Neurodegenerative diseases affect millions of people worldwide. Neurodegenerative diseases result from progressive damage to nerve cells in the brain or peripheral nervