The long-term goal of our research is to understand the cellular and molecular mechanisms underlying plastic changes of the brain during development and memory formation. We use three complimentary systems in our research: the mammalian nervous system preparations, the Drosophila neuromuscular junction (NMJ), and the neuron/non-neuronal cell hemi-synapse system. The mammalian nervous system preparation is used to study the molecular mechanisms of known processes involved in synaptic plasticity (i.e. dendritic translational regulation in homeostatic synaptic plasticity). The Drosophila NMJ has robust synaptic plasticity and is an ideal genetic screen system to identify novel molecular players mediating synaptic plasticity. Finally, our hemi-synapse system allows us to directly test what we’ve learned in the above two systems through synapse (and plasticity) reconstitution.
I. Molecular Mechanisms of the Postsynaptic Regulation of Translation
Local protein synthesis in dendrites has emerged as a key mechanism that contributes to long-lasting synaptic modifications. However, the molecular mechanisms by which altered synaptic activities regulate dendritic translation of specific subsets of mRNAs remain largely unknown.
Our recent work provided the first evidence that all-trans retinoic acid (RA), a well-known morphogen during development, mediates activity blockade-induced synaptic scaling by activating dendritic protein translation. This novel form of RA signaling acts through a translation-dependent but transcription-independent mechanism, leading to the up-regulation of glutamate receptors in the dendrite. Specifically, we found that RA synthesis is activated by reduced global neural network activity, which has been shown to induce robust synaptic scaling. Activation of dendritically localized retinoic acid receptor RARalpha by RA leads to accumulation of RARalpha into dendritic RNA granules enriched with the Fragile-X mental retardation protein (FMRP), ribosomal proteins and other translation factors. The accumulation of RARalpha into RNA granules proceeds the activation of protein translation, indicated by the recruitment of additional translation factors such as eEIF4E, phosphorylation of ribosomal protein S6, and an increase in GluR1 protein level in the RNA granule. Newly synthesized homomeric (GluR1-containing) glutamate receptors are inserted into synapses, leading to up-regulation of synaptic transmission.
The unexpected role of retinoic acid and its receptor RARalpha in translational regulation is, to our knowledge, the first ligand-gated mechanism identified for translational regulation. We plan to investigate the molecular underpinnings of RA-mediated translational regulation in studies that range from an analysis of the atomic basis of the interaction of RARalpha with target mRNAs to a definition of the mRNA population that is targeted by RARalpha. Moreover, we plan to study the mechanisms that regulate RARalpha nuclear export and that control its effect on mRNA translation. Finally, we will investigate the functional interactions between RA signaling and other known translational regulatory pathways, such as FMRP-mediated translational regulation.
In a second set of projects, we will exploit the fact that our findings represent the first known mechanism for translational activation during homeostatic synaptic plasticity. This observation will potentially allow us to manipulate homeostatic synaptic plasticity in vivo, and make it possible for us to examine the functional significance of dendritic translation-dependent homeostatic plasticity for systems functions. Specifically, we are interested in the potential interplay between homeostatic plasticity and Hebbian-type synaptic plasticity, and its role in the development and plasticity of sensory cortical maps. Using various genetically manipulated mice models related to the RA signaling pathway, we will investigate whether and how homeostatic plasticity, acting as a form of metaplasticity, modulates neurons’ capacity for undergoing Hebbian-type plasticity in both brain slices and in vivo.
II. Structural and Functional Analysis of the Synapse
In addition to modifying existing synapses, long-term synaptic changes can also be mediated by regulating synaptogenesis. To delineate the molecular players underlying glutamatergic synapse formation, we are studying the functions of synaptic cell-adhesion molecules.
