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Isobaric tagging-based quantification by mass spectrometry of differentially regulated proteins in synaptosomes of HIV/gp120 transgenic mice: implications for HIV-associated neurodegeneration

Reputable Mentor II
Reputable Mentor II
Banerjee S, Liao L, Russo R, Nakamura T, McKercher SR, Okamoto S, Haun F, Nikzad R, Zaidi R, Holland E, Eroshkin A, Yates JR 3rd, Lipton SA.
Exp Neurol. 2012 Aug;236(2):298-306.
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 13 month-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.
Del E. Webb Center for Neuroscience, Aging, and Stem Cell Research, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA.
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