Intracellular mechanisms of gp120 neurotoxicity: role of microtubules

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PROJECT SUMMARY/ABSTRACT Even in the era of combined antiretroviral therapy, up to 50% of HIV-positive patients will demonstrate neurocognitive impairments in their lifetimes. These impairments are collectively known as HIV-associated neurocognitive disorders (HAND). While the neuropathology of HAND has been well-characterized, the specific mechanism by which HAND occurs remains to be clarified. Considerable experimental evidence indicates that HIV proteins, including the envelope protein gp120, cause neurological damage to a similar extent as the full virus. Thus, gp120 has emerged as potential agent underlying HIV neurotoxicity. However, the full mechanism of gp120- mediated neurotoxicity is still unknown. Therefore, it is imperative to investigate these mechanisms of neurotoxicity and elucidate targets for potential therapeutic intervention. I have established that gp120 is internalized into neurons via dynamin-dependent endocytosis and that internalized gp120 can bind to class-III β-tubulin, a component of neuronal microtubules. Moreover, gp120 causes the deacetylation of tubulin, a post-translational modification that impairs the functionality of microtubules. Furthermore, tubulin deacetylation causes a dissociation of the motor proteins kinesin-1 and dynein from microtubules, which impairs axonal transport. Preliminary data indicate that intracellular trafficking of essential organelles, such as mitochondria, is greatly diminished in the presence of gp120. Therefore, I hypothesize that gp120 impairs axonal transport of organelles and cargo-containing vesicles through the deacetylation of tubulin. To confirm whether this deacetylation of tubulin underlies the neurotoxic effect of gp120, I first will inhibit the regulatory enzyme HDAC6 pharmacologically with tubacin (AIM 1) to prevent deacetylation of tubulin. I will confirm these results by utilizing siRNA for HDAC6. Using primary rat cortical neurons, I hypothesize that inhibition of HDAC6 will be neuroprotective, as shown in other neurodegenerative diseases. Secondly, I propose to establish whether gp120 causes decreased binding of kinesin-1 and dynein to microtubules (AIM 2A). To examine this, I will evaluate the binding of kinesin-1/dynein to tubulin using co-immunoprecipitation and sub-cellular fractionation to isolate microtubule associated proteins. Finally, using rat primary cortical neurons grown in a microfluidic chamber to isolate axons, I will evaluate axonal transport in the presence of gp120 (AIM 2B) using live imaging of quantum dot labeled brain-derived neurotrophic factor (BDNF). I hypothesize that gp120 will cause a decrease in kinesin-1/dynein binding to microtubules and therefore will impair both velocity and total distance travelled by the labeled BDNF. These studies aim to establish a new mechanism of gp120-mediated neurotoxicity that impairs axonal transport through tubulin deacetylation. Moreover, throughout the proposed training, I will gain expertise in a variety of molecular experimental approaches with emphasis on motor proteins and axonal transport.