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Abstract: HIV/gp120 transgenic mice manifest neuropathological features similar to HIV-associated neurocognitive disorders (HAND) in humans, including astrogliosis, microglia activation, and decreased neuronal synapses. Here, proteomic screening of synaptosomes from HIV/gp120 transgenic mice was conducted to determine potential neuronal markers and drug targets associated with HAND. Synaptosomes from 13month-old wild-type (wt) and HIV/gp120 transgenic mouse cortex were subjected to tandem mass tag (TMT) labeling and subsequent analysis using an LTQ-Orbitrap mass spectrometer in pulsed-Q dissociation (PQD) mode for tandem mass spectrometry (MS/MS). A total of 1301 proteins were identified in both wt and HIV/gp120 transgenic mice. Three of the most differentially-regulated proteins were validated by immunoblotting. To elucidate putative pathways associated with the proteomic profile, 107 proteins manifesting a ≥1.5 fold change in expression were analyzed using a bioinformatics pathway analysis tool. This analysis revealed direct or indirect involvement of the phosphotidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway, a well-known neuronal survival pathway. Immunoblots confirmed a lower phospho (p)Akt/Akt ratio in synaptosomes from HIV/gp120 transgenic animals compared to wt, suggesting that this neuroprotective pathway was inactivated in the HIV/gp120 transgenic brain. Based on this information, we then compared immunoblots of pAkt/Akt in the forebrains of these mice as well as in human postmortem brain. We observed a significant decrease in the pAkt/Akt ratio in synaptosomes and forebrain of HIV/gp120 transgenic compared to wt mice, and a similar decrease in human forebrain from HAND patients compared to neurologically unimpaired HIV+ and HIV− controls. Moreover, mechanistic insight into an additional pathway for decreased Akt activity in HIV/gp120 mouse brains and human HAND brains was shown to occur via S-nitrosylation of Akt protein, a posttranslational modification known to inhibit Akt activity and contribute to neuronal cell injury and death. Thus, MS proteomic profiling in the HIV/gp120 transgenic mouse predicted dysregulation of the PI3K/Akt pathway observed in human brains with HAND, providing evidence that this mouse is a useful disease model and that the Akt pathway may provide multiple drug targets for the treatment of HIV-related dementias. [Copyright &y& Elsevier]

Mass spectrometry-based proteomics is increasingly used to address basic and clinical questions in biomedical research through studies of differential protein expression, protein-protein interactions, and posttranslational modifications. The complex structural and functional organization of the human brain warrants the application of high-throughput, systematic approaches to understand the functional alterations under normal physiological conditions and the perturbations of neurological diseases. This primer focuses on shotgun-proteomics-based tandem mass spectrometry for the identification of proteins in a complex mixture. It describes the basic concepts of protein differential expression analysis and posttranslational modification analysis and discusses several strategies to improve the coverage of the proteome. [Copyright &y& Elsevier]

Large-scale proteomic analysis of the mammalian brain has been successfully performed with mass spectrometry techniques, such as Multidimensional Protein Identification Technology (MudPIT), to identify hundreds to thousands of proteins. Strategies to efficiently quantify protein expression levels in the brain in a large-scale fashion, however, are lacking. Here, we demonstrate a novel quantification strategy for brain proteomics called SILAM (Stable Isotope Labeling in Mammals). We utilized a 15N metabolically labeled rat brain as an internal standard to perform quantitative MudPIT analysis on the synaptosomal fraction of the cerebellum during post-natal development. We quantified the protein expression level of 1138 proteins in four developmental time points, and 196 protein alterations were determined to be statistically significant. Over 50% of the developmental changes observed have been previously reported using other protein quantification techniques, and we also identified proteins as potential novel regulators of neurodevelopment. We report the first large-scale proteomic analysis of synaptic development in the cerebellum, and we demonstrate a useful quantitative strategy for studying animal models of neurological disease. [ABSTRACT FROM AUTHOR]