Fluorescent probes for monitoring mitochondrial membrane potential are frequently used for assessing mitochondrial function, particularly in the context of cell fate determination in biological and biomedical research. However, valid interpretation of results obtained with such probes requires careful consideration of numerous controls, as well as possible effects of non-protonic charges on dye behavior. In this context, we provide an overview of some of the important technical considerations, controls, and parallel complementary assays that can be employed to help ensure appropriate interpretation of results, thus providing a practical usage guide for monitoring mitochondrial membrane potentials with cationic probes. In total, this review will help illustrate both the strengths and potential pitfalls of common mitochondrial membrane potential dyes, and highlight best-usage approaches for their efficacious application in life sciences research.

Neurologic disease caused by human immunodeficiency virus type 1 (HIV-1) is ultimately refractory to highly active antiretroviral therapy (HAART) because of failure of complete virus eradication in the central nervous system (CNS), and disruption of normal neural signaling events by virally induced chronic neuroinflammation. We have previously reported that HIV-1 Tat can induce mitochondrial hyperpolarization in cortical neurons, thus compromising the ability of the neuron to buffer calcium and sustain energy production for normal synaptic communication. In this report, we demonstrate that Tat induces rapid loss of ER calcium mediated by the ryanodine receptor (RyR), followed by the unfolded protein response (UPR) and pathologic dilatation of the ER in cortical neurons in vitro. RyR antagonism attenuated both Tat-mediated mitochondrial hyperpolarization and UPR induction. Delivery of Tat to murine CNS in vivo also leads to long-lasting pathologic ER dilatation and mitochondrial morphologic abnormalities. Finally, we performed ultrastructural studies that demonstrated mitochondria with abnormal morphology and dilated endoplasmic reticulum (ER) in brain tissue of patients with HIV-1 inflammation and neurodegeneration. Collectively, these data suggest that abnormal RyR signaling mediates the neuronal UPR with failure of mitochondrial energy metabolism, and is a critical locus for the neuropathogenesis of HIV-1 in the CNS.

Despite the efficacy of highly active antiretroviral therapy in reducing viral burden, neurologic disease associated with HIV-1 infection of the CNS has not decreased in prevalence. HIV-1 does not induce disease by direct infection of neurons, although extensive data suggest that intra-CNS viral burden correlates with both the severity of virally induced neurologic disease, and with the generation of neurotoxic metabolites. Many of these molecules are capable of inducing neuronal apoptosis in vitro, but neuronal apoptosis in vivo does not correlate with CNS dysfunction, thus prompting us to investigate cellular and synaptic events occurring before cell death that may contribute to HIV-1-associated neurologic disease. We now report that the HIV-1 regulatory protein transactivator of transcription protein (Tat) increased oxidative stress, ATP levels, and mitochondrial membrane potential in primary rodent cortical neurons. Additionally, a proinflammatory cellular metabolite up-regulated by Tat, platelet-activating factor, also induced oxidative stress and mitochondrial hyperpolarization in neurons, suggesting that this type of metabolic dysfunction may occur on a chronic basis during HIV-1 infection of the CNS. Tat-induced mitochondrial hyperpolarization could be blocked with a low dose of the protonophore FCCP, or the mitochondrial KATP channel antagonist, tolbutamide. Importantly, blocking the mitochondrial hyperpolarization attenuated Tat-induced neuronal apoptosis, suggesting that increased mitochondrial membrane potential may be a causal event in precipitating neuronal apoptosis in cell culture. Finally, Tat and platelet-activating factor also increased neuronal vesicular release, which may be related to increased mitochondrial bioenergetics and serve as a biomarker for early damage to neurons.

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.

Methods for growing primary neuronal cultures rely on the inclusion of antioxidants in the culture medium, but no studies have determined precisely if or when antioxidants are required for neuronal survival, despite the significance this information would have for understanding neurodevelopment and studying oxidative stress. We show that cortical neurons grown in Neurobasal media with B27 supplement required antioxidants for only the first 24 hr post-explantation, after which the antioxidants could be removed permanently without noticeable loss of neuronal survival over the normal lifespan. Cortical cultures never exposed to antioxidants did not survive. These findings represent a novel method for substantially antioxidant-free neuronal culture, whereby antioxidants can be removed permanently from the cultures after only 1 day. This method may prove critical for studies of oxidative stress, because B27 antioxidants significantly diminished pro-oxidative effects of the excitatory neurotransmitter glutamate and hydrogen peroxide on cortical cultures, even if antioxidants were removed before the oxidizing treatment. Together, these findings suggest a brief window of high vulnerability to reactive oxygen species, and have important implications for studies of oxidative stress and developmental neuroscience.