Abstract
Human immunodeficiency virus-associated neurological disorders (HANDs) affect the majority of AIDS patients and are a significant problem among HIV-1-infected individuals who live longer because of combined anti-retroviral therapies. HIV-1 utilizes a number of viral proteins and subsequent cytokine inductions to unleash its toxicity on neurons. Among HIV-1 viral proteins, Nef is a small protein expressed abundantly in astrocytes of HIV-1-infected brains and has been suggested to have a role in the pathogenesis of HAND. In order to explore its effect in the central nervous system, HIV-1 Nef was expressed in primary human fetal astrocytes (PHFAs) using an adenovirus. Our results revealed that HIV-1 Nef is released in extracellular vesicles (EVs) derived from PHFA cells expressing the protein. Interestingly, HIV-1 Nef release in EVs was enriched significantly when the cells were treated with autophagy activators perifosine, tomaxifen, MG-132, and autophagy inhibitors LY294002 and wortmannin suggesting a novel role of autophagy signaling in HIV-1 Nef release from astrocytes. Next, Nef-carrying EVs were purified from astrocyte cultures and neurotoxic effects on neurons were analyzed. We observed that HIV-1 Nef-containing EVs were readily taken up by neurons as demonstrated by immunocytochemistry and immunoblotting. Furthermore, treatment of neurons with Nef-carrying EVs induced oxidative stress as evidenced by a decrease in glutathione levels. To further investigate its neurotoxic effects, we expressed HIV-1 Nef in primary neurons by adenoviral transduction. Intracellular expression of HIV-1 Nef caused axonal and neurite degeneration of neurons. Furthermore, expression of HIV-1 Nef decreased the levels of phospho-tau while enhancing total tau in primary neurons. In addition, treatment of primary neurons with Nef-carrying EVs suppressed functional neuronal action potential assessed by multielectrode array studies. Collectively, these data suggested that HIV-1 Nef can be a formidable contributor to neurotoxicity along with other factors, which leads to HAND in HIV-1-infected AIDS patients.
Highlights
In order to investigate whether Human immunodeficiency virus-1 (HIV-1) Nef is released in extracellular vesicles (EVs) from astrocytes, primary human fetal astrocytes (PHFAs) cells were transduced with an adenovirus construct expressing Nef as described in Materials and Methods section
EVs derived from these cells contained HIV-1 Nef protein indicating that Nef protein is associated with these EVs
We have elucidated some of the neurotoxic effects of HIV-1 Nef on human neurons using a two-pronged approach: first to deliver the Nef protein to neurons by means of astrocytic exosomes and second to transduce the neuronal cells with AdNef to express Nef protein intracellularly
Summary
Human immunodeficiency virus-1 (HIV-1), the etiological agent of AIDS, wreaks havoc on the immune system,[1,2,3] and inflicts the central nervous system (CNS)[4] leading to HIV-associated neurological disorders (HANDs).[5,6,7,8,9,10] Before combined anti-retroviral therapies (cARTs), HIV-1 infections were responsible for neuroinflammation leading to encephalitis (HIVE) as evidenced by astrocytosis, neuronal loss, activated microglia and infiltration of macrophages in to infected brains.[7,11] The introduction of cART reduced the neuronal damage inflicted by HIV infection.[12]. MVBs are in turn released as EVs.[26] HIV-1 Nef appears to be responsible for many events leading to neurological impairments in the HIV-1-infected brain such as neuronal degeneration by inducing IP-10 release,[28] cytokine production and negatively affecting cellular pathways.[29,30] Neurotoxic effects of HIV-1 Nef were shown using recombinant Nef on human glial cells and neurons.[31] animal studies revealed that HIV-1 Nef-induced neurocognitive impairments in rats.[32,33] The effect of HIV-1 infection on the brain depends on the subtype of virus. We elucidate the neurotoxic effects of HIV-1 Nef utilizing both EVs and by directly expressing this viral protein in primary human neurons
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