By Jamshed Arslan, Pharm. D., PhD.
December 1 is the World AIDS Day. Despite the combination antiretroviral therapy, 10-25% of Human Immunodeficiency Virus (HIV)-positive individuals report neurocognitive impairments, especially those taking addictive stimulants like methamphetamine. HIV disrupts mitophagy and mitochondrial dynamics (fission and fusion), and the use of methamphetamine is thought to exacerbate this damage. Since dysfunctional mitochondria are known to generate excess reactive oxygen species (ROS), researchers in California, USA, hypothesized that disruption of mitophagy and mitochondrial dynamics, as seen in HIV infection with methamphetamine use, increases oxidative stress and neuronal injury. The team used two viral proteins, gp120 and Tat, to represent HIV infection in human primary neurons, and found that methamphetamine disrupted mitochondrial network and caused oxidative stress-induced neuronal degeneration. They were able to restore mitochondrial dynamics and abrogate degeneration of these neurons by using a potent antioxidant N-acetylcysteine. This shows that oxidative stress is at the center of HIV-associated neurocognitive disorders (HAND) in individuals who abuse methamphetamine.
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Methamphetamine disrupts mitophagy and promotes mitochondrial fragmentation in HIV exposed neurons
To determine the effect of methamphetamine-HIV combination on mitophagy, human primary neurons were treated with HIV proteins (gp120, Tat) and methamphetamine. Western blot of the extracted proteins revealed an increase in the lapidated form of microtubule-associated protein 1 LC3B-II, which is an autophagosomal marker in mammals. Expression and association of ubiquitin-binding protein p62 (sequestosome-1) to TOM20-labelled mitochondria was also enhanced with methamphetamine-HIV combination. P62 is an autophagosome cargo protein that targets other proteins for autophagy. Furthermore, confocal microscopy revealed mitochondrial fragmentation with methamphetamine-HIV duo, which served as a prelude to neuronal degeneration. That is, neurite staining with Microtubule Associated Protein 2 (MAP2) somatodendritic marker showed substantial decline in neuritic network. Likewise, the expression and translocation of Dynamin-Related Protein 1 (DRP1) to mitochondria increased with HIV proteins and methamphetamine. DRP1 is a GTPase that controls mitochondrial fragmentation. These results indicate disruption in mitophagy and mitochondrial dynamics with methamphetamine-HIV combination.
The next step was to study the role of reactive oxygen species (ROS) in this phenomenon.
(Left) Immunofluorescent analysis of cortical neuron-glial cell culture from E20 rat stained with chicken polyclonal antibody to microtubule associated protein 2 (MAP2) [NB300-213] at 1:10,000 dilution in red, and antibody to MAP-tau in green. The blue is DAPI staining of nuclear DNA. MAP2 antibody stains dendrites and perikarya of neurons, while MAP-tau antibody labels neuronal perikarya, dendrites and axonal process. As a result, perikarya and dendrites appears orange-yellow, since they contain both MAP2 and tau, while axons are green. (Right) Analysis of FFPE tissue section of mouse kidney using DRP1 antibody [NB110-55288] at 1:300. The primary antibody bound to DRP1 protein in the tissue section was detected using a HRP labeled secondary antibody and DAB reagent. Nuclei of the cells were counterstained with hematoxylin. This antibody generated a diffused cytoplasmic staining of DRP1 in the epithelial cells of various tubules and in the cells of glomeruli.
Effects of methamphetamine-HIV combination are mediated by oxidative stress
To measure oxidative stress, the researchers used a cell permeable dye that fluoresces upon oxidation by ROS. Enhanced staining of HIV-methamphetamine-treated neurons was observed, signifying increased ROS production. Not surprisingly, neurons pre-treated with antioxidant N-acetylcysteine (NAC) showed basal levels of ROS production. Likewise, pre-treatment with NAC diminished p62 expression, reversed p62-TOM20 association, abrogated DRP1 expression and translocation, reduced mitochondrial fragmentation, and restored neuritic network.
All in all, methamphetamine causes oxidative stress-induced disruption of mitophagy, mitochondrial dynamics and neuronal degeneration in HIV positive individuals.
Antioxidants for mitochondrial health and neuroprotection
This study proposes a novel approach of using antioxidants against neuronal degeneration, especially in the HIV-associated neurocognitive disorders (HAND) seen in the amphetamine users. However, the question of the extent to which mitochondrial morphology dictates mitochondrial function needs further elaboration.
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Jamshed Arslan, Pharm D, PhD
Dr Arslan is an Assistant Professor at Barrett Hodgson University, Pakistan,
where he uses various pedagogical methods to teach Pharm D students.
Teodorof-Diedrich, C., & Spector, S.A. (2020). Human immunodeficiency virus type-1 and methamphetamine mediated mitochondrial damage and neuronal degeneration in human neurons. Journal of Virology. https://doi.org/10.1128/JVI.00924-20.