Mechanisms of Tat neurotoxicity: the role of mitochondria

Georgetown University, Washington DC

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Abstract

? DESCRIPTION (provided by applicant): Perturbations to the mitochondrial life cycle have been implicated in a causal role of neurodegenerative diseases, including Parkinson's, Alzheimer's, and Huntington's diseases. Moreover, aberrant mitochondria have also been observed in the post-mortem tissue of HIV associated dementia (HAD) subjects who additionally exhibit dendritic pruning, neuronal apoptosis, and synaptic abnormalities, indicating that mitochondrial dysfunction may play a role in disease progression. However, as yet this hypothesis has no direct support. My overall goal is to demonstrate that Tat impairs mitochondrial homeostasis in neurons, which can contribute to neuronal impairment in HAD. The specific aims of the proposed research are to: 1) determine in vitro how Tat impairs mitochondrial dynamics (i.e. fusion, fission, and trafficking), 2) assess Tat-induced alterations t the activity of dynamin- related protein 1 (Drp1), and 3) determine how Tat impairs mitophagy. It has been established that Tat causes synaptic simplification and neuronal apoptosis, preceded by significant impairment to mitochondria as determined by a decrease in mitochondrial membrane potential and increased reactive oxygen species (ROS) release. Furthermore, my preliminary data show that Tat impairs mitochondrial subcellular localization in neurons in vitro. Thus, Aim 1 will consist of studies in which primary neuronal cultures containing fluorescently labeled mitochondria will be exposed to Tat. From these experiments, mitochondrial dynamics will be observed by live-cell imaging and mitochondria ultrastructure by stochastic optical reconstruction microscopy (STORM) and scanning electron microscopy (EM). In preliminary studies, I have observed mitochondrial impairment as well as somal accumulation of mitochondria. Therefore, I expect to see an increase in retrograde transport of mitochondria and increased mitochondrial fragmentation. Aim 2 will assess a mechanism by which Tat causes mitochondrial fragmentation. Primary neuronal cultures will be exposed to Tat and then probed for levels of total and phosphorylated Drp1 as well as calcineurin activity. Since Tat is known previously to activate calcineurin, I expect to see increased calcineurin activity and decreased levels of phosphorylated Drp1. Aim 3 will assess the impact of Tat on mitophagy. To fulfill this Aim, I will assess mitophagy within primary neuronal cultures following Tat exposure utilizing three complementary techniques: 1) subcellular fractionation to isolate autophagic vacuoles, 2) proximity ligation assay, and 3) EM to examine autophagosomal ultrastructure. Taken together, I expect to observe an increase in mitochondrial localization within autophagosomes following Tat exposure but do not expect to see a decrease in whole mitochondria levels due to inefficiency in the mitophagic process. This proposal will enhance my training in mitochondria neurobiology and HIV-1 neurotoxicity. Moreover, the mechanistic studies proposed will likely present a clearer way to target this damage with newly developed therapies.