Project 1: Molecular mechanisms of shaft glutamatergic synapse formation
Whereas the majority of excitatory synapses is formed on dendritic spines, a subpopulation of is formed on dendritic shafts. Although relatively rare, shaft synapses may have a greater influence on dendritic signal integration than spine synapses, as suggested by some well-characterized pairs of spine vs. shaft synapses, such as parallel fiber vs. climbing fiber synapses in the cerebellum. The percentage of glutamatergic contacts on dendritic shafts is high in young animals, but drops during development, and most glutamatergic synaptic contacts are found on spines in the adult brain. As a result, a central dogma of synapse formation is that most shaft synapses represent an intermediate stage, and eventually become spine synapses. This view, however, is challenged by several observations. First, well-characterized specific shaft synapses exist, as exemplified by the climbing fiber synapses mentioned above. Second, the percentage of shaft synapses in adult brain can vary from less than 10% in the hippocampus to nearly 30% in layer I of the somatosensory cortex. Third, in the adult neocortex spine growth precedes synaptic contact. Fourth, the shaft-to-spine synapse formation hypothesis predicts that the curvature of axons should change due to spines emerging from existing shaft synapses. Such changes have never been observed. Fifth, increasing evidence indicates that spine and shaft glutamatergic synapses can be differentially regulated by activity, plasticity, and behavioral learning. Thus far, research on shaft synapses has been limited to anatomical analysis, largely because the mechanisms for shaft synapse modulation were unknown. Identifying the molecular basis of shaft vs. spine synapse formation is a critical first step to further understanding shaft synapse-related synaptic functions.
We focused on the ephrins and Eph receptors, families of interacting cell-adhesion and signaling molecules that play important roles in CNS development and plasticity. Our work revealed an unexpected function of postsynaptic ephrinB3 in shaft glutamatergic synapse formation. We showed that reduction of ephrinB3 levels in postsynaptic neurons via siRNA knockdown reduces glutamatergic synapse density as well as synaptic transmission. These changes, quantified with both pre- and postsynaptic markers, occur solely via changes in shaft synapse numbers without detectable changes in spine synapse numbers. We also observed a reduced shaft glutamatergic synapse abundance in the CA1 region of the ephrinB3-null mouse at the ultrastructural level. Moreover, GRIP1, an ephrinB3-interacting PDZ protein, is required for ephrinB3-mediated shaft synapse formation. Our results identify a novel function of postsynaptic ephrinB3 in excitatory synapse formation, provide the first evidence that shaft and spine synapses are different entity molecularly, and reveal a molecular mechanism by which shaft synapse formation may occur independently from spine synapses.
Project 2: Dissecting synapses using neuron/non-neuronal cell co-cultures
Interactions between synaptic proteins form the structural basis of synaptic function. These interactions are sometimes difficult to pinpoint, primarily because of interference from other proteins in the synapse. To solve this problem, we have established a “hemi-synapse” system in which either the pre- or the post-synaptic compartment is replaced by non-neuronal cells that lack neuronal proteins, providing a “clean” physiological recipient. Using these methods, we are discovering the intra-synaptic signals that initiate synapse formation and the protein interactions mediating receptor localization to the synapse.
There are two steps in postsynaptic differentiation: clustering of scaffolding proteins at the postsynaptic site, and recruiting receptors and other regulatory proteins to the synaptic scaffold. The first step appears to be guided by presynaptic cues because it occurs only at dendritic sites contacting presynaptic terminals. To identify these cues, we have expressed candidate presynaptic proteins in non-neuronal cells and placed them in neuronal cultures, looking for proteins that can “fool” neurons into making postsynaptic contact with the transfected non-neuronal cells by forming a “hemi-synapse”. We have discovered that a presynaptic neuronal adhesion molecule, b-neurexin, induces clustering of postsynaptic scaffold proteins through its direct interaction with postsynaptic neuroligins. However, only certain types of glutamate receptors (i.e. NMDA receptors) can be recruited to the postsynaptic site by b-neurexin, rendering the newly formed synapse non-functional. Remarkably, by activating NMDA receptors with the neurotransmitter glutamate (mimicking presynaptic release) at these contact sites, another type of glutamate receptor, the AMPA-type receptor, is inserted into the induced synaptic sites, a process that converts these non-functional synapses into functional ones. Results from this study demonstrate that post-synaptic differentiation is a multi-step process. The interaction between pre- and post-synaptic adhesion molecules such as neurexin and neuroligin initiates synaptogenesis in two fundamental ways: 1) by inducing pre-synaptic transmitter release and 2) by clustering post-synaptic proteins such as scaffold proteins and NMDA receptors. Activation of NMDA receptors completes post-synaptic maturation by inserting AMPA receptors into the synapse.
