Methamphetamine (METH) is the most commonly synthesized and heavily abused illicit drug in the United States and around the world. Significant populations of METH abusers are also infected with the human immunodeficiency virus (HIV)-1 or of high risk of developing HIV-1 infection. As a highly addictive phychostimulant, METH can cause cognitive damage by exerting its actions on neurons, and enhance the damage of HIV on the CNS by compromising the integrity of the blood-brain barrier (BBB). The BBB consists of brain microvascular endothelial cells (BMVECs), astrocytes, and pericytes. BMVECs are specialized endothelial cells that line the capillaries within the brain and control the flow of substances between the blood circulation and the brain parenchyma. METH is well known to exert detrimental effects on the BMVECs, and to compromise the BBB. HIV-1 infection can accelerate endothelial cell death through elevated tumor necrosis factor (TNF)-?-induced apoptotic pathway. Phosphoprotein enriched in astrocytes, 15 KDa (PEA-15) plays a neural protective role by inhibiting TNF?-induced cell death, and its functions are tightly regulated by the phosphorylation states on the irregularly structured C-terminal tail. It has been reported that substance abuse, including METH, can greatly alter the phosphorylation states of PEA-15 while the overall PEA-15 expression level is relatively constant. Recently, we first proposed a phosphorylation homeostasis model, in which various phosphorylation states of PEA-15 must be balanced in different cell types and tissues, and any disruption of this delicate balance could be harmful to the cells and tissues. Despite the importance of this phosphorylation homeostasis in the neuroprotection, little is known regarding the underlying mechanism and regulation of PEA-15 in preventing apoptosis and how METH and HIV-1 can affect PEA-15's protective effect by altering its phosphorylation homeostasis. Particularly the structures and regulations of PEA-15 in the formation of death-inducing signaling complex (DISC) initiated by TNF? are yet to be elucidated. Based on the recent literature and our preliminary studies, we hypothesize that the conformation of PEA-15 DED, and consequently its binding specificity, is allosterically modulated by the status of the C-terminal tail, and METH and HIV-1 target the allostery of PEA-15 to promote apoptosis at the endothelium. To test our hypothesis, we propose the following two specific aims: (1) To elucidate the mechanistic connection between METH and HIV-induced endothelial cell death and phosphorylation homeostasis of PEA-15.; and (2) To delineate the relevant conformations and allosteric control of PEA-15 within cellular contents and the mechanism of PEA-15 in alleviation of the detrimental effects of METH and HIV-1 Tat on primary BMVECs using novel in-cell NMR techniques. In this Cutting-Edge Basic Research Awards (CEBRA) application, we will substantiate the novel concept of phosphorylation homeostasis of PEA-15 and its important prohibition of apoptosis and crucial protection of the BBB in the context of METH abuse and HIV infection. We will examine the allosteric control of DISC formation and caspase-8 activation by phosphorylation of PEA- 15 using novel in-cell NMR techniques. This study is innovative because, to our knowledge, this will be the first study of potential antiapoptotic effects of PEA-15 in the context of METH/HIV-1 induced endothelial cell death and BBB disruption. In addition, there is no structural information is currently available for DISC with and without PEA-15 at atomic level, and it is unclear how cellular environment and extracellular stimuli could affect the intersection of death and survival, which PEA-15 plays a critical role in the regulation. Our study is highly significant and clinically relevant because it will provide valuable information on the mechanisms underlying the detrimental effects on BBB of METH abuse in the context of HIV-1 infection, which can help to develop therapeutic strategies to protect the BBB integrity from METH-induced dysregulation of immune responses with HIV-1 infection.
Methamphetamine (METH) is a widely abused illicit drug with strong effects on behavior and physiological functions, including inflammation and other immune responses, particularly among HIV-1 infected individuals. Both METH and HIV-1 virus are detrimental to the protective blood-brain barrier (BBB), but the means by which METH and HIV-1 virus damage the BBB is poorly understood. As a result, there are no effective means to treat or prevent the epidemics of METH abuse and HIV-1 infection. In this application, we propose to study the protective functions of PEA-15 in preserving the integrity of the BBB and alleviating the deleterious effects of MET at the BBB in the presence of HIV-1 viral proteins. We will also examine the regulatory mechanism of PEA-15 in preventing METH and HIV-1 induced endothelial cell death at the BBB, which will inform new drug development. This research is expected to shed light on the underlying mechanisms through which METH affects the barrier's functions that allow HIV-1 to penetrate into the central nervous system, and provide information that could be used to develop therapeutic strategies to prevent or reverse the adverse effects of METH abuse, particularly among HIV- 1 infected individuals.