Date of Award

8-1-2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Field of Study

Microbiology and Immunology

Department

Graduate School of Biomedical Sciences

First Advisor

Johnny J. He

Abstract

Human immunodeficiency virus invades the central nervous system (CNS) soon after the initial infection, often leading to neurological complications including cognitive and motor dysfunction, which have been collectively termed HIV/neuroAIDS. The introduction of combination antiretroviral therapy in the mid-1990’s led to reduced viral replication, improved immune function and increased life expectancy among HIV-infected individuals. As a result, the incidence of the most severe form of cognitive impairment due to HIV, so called HIV-associated dementia, reduced dramatically. However, the treatment regimen was not successful in protecting the patients from neuroAIDS as more discrete forms of CNS dysfunction, so-called minor cognitive motor disorders, have become more common.

HIV-1 Tat protein is an indispensable factor for successful transcription and replication of the viral genome. Aside from nucleus-bound functions, Tat is diffusely and unconventionally secreted outside of infected cells and contributes immensely to the pathology of neuroAIDS as a potent neurotoxin. The presence of Tat in the CNS despite the implementation of combination anti-retroviral therapy and the strong correlation of pathological hallmarks of neuroAIDS with continued Tat expression in CNS cells warrant a thorough understanding of the partially explained unconventional secretion mechanism(s) by Tat. Exosomal secretion of cargo has been established as an extremely efficient pathway of glia-neuron communications and astrocytes have been shown to utilize this delivery mechanism for the provision of neurotrophic factors and danger-associated molecular patterns to neurons.

My dissertation research consisted of two parts. In the first part, we investigated the possibility of exosomal association and distribution of Tat protein from astrocytes and its delivery to neurons. We demonstrated significant presence of HIV-1 Tat in exosomes derived from Tat-expressing primary astrocytes, astrocytoma cell lines, and HIV-infected T cells. We further showed that exosome-associated Tat from Tat-expressing astrocytes was capable of causing neurite shortening and neuron death, further supporting that this new form of extracellular Tat is biologically active. Lastly, we constructed a Tat mutant deleted of its basic domain and determined the role of the basic domain in Tat trafficking into exosomes. Basic domain-deleted Tat exhibited no apparent effects on Tat trafficking into exosomes, while maintained its dominant negative function in Tat-mediated LTR transactivation. Taken together, these results show a significant fraction of Tat is secreted and present in the form of exosomes and may contribute to the stability of extracellular Tat and broaden the spectrum of its target cells.

In the second part, we investigated the mechanism of neurite shortening by Tat. Dendritic pruning and synaptic loss of neurons are the most prominent pathological hallmarks of neuroAIDS in the cART era. Although Tat has been implicated in the synaptodendritic damage to neurons, the exact mechanisms of this injury by Tat have not yet been elucidated. Several important controllers of dendritic plasticity have been shown to be post-transcriptionally regulated by a brain-enriched microRNA, miR-132, which is abundantly expressed in the brains of the HIV-infected individuals with cognitive impairment. We found significant induction of miR-132 in both astrocytic and neuronal cell lines following Tat transfection. Tat expression in primary astrocytes from our doxycycline-inducible Tat transgenic mice (iTat) and HIV-infected primary human astrocytes also led to significant upregulation of this microRNA. We confirmed the repression of miR-132 target genes involved in the regulation of dendritic length following Tat expression. Using a basic-domain-deletion mutant of Tat we further demonstrated that Tat-induced miR-132 expression involved CREB phosphorylation. Lastly, we showed that following Tat expression in astrocytes, exosome-associated miR-132 was significantly increased and caused neurite shortening in primary mouse cortical neurons. Taken together, these results demonstrate for the first time the role of miR-132 in Tat-induced damage of the dendritic arbor.

Comments

Available August 2017.

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