Kazumi Fukatsu

An RNA-interacting protein, SYNCRIP (heterogeneous nuclear ribonuclear protein Q1/NSAP1) is a component of mRNA granule transported with inositol 1,4,5-trisphosphate receptor type 1 mRNA in neuronal dendrites

mRNA transport and local translation in the neuronal dendrite is implicated in the induction of synaptic plasticity. Recently, we cloned an RNA-interacting protein, SYNCRIP (heterogeneous nuclear ribonuclear protein Q1/NSAP1), that is suggested to be important for the stabilization of mRNA. We report here that SYNCRIP is a component of mRNA granules in rat hippocampal neurons. SYNCRIP was mainly found at cell bodies, but punctate expression patterns in the proximal dendrite were also seen. Time-lapse analysis in living neurons revealed that the granules labeled with fluorescent protein-tagged SYNCRIP were transported bi-directionally within the dendrite at ∼0.05 μm/s. Treatment of neurons with nocodazole significantly inhibited the movement of green fluorescent protein-SYNCRIP-positive granules, indicating that the transport of SYNCRIP-containing granules is dependent on microtubules. The distribution of SYNCRIP-containing granules overlapped with that of dendritic RNAs and elongation factor 1α. SYNCRIP was also found to be co-transported with green fluorescent protein-tagged human staufen1 and the 3′-untranslated region of inositol 1,4,5-trisphosphate receptor type 1 mRNA. These results suggest that SYNCRIP is transported within the dendrite as a component of mRNA granules and raise the possibility that mRNA turnover in mRNA granules and the regulation of local protein synthesis in neuronal dendrites may involve SYNCRIP.

DNA methylation-dependent epigenetic regulation of dimethylarginine dimethylaminohydrolase 2 gene in trophoblast cell lineage

Trophoblast cell lineage is established through the first cellular differentiation in mammalian embryogenesis, and its developmental potential is restricted to the extraembryonic tissues contributing solely to the placenta. Several lines of evidence suggest a relative lack of importance of DNA methylation in gene regulation in the extraembryonic tissues when compared with embryonic ones. Here we analyzed the dynamics of epigenetic status in the upstream region of mouse Ddah2 gene, which was found to be specifically repressed in a stem cell population of trophoblast cell lineage. We found a tissue-dependent differentially methylated region in the regulatory region of the Ddah2 gene. This region was hypermethylated in trophoblast stem cells and was hypomethylated in differentiated cells both in vivo and in vitro. This change was well correlated with Ddah2 expression. In addition, in vitro methylation confined to the differentially methylated region was sufficient to repress promoter activity in the reporter assay. Furthermore, a repressive pattern of histone modifications was formed around the differentially methylated region in undifferentiated trophoblast stem cells with repressed Ddah2. Our data suggest that DNA methylation-mediated chromatin remodeling is involved in the regulation of the Ddah2 gene expression and thus is important even in trophoblast cell lineage.

Gephyrin-Independent GABAAR Mobility and Clustering during Plasticity

The activity-dependent modulation of GABA-A receptor (GABAAR) clustering at synapses controls inhibitory synaptic transmission. Several lines of evidence suggest that gephyrin, an inhibitory synaptic scaffold protein, is a critical factor in the regulation of GABAAR clustering during inhibitory synaptic plasticity induced by neuronal excitation. In this study, we tested this hypothesis by studying relative gephyrin dynamics and GABAAR declustering during excitatory activity. Surprisingly, we found that gephyrin dispersal is not essential for GABAAR declustering during excitatory activity. In cultured hippocampal neurons, quantitative immunocytochemistry showed that the dispersal of synaptic GABAARs accompanied with neuronal excitation evoked by 4-aminopyridine (4AP) or N-methyl-D-aspartic acid (NMDA) precedes that of gephyrin. Single-particle tracking of quantum dot labeled-GABAARs revealed that excitation-induced enhancement of GABAAR lateral mobility also occurred before the shrinkage of gephyrin clusters. Physical inhibition of GABAAR lateral diffusion on the cell surface and inhibition of a Ca2+ dependent phosphatase, calcineurin, completely eliminated the 4AP-induced decrease in gephyrin cluster size, but not the NMDA-induced decrease in cluster size, suggesting the existence of two different mechanisms of gephyrin declustering during activity-dependent plasticity, a GABAAR-dependent regulatory mechanism and a GABAAR-independent one. Our results also indicate that GABAAR mobility and clustering after sustained excitatory activity is independent of gephyrin.

Lateral diffusion of inositol 1,4,5-trisphosphate receptor type 1 in Purkinje cells is regulated by calcium and actin filaments

J. Neurochem. (2010) 114, 1720–1733.