Dolutegravir Disrupts Mouse Blood-Brain Barrier by Inducing Endoplasmic Reticulum Stress

Abstract

Background Dolutegravir (DTG) - based antiretroviral therapy is the contemporary first-line therapy to treat HIV infection. Despite its efficacy, mounting evidence has suggested a higher risk of neuropsychiatric adverse effect (NPAE) associated with DTG use with a limited understanding of the underlying mechanisms. Our laboratory has previously reported a toxic effect of DTG comparable to efavirenz in disrupting the blood-brain barrier (BBB) integrity in vitro and in vivo. The current study aimed to investigate, in vitro, the potential mechanisms involved in DTG toxicity. Methods Primary cultures of mouse brain microvascular endothelial cells were used as a robust rodent BBB cell model. The cells were treated with DTG at therapeutic relevant concentrations (2500, 3500, 5000 ng/ml) for 3–48 h with or without the presence of three endoplasmic reticulum (ER) sensor inhibitors (GSK2606414, 4µ8c, 4PBA). RNA-sequencing, qPCR, western blot analysis and cell stress assays (Ca²⁺ flux, H₂DCFDA, TMRE, MTT) were performed. Results Our initial Gene Ontology (GO) analysis of RNA-Sequencing data revealed an enriched transcriptome signature of ER stress pathway in DTG treated cells. We further demonstrated that therapeutic concentrations of DTG significantly activated the ER stress sensor proteins (PERK, IRE1, p-IRE1) and downstream ER stress markers (eIF2α, p-eIF2α, Hspa5, Atf4, Ddit3, Ppp1r15a, Xbp1, spliced-Xbp1). In addition, DTG treatment resulted in a transient Ca²⁺ flux, an aberrant mitochondrial membrane potential, and a significant increase in reactive oxygen species in primary cultures of mouse brain microvascular endothelial cells. Furthermore, we found that prior cell treatment with 4PBA (a broad-spectrum ER stress inhibitor) significantly rescued DTG-induced downregulation of tight junction proteins (Zo-1, Ocln, Cldn5), whereas GSK2606414 (a PERK inhibitor) elicited the greatest protective effect on DTG-induced elevation of pro-inflammatory cytokines and chemokines (Il6, Il23a, Il12b, Cxcl1, Cxcl2). Conclusions The current study provides valuable insights into DTG toxicological cell mechanisms, which may serve as a potential explanation of DTG-associated NPAEs in the clinic.