Moving molecules: mRNA trafficking in mammalian oligodendrocytes and neurons

Numerous mRNA molecules are localized in regions of the dendrites of neurons, some moving along dendrites in response to synaptic activity. The proteins encoded by these RNAs have diverse functions, including participation in memory formation and long-term potentiation. Recent experiments have shown that a cytoplasmic RNA trafficking pathway described for oligodendrocytes also operates in neurons. Transported RNAs possess a cis-acting element that directs them to granules, which are transported along microtubules by the motor proteins kinesin and dynein. These RNA molecules are recruited to the cytoplasmic transport granules by cooperative interaction with a cognate trans-acting factor. mRNAs containing the 11-nucleotide A2RE11 or 21-nucleotide A2RE sequences bind heterogeneous nuclear ribonucleoproteins A2 and A3, which are abundant in the brain. Mutations in this cis-acting element that weaken its interaction with hnRNP A2 also interfere with RNA trafficking. Several dendritically localized mRNAs, including those encoding calcium-calmodulin-dependent protein kinase 11 a subunit and neurogranin, possess A2RE-like sequences, suggesting that they may be localized by interaction with these heterogeneous nuclear ribonucleoproteins. Calcium-calmodulin-dependent protein kinase 11 a subunit is of particular interest: Its RNA is transported in depolarized neurons, and the protein it encodes is essential for establishing long-term memory. Several other cis-acting sequences and trans-acting factors that participate in neuronal RNA localization have been discovered.

Increased expression of the MBP mRNA binding protein HnRNP A2 during oligodendrocyte differentiation

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a trans-acting factor that mediates intracellular trafficking of myelin basic protein (MBP) mRNA to the myelin compartment in oligodendrocytes, is most abundant in the nucleus, but shuttles between the nucleus and cytoplasm. In the cytoplasm, it is associated with granules that transport mRNA from the cell body to the processes of oligodendrocytes. We found that the overall level of hnRNP A2 increased in oligodendrocytes as they differentiated into MBIP-positive cells, and that this augmentation was reflected primarily in the cytoplasmic pool of hnRNP A2 present in the form of granules. The extranuclear distribution of hnRNP A2 was also observed in brain during the period of myelination in vivo. Methylation and phosphorylation have been implicated previously in the nuclear to cytoplasmic distribution of hnRNPs, so we used drugs that block methylation and phosphorylation of hnRNPs to assess their effect on hnRNP A2 distribution and mRNA trafficking. Cultures treated with adenosine dialdehyde (AdOx), an inhibitor of S-adenosyl-L-homocysteine hydrolase, or with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a drug that inhibits casein kinase 2 (CK2), maintained the preferential nuclear distribution of hnRNP A2. Treatment with either drug affected the transport of RNA trafficking granules that remained confined to the cell body. (C) 2004 Wiley-Liss, Inc.

Differential expression of the GABA transporters GAT-1 and GAT-3 in brains of rats, cats, monkeys and humans

The homeostasis of GABA is critical to normal brain function. Extracellular levels of GABA are regulated mainly by plasmalemmal gamma-aminobutyric acid (GABA) transporters. Whereas the expression of GABA transporters has been extensively studied in rodents, validation of this data in other species, including humans, has been limited. As this information is crucial for our understanding of therapeutic options in human diseases such as epilepsy, we have compared, by immunocytochemistry, the distributions of the GABA transporters GAT-1 and GAT-3 in rats, cats, monkeys and humans. We demonstrate subtle differences between the results reported in the literature and our results, such as the predominance of GAT-1 labelling in neurons rather than astrocytes in the rat cortex. We note that the optimal localisation of GAT-1 in cats, monkeys and humans requires the use of an antibody against the human sequence carboxyl terminal region of GAT-1 rather than against the slightly different rat sequence. We demonstrate that GAT-3 is localised mainly to astrocytes in hindbrain and midbrain regions of rat brains. However, in species such as cats, monkeys and humans, additional strong immunolabelling of oligodendrocytes has also been observed. We suggest that differences in GAT distribution, especially the expression of GAT-3 by oligodendrocytes in humans, must be accommodated in extrapolating rodent models of GABA homeostasis to humans.

The pathophysiology of 'brain shrinkage' in alcoholics - Structural and molecular changes and clinical implications

This article represents a symposium of the 2004 ISBRA Congress held in Mannheim. The presentations were: Review of the neuropathological and neurochemical changes seen in alcohol-related ' brain shrinkage ' by Clive Harper; In Vivo Detection of Macrostructural and Microstructural Markers of Brain Integrity in Human Alcoholism and a Rodent Model of Alcoholism by Adolf Pfefferbaum, Elfar Adalsteinsson and Edith Sullivan; Gene and Protein Changes in the Brains of Alcoholics with ' Brain Shrinkage ' by Joanne Lewohl and Peter Dodd; Cross sectional and longitudinal MR spectroscopy studies of chronic adult alcoholics by Michael Taylor; Brain Atrophy Associated with Impairment on a Simulated Gambling Task in Long-Term Abstinent Alcoholics by George Fein and Bennett Landman.