III. Regulation of Synaptic Plasticity in Drosophila
In addition to studying the functions of molecules known to be involved in synaptic plasticity and synaptogenesis, we hope to further expand this molecular repertoire by identifying new players using Drosophila melanogaster as a genetic model organism. The overall goal of this work is to eventually combine the powerful physiological approaches available in mammalian systems with the amazing genetic tools available in Drosophila to identify evolutionarily conserved pathways that control and modify synaptic strength.
Poon, M. and Chen, L. (2008). Retinoic acid-gated sequence-dependent translational control by RARa. Proceedings of the National Academy of Sciences of the United States of America, (in press).
Maghsoodi, B., Poon, M., Nam, C.I., Aoto J., Ting, P. and Chen, L. Retinoic acid regulates RARalpha-mediated control of translation in dendritic RNA granules during homeostatic synaptic plasticity.Proceedings of the National Academy of Sciences of the United States of America, 105, 16015-20.
Aoto J., Maghsoodi, B., Nam, C.I., Poon, M., Ting, P. and Chen, L. Aoto, J., Nam, C.I., Poon, M., Ting, P. and Chen, L. (2008). Synaptic signaling by all-trans retinoic acid in homeostatic synaptic plasticity. Neuron, 60, 308-320.
Hastie, P. and Chen, L. (2007). Synaptic trapping of AMPA receptors. Cell Science Reviews, 4(1).
Aoto, J. and Chen, L. (2007). Bidirectional ephrin/Eph signaling in synaptic function. Brain Research Reviews, 1184, 72-80.
Aoto, J., Ting, P., Maghsoodi, B., Xu, N., Henkemeyer, M. and Chen, L. (2007). Postsynaptic ephrinB3 promotes shaft glutamatergic synapse formation. Journal of Neurosicence, 27(28), 7508-19.
Chen, L., Tracy, T., and Nam, C.I. (2007). Dynamics of postsynaptic glutamate receptor targeting. Current Opinion in Neurobiology,17(1), 53-58.
Chen, L., and Maghsoodi, B., (2006). Synaptic trafficking of AMPA receptors. [In Protein Trafficking in the Neuron (Ed. Bean, A. J.). Elsevier Academic Press, Oxford, UK].
Nam, C. I. and Chen, L. (2005). Postsynaptic Assembly Induced by Presynaptic Neurexin and Neurotransmitter. Proceedings of the National Academy of Sciences of the United States of America, 102, 6137-42.
Chen, L., El-Husseini, A., Tomita, A., Bredt, D. S., and Nicoll, R. A. (2003). Stargazin differentially controls the trafficking of AMPA and kainate receptors. Molecular Pharmacology, 64, 703-706.
Tomita, S., Chen, L., Kawasaki, Y., Petralia, R. S., Wenthold, R. J., Nicoll, R. A., and Bredt, D. S. (2003). Functional studies and distribution define a family of transmembrane AMPA receptor regulatory proteins. Journal of Cell Biology, 161, 805-816.
Chen, L., Chetkovich, D. M., Petralia, R. S., Sweeney, N., Kawaski, Y., Wenthold R. J., Bredt, D. S., and Nicoll, R. A. (2000). Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms. Nature, 408, 936-943.
Last Updated 2008-11-18