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HIV-1 Trans Activator of Transcription Protein Elicits Mitochondrial Hyperpolarization and Respiratory Deficit, with Dysregulation of Complex IV and Nicotinamide Adenine Dinucleotide Homeostasis in Cortical Neurons¹

John P. Norman^2,*,, Seth W. Perry^*, Karl A. Kasischke^¶, David J. Volsky^|| and Harris A. Gelbard^*,,

^* Department of Neurology (Child Neurology Division), Center for Aging and Developmental Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642; Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642; Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642; Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642; ^¶ Department of Neurosurgery, Center for Aging and Developmental Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642; and ^|| Molecular Virology Division, St. Luke’s-Roosevelt Hospital Center and College of Physicians and Surgeons, Columbia University, New York, NY 10019

HIV-1 causes a common, progressive neurological disorder known as HIV-associated dementia (HAD). The prevalence of this disorder has increased despite the use of highly active antiretroviral therapy, and its underlying pathogenesis remains poorly understood. However, evidence suggests that some aspects of HAD may be reversible. To model the reversible aspects of HAD, we have used the HIV-1 neurotoxin trans activator of transcription protein (Tat) to investigate nonlethal changes in cultured neurons. Exposure of rodent cortical neurons to sublethal concentrations of Tat elicits mitochondrial hyperpolarization. In this study, we used the cationic lipophilic dye rhodamine 123 to confirm this observation, and then performed follow-up studies to examine the mechanism involved. In intact neurons, we found Tat elicited a rapid drop in internal mitochondrial pH, and addition of Tat to purified mitochondrial extracts inhibited complex IV of the electron transport chain. To correlate enzyme activity in mitochondrial extracts with results in intact cells, we measured neuronal respiration following Tat exposure. Cortical neurons demonstrated decreased respiration upon Tat treatment, consistent with inhibition of complex IV. We examined mitochondrial Ca²⁺ homeostasis using a mitochondrial targeted enhanced yellow fluorescent protein-calmodulin construct. We detected a decrease in mitochondrial calcium concentration following exposure to Tat. Finally, we measured the energy intermediate NAD(P)H after Tat treatment, and found a 20% decrease in the autofluorescence. Based on these findings, we suggest that decreased NAD(P)H and calcium concentration contribute to subsequent respiratory decline after exposure to Tat, with detrimental effects on neuronal signaling.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the Geoffrey Waasdorp Pediatric Neurology Fund and National Institutes of Health Grants PO1MH64570, R01MH56838, and R01MH071176 (to H.A.G.); T32ES07026 (to J.P.N.); and PO1NS31492 (to D.J.V. and H.A.G.). This work was also supported by the American Heart Association Grant 0635595T (to K.A.K.) and by the ALS Association Grant 1112 (to K.A.K.).

² Address correspondence and reprint requests to John P. Norman, Center for Aging and Developmental Biology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Box 645, Rochester, NY 14642. E-mail address: John_Norman{at}urmc.rochester.edu

³ Abbreviations used in this paper: HAD, HIV-1-associated dementia; _m, mitochondrial membrane potential; p, proton motive force; ETC, electron transport chain; FCCP, carbonyl cyanide-4(trifluoromethoxy)phenylhydrazone; NMDA, N-methyl-D-asparate; pH_m, mitochondrial pH; PTD, protein transduction domain; rhod123, rhodamine 123; Tat, trans activator of transcription protein; EYFP, enhanced yellow fluorescent protein; CFP, cyan fluorescent protein; NAD(P)H, signifies both NADH and NADPH.