Local protein synthesis in neuronal dendrites is critical for synaptic plasticity. However, the signaling cascades that couple synaptic activation to dendritic protein synthesis remain elusive. The purpose of this study is to determine the role of glutamate receptors and the mammalian target of rapamycin (mTOR) signaling in regulating dendritic protein synthesis in live neurons. We first characterized the involvement of various subtypes of glutamate receptors and the mTOR kinase in regulating dendritic synthesis of a green fluorescent protein (GFP) reporter controlled by ␣CaMKII 5 and 3 untranslated regions in cultured hippocampal neurons. Specific antagonists of N-methyl-D-aspartic acid (NMDA), ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and metabotropic glutamate receptors abolished glutamate-induced dendritic GFP synthesis, whereas agonists of NMDA and metabotropic but not AMPA glutamate receptors activated GFP synthesis in dendrites. Inhibitions of the mTOR signaling, as well as its upstream activators, phosphatidylinositol 3-kinase and AKT, blocked NMDA receptor-dependent dendritic GFP synthesis. Conversely, activation of mTOR signaling stimulated dendritic GFP synthesis. In addition, we also found that inhibition of the mTOR kinase blocked dendritic synthesis of the endogenous ␣CaMKII and MAP2 proteins induced by tetanic stimulations in hippocampal slices. These results identify critical roles of NMDA receptors and the mTOR signaling pathway for control of synaptic activity-induced dendritic protein synthesis in hippocampal neurons.

Gene transcription is required for establishing and maintaining the enduring form of long term potentiation (LTP). However, the transcriptome and its associated molecular programs that support LTP are not well understood. The purpose of this study was to identify activity-regulated genes (ARGs) and their molecular pathways that are modulated by LTP induction and to investigate the genomic mechanism for coordinating the transcription of ARGs. We performed time course DNA microarray analyses on the mouse dentate gyrus to determine the temporal genomic expression profiles of ARGs in response to LTP-inducing tetanic stimulation. Our studies uncovered ARGs that regulate various cellular processes, including the structure and function of the synapse, and offered an overview of the dynamic molecular programs that are probably important for LTP. Surprisingly, we found that ARGs are clustered on chromosomes, and ARG clusters are conserved during evolution. Although ARGs in the same cluster have apparently different molecular properties, they are functionally correlated by regulating LTP. In addition, ARGs in specific clusters are co-regulated by the cAMP-response element-binding protein. We propose that chromosomal clustering provides a genomic mechanism for coordinating the transcription of ARGs involved in LTP.

Growth-arrest and DNA-damage inducible (GADD) genes and Myeloid differentiation primary response (MyD) genes represent a family of genes that play a key role in negative control of cell growth. In the present study, following clone and location of human GADD45 gamma (MyDL) gene, we have found that its mRNA expression level was down-regulated in 15/23 cases of clinic hepatocellular carcinoma (HCC) by comparing the northern hybridization results between the tumor tissues and adjacent normal tissues. Transient transfection of GADD45 gamma cDNA with intact open reading frame sequence into the human hepatoma cells Hep-G2 resulted in dramatic growth suppression in colony formation assays. Furthermore, flow cytometry analysis indicated that GADD45 y caused cell cycle arrest at G2/M transition when transfected into Hep-G2 cells. Therefore, the possible role of GADD45 gamma in cell growth control was further confirmed in this paper.

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The expression of long-lasting synaptic plasticity requires synthesis of new proteins. A critical locus for protein synthesis to support synaptic plasticity is the dendrites. Previous studies demonstrate that synaptic activity activates dendritic protein synthesis. The mechanism by which synaptic activity stimulates protein synthesis in dendrites is, however, poorly understood. This study is to determine the role of the mitogen-activated protein kinase signaling pathway in activity-dependent dendritic protein synthesis. Using a green fluorescent protein reporter with CaMKII 5' and 3'untranslated regions, we show that dendritic synthesis of the green fluorescent protein induced by N-methyl-D-aspartate stimulation is abolished by U0126, a specific inhibitor of mitogen-activated protein kinase signaling. Our results suggest an important role of the mitogen-activated protein kinase signaling in dendritic protein synthesis induced by N-methyl-D-aspartate receptor activation.

A full-length cDNA of 595 bp was isolated from a human fetal brain cDNA library. It contains an open reading frame encoding 153 amino acids, with an 18-bp 5'UTR and a 118-bp 3'UTR in which there is an atypical polyadenylation signal (ATTAAA). The calculated molecular weight of the deduced protein is 17.3 kU. The predicted isoelectric point is 4.89. On account of its high homology to mouse neuronal protein NP15.6 (81.2% identity), the deduced protein was named neuronal protein 17.3 (NP17.3). When its secondary structure was examined by the GGBSM program of PCGENE software, it was found that 32.6 and 15.0% of its amino acids are involved in forming alpha-helices and beta-sheets, respectively. Examined with the PESTFIND program, a typical PEST region found in rapidly degraded proteins was found between residue 48 and residue 68.

In the present study, a brain abundant member of beta 4-galactosyltransferase gene family with an open reading frame encoding 343 amino acids was cloned and identified from a human leukemia cell cDNA library. The putative protein sequence is about 94.8 and 94.2% identical to the 382-amino-acid mouse and rat beta 4-galactosyltransferase respectively and also contains cysteine residues previously shown to be important for the function of the gene family members. This cDNA (tentatively termed beta 4GalT-VIb) is identical to a recently reported cDNA (tentatively termed beta 4GalT-VIa) of human beta 4-galactosyltransferase except lacking one exon, suggesting that these two cDNAs are two different alternative transcripts of the same gene. Northern hybridization showed that the new alternative transcript, beta 4GalT-VIb, is expressed in all 16 human tissues tested with highest level in brain and rich level in testis, thymus and pancreas, whereas weak expression was detected in lung. The beta 4GalT-VIb gene was located to human chromosome 18q12.1 between markers WI-9180 and SGC35630 by radiation hybrid mapping. The genomic organization and adjacent gene content of beta 4GalT-VIb were identified by comparing its cDNA sequence with three genomic sequences AC017100, AP002474 and AP001336, which showed that beta 4GalT-VIb spans an approximately 58 kb region and is composed of 8 exons. In addition, the most conserved motif composed of 41 residues, LXYX3FGGVSXL(T/S)X2 QFX2INGFPNX(Y/F)WGWGGEDDDX2NR, was defined according to 17 sequences of beta 4GalTs from seven different organisms for the first time.

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