Molecular mechanisms of synaptic differentiation and selective synapse assembly

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an enhanced biologic stress–response mechanism, especially a hyperactive hypothalamic-pituitary-adrenal (HPA) axis and high levels of circulating cortisol. Although dysregulation of the HPA axis by chronic stress is indicative of major depression, the molecular mechanisms and functional changes in the brain underlying depression are largely unknown. Thus, we first developed chronically stressed mice by water immersion and resistance method and identified serum- and glucocorticoid-inducible kinase 1 (SGK1) by a comprehensive analysis of the variation in the gene expression levels in the brains of stressed mice compared to that in the brains of unstressed mice. Furthermore, it was confirmed that in the chronically stressed mice, SGK1 mRNA and protein were upregulated, and that SGK1 phosphorylation level was also higher in the oligodendrocytes at bundles of nerve fibers such as corpus callosum. It is well known that SGK1 receives upstream signal from phosphatidylinositol 3-kinase, but the downstream targets of SGK1 in the brain are yet unknown. Next, we investigated the factors that interact with SGK1 in the oligodendrocytes. Our study showed that N-myc downstreamregulated gene 1 (NDRG1) interacted with SGK1 and was phosphorylated by SGK1 in the oligodendrocytes. These results indicate that phosphorylation of NDRG1 possibly plays a key role in the upregulation of adhesion molecules and causing changes in the morphology of oligodendrocytes. Moreover, the chronic stress-induced dysregulation the oligodendrocytes is suggested to be closely associated with the development of major depression. doi:10.1016/j.neures.2010.07.390

S3-2-1-1 Post-translational palmitoylation and regulation of AMPA receptors and NMDA receptors Takashi Hayashi Dept Mol Neurobiol and Pharmacol, Grad Sch Med, Univ of Tokyo, Tokyo Modification of glutamate receptor function and trafficking contributes to the regulation of synaptic transmission and is important for several forms of synaptic plasticity. Like phosphorylation, post-translational palmitoylation is a labile and reversible modification that regulates localization of many proteins. We demonstrate that all AMPA receptor subunits (GluRalpha1-4) and NMDA receptor subunits GluRepsilon1/NR2A and GluRepsilon2/NR2B are palmitoylated in neuron. GluRalpha1-4 have two distinct palmitoylation sites in their transmembrane domain (TMD) 2 channel pore region and in their C-terminal domain. GluRepsilon1/NR2A and GluRepsilon2/NR2B have two distinct clusters of palmitoylation sites in their C-terminal region. Palmitoylation of the first cysteine cluster (Cys cluster I) controls stable expression and constitutive internalization of surface NMDA receptor. The second cysteine cluster (Cys cluster II) is palmitoylated by Golgi-localizing palmitoyl acyl transferase (PAT) GODZ and depalmitoylation of the site regulates surface delivery of NMDA receptors. Similar to GODZ-mediated palmitoylation of cluster II in the GluRepsilon/NR2 subunits, the palmitoylation of TMD 2 by GODZ retains AMPA receptors in the Golgi apparatus. Depalmitoylation of the TMD 2 site is presumably a trigger for the receptor surface delivery. In contrast, C-terminal depalmitoylation of the AMPA receptor GluRalpha1 subunit stabilizes the receptor surface expression through the interacting molecule such as protein 4.1N. While amino acid sequences around these palmitoylation sites are not conserved between GluRalpha14 and GluRepsilon/NR2, the trafficking of AMPA receptors and NMDA receptors both occur in a palmitoylation-dependent two-step manner. These data indicate that palmitoylation of GluRalpha1-4 and GluRepsilon/NR2 subunits play critical roles in the trafficking of both AMPA and NMDA receptors and may regulate glutamate receptors dependent synaptic plasticity. doi:10.1016/j.neures.2010.07.391

