Shao Jun Tang

A rapamycin-sensitive signaling pathway contributes to long-term synaptic plasticity in the hippocampus

Many forms of long-lasting behavioral and synaptic plasticity require the synthesis of new proteins. For example, long-term potentiation (LTP) that endures for more than an hour requires both transcription and translation. The signal-transduction mechanisms that couple synaptic events to protein translational machinery during long-lasting synaptic plasticity, however, are not well understood. One signaling pathway that is stimulated by growth factors and results in the translation of specific mRNAs includes the rapamycin-sensitive kinase mammalian target of rapamycin (mTOR, also known as FRAP and RAFT-1). Several components of this translational signaling pathway, including mTOR, eukaryotic initiation factor-4E-binding proteins 1 and 2, and eukaryotic initiation factor-4E, are present in the rat hippocampus as shown by Western blot analysis, and these proteins are detected in the cell bodies and dendrites in the hippocampal slices by immunostaining studies. In cultured hippocampal neurons, these proteins are present in dendrites and are often found near the presynaptic protein, synapsin I. At synaptic sites, their distribution completely overlaps with a postsynaptic protein, PSD-95. These observations suggest the postsynaptic localization of these proteins. Disruption of mTOR signaling by rapamycin results in a reduction of late-phase LTP expression induced by high-frequency stimulation; the early phase of LTP is unaffected. Rapamycin also blocks the synaptic potentiation induced by brain-derived neurotrophic factor in hippocampal slices. These results demonstrate an essential role for rapamycin-sensitive signaling in the expression of two forms of synaptic plasticity that require new protein synthesis. The localization of this translational signaling pathway at postsynaptic sites may provide a mechanism that controls local protein synthesis at potentiated synapses.

Specific Activation of Smad1 Signaling Pathways by the BMP7 Type I Receptor, ALK2

BMP7 and activin are members of the transforming growth factor β superfamily. Here we characterize endogenous activin and BMP7 signaling pathways in P19 embryonic carcinoma cells. We show that BMP7 and activin bind to the same type II receptors, ActRII and IIB, but recruit distinct type I receptors into heteromeric receptor complexes. The major BMP7 type I receptor observed was ALK2, while activin bound exclusively to ALK4 (ActRIB). BMP7 and activin elicited distinct biological responses and activated different Smad pathways. BMP7 stimulated phosphorylation of endogenous Smad1 and 5, formation of complexes with Smad4 and induced the promoter for the homeobox gene,Tlx2. In contrast, activin induced phosphorylation of Smad2, association with Smad4, and induction of the activin response element from the Xenopus Mix.2gene. Biochemical analysis revealed that constitutively active ALK2 associated with and phosphorylated Smad1 on the COOH-terminal SSXS motif, and also regulated Smad5 and Smad8 phosphorylation. Activated ALK2 also induced the Tlx2promoter in the absence of BMP7. Furthermore, we show that ALK1 (TSRI), an orphan receptor that is closely related to ALK2 also mediates Smad1 signaling. Thus, ALK1 and ALK2 induce Smad1-dependent pathways and ALK2 functions to mediate BMP7 but not activin signaling.

Activity-dependent Synaptic Wnt Release Regulates Hippocampal Long Term Potentiation

Wnts are important for various developmental and oncogenic processes. Here we show that Wnt signaling functions at synapses in hippocampal neurons. Tetanic stimulations induce N-methyl-d-aspartate receptor-dependent synaptic Wnt3a release, nuclear β-catenin accumulations, and the activation of Wnt target genes. Suppression of Wnt signaling impairs long term potentiation. Conversely, activation of Wnt signaling facilitates long term potentiation. These findings suggest that Wnt signaling plays a critical role in regulating synaptic plasticity.

A Role for a Rat Homolog of Staufen in the Transport of RNA to Neuronal Dendrites

RNAs are present in dendrites and may be used for local protein synthesis in response to synaptic activity. To begin to understand dendritic RNA targeting, we cloned a rat homolog of staufen, a Drosophila gene that participates in mRNA targeting during development. In hippocampal neurons, rat staufen protein displays a microtubule-dependent somatodendritic distribution pattern that overlaps with dendritic RNAs. To determine whether r-staufen is required for dendritic RNA targeting, we constructed a mutant version containing the RNA binding domains (stau-RBD) but lacking the C-terminal portion potentially involved in dendritic targeting. Stau-RBD expression was restricted to the cell bodies and proximal dendrites. Expression of stau-RBD significantly decreased, while overexpression of wild-type r-staufen increased, the amount of dendritic mRNA. Taken together, these results suggest that the rat staufen protein plays an important role in the delivery of RNA to dendrites.

Roles of Glutamate Receptors and the Mammalian Target of Rapamycin (mTOR) Signaling Pathway in Activity-dependent Dendritic Protein Synthesis in Hippocampal Neurons

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 byαCaMKII 5′ and 3′ untranslated regions in cultured hippocampal neurons. Specific antagonists of N-methyl-d-aspartic acid (NMDA), α-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 αCaMKII 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.

Chronic-Pain-Associated Astrocytic Reaction in the Spinal Cord Dorsal Horn of Human Immunodeficiency Virus-Infected Patients

Studies with animal models have suggested that reaction of glia, including microglia and astrocytes, critically contributes to the development and maintenance of chronic pain. However, the involvement of glial reaction in human chronic pain is unclear. We performed analyses to compare the glial reaction profiles in the spinal dorsal horn (SDH) from three cohorts of sex- and age-matched human postmortem tissues: (1) HIV-negative patients, (2) HIV-positive patients without chronic pain, and (3) HIV patients with chronic pain. Our results indicate that the expression levels of CD11b and Iba1, commonly used for labeling microglial cells, did not differ in the three patient groups. However, GFAP and S100β, often used for labeling astrocytes, were specifically upregulated in the SDH of the “pain-positive” HIV patients but not in the “pain-negative” HIV patients. In addition, proinflammatory cytokines, TNFα and IL-1β, were specifically increased in the SDH of pain-positive HIV patients. Furthermore, proteins in the MAPK signaling pathway, including pERK, pCREB and c-Fos, were also upregulated in the SDH of pain-positive HIV patients. Our findings suggest that reaction of astrocytes in the SDH may play a role during the maintenance phase of HIV-associated chronic pain.

Characterization and developmental expression of Tlx-1, the murine homolog of HOX11

HOX11, a human homeobox gene with putative oncogenic potential, was originally discovered at the chromosome 10 breakpoint in T-cell acute lymphoblastic leukemias bearing the chromosomal translocation t(10;14)(q24;q11). To provide insight into the possible roles of this gene in development, we isolated and characterized its murine homolog, Tlx-1, and examined its profile of expression. Tlx-1 transcripts are first detected at E8.5 in the surface ectoderm and central mesenchyme of the first branchial arch. This expression subsequently extends to the 2nd, 3rd, and 4th branchial arches, as well as the presumptive pharynx, as these structures develop. Between E12.5 and E15.5, the profile of Tlx-1 expression becomes more complex; expression is observed in the developing pancreas and salivary glands, as well as in several components of the nervous system, including the trigeminal, glossopharyngeal and vestibulocochlear ganglia, the spinal cord, and the curvature of the pons-medulla. In addition, expression is seen in the pinna and external auditory meatus of the outer ear, the tooth primordia, and specific cell populations of the mandible and tongue. These complex patterns of expression are consistent with multiple and varied roles for Tlx-1 in development and suggest that Tlx-1 marks, amongst other cell populations, structures derived from cranial neural crest cells and migratory paraxial mesoderm that arise at corresponding levels along the rostral-caudal axis of the developing embryo.

Regulation of microRNA Expression by Induction of Bidirectional Synaptic Plasticity

Activity-induced protein synthesis is critical for long-lasting synaptic plasticity and subject to tight controls. MicroRNAs (miRNAs) are negative regulators of mRNA translation, but their role during synaptic plasticity is not clear. In this study, we have investigated how induction of long-term potentiation (LTP) and long-term depression (LTD) regulates the expression of miRNAs. Using miRNA arrays, we determined the temporal expression profiles of 62 hippocampal miRNAs following induction of chemical LTP (C-LTP) and metabotropic glutamate receptor-dependent LTD (mGluR-LTD). Several striking features were observed. First, C-LTP or mGluR-LTD induction changed the expression levels of most hippocampal miRNAs. Second, the majority of miRNAs regulated by C-LTP or mGluR-LTD induction followed a similar temporal expression profile. Third, most miRNAs were regulated by both C-LTP and mGluR-LTD induction, but displayed distinct expression dynamics. Fourth, many miRNAs were upregulated at specific time points C-LTP and mGluR-LTD induction, suggesting that C-LTP and mGluR-LTD induction elicits miRNA-mediated suppression of mRNA translation. We propose that the upregulated miRNA expression provides a mechanism to prevent excess protein synthesis during the expression of synaptic plasticity. The extensive regulation of miRNA expression by C-LTP and mGluR-LTD induction suggests a critical role of miRNAs in synaptic plasticity.

NMDA receptor activation stimulates transcription-independent rapid wnt5a protein synthesis via the MAPK signaling pathway

Wnt proteins are emerging key regulators of the plasticity and functions of adult brains. However, the mechanisms by which the expression of Wnt proteins is regulated in neurons are unclear. Using cortical primary cultures, we show here that activation of NMDA receptors (NMDARs) induces rapid Wnt5a protein synthesis and secretion. This NMDAR-regulated Wnt5a synthesis does not require transcription and is a result of activity-dependent translation. We also show that NMDAR-regulated Wnt5a translation depends on MAPK signaling but not mTOR signaling. Our findings suggest that the synaptic activity of CNS neurons activates NMDARs, which in turn stimulate translation from stored Wnt5a mRNA via the MAPK signaling pathway.

Molecular Network and Chromosomal Clustering of Genes Involved in Synaptic Plasticity in the Hippocampus

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.