A Switch from Glial to Neuronal Gene Expression Alterations in the Spinal Cord of SIV-infected Macaques on Antiretroviral Therapy

Despite antiretroviral therapy (ART), HIV-associated peripheral neuropathy remains one of the most prevalent neurologic manifestations of HIV infection. The spinal cord is an essential component of sensory pathways, but spinal cord sampling and evaluation in people with HIV has been very limited, especially in those on ART. The SIV/macaque model allows for assessment of the spinal cord at key time points throughout infection with and without ART. In this study, RNA was isolated from the spinal cord of uninfected, SIV+, and SIV+ART animals to track alterations in gene expression using global RNA-seq. Next, the SeqSeek platform was used to map changes in gene expression to specific cell types. Pathway analysis of differentially expressed genes demonstrated that highly upregulated genes in SIV-infected spinal cord aligned with interferon and viral response pathways. Additionally, this upregulated gene set significantly overlapped with those expressed in myeloid-derived cells including microglia. Downregulated genes were involved in cholesterol and collagen biosynthesis, and TGF-b regulation of extracellular matrix. In contrast, enriched pathways identified in SIV+ART animals included neurotransmitter receptors and post synaptic signaling regulators, and transmission across chemical synapses. SeqSeek analysis suggested that upregulated genes were primarily expressed by neurons rather than glia. These findings propose that pathways activated in the spinal cord of SIV + ART macaques are involved in neuronal signaling rather than proinflammatory pathways. These findings provide the basis for further evaluation of mechanisms of SIV infection + ART within the spinal cord with a focus on therapeutic interventions to maintain synaptodendritic homeostasis. Overall design: SIV+ animals were inoculated intravenously with molecular clone SIV/17E-Fr and the immunosuppressive swarm SIV/DeltaB670. SIV+ART animals were inoculated with the same SIV combination and received ART beginning 12 days post-inoculation until study end. RNA was isolated from 100 mg frozen lumbar spinal cord samples. For transcriptome sequence sample preparation, isolated RNA samples were purified and cDNA libraies were prepared. SIV viral RNA loads in plasma were measured at serial time points by quanitative RT-PCR targeting the SIV gag gene. DNA was isolated from frozen lumbar spinal cord samples of animals receiving ART. Viral DNA was measured in tissues by ddPCR using primers in the SIV gag region and macaque beta interferon for sample normalization and cellular quantitation. Transcriptomic data was collected by RNA sequencing from spinal cord samples of pigtailed macaques and read alignment was performed, followed by quality checking with Fastqc. Gene and transcript expression levels were computed and pathway analysis of differentially expressed genes was performed. Generated cDNA was amplified and diluted. qPCR was performed.