S3-2-1-2 Experience dependent synaptic delivery of AMPA receptors Takuya Takahashi Department of Physiology, Yokohama City University, Yokohama The molecular and cellular mechanisms underlying experience-dependent plasticity of brain function are poorly understood. Recent in vitro studies have identified the regulated trafficking of AMPA receptors (-Rs) into synapses as a major molecular component of neural plasticity. Here we ask if experiencedriven plasticity in the developing rat barrel cortex is accompanied by and/or requires AMPA-R delivery to synapses. By combining in vivo gene delivery with in vitro recordings, we show that experience drives recombinant GluR1, an AMPA-R subunit, into synapses formed between layer 4 and layer 2/3 neurons. These studies show that synaptic delivery of AMPA-Rs contributes to plasticity driven by natural stimuli in the mammalian brain. Here, how

environment regulates experience-dependent synaptic delivery of AMPA receptors will be presented. doi:10.1016/j.neures.2010.07.392

S3-2-1-3 Cbln1 and its receptor: A unique and essential bidirectional synaptic organizer complex Keiko Matsuda , Michisuke Yuzaki School of Medicine, Keio University Cbln1, which belongs to the C1q/tumor necrosis factor (TNF) superfamily, is a unique molecule that is not only required for maintaining normal parallel fiber (PF)-Purkinje cell synapses, but is also capable of rapidly inducing new PF synapses, in adult cerebellum. Although Cbln1 is secreted from granule cells, where and how Cbln1 binds in the cerebellum has remained largely unclear. Here, we identified a receptor for Cbln1, which specifically recognized a hexameric form of Cbln1 at the postsynaptic sites of PF-Purkinje cell synapses. Expression of this receptor in postsynaptic cells, combined with exogenously applied Cbln1, was necessary and sufficient to induce new synapses in vitro and in the adult cerebellum in vivo. Further, beads coated with recombinant Cbln1 directly induced presynaptic differentiation and indirectly caused clustering of postsynaptic molecules via this receptor. These results indicate that the Cbln1 and its receptor serve as a unique synapse organizer that acts bidirectionally on both pre- and postsynaptic components. Cbln1 are also expressed in various brain regions other than the cerebellum. Cbln1 were sufficient to induce presynaptic differentiation, not only at PF terminals but also at nerve terminals of various types of neurons, including cortical and hippocampal neurons. There results suggest that Cbln1 may serve as a general presynaptic organizer and that its target molecule is ubiquitously expressed. Furthermore, other Cbln subfamily members, Cbln2 and Cbln4, are expressed in various neurons in developing and mature brains. Therefore, we are currently examining whether these Cbln subfamily members share a common presynaptic target molecule and regulate presynaptic differentiation in CNS. doi:10.1016/j.neures.2010.07.393

S3-2-1-4 Wiring the functional brain Hisashi Umemori University of Michigan Neurons analyze and transmit information in the brain. Information is transferred from one neuron to another at functional contact sites called synapses. Precise assembly of synapses is critical for proper functioning of the brain; abnormal synapse formation is involved in various neurological and psychiatric disorders. Synapses are formed through the communication between the appropriate synaptic partners. There are two important steps in order for synapses to form properly: a neural activity-independent step and an activity-dependent step. In the first step, pre- and postsynaptic cells recognize and differentiate each other by exchanging molecular cues to form specific synaptic connections. In the second step, activity-dependent signals either stabilize or destabilize the synapse to establish appropriate synaptic connections. The goal of our research is to understand the precise mechanisms underlying these steps that organize functional neural circuits in the brain. In this symposium, I will show that specific target-derived molecules organize specific presynaptic terminals and help specify synaptic type in the mammalian brain. Furthermore, I will show the role of neural activity in the refinement of neural circuits in the hippocampus, the structure known to be critical for long-term memory formation. These studies contribute significantly to understanding of mechanisms underlying the establishment of specific and functional neural circuits in the brain and provide important clues for the etiology of neurological disorders associated with abnormal circuit formation. doi:10.1016/j.neures.2010.07.394

S3-2-1-5 Molecular mechanisms of synaptic differentiation and selective synapse assembly Peter Scheiffele Biozentrum of the University of Basel The assembly of functional neuronal circuits during development relies on an intricate interplay of cellular interactions, molecular recognition signals, and neuronal activity-dependent processes. The goal of our work is to understand the molecular mechanisms underlying the differentiation of synaptic junctions and the signaling systems that restrict synapse forma-

