Methamphetamine Increases the Proportion of SIV-Infected Microglia/Macrophages, Alters Metabolic Pathways, and Elevates Cell Death Pathways: A Single-Cell Analysis

Abstract

Both substance use disorder and HIV infection continue to affect many individuals. Both have untoward effects on the brain, and the two conditions often co-exist. In the brain, macrophages and microglia are infectable by HIV, and these cells are also targets for the effects of drugs of abuse, such as the psychostimulant methamphetamine. To determine the interaction of HIV and methamphetamine, we isolated microglia and brain macrophages from SIV-infected rhesus monkeys that were treated with or without methamphetamine. Cells were subjected to single-cell RNA sequencing and results were analyzed by statistical and bioinformatic analysis. In the animals treated with methamphetamine, a significantly increased proportion of the microglia and/or macrophages were infected by SIV. In addition, gene encoding functions in cell death pathways were increased, and the brain-derived neurotropic factor pathway was inhibited. The gene expression patterns in infected cells did not cluster separately from uninfected cells, but clusters comprised of microglia and/or macrophages from methamphetamine-treated animals differed in neuroinflammatory and metabolic pathways from those comprised of cells from untreated animals. Methamphetamine increases CNS infection by SIV and has adverse effects on both infected and uninfected microglia and brain macrophages, highlighting the dual and interacting harms of HIV infection and drug abuse on the brain.

Cryopreservation of microglia enables single-cell RNA sequencing with minimal effects on disease-related gene expression patterns

“…We routinely isolate enriched populations of microglia and macrophages from the brains of nonhuman primates in our studies on SIV as a model for the neurological effects of HIV and cryopreserve them for use in our future experiments ( Bortell et al., 2018 ; Chaudhuri et al., 2013 ; Marcondes et al., 2001 ; Watry et al., 1995 ). Recently we reported the use of these cryopreserved preparations in an scRNA-seq study ( Niu et al., 2020 ). In order to investigate the effect of cryopreservation on scRNA-seq of microglia, we compared scRNA-seq results from freshly isolated and cryopreserved cells from two rhesus monkeys.…”

“…Despite an increase in expression of IEGs due to cryopreservation, they still may be responsive to changes in experimental conditions. To assess this possibility, we examined IEG expression in our prior study of the effect of drug abuse on a model of neuroHIV in monkeys, which utilized cryopreserved microglia for scRNA-seq ( Niu et al., 2020 ). In fact, a number of the IEGs were altered between the saline and methamphetamine conditions, with some IEGs increased (e.g., IL1B and DUSP2 ), and others decreased (e.g., CCL3 and CCL5 ), in expression by methamphetamine compared with saline treatment of the animals ( Figure 3 ).…”

“… (B) Overlaid dot and violin plots indicate expression levels of the selected IEGs in the two conditions, with fold change of the methamphetamine relative to the saline condition indicated. All data from ( Niu et al., 2020 ). …”

“…Whether cryopreservation may introduce a bias in the cells analyzed, as with any isolation and purification procedure, remains to be determined, and future studies are needed to assess such a potential bias. We have successfully performed scRNA-seq from microglia samples that have been cryopreserved for over 7 years ( Niu et al., 2020 ). Future studies can also address any limits on storage time on cryopreserved microglia, and changes that may occur due to different storage times.…”

“…A comparison of freshly isolated cells with cells cryopreserved in DMSO or fixed under two different conditions concluded that cryopreservation in DMSO was optimal for scRNA-seq ( Wohnhaas et al., 2019 ). We have recently described the use of scRNA-seq on DMSO-cryopreserved microglia from Simian immunodeficiency virus (SIV)-infected rhesus monkeys, illuminating the effects of methamphetamine on SIV-induced CNS disease and modeling neuroHIV (in animals with SIV encephalitis [SIVE]) in those with and without a methamphetamine substance use disorder ( Niu et al., 2020 ). Here we examined the effects of microglial cryopreservation on gene expression in microglia to determine whether this is a suitable method to archive cells for later scRNA-seq analysis or whether cryopreservation may also have an untoward effect on detecting activation as well as other disease-associated differentially expressed genes.…”