Dysregulation of dopamine neurotransmission has been linked to the development of human immunodeficiency virus (HIV)associated neurocognitive disorder (HAND). To investigate the mechanisms underlying this phenomenon, this study used an inducible HIV-1 transactivator of transcription (Tat) transgenic (iTat-tg) mouse model, which demonstrates brain-specific Tat expression induced by administration of doxycycline. We found that induction of Tat expression in the iTat-tg mice for either 7 or 14 days resulted in a decrease (∼30%) in the V max of [ 3 H] dopamine uptake via both the dopamine transporter (DAT) and norepinephrine transporter (NET) in the prefrontal cortex (PFC), which was comparable to the magnitude (∼35%) of the decrease in B max for [ 3 H]WIN 35,428 and [ 3 H]nisoxetine binding to DAT and NET, respectively. The decreased V max was not accompanied by a reduction of total or plasma membrane expression of DAT and NET. Consistent with the decreased V max for DAT and NET in the PFC, the current study also found an increase in the tissue content of DA and dihydroxyphenylacetic acid in the PFC of iTattg mice after 7 days' administration of doxycycline. Electrophysiological recordings in layer V pyramidal neurons of the prelimbic cortex from iTat-tg mice found a significant reduction in action potential firing, which was not sensitive to selective inhibitors for DAT and NET, respectively. These findings provide a molecular basis for using the iTat-tg mouse model in the studies of NeuroHIV. Determining the mechanistic basis underlying the interaction between Tat and DAT/NET may reveal novel therapeutic possibilities for preventing the increase in comorbid conditions as well as HAND. SIGNIFICANCE STATEMENTHuman immunodeficiency virus (HIV)-1 infection disrupts dopaminergic neurotransmission, leading to HIV-associated neurocognitive disorders (HANDs). Based on our in vitro and in vivo studies, dopamine uptake via both dopamine and norepinephrine transporters is decreased in the prefrontal cortex of HIV-1 Tat transgenic mice, which is consistent with the increased dopamine and dihydroxyphenylacetic acid contents in this brain region. Thus, these plasma membrane transporters are an important potential target for therapeutic intervention for patients with HAND.

No abstract

Dysregulation of dopaminergic system induced by HIV-1 Tat protein-mediated direct inhibition of the dopamine transporter (DAT) has been implicated as a mediating factor of HIV-1 associated neurocognitive disorders. We have reported that single point mutations on human DAT (hDAT) at tyrosine88 (Y88F), lysine92 (K92M), and histidine547 (H547A) differentially regulate basal dopamine uptake but diminish Tat-induced inhibition of dopamine uptake by changing dopamine transport process. This study evaluated the effects of double (Y88F/H547A) and triple (Y88F/K92M/H547A) mutations on basal dopamine uptake, Tat-induced inhibition of DAT function, and dynamic transport process. Compared to wild-type hDAT, the V max values of [ 3 H]Dopamine uptake were increased by 96% in Y88F/H547A but decreased by 97% in Y88F/K92M/H547A. [ 3 H]WIN35,428 binding sites were not altered in Y88F/H547A but decreased in Y88F/K92M/H547A. Y88F/H547A mutant attenuated Tat-induced inhibition of dopamine uptake observed in wild-type hDAT. Y88F/H547A displayed an attenuation of zinc-augmented [ 3 H]WIN35,428 binding, increased basal dopamine efflux, and reduced amphetamine-induced dopamine efflux, indicating this mutant alters transporter conformational transitions. These findings further demonstrate that both tyrosine88 and histidine547 on hDAT play a key role in stabilizing basal dopamine transport and Tat-DAT integration. This study provides mechanistic insights into developing small molecules to block multiple sites in DAT for Tat binding.