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 Neurons

Abstract

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.

Nitric oxide, mitochondrial hyperpolarization, and T cell activation☆

“…Transient MHP is an early event preceding caspase activation, phosphatidylserine (PS) externalization, and disruption of Δψ m in Fas- [6] and H 2 O 2 -induced apoptosis of Jurkat human leukemia T cells and normal human peripheral blood lymphocytes (PBL) [8]. These observations were widely confirmed and extended to other apoptosis pathways [9,10,11,12,13,14]. Transient MHP is also triggered by activation of T cells by Con A [6] and CD3/CD28 costimulation [15] via ROI-and Ca 2+ -dependent production of NO [16].…”

Role of Tat Protein in HIV Neuropathogenesis

“…There is an initial brief burst of intracellular calcium release through IP-3 sensitive pools followed by prolonged increases in cytoplasmic calcium resulting from an influx of extracellular calcium (Haughey et al 1999). This is followed by mitochondrial calcium uptake, inhibition of complex IV of the electron transport chain, generation of reactive oxygen species, activation of caspases, and eventually results in apoptosis (Kruman et al 1998;New et al 1997;Norman et al 2007). Tat-induced neuronal cell death can be prevented by excitatory amino acid receptor antagonists (Brailoiu et al 2008;Magnuson et al 1995), inhibitors of nitric oxide synthase and caspases, antioxidants, and agents that stabilize mitochondrial membrane permeability and IP-3 pools of intracellular calcium (Haughey et al 1999;Kruman et al 1998;Perry et al 2005) and inhibition of glycogen synthase kinase3beta by lithium (Maggirwar et al 1999;Sui et al 2006).…”

Neurological and Motor Disorders: Neuronal Store-Operated Ca2+ Signaling: An Overview and Its Function

“…Activation of immune cells by HIV results in the secretion of soluble factors that destabilize neuronal Ca 2+ homeostasis, encourage oxidative stress and result in neural damage, which is thought to underlie the cognitive-motor dysfunction that develops in many HIV-infected patients. The HIV-1 transactivator protein (Tat) has been reported to be involved in cognitive impairment (Li et al 2004) and promotes neuronal death by inducing mitochondrial dysfunction and oxidative stress (Norman et al 2007; Self et al 2004). Additionally, evidence also suggests a role of HIV-1 Tat in altering cellular Ca 2+ homeostasis (Bonavia et al 2001; Self et al 2004), presumably interfering with molecular components of Ca 2+ stores (Haughey et al 1999; Norman et al 2008).…”