We are applying proteomic methodology to unresolved problems in neuropathologic diseases. Progress continues in studies on the structure of the postsynaptic density and its remodeling by drugs used in the treatment of mood disorders, on protein complexes implicated by genomic studies of schizophrenia, and synaptic protein changes accompanying long term depression (LTD). We are testing the hypothesis that bipolar disorder arises from abnormalities in cellular plasticity cascades, leading to aberrant information processing in synapses and circuits. The mood stabilizers lithium and valproate are thought to exert their therapeutic effects via actions on systems involved in synaptic plasticity. The postsynaptic density (PSD) is an elaborate cytoskeletal and signaling complex that provides anchors for synaptic proteins close to the region of presynaptic neurotransmitter release, and therefore mediates signaling in divergent signal transduction pathways. Collaborative studies with NINDS focused on defining the composition and stoichiometry of the post-synaptic density complex. We chose thirty two signature peptides observed consistently in LC/MS/MS analyses, representing the C-and N-terminal regions from 16 prominent proteins isolated with the PSD signaling complex. Work on this project is on-going. The goal of a second PSD project is to understand the temporal and spatial dynamics of the PSD in the treatment of mood disorders. Bipolar disorder is a devastating illness that is marked by recurrent episodes of mania and depression. There is growing evidence that the disease is correlated with disruptions in synaptic plasticity cascades involved in cognition and mood regulation. Alleviating the symptoms of bipolar disorder involves chronic treatment with mood stabilizers like lithium or valproate. These two structurally dissimilar drugs are known to alter prominent signaling cascades in the hippocampus, but their effects on the postsynaptic density complex remain undefined. In this work, we utilized mass spectrometry for quantitative profiling of the rat hippocampal postsynaptic proteome to investigate the effects of chronic mood stabilizer treatment. Our data shows that in response to chronic treatment of mood stabilizers there were not gross qualitative changes but rather subtle quantitative perturbations in PSD proteome linked to several key signaling pathways. Our data specifically support the changes in actin dynamics on valproate treatment. Using label free quantification methods, we report that lithium and valproate significantly altered the abundance of 21 and 43 proteins, respectively. Seven proteins were affected similarly by both lithium and valproate: Ank3, Grm3, Dyhc1, and four isoforms of the 14-3-3 family. Immunoblotting the same samples confirmed the changes in Ank3 and Grm3 abundance. Our findings support the hypotheses that BPD is a synaptic disorder and that mood stabilizers modulate the protein signaling complex in the hippocampal PSD. Calcium is a ubiquitous and fundamental signaling component that is utilized by cells to regulate a diverse range of critical cellular functions. The transient receptor potential (TRP) superfamily of ion channels are involved in calcium signaling mechanisms which regulate critical sensory functions in cells as well as other processes such as secretion, proliferation, neuronal guidance, cell death, and development. We have characterized the proteome of TRPCs and observed many of the same proteins in both the TRPC1 and TRPC3 proteome (approximately 50%). Moreover, when we analyzed the functional similarities among common proteins, a very asymmetrical distribution was evident. The functional comparison between the two reveals a greater overlap in certain functional groups, e.g. protein involved in endocytosis. Hybrid TRPC1/TRPC3 channels have been proposed to exist, and, consistent with this observation, TRPC3 co-immunoprecipitates with TRPC1-IP from rat brain extracts. In certain functional areas TRPC1 and TRPC3 may co-exist as a heterodimer and thus share the same protein partners. In contrast, the functional areas where little overlap in binding partners is observed could indicate independent roles for TRPC1 and TRPC3. Further studies will be needed to elucidate the complexes where TRPC1 and TRPC3 interact independently as opposed to complexes where both are present. The ErbB4 and Dystrobrevin binding protein 1 (dysbindin) protein signaling complexes have both been implicated by genomic studies on schizophrenia and have remained difficult to analyze in brain. We are continuing a project to identify, characterize and validate complexes associated with both proteins. ErbB4 is a receptor tyrosine-protein kinase, and we have used antibodies to covalently link it to magnetic beads. Comparative LC/MS/MS proteomic studies using on-bead digestion procedures developed for this project have failed to provide the enrichment factor required for ErbB4 recovery and mass spectrometric detection. However, new peptide targeting strategies are planned using labeled proteins from synthetic genes to allow better tracking of enrichment and recovery of this complex. Protein degradation/modification by proteases involves many physiological processes such as apoptosis. Caspase-3 is a cysteine protease best known as an executioner protease in apoptosis. Recent studies suggest that caspase-3 plays an important non-apoptotic function in synapses as well. In hippocampal neurons, caspase-3 activity is specifically required for NMDA receptor-dependent long-term depression (LTD), but not for long-term potentiation. More importantly, activation of casapse-3 in LTD promotes AMPA receptor endocytosis instead of cell death. To understand the molecular mechanism by which caspase-3 activation leads to LTD, we applied a published method developed to selectively capture emergent peptides derived from proteolytic cleavage upon NMDA receptor activation and apoptosis induction. Briefly, the newly formed free, unblocked protein N-termini produced from protease cleavage events are tagged with a biotinylated peptide using an engineered enzyme subtiligase. The labeled proteins are enriched by immobilized avidin and analyzed by mass spectrometry. The substrate degradomes of control (untreated neurons), NMDA treated, and staurosporine treated samples are compared. Currently, we are focusing on the substrates with putative caspase-cleavage sites and testing their functions in LTD.
