neurons improved the DNA uptake and/or increased the expression of the reporter genes.

Guardian of Genetic Messenger-RNA-Binding Proteins

RNA in cells is always associated with RNA-binding proteins that regulate all aspects of RNA metabolism including RNA splicing, export from the nucleus, RNA localization, mRNA turn-over as well as translation. Given their diverse functions, cells express a variety of RNA-binding proteins, which play important roles in the pathologies of a number of diseases. In this review we focus on the effect of alcohol on different RNA-binding proteins and their possible contribution to alcohol-related disorders, and discuss the role of these proteins in the development of neurological diseases and cancer. We further discuss the conventional methods and newer techniques that are employed to identify RNA-binding proteins.

Urea can inhibit efficient reduction and alkylation of protein dimers in solution demonstrated by the beta subunit of alpha glucosidase II

Protein reduction and alkylation is routinely used for analysis of protein dimers and protein complexes in cell fractions using two dimensional gel electrophoresis and mass spectrometry. To resolve the heterogeneity of a high molecular weight protein band that is highlighted by an antibody to the beta subunit of alpha glucosidase II (GIIβ), we performed reduction and alkylation of cytosolic proteins extracted from mouse brain. The presence of urea in the reduction/alkylation buffer inhibited the chemical processes. It is thus recommended that protein reduction/alkylation be performed both in the presence and absence of urea for the separation of mono-/hetero-mers.

Expression of alpha subunit of alpha glucosidase II in adult mouse brain regions and selective organs

α‐Glucosidase II (GII), a resident of endoplasmic reticulum (ER) and an important enzyme in the folding of nascent glycoproteins, is heterodimeric, consisting of α (GIIα) and β (GIIβ) subunits. The catalytic GIIα subunit, with the help of mannose 6‐phosphate receptor homology domain of GIIβ, sequentially hydrolyzes two α1‐3‐linked glucose residues in the second step of N‐linked oligosaccharide‐mediated protein folding. The soluble GIIα subunit is retained in the ER through its interaction with the HDEL‐containing GIIβ subunit. N‐glycosylation and correct protein folding are crucial for protein stability and trafficking and cell surface expression of several proteins in the brain. Alterations in N‐glycosylation lead to abnormalities in neuronal migration and mental retardation, various neurodegenerative diseases, and invasion of malignant gliomas. Inhibitors of GII are used to inhibit cell proliferation and migration in a variety of different pathologies, such as viral infection, cancer, and diabetes. Despite the widespread use of GIIα inhibitory drugs and the role of GIIα in brain function, little is known about its expression in brain and other tissues. Here, we report generation of a highly specific chicken antibody to the GIIα subunit and its characterization by Western blotting and immunoprecipitation using cerebral cortical extracts. By using this antibody, we showed that the GIIα protein is highly expressed in testis, kidney, and lung, with the lowest amount in heart. GIIα polypeptide levels in whole brain were comparable to those in spleen. However, a higher expression of GIIα protein was detected in the cerebral cortex, reflecting its continuous requirement in correct folding of cell surface proteins.