title: Isobaric tagging-based quantification by mass spectrometry of differentially regulated proteins in synaptosomes of HIV/gp120 transgenic mice: implications for HIV-associated neurodegeneration.
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 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.
authors.affiliation: Del E. Webb Center for Neuroscience, Aging, and Stem Cell Research, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA.
authors.forename: Sugato
authors.initials: S
authors.lastname: Banerjee
authors.forename: Lujian
authors.initials: L
authors.lastname: Liao
authors.forename: Rossella
authors.initials: R
authors.lastname: Russo
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Nakamura
authors.forename: Scott R
authors.initials: SR
authors.lastname: McKercher
authors.forename: Shu-ichi
authors.initials: S
authors.lastname: Okamoto
authors.forename: Florian
authors.initials: F
authors.lastname: Haun
authors.forename: Rana
authors.initials: R
authors.lastname: Nikzad
authors.forename: Rameez
authors.initials: R
authors.lastname: Zaidi
authors.forename: Emily
authors.initials: E
authors.lastname: Holland
authors.forename: Alexey
authors.initials: A
authors.lastname: Eroshkin
authors.forename: John R
authors.initials: JR
authors.lastname: Yates
authors.forename: Stuart A
authors.initials: SA
authors.lastname: Lipton
mesh_terms: D000328:Adult; D000368:Aged; D000818:Animals; D005260:Female; D015699:HIV Envelope Protein gp120 / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000502:physiology; D015658:HIV Infections / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008875:Middle Aged; D009434:Neural Pathways / Q000737:chemistry / Q000473:pathology / Q000821:virology; D019636:Neurodegenerative Diseases / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D018696:Neuroprotective Agents / Q000037:antagonists & inhibitors / Q000737:chemistry; D013574:Synaptosomes / Q000737:chemistry / Q000502:physiology* / Q000821:virology; D053719:Tandem Mass Spectrometry / Q000379:methods*; D054334:Viral Regulatory and Accessory Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*; D038301:Virus Inactivation
chemical_list: D015699:HIV Envelope Protein gp120; D018696:Neuroprotective Agents; D054334:Viral Regulatory and Accessory Proteins