HIV-associated neurocognitive disorder (HAND) is a neurological syndrome consisting of cognitive, motor and behavioral deficits. Neuronal loss in HAND follows the infiltration of the central nervous system by HIV-infected/activated monocytes-derived macrophages. These macrophages and subsequently activated resident microglia secrete a collection of soluble factors that alter the extracellular environment and determine neuronal viability. The neuronal response to this complex signaling environment involves activation of the cell cycle regulatory machinery, notably increased expression of the transcription factor E2F1, which is known to activate transcription of genes necessary for G1 to S phase progression of the cell cycle. This project proposal aims to determine the role of E2F1 in HIV-mediated neuronal loss with the long-term goal of producing a therapeutic strategy for HAND that involves blocking E2F1 pathological function. Preliminary data for this proposal shows that E2F1 is cleaved by calpain in primary neuronal cultures following an HIV insult. Furthermore, both inhibition of calpain activation and deletion of E2F1 attenuate HIV-induced neuronal death in primary neuronal cultures. Finally, treatment of primary neurons with HIV insult leads to reduced levels of pro-survival protein MDMx and accumulation of pro-death protein p73. These results suggest the hypothesis that cleaved neuronal E2F1 plays a contributing role in HIV-induced neuronal death, possibly by destabilizing the pro-survival protein MDMx and/or stabilizing the pro-death protein p73. To determine whether cleaved E2F1 contributes to HIV-induced neurotoxicity, the calpain cleavage site in E2F1 will be identified using an immunoprecipitation approach combined with protein sequencing and mass spectrometry analyses. Stability of cleaved E2F1 protein will be compared to stability of full length E2F1 by pulse-chase. A lentiviral approach will then be used to infect primary cortical cultures with a construct encoding the cleaved E2F1 sequence, allowing for subsequent analysis of the effects of cleaved E2F1 expression on neuronal viability by a MAP2 cell-based ELISA and hand counts of infected cells. To determine whether observe alterations in MDMs and p73 stability are due to cleaved E2F1, stability of each protein will be compared between wildtype and E2F1-/- cortical cultures treated with HIV insult using a pulse-chase approach. Sensitivity of MDMx-E2F1 binding to calpain activation will be assessed through a co-immunoprecipitation approach utilizing pharmacological inhibitors to calpain. Finally, the contribution of MDMx and p73 protein level alterations to HIV- induced neurotoxicity will be determined by assessing the effects of pharmacological inhibition of p73 and siRNA-mediated knockdown of MDMx on HIV-induced neuronal death. Together, the results from these experiments will define the role of calpain-cleaved E2F1 in HIV-induced neurotoxicity and indicate its potential as a therapeutic target in HAND.

Public Health Relevance

Patients with HIV-associated dementia exhibit increased expression of cell cycle-related protein E2F1. However, its pathologic role in neurodegeneration is unclear. We believe that defining its contribution to neuronal loss will lead to novel therapeutic strategy of targeting E2F1 to prevent the neuronal damage and subsequent cognitive decline characterized by HIV-associated dementia.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1-AARR-J (22))
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Wong, May
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University of Pennsylvania
Schools of Medicine
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Zyskind, Jacob W; Wang, Ying; Cho, Giyong et al. (2015) E2F1 in neurons is cleaved by calpain in an NMDA receptor-dependent manner in a model of HIV-induced neurotoxicity. J Neurochem 132:742-55
Colacurcio, Daniel J; Zyskind, Jacob W; Jordan-Sciutto, Kelly L et al. (2015) Caspase-dependent degradation of MDMx/MDM4 cell cycle regulatory protein in amyloid ?-induced neuronal damage. Neurosci Lett 609:182-8
Wang, Ying; Zyskind, Jacob W; Colacurcio, Daniel J et al. (2012) Differential roles for caspase-mediated and calpain-mediated cell death in 1- and 3-week-old rat cortical cultures. Neuroreport 23:1052-8