acase@tulane.edu / Objectives Human Immunodeficiency Virus (HIV) infects immune cells and lowers cell-mediated immunity leading to acquired immune deficiency syndrome or AIDS. The virus causes damage/dysfunction of helper T cells, macrophages, and dendritic cells. One of the long-term complications of untreated AIDS is severe cognitive impairment caused by HIV-associated dementia (HAD). Highly Active Antiretroviral therapy, the HAART regimen, which inhibits virus replication has been shown to reduce the incidence of HAD. HAART includes several mechanistically diverse classes of drugs such as protease inhibitors, nucleoside inhibitors of reverse transcriptase, and non-nucleoside reverse transcriptase inhibitors. The protease inhibitor, nelfinavir, is used in HIV therapy in order to mitigate the effects of the HIV virus by breaking down HIV-1 and HIV-2 proteases, which are essential to the replication of the virus within the host cell. HAART has decreased the incidence of HAD yet milder cognitive dysfunction, considered to be a consequence of anti-HIV drug toxicity, often manifests. Previous studies have shown that protease inhibitors may play a role in causing oxidative stress in endothelial cells. The present study involves understanding the effect of nelfinavir on mitochondrial oxidative stress and its role in the injury to human brain microvascular endothelial cells that form the blood-brain barrier. Methods and Results Our studies utilized primary human brain microvascular endothelial cells (hBMECs). We performed measurements of mitochondrial superoxide levels (ESR Spectrscopy), oxygen consumption rates or OCR (Seahorse XFe Extracellular Flux Analyzer and MitoStress Test Assay), cell viability/proliferation (CCK8 based Cellular Viability Assay). Sub-therapeutic doses of nelfinavir (1 µmol/L) increased the cell proliferation whereas therapeutic (3-5 µmol/L) and supra-therapeutic (10 µmol/L) doses of nelfinavir reduced the cell viability. In addition, treatment with sub-therapeutic levels of nelfinavir has no effect on the levels of mitochondrial superoxide in hBMECs but therapeutic and supra-therapeutic levels of nelfinavir increased mitochondrial superoxide levels. Measurements of OCR showed that sub-therapeutic doses of nelfinavir enhanced the basal and maximal respiration in hBMECs. In contrast, therapeutic concentration of nelfinavir reduced ATP production and spare respiratory capacity although basal respiration, proton leak, and non-mitochondrial respiration were unchanged. However, supra-therapeutic dose of nelfinavir significantly reduced basal respiration, ATP production, and spare respiratory capacity accompanied by reduced non-mitochondrial respiration and proton leak. Conclusions We identified that nelfinavir treatment was associated with a decrease in cellular proliferation at therapeutic and supra-therapeutic levels. Furthermore, we identified an increase in mitochondrial superoxide species in cells treated with nelfinavir in concentrations beyond therapeutic levels which was accompanied by a decrease in basal respiration, ATP production, and mitochondrial spare capacity. These results are indicative of nelfinavir causing cellular cytotoxicity in BMECs that are likely mediated by mitochondrial oxidative stress and impaired mitochondrial respiration. / 1 / Gowthamram Rajaprabhakaran