Abstracts / Neuroscience Research 68S (2010) e4–e52

tion and/or stability to the appropriate target cells in vivo. We screened for cell adhesion and signaling molecules that can either stabilize or destabilize synaptic junctions. In this effort, we identified Bone Morphogentic Proteins as novel inhibitory regulators of synapse formation in the mouse cerebellum. A second focus of our studies has been on the neuroligin–neurexin protein complex, a heterophilic adhesion system at central synapses with synaptogenic activities. Neuroligins and neurexins are encoded by multiple genes and substantial molecular diversity is generated at the level of alternative splicing. We have characterized isoform-specific functions of individual neuroligin and neurexin isoforms. Moreover, we have uncovered a signal transduction pathway which dynamically regulates alternative splicing of the neurexin mRNAs in response to neuronal activity. Copy number variations and mutations in the human neuroligin and neurexin genes have been identified in patients with autism-spectrum disorders. Therefore, our insights into the basic molecular mechanisms of neuroligin and neurexin functional regulation may be helpful with respect to understanding the neuronal abnormalities underlying these disorders. doi:10.1016/j.neures.2010.07.395

S3-2-2-1 Newly identified neuronal growth-associated proteins (nGAPs) using proteomics Michihiro Igarashi Div Mol Cell Biol, Grad Sch Med Dent Sci, Niigata Univ The neuronal growth cone is definitely important structure for the neural wiring, synapse formation and axonal regeneration. Continuous rearrangement of cytoskeletons and recruitment of transport vesicles are essential to the growth cone motility, however, it is unclear how the proteins are directly involved in processes of axonal growth. We successfully used a proteomic approach to identify 945 proteins present in developing rat forebrain growth cones, including highly abundant, membrane-or actin-associated proteins. Almost one hundred of the proteins appear to be highly enriched in the growth cone, and for 18 proteins, the results of RNAi suggest a role in axon growth. Few of the proteins we identified have previously been implicated in axon growth and thus, their identification presents a big step forward, providing new marker proteins and candidates (neuronal growth-associated proteins; nGAPs) for control of the many important functions of growth cones1). We will report and discuss about roles of identified nGAPs for the growth cone function and morphology. Reference Nozumi et al., 2009. PNAS 106, 17211–17216. doi:10.1016/j.neures.2010.07.396

S3-2-2-2 Identification of spatially regulated phosphoprotein networks controlling neuritogenesis Richard L. Klemke 1,2 , Yingchun Wang 1,2 , Jon M. Jacobs 3 , Feng Yang 3 , Wei Wang 1,2 , David G. Camp II 3 , Richard D. Smith 3 1

Department of Pathology, University of California, San Diego, La Jolla USA Moores Cancer Center, University of California, San Diego, La Jolla USA 3 Biological Sciences Division, Pacific Northwest National Laboratory, Richland, USA 2

Neurite formation and navigation are highly polarized processes modulated by extracellular gradients of cytokines, chemokines, and matrix proteins. These directional cues precisely regulate neurite growth and retraction dynamics through poorly understood G-protein and phosphoprotein signaling networks. Here we use IMAC-mass spectrometry combined with microporous filter technology, which facilitates the large-scale fractionation of extending or retracting neurites from the cell bodies of polarized cells, to spatially map the phosphoprotein, kinase, and G-protein signaling networks that mediate these processes. Greater than 4000 phosphorylation sites on more than 3000 phosphoproteins have been annotated and assigned to signaling pathways that control cytoskeletal remodeling, integrin signaling, kinase/phosphatase activation, and dendrite/axon specification. Interestingly, bioinformatic analyses and functional studies revealed a RasErk activation/deactivation mechanism as a critical phosphoprotein switch controlling neurite growth and retraction kinetics. Our findings provide novel insight into how phosphoprotein signaling networks are spatially organized to regulate neuritogenesis, and reveal a spatially regulated RAS-ERK signaling switch that controls neurite formation and retraction. doi:10.1016/j.neures.2010.07.397

