Proteomics in neurotoxicology

National Institute Of Mental Health

Investigators

Linked publications, trials & patents

Abstract

We are applying proteomic methodology to unresolved problems in neuropathologic diseases. In the past year, progress has been made in studies on the postsynaptic density (published and in progress), on Huntingtons disease-related proteins (published), and on Parkinson disease-related proteins (published).[unreadable] [unreadable] Increasing evidence supports the hypothesis that bipolar disorder arises from abnormalities in cellular plasticity cascades, leading to aberrant information processing in synapses and circuits. In the context of this hypothesis, mood stabilizers such as lithium and valproic acid 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 a host of divergent signal transduction pathways. Collaborative studies with NINDS have focused on defining the composition of the post-synaptic density complex. The goal of a second PSD project is to understand the temporal and spatial dynamics of the PSD in the treatment of mood disorders; it is our contention that in addition to manipulating key candidate molecules these studies are critical to elucidate the mechanisms of synaptic regulation and of mood stabilizer action. Previous studies have demonstrated that multiple signaling molecules, including AMPA and NMDA glutamate receptor subunits, and downstream signaling molecules such as glycogen synthase kinase-3 (GSK-3), are regulated by mood stabilizers such as lithium and valproic acid. We are using proteomic techniques to identify and quantify proteins in PSDs subjected to mood stabilizer treatment. These experiments identify brain region-specific changes in PSD protein levels in response to treatment, focusing on brain areas that have been implicated in bipolar disorder, including prefrontal cortex, hippocampus, and striatum. The protein composition of PSD preparations from these areas will be determined by 2-D liquid chromatography and tandem mass spectrometry. [unreadable] [unreadable] Huntington's disease is a dominant autosomal neurodegenerative disorder caused by an expansion of polyglutamines in the huntingtin (Htt) protein, whose cellular function remains controversial. To gain insight into Htt function in collaborative studies with NYU investigators, purified epitope-tagged Htt was used to identify Argonautes among the associated proteins using mass spectrometry. Colocalization studies demonstrated Htt and Ago2 to be present in P bodies, and depletion of Htt showed compromised RNA-mediated gene silencing. Mouse striatal cells expressing mutant Htt showed fewer P bodies and reduced reporter gene silencing activity compared with wild-type counterparts. These data suggest that the previously reported transcriptional deregulation in HD may be attributed in part to mutant Htt's role in post-transcriptional processes. The comparative biochemical purifications uncovered previously undescribed interaction partners for Htt and confirmed several previously reported. The association of Htt with Ago2 and its localization to P bodies provides evidence connecting Htt with the post-transcriptional control of gene expression and P body integrity. It is thought that P bodies are composed of a collection of subcomplexes. Htt can now be added to the list of proteins that interact with Agos, which include the Dcp1-Dcp2 decapping complex, GW182, and Rck/p54. Recent studies have shown that P body formation is triggered as a consequence of gene silencing, but silencing can occur in the absence of microscopically visible P bodies. Although this data does not illuminate the precise molecular role Htt plays in gene silencing, it provides compelling evidence that Htt functions as an Ago accessory factor involved in RNAi-mediated mechanisms and/or other translation repression/mRNA decay pathways associated with P bodies. Interestingly, ataxin-2, another protein subject to polyQ expansion and implicated in pathogenesis of SCA2, was reported to localize to and affect the assembly of P bodies and stress granules through an interaction with a P body component DDX6. Thus, altered post-transcriptional regulation may be a common feature of polyQ expansion diseases. [unreadable] [unreadable] In collaborative studies with NHGRI, the mitochondrial electron transport complex-I proteomics were characterized in order to test the hypothesis that deficiency in this complex results in Parkinson Disease. Mutations and copy number variation in the SNCA gene encoding the neuronal protein alpha-synuclein have been linked to familial Parkinson disease. The C-terminus of alpha-synuclein can be phosphorylated at tyrosine 125 and serine 129, although only a small fraction of the protein is phosphorylated under normal conditions. Under pathological conditions, such as in Parkinson disease, alpha-synuclein is a major component of Lewy bodies, a pathological hallmark of Parkinson disease, and is mostly phosphorylated at S129. Controversy exists over the extent to which phosphorylation of alpha-synuclein and/or the visible protein aggregation in Lewy bodies are steps in disease pathogenesis, are protective, or are neutral markers for the disease process. Here we use the combination of peptide pull-down assays and mass spectrometry to identify and compare protein-protein interactions of phosphorylated and non-phosphorylated alpha-synuclein. We show that non-phosphorylated alpha-synuclein C-terminus pulls down protein complexes that are highly enriched for mitochondrial electron transport proteins while alpha-synuclein C-terminus phosphorylated on either S129 or Y125 does not. Instead, the set of proteins pulled down by phosphorylated alpha- synuclein is highly enriched in certain cytoskeletal proteins, in vesicular trafficking proteins, and in a small number of enzymes involved in protein serine phosphorylation. This targeted comparative proteomic approach for unbiased identification of protein-protein interactions suggests there are functional consequences when alpha-synuclein is phosphorylated.[unreadable] [unreadable] A new project has been initiated to identify, characterize and validate proteins associated with the receptor tyrosine-protein kinase erbB-4, a protein signaling complex implicated by genomic studies on schizophrenia. Developmental, anatomical, and pharmacological changes in neuregulin-erbB4 signaling will be studied in mice using proteomics in order to monitor changes in protein expression levels and phosphorylation conditions of the ErbB4 receptor and its binding partners to allow for a more thorough understanding of the neuregulin/ErbB signaling pathway, and establish its importance in neuronal functions.