HIV Nef and Antiretroviral Therapy Have an Inhibitory Effect on Autophagy in Human Astrocytes that May Contribute to HIV-Associated Neurocognitive Disorders

Abstract

A significant number of people living with HIV (PLWH) develop HIV-associated neurocognitive disorders (HAND) despite highly effective antiretroviral therapy (ART). Dysregulated macroautophagy (autophagy) is implicated in HAND pathogenesis. The viral protein Nef, expressed even with suppressive ART, and certain antiretrovirals affect autophagy in non-CNS cells. Astrocytes, vital for CNS microenvironment homeostasis and neuronal health, require autophagy for their own homeostasis. We hypothesized that extracellular Nef and/or ART impact astrocyte autophagy, thus contributing to HAND. We studied in-bulk and selective autophagic flux in primary human astrocytes treated with extracellular Nef and/or a combination of tenofovir+emtricitabine+raltegravir (ART) using Western blotting, a tandem fluorescent LC3 reporter, and transmission electron microscopy/morphometry. We show that after 24 h treatment, Nef and ART decrease autophagosomes through different mechanisms. While Nef accelerates autophagosome degradation without inducing autophagosome formation, ART inhibits autophagosome formation. Combination Nef+ART further depletes autophagosomes by inducing both abnormalities. Additionally, extracellular Nef and/or ART inhibit lysosomal degradation of p62, indicating Nef and/or ART affect in-bulk and selective autophagy differently. Dysregulation of both autophagic processes is maintained after 7 days of Nef and/or ART treatment. Persistent autophagy dysregulation due to chronic Nef and/or ART exposure may ultimately result in astrocyte and neuronal dysfunction, contributing to HAND.

Diurnal Variation of Plasma Extracellular Vesicle Is Disrupted in People Living with HIV

“…In particular, the HIV proteins TAT, NEF, and ENV are involved in this regulation by either blocking or stimulating autophagy through direct interaction with autophagy proteins and/or modulation of the mTOR pathway [ 59 , 60 , 61 , 62 ]. Likewise, ART drugs [ 63 , 64 , 65 ] and aging [ 66 , 67 ] have been associated with dysregulation of autophagy. These different factors associated with dysregulation of autophagy may contribute to autophagosome and multivesicular bodies fusion [ 68 ] and induce larger EV release.…”

Antiretroviral Drugs Impact Autophagy with Toxic Outcomes

“…Cells respond to mitochondrial or ER stress by inducing autophagy. However, as highlighted in these reviewed studies, autophagy upregulation can also lead to autophagy exhaustion [ 7 ], may be toxic by itself [ 17 , 18 , 27 ], or autophagy may fail to be upregulated or is inhibited [ 11 , 12 , 13 , 14 , 15 , 20 , 25 , 26 , 27 ], subsequently leading to decreased cell viability and death ( Table 1 ). Thus, these studies reinforce the major significance of autophagy dysregulation as a mechanism underlying drug toxicity.…”

“…Three comprehensive studies showed that three-drug regimens containing two NRTI plus an ISTI inhibit autophagy, and may contribute to HAND in PLWH [ 25 , 26 , 27 ]. A two-part study addressed the effects of tenofovir disoproxil fumarate (TDF), emtricitabine (FTC), and dolutegravir (DTG) on autophagy and inflammatory genes/proteins in primary rat microglia.…”

“…The third study, from our laboratory, demonstrated that a combination of tenofovir (TEN) + FTC + RAL inhibits autophagy in primary human astrocytes at concentrations consistent with CSF levels [ 27 , 64 , 65 ]. Using several techniques, including two different flux assays, we showed that APG biogenesis was inhibited after 24 h as well as after 7 days of daily treatment with the cocktail.…”

“…Using several techniques, including two different flux assays, we showed that APG biogenesis was inhibited after 24 h as well as after 7 days of daily treatment with the cocktail. We also showed increased p62 protein and decreased p62 flux without an increase in p62 transcription at multiple time points, suggesting that p62-mediated selective autophagy is inhibited [ 27 ]. The mechanisms that mediate decreased selective autophagy may be related to inhibited APG biogenesis, which decreases the amount of p62 available for degradation by autophagy.…”