About 34 million people are currently living with human immunodefiency virus (HIV) worldwide. The gastrointestinal (GI) tract is a major target and reservoir of the HIV virus containing a huge number of lymphocytes and having a higher level of CD4 T cell depletion during acute and chronic HIV infection compared to peripheral blood. The enteric nervous system regulates GI processes such as motility,and digestion. These processes are significantly affected in HIV infected patients. The HIV virus does not infect neurons. The majority of HIV effects are not achieved by lytic propagation of the virus but by viral proteins. HIV-1 Tat, a regulatory protein between 86 to 101 amino acids in length is one of such proteins. Tat is released by infected cells and is able to penetrate and modulate neuronal function. Additionally, Tat plays an important role in the escape from latency which is a hallmark of acquired immunodeficiency syndrome (AIDS) pathogenesis. Recent experimental and clinical studies suggest that the enteric nervous system is affected during HIV infection thereby contributing to HIV mediated GI neurogastroenteropathies. A bulk of the studies on the effects of Tat on neurons has been done on central nervous system (CNS) neurons. In the CNS, Tat has been shown to increase neuronal excitability but the mechanism by which this occurs is still not yet well understood. Based on our preliminary data, we hypothesize that Tat increases enteric neuronal excitability by modulating sodium channel properties. We will test the hypothesis that Tat increases neuronal excitability in both an in vitro system and an animal model (Doxycycline-inducible Tat transgenic mouse) using whole cell patch clamp studies in single isolated neurons from the adult mouse ileum. We will then determine the mechanism of enhanced enteric neuronal excitability by Tat by examining the effects of Tat on sodium channels which play an important role in neuronal excitability. We will build on our preliminary data which show that Tat (1) increases the sodium current density, (2) shifts the Boltzmann's activation curve to the left and (3) increases the transcription of Nav1.7 and Nav1.8 isoforms of the sodium channel. We will determine if this leftward shift in the activation curve is an acute, direct or allosteric effect of Tat. We will use voltage clamp experiments and a double pulse inactivation protocol on enteric neurons treated with Tat for up to 30min to determine if it is an acute effect. We will use co-immunoprecipitation to determine if it is a direct effect of Tat and a IP/Immunoblot technique to assess whether the effect is an indirect and/or post translational modification. We will then elucidate the mechanism by which Tat transcriptionally modulates the Nav1.7 and Nav1.8 isoforms, and whether this increase in Nav1.7 and Nav1.8 transcription is translated into proteins and if Tat is also modulating translation of these isoforms.
HIV AIDS is a global pandemic with more than 1.1 million cases in the USA and about 50-93 % of these patients have marked gastrointestinal disturbances during the course of their disease. Currently, the mechanism that underlies HIV mediated neurogastroenteropathies is not well understood. The proposed study will determine the mechanism by which HIV-1 Tat modulates enteric neuronal excitability thereby contributing to HIV mediated gastrointestinal disorders.
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