| HIV Associated Dementia (HAD) is a progressive disease with characteristics traditionally associated with Parkinson's and Alzheimer's disease. The neurological phenotype of this disease results in part from acute bioenergetic stress in neurons that are vulnerable to viral and cellular toxins elaborated from infected mononuclear phagocytes in the CNS. Our laboratory has described the phenomenon of mitochondrial hyperpolarization in cortical neurons exposed to the HIV-1 protein Tat as one aspect of this stress response. In addition to mitochondrial hyperpolarization, we have observed the induction of the unfolded protein response (UPR), further compounding stress to the CNS. The studies presented were designed to describe the underlying mechanism responsible for mitochondrial hyperpolarization and UPR induction caused by HIV-1 Tat.;The first investigations focused on several mitochondrial systems including: pH, electron transport chain (ETC) complex activity, ion homeostasis, and energy intermediates (NADH). Application of Tat to cortical neurons elicited a rapid drop in internal mitochondrial pH in concordance with hyperpolarization. Addition of Tat to purified mitochondrial protein not only caused an inhibition of complex IV of the electron transport chain, but also decreased cortical neuron respiration, corroborating the physiological inhibition of complex IV. Using a mitochondrial targeted CFP:EITFP-calmodulin construct, we observed a decrease in [Ca2+]mit. Finally, we measured [Ca 2+]mit and observed a rapid decrease that coincided with the mitochondrial hyperpolarization. The results lead us to speculate that the decrease in calcium contributes to mitochondrial hyperpolarization after exposure to Tat, a hypothesis that was borne out in later studies. The involvement of [Ca2+] mit in Tat neurotoxicity necessitated investigations of the endoplasmic reticulum, an important sub-cellular organelle for mitochondrial function and a major site for Ca2+ storage in neurons.;Our investigations into ER function resulted in the observation that HIV-1 Tat induces rapid loss of ER calcium through activation of the ryanodine receptor (RyR), as well as initiation of the unfolded protein response (UPR). This phenomenon induced a pathological dilatation of the ER that could be observed at the ultra-structural level using electron microscopy. In our paradigm of mitochondrial hyperpolarization, the use of the RyR inhibitor dantrolene and inhibitory concentrations of ryanodine reversed the hyperpolarization of mitochondrial membrane potential, leading us to speculate on the presence of a functional mitochondrial RyR. The resulting data indicates that RyR activation is the common mechanism through which HIV-1 Tat elicits, at least in part, its neurotoxic effect on mitochondrial and ER function. In addition to the identification of the RyR as a novel therapeutic target, we also identified the drug dantrolene as a possible pharmaceutical agent for use in HAD therapy. |