Molecular recognition of an RNA trafficking element by heterogeneous nuclear ribonucleoprotein A2

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a multitasking protein involved in RNA packaging, alternative splicing of pre-mRNA. telomere maintenance, cytoplasmic RNA trafficking, and translation. It binds short segments of single-stranded nucleic acids, including the A2RE11 RNA element that is necessary and sufficient for cytoplasmic transport of a subset of rnRNAs in oligodendrocytes and neurons. We have explored the structures of hnRNP A2, its RNA recognition motifs (RRMs) and Gly-rich module, and the RRM complexes with A2RE11. Circular dichroism spectroscopy showed that the secondary structure of the first 189 residues of hnRNP A2 parallels that of the tandem beta alpha beta beta alpha beta RRMs of its paralogue, hnRNP A1, previously deduced from X-ray diffraction studies. The unusual GRD was shown to have substantial beta-sheet and beta-turn structure. Sedimentation equilibrium and circular dichroism results were consistent with the tandem RRM region being monomeric and supported earlier evidence for the binding of two A2RE11 oligoribonucleotides to this domain, in contrast to the protein dimer formed by the complex of hnRNP A1 with the telomeric ssDNA repeat. A three-dimensional structure for the N-terminal, two-RRM-containing segment of hnRNP A2 was derived by homology modeling. This structure was used to derive a model for the complex with A2RE11 using the previously described interaction of pairs of stacked nucleotides with aromatic residues on the RRM beta-sheet platforms, conserved in other RRM-RNA complexes, together with biochemical data and molecular dynamics-based observations of inter-RRM mobility.

Suppressor of cytokine signaling 3 limits protection of leukemia inhibitory factor receptor signaling against central demyelination

Enhancement of oligodendrocyte survival through activation of leukemia inhibitory factor receptor (LIFR) signaling is a candidate therapeutic strategy for demyelinating disease. However, in other cell types, LIFR signaling is under tight negative regulation by the intracellular protein suppressor of cytokine signaling 3 (SOCS3). We, therefore, postulated that deletion of the SOCS3 gene in oligodendrocytes would promote the beneficial effects of LIFR signaling in limiting demyelination. By studying wild-type and LIF-knockout mice, we established that SOCS3 expression by oligodendrocytes was induced by the demyelinative insult, that this induction depended on LIF, and that enclogenously produced LIF was likely to be a key determinant of the CNS response to oligodendrocyte loss. Compared with wild-type controls, oligo-dendrocyte-specific SOCS3 conditional-knockout mice displayed enhanced c-fos activation and exogenous LIF-induced phosphorylation of signal transducer and activator of transcription 3. Moreover, these SOCS3-deficient mice were protected against cupri-zone-induced oligodendrocyte loss relative to wild-type animals. These results indicate that modulation of SOCS3 expression could facilitate the endogenous response to CNS injury.

Binding of an RNA trafficking response element to heterogeneous nuclear ribonucleoproteins A1 and A2

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 binds a 21-nucleotide myelin basic protein mRNA response element, the A2RE, and A2RE-like sequences in other localized mRNAs, and is a trans-acting factor in oligodendrocyte cytoplasmic RNA trafficking. Recombinant human hnRNPs A1 and A2 were used in a biosensor to explore interactions with A2RE and the cognate oligodeoxyribonucleotide. Both proteins have a single site that bound oligonucleotides with markedly different sequences but did not bind in the presence of heparin. Both also possess a second, specific site that bound only A2RE and was unaffected by heparin, hnRNP A2 bound A2RE in the latter site with a K-d near 50 nM, whereas the K-d for hnRNP A1 was above 10 muM. UV cross-linking assays led to a similar conclusion. Mutant A2RE sequences, that in earlier qualitative studies appeared not to bind hnRNP A2 or support RNA trafficking in oligodendrocytes, had dissociation constants above 5 muM for this protein. The two concatenated RNA recognition motifs (RRMs), but not the individual RRMs, mimicked the binding behavior of hnRNP A2. These data highlight the specificity of the interaction of A2RE with these hnRNPs and suggest that the sequence-specific A2RE-binding site on hnRNP A2 is formed by both RRMs acting in cis.

The effects of ethanol on PPARS in MCF-7 human breast cancer cells

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors involved in various metabolic diseases. In the liver, PPARα is involved in alcohol metabolism and may lead to the development of alcoholic fatty liver and other alcohol mediated liver injuries. PPARβ modulation by ethanol induces abnormal myelin production by oligodendrocytes. PPARα and PPARβ are PPAR isoforms expressed in the human breast cell lines. Epidemiological studies show a positive correlation between alcohol intake and breast cancer risk, however, the molecular mechanisms involved are unclear. We hypothesized that ethanol would affect the expression and transactivation of human PPAR isoforms in estrogen receptor (ER) positive and ER negative breast cancer cells. Using real time RT-PCR we looked at the transcription of PPAR isoforms in the presence of increasing concentrations of ethanol and saw isoform and time dependent specific effects. Gene reporter assays enabled us to ascertain the effects of ethanol on ligand-mediated activation of human PPARα and PPARβ at concentrations equivalent to both moderate and chronic alcohol consumption. Ethanol differentially blocked the ligand-mediated activation of both PPARα and PPARβ. Since PPARα and PPARβ are involved in the differentiation and proliferation of breast cancer cells, PPARs may be a possible mechanism involved in the effect of ethanol in breast cancer.