Mechanisms of HIV Tat neurotoxicity and CCL2 protection

[unreadable] DESCRIPTION (provided by applicant): HIV infection of the CNS can result in neurologic dysfunction that has devastating consequences in a significant number of individuals with AIDS, including motor impairment and cognitive deficits. NeuroAIDS is characterized by neuronal injury and loss, yet there is no evidence of HIV positive neurons. Therefore, the altered function and apoptosis seen in neurons must be due to indirect effects of HIV infection of other CNS cells, including the elaboration of neurotoxic viral proteins, such as tat. Others and we demonstrated that HIV tat is toxic to human neurons. The first goal of this proposal is to identify the signaling pathways that mediate tat-induced apoptosis. Our data suggest that the mechanisms that mediate tat induced apoptosis can be divided into early and late stages. Our hypothesis is that the early events leading to apoptosis involve LRP-mediated tat endocytosis and immediate activation of NMDA channels. These activities are not sufficient to induce cell death. These early events subsequently result in later activation of signaling pathways and additional NMDA channels, leading to the dysregulation of glutamate and NO production, resulting in neuronal apoptosis. Chemokines play an important role in CNS inflammation. While their function in chemotaxis is essential, we identified a novel role for one chemokine, CCL2 (MCP-1), demonstrating that it protects human mixed cultures of neurons and astrocytes from tat-induced apoptosis. Our second goal is to determine the mechanisms by which CCL2 mediates its protection. Our data indicate that CCL2 reduces tat internalization into neurons. While cotreatment of cultures with tat and CCL2 inhibits apoptosis, addition of CCL2 2h after tat treatment does not abrogate tat induced cell death. We propose that CCL2 acts on the early phase of tat-induced apoptosis by altering endocytosis of tat and the intracellular signaling pathways activated during tat internalization. We will: 1. Determine the mechanisms that mediate tat internalization into neurons and the immediate early consequences of this uptake, including changes in protein-protein interactions involved in tat internalization (LRP, PSD-95), and the activation of signaling molecules (PSD-95, Fyn, Pyk2) and effector proteins (NMDA channels, nNOS) that may be involved in regulation of tat induced neuronal apoptosis. 2.Determine the mechanisms by which CCL2 alters tat endocytosis and the signaling pathways responsible for tat-induced apoptosis. 3. Analyze the late pathways involved in tat-induced apoptosis, including expression and activity of NMDA channels, dysregulation of extracellular glutamate and production of NO. [unreadable] [unreadable]