Proteomics in neurotoxicology

National Institute Of Mental Health

Investigators

Linked publications & trials

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 (in press), on Huntingtons disease-related proteins (in review), on Parkinson disease-related proteins (in preparation), and on the analysis of proteins during brain developmental stages (in press).[unreadable] [unreadable] Collaborative studies with NINDS have focused on defining the composition of the post-synaptic density complex. Postsynaptic density (PSD)-95, a specialized scaffold protein with multiple protein interaction domains, forms the backbone of an extensive postsynaptic protein complex that organizes receptors and signal transduction molecules at the synaptic contact zone. Large, detergent-insoluble PSD-95-based postsynaptic complexes can be affinity-purified from conventional PSD fractions using magnetic beads coated with a PSD-95 antibody. In the present study purified PSD-95 complexes were analyzed by LC/MS/MS. A semiquantitative measure of the relative abundances of proteins in the purified PSD-95 complexes and the parent PSD fraction was estimated based on the cumulative ion current intensities of corresponding peptides. The affinity-purified preparation was largely depleted of presynaptic proteins, spectrin, intermediate filaments, and other contaminants prominent in the parent PSD fraction. We identified 525 of the proteins previously reported in parent PSD fractions, but only 288 of these were detected after affinity purification. There are 26 proteins that are major components in the PSD-95 complex based upon abundance ranking and affinity co-purification with PSD-95. This subset represents a minimal list of constituent proteins of the PSD-95 complex and includes,in addition to the specialized scaffolds and N-methyl-D-aspartate receptors, an abundance of AMPA receptors, small G-protein regulators, cell adhesion molecules, and hypothetical proteins. The identification of two Arf regulators, BRAG1 and BRAG2b, as co-purifying components of the complex implies pivotal functions in spine plasticity such as the reorganization of the actin cytoskeleton and insertion and retrieval of proteins to and from the plasma membrane. Another co-purifying protein, the hypothetical protein (Q8BZM2) with two sterile motif domains, may represent a novel structural core element of the PSD.[unreadable] [unreadable] Huntingtons disease is a dominant autosomal neurodegenerative disorder caused by an expansion of poly-glutamines in the huntingtin (Htt) protein, whose cellular function remains controversial. In collaborative studies with NYU, we are performing proteome analyses of several affinity purified protein extracts from HeLa cells expressing the amino terminus of the Huntingtons Disease protein with different numbers of glutamines. The objective of this project is to test the hypothesis that there is transcriptional dysregulation associated with Huntingtons Disease. To gain insight into Htt function, we purified epitope-tagged Htt and identified Argonaute proteins. Co-localization 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 Htts role in post-transcriptional processes.[unreadable] [unreadable] In collaborative studies with NHGRI, the mitochondrial electron transport complex-I proteomics are being characterized. The hypothesis that deficiency in this complex results in Parkinson Disease is being tested. Preliminary work suggested phosphorylation dependent differences in proteins interacting with the C-terminus of alpha-synuclein. Of particular interest is the interaction of only the non-phosphorylated alpha-synuclein form with mitochondrial electron transport chain proteins. This data suggests that the function and localization of alpha-synuclein might be regulated by kinases in the synapse and that phosphorylation may be an important factor in protein aggregation and Lewy body formation. Although overexpression of alpha-synuclein induces signs of neurodegeneration, alpha-synuclein knockout mice show little to no obvious phenotype. Therefore, our proposal to identify proteins interacting with the alpha-synuclein tail under a variety of conditions will be of importance to understanding the function and localization of alpha-synuclein, possible physiological pathways of regulation and insight into its role in Parkinson Disease.[unreadable] [unreadable] We have investigated whether data from matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be used to characterize brain developmental stages in animals. A bootstrap method for point-based detection of candidate biomarker peaks has been developed from pattern classifiers. Point-based detection methods are advantageous in comparison to peak-based methods. Peak determination and selection is problematic when spectral peaks are not baseline resolved or on a varying baseline. The benefit of point-based detection is that peaks can be globally determined from the characteristic features of the entire data set (i.e., subsets of candidate points) as opposed to the traditional method of selecting peaks from individual spectra and then combining the peak list into a data set. The point-based method is demonstrated to be more effective and efficient using a synthetic data set when compared to using Mahalanobis distance for feature selection. In addition, probabilities that characterize the uniqueness of the peaks are determined. This method was applied for detecting peaks that characterize age-specific patterns of protein expression of developing and adult mouse cerebella from MALDI-MS data. A fuzzy rule-building expert system (FuRES) was applied to investigate the correlation of age with features in the MS data. FuRES detected two outlier pup-14 spectra. Prediction was evaluated using 100 bootstrap samples of 2 Latin-partitions (i.e., 50:50 split between training and prediction set) of the mice. The FuRES predictions were consistent with those obtained by discriminant partial least squares.