Servio H. Ramirez, Ph.D - US grants

Overexpression of glycogen synthase kinase 3-beta (GSK-3b) can lead to neuronal apoptosis, and work in the sponsor's lab has demonstrated that GSK-3b activity is upregulated in neurons, following exposure to the candidate HIV-1 neurotoxins Tat and PAF. Furthermore, lithium and valproate treatment both resulted in protection from Tat- and PAF- mediated neurotoxicity. Since lithium and sodium valproate are selective inhibitors of GSK-3b, this suggests that GSK-3b activation may play a role in Tat- and PAF- mediated neurotoxicity. However, lithium and valproate have pleiotropic effects on neurons. Thus, additional experiments are needed to test whether GSK-3b activation may contribute to the neurotoxicity of Tat and PAR Three specific aims are proposed. First, studies will be conducted to test whether other inhibitors of GSK-3b can also protect neurons from PAF- and Tat- induced toxicity. Second, an endogenous inhibitor of GSK-3b (Frat) will be overexpressed in neurons, and cell survival in the presence and absence of PAF and Tat will be quantitated. Third, new inhibitors of GSK-3b will be generated using phage display technology, and the effects of these informational molecules on PAF- and Tat- mediated neurotoxicity will be examined.

DESCRIPTION (provided by applicant): Current treatment of HIV-infected patients with combined antiretroviral therapy (cART) has provided effective suppression of virus replication, prevented immunosuppression and increased lifespan. Despite these advances, even in the presence of cART, chronic neuroinflammation cannot be fully prevented, leading to development of HIV-associated neurocognitive disorders (HAND). The above clinical observation has prompted the necessity for quantitative biomarkers to help in detection and monitoring of central nervous system (CNS) complications resulting from HIV infection. Dysfunction of the blood-brain barrier (BBB) is a well-known phenomenon in HIV-1 neuropathogenesis potentially leading to neurodegeneration. We propose novel biomarkers reflecting BBB injury, namely detection of extracelluar microvesicles (eMVs) containing tight junction proteins (TJP) released from brain endothelium. We discovered that eMV release from the brain endothelium occurs as a result of inflammation and vascular remodeling. Importantly, the same pro- inflammatory insult on brain endothelial cells (EC) induces barrier permeability and biochemical changes to tight junction complexes. This project is unique in both the development of innovative tools for biomarker discovery and identification of a novel pathophysiologic phenomenon occurring at the BBB as consequence of chronic HIV-associated neuroinflammation. In the first aim, we will profile and chronologically define the presence of TJPs in eMV secreted as a consequence of relevant inflammatory stimuli and HIV virotoxins. These determinations will be made in parallel with measurement of barrier integrity, thus allowing for correlations between eMV shedding and BBB dysfunction. Because structural proteins identify the cellular origin of the eMV, the second aim explores the notion of eMV carrying TJP as the basis for a serological biomarker for BBB dysfunction. Using the most sensitive ELISA-based available technology, we have built prototype ELISAs to measure the level of blood circulating eMVs containing TJP. Using patient serum from HIV-1 infected individuals, we provide proof-of concept results that this novel biomarker correlates with diagnosis of HAND. Experiments outlined in the third aim dissect plausible molecular mechanisms involved in the biogenesis of brain EC microvesicle formation and packaging of TJP. It will offer, not only an understanding of how eMV formation is triggered, but also will reveal targets within the cell for pharmacological intervention that promote BBB protection.

The Cell and Immunology Core will to continue to offer assays to extend the work of laboratories at Temple University and of outside collaborators who want to add immune or histological/pathological end-points to their studies. Dr. Eisenstein will supervise the work to be carried out in Aims 1, 2, and 4. A major addition to the Core in this renewal is Dr. Yuri Persidsky. Trained as a pathologist, he will supervise new services offered by the Cell and Immunology Core as related to analysis of tissue as described in Aim 3. In addition, he will bring his expertise in HIV-1 infection of relevant human cells and the blood-brain barrier (BBB) to the Core, as well as projects using the HIV-1-infected humanized NOD.Cg-Prkdcscid ll2rgtm1Wjl /SzJ (NSG mice or huNSG) mouse model that recapitulates key features of infection in humans.

Treatment of HIV-infected patients with antiretroviral therapy (ART) has effectively suppressed viral replication; however, the central nervous system (CNS) is still a major target and reservoir of the virus leading to the development of HIV-1-associated neurocognitive disorders (HAND). Importantly, since the beginning of the HIV epidemic, drug use has remained a primary risk factor for contracting, transmitting, and worsening the outcomes of HIV. In fact, regarding pathogenesis, psychostimulants can induce higher viral loads, reduce CD4 counts, and increase rates of ART resistance. Furthermore, neuropsychological decline is greater in individuals that are cocaine users and HIV-seropositive. Extracellular microvesicles (EVs), which includes microvesicles (MVs) and exosomes, have emerged as a novel biological phenomenon, released by virtually every cell type in the body. We have shown that brain microvasculature endothelial cell (BMVEC)-derived EVs contain tight junction proteins (TJPs) and transporter proteins, which are main constituents of the BBB. Furthermore, a hallmark feature of HAND is the disruption of the BBB and loss of TJ complexes. In this proposal, we show that BMVECs shed EVs in response to HIV virotoxins and psychostimulants. Thus, we hypothesize that HIV infection and/or drugs of abuse triggers EV release leading to BBB instability and facilitation of neuroinvasion by infected immune cells. The innovative nature of this proposal is featured in three independent aims. In the first aim, we will characterize the degree of EV production (MVs and exosomes) as a function of psychostimulant type using a novel microfluidic model of the neurovascular unit with primary human cells. We will also investigate the effects of exposure to HIV virotoxins and ART pharmacologic agents on BMVEC-EV production. Furthermore, we will correlate EC-EV release to the phases of addiction in an in-vivo self-administration model as well as in a humanized mouse model of HIV infection with or without drug administration. In the second aim, we introduce the novel concept that BMVEC-derived EVs bind to activated or infected monocytes, which triggers increased monocytic transendothelial migration. Thus, we hypothesize that monocytes utilize TJPs on EVs to engage endothelial tight junction complexes and facilitate immune infiltration of the CNS. In the third aim, we will explore a therapeutic strategy that could minimize EV production and thus rescue BBB integrity. The above will be accomplished by targeted inhibition of ARF6, which is involved in MV and exosome biogenesis. Using new pharmacological tools to inhibit ARF6, we aim to stabilize the BBB to prevent BBB barrier dysfunction during neuroinflammation. The studies proposed herein will offer crucial insight to brain endothelial EV production, mechanisms of immune cell infiltration into the CNS, and also reveal targets for pharmacological interventions that promote BBB protection in HIV and substance use disorder.