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S3-2-2-3 A proteomic approach for comprehensively screening substrates of protein kinases Mutsuki Amano 1 , Kozo Kaibuchi 1,2 1 Department of Cell Pharmacology, Nagoya University, Graduate School of Medicine 2 JST, CREST

Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored. We combined shotgun liquid chromatography tandem–mass spectrometry (LC–MS/MS) analysis and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of protein kinases including Rho-kinase/ROCK/ROK, we obtained numerous interacting proteins from the rat brain lysate, which included the proteins previously reported as their substrates. Several novel interacting proteins were phosphorylated by these kinases. This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity. doi:10.1016/j.neures.2010.07.398

S3-2-2-4 Proteomic analyses of TDP-43 proteinopathy Masato Hasegawa 1 , Tetsuaki Arai 2 , Takashi Nonaka 1 , Fuyuki Kametani 1 , Mari Yoshida 3 , Kenji Ikeda 4 , Haruhiko Akiyama 5 1

Molecular Neurobiology, Tokyo Institute of Psychiatry, Tokyo 2 Tsukuba University, Tsukuba 3 Aichi Medical University, Aichi 4 Jikei Hospital, Okayama 5 Psycogeriatrics, Tokyo Institute of Psychiatry, Tokyo

Ubiquitin-positive tau-negative cytoplasmic inclusions are common pathological features in frontotemporallobar degeneration with ubiquitin-positive inclusions (FTLD-U) and in amyotrophic lateral sclerosis (ALS). Using proteomic and immunohistochemical analyses, we have identified a TAR DNA-binding protein of 43 kDa (TDP-43), a nuclear factor that functions in regulating transcription and alternative splicing, as a component of these structures. Biochemical analyses suggested that abnormal phosphorylation takes place in accumulated TDP-43. To identify the phosphorylation sites, we raised antibodies to phosphopeptides representing 36 out of 64 candidate phosphorylation sites of human TDP-43. The antibodies to pS379, pS403/404, pS409, pS410 and pS409/410 labeled the inclusions but not the nuclei. Immunoblot analyses demonstrated that the antibodies recognized TDP-43 at ∼45 kDa, smearing substances and the ∼25 kDa fragment, all of which were present in the brains of FTLD-U and ALS but not controls. The results clearly indicate that abnormally phosphorylated full-length TDP-43 and the C-terminal fragments are the major component of the inclusions. Expression of TDP-43 C-terminal fragments as GFP fusions in SH-SY5Y cells resulted in the formation of abnormally phosphorylated and ubiquitinated inclusions similar to those in FTLD-U and ALS. When DsRed-full-length TDP43 was co-expressed with the C-terminal fragment, cytoplasmic aggregates positive for both GFP and DsRed are formed, suggesting that full-length TDP-43 is recruited to cytoplasmic aggregates of TDP-43 C-terminal fragments. Furthermore, we identified the cleavage sites of TDP-43 C-terminal fragments deposited in FTLD-U brains. These findings together with recent discovery of mutations in the TDP-43 gene in ALS strongly suggest that TDP43 is the key molecule responsible for neurodegeneration in FTLD-U and ALS. doi:10.1016/j.neures.2010.07.399

S3-2-2-5 Comprehensive elucidation of enzyme-substrate relationship by proteomics: Say good-bye to Western blotting Keiichi Nakayama Depart Mol Cell Biol, Med Inst Bioreg, Kyushu Univ A bottleneck of the modern biology is a lack of universal method to identify substrates from enzymes. It is further difficult to find substrates of ubiquitin ligases, given that substrates are unstable when recognized by the enzymes. Here we show development of new technology designated DiPIUS (differential proteomics-based identification of ubiquitylation substrates) to discover substrates for Skp1-Cul1-F-box protein (SCF) complex. We applied DiPIUS to Skp2 and Fbw7, two of the most well-characterized F-box proteins, and identified candidates of their substrates including p27 and c-Myc, respectively. Among the candidates, three transcription factors were identified as targets of SCF/Fbw7 and subsequently subjected to validation studies.