This year we have made a significant progress in revealing molecular mechanisms underlying long-term synaptic modulation by neurotrophins. ? ? 1) Regulation of the development of the neuromuscular junction ? Neurotrophins elicit both structural and functional changes of synapses. Whether these changes are mediated by the same or different mechanisms are not known. We report the mechanistic separation of functional and structural synaptic regulation by neurotrophin 3 (NT-3), using the neuromuscular junction (NMJ) as a model. Inhibition of cAMP response element (CRE)-binding protein (CREB)-mediated transcription blocks the enhancement of transmitter release elicited by NT-3, without affecting the synaptic varicosity of the presynaptic terminals. Further analysis indicates that CREB is activated through Ca(2)calmodulin-dependent kinase IV (CaMKIV) pathway, rather than the mitogen-activated protein kinase (MAPK) or cAMP pathway. In contrast, inhibition of MAPK prevents the NT-3-induced structural, but not functional, changes. Genetic and imaging experiments indicate that the small GTPase Rap1, but not Ras, acts upstream of MAPK activation by NT-3. Thus, NT-3 initiates parallel structural and functional modifications of synapses through the Rap1-MAPK and CaMKIV-CREB pathways, respectively. These findings may have implications in the general mechanisms of long-term synaptic modulation by neurotrophins.? Long-term synaptic modulation by neurotrophins requires protein synthesis, but the role of protein degradation has not been studies. In this study, we investigated whether ubiquitin-proteasome pathway is involved in the development of NMJ. We have identified a PDZ domain containing RING finger 3 (PDZRN3) as a synapse-associated E3 ubiquitin ligase and have demonstrated that it regulates the surface expression of muscle-specific receptor tyrosine kinase (MuSK), the key organizer of postsynaptic development at the NMJ. PDZRN3 binds to MuSK and promotes its ubiquitination. Regulation of cell surface levels of MuSK by PDZRN3 requires the ubiquitin ligase domain and is mediated by accelerated endocytosis. Gain- and loss-of-function studies in cultured myotubes show that regulation of MuSK by PDZRN3 plays an important role in MuSK-mediated ACh receptor clustering. Furthermore, overexpression of PDZRN3 in skeletal muscle of transgenic mice perturbs the growth and maturation of the NMJ. These results identify a synapse-associated E3 ubiquitin ligase as an important regulator of synapse development.? ? 2) Neurotrophic regulation of adult neurogenesis, memory and mood disorders? While adult neurogenesis is thought to play a role in learning and memory as well as in depression, how newly generated neurons contribute to the cognitive process remains unknown. Fibroblast growth factor 2 (FGF-2) is known to stimulate the proliferation of neuronal progenitor cells (NPCs) in adult brain. Using conditional knockout mice that lack brain expression of FGFR1, a major receptor for FGF-2, we have investigated the role of adult neurogenesis in hippocampal synaptic plasticity and learning and memory. Bromodeoxyuridine labeling experiments demonstrate that FGFR1 is required for the proliferation of NPCs as well as generation of new neurons in the adult dentate gyrus (DG). Moreover, deficits in neurogenesis in Fgfr1 mutant mice are accompanied by a severe impairment of LTP at the medial perforant path (MPP)-granule neuron synapses in the hippocampal dentate. Finally, the Fgfr1 mutant mice exhibit significant deficits in memory consolidation but not spatial learning. Our study suggests a critical role of FGFR1 in adult neurogenesis in vivo, provides a potential link between proliferative neurogenesis and dentate LTP, and raises the possibility that adult neurogenesis might contribute to memory consolidation. The role of FGFR1 in depression remains to be further investigated.? The neurotrophin hypothesis of depression is based largely on correlations between stress or antidepressant treatment and down- or up-regulation, respectively, of BDNF. The current status of research on BDNF and depression is reviewed. Genetic disruption of the signaling pathways involving BDNF and its TrkB receptor does not seem to cause depressive behaviors, but does hamper the effect of antidepressant drugs. Thus, BDNF may be a target of antidepressants, but not the sole mediator of depression or anxiety. Advances in BDNF cell biology, including its transcription through multiple promoters, trafficking and secretion, may provide new insights into its role in mood disorders. Moreover, as the precursor proBDNF and the mature protein mBDNF can elicit opposite effects on cellular functions, the impact of proBDNF and its cleavage on mood should be considered. Opposing influences of mBDNF and proBDNF on LTP and LTD might contribute to the dichotomy of BDNF actions on behaviors mediated by the brain stress and reward systems.? ? 3) Studies of genes involved in schizophrenia? A new line of research in this lab is to study genes involved in cognition and schizophrenia. We have recently collaborated with Dr. Hong-Jun Songs group at Johns Hopkins University to investigate the role of Disrupted-In-Schizophrenia 1 (disc1), a susceptibility gene for schizophrenia. DISC1 expression is broad in many brain regions during embryonic development and fairly restricted in the adult brain with particularly high expression in dentate granule cells of the hippocampus and interneurons of the olfactory bulb, two neuronal types that are continuously generated through adult neurogenesis. A role of DISC1 in neuronal development was first suggested by biochemical identification of interacting proteins. For example, DISC1 binds Ndel1 (NUDEL), a molecule involved in embryonic neuronal development including migration. In vitro studies with PC12 cells and primary neurons showed that blocking DISC1 function impairs neurite outgrowth. Furthermore, in utero electroporation-mediated expression of DISC1 shRNAs in embryos leads to retarded migration. The finding that DISC1 promotes migration in the embryonic cortex and neurite outgrowth in vitro, as well as its restricted expression in neurons produced during adult neurogenesis, raise a tantalizing possibility that DISC1 may play an important role in regulating the process of adult neurogenesis.? To ascertain the in vivo function of DISC1 in adult neurogenesis, we employed an oncoretrovirus-mediated RNA interference approach to genetically manipulate DISC1 expression within individual cells in specific brain regions. Such in vivo single-cell genetic approach allows characterization of cell-autonomous roles of DISC1 specifically in adult neurogenesis, without the complication of potential developmental defects andor compensations in traditional germ-line knockout animals. We demonstrated that DISC1 regulates almost all essential steps of neuronal integration in adult neurogenesis. In contrast to what has been found in embryonic cortical development and cultured neuronal cells, DISC1 knockdown in newborn dentate granule cells of the adult hippocampus leads to soma hypertrophy, accelerated dendritic outgrowth with appearance of ectopic dendrites, mis-positioning from over-extended migration, enhanced intrinsic excitability and accelerated synapse formation of new neurons. These findings indicate that DISC1, a schizophrenia susceptibility gene, serves as a key regulator that controls the tempo of neuronal development and therefore keeps the progress of new neuron integration in the adult brain in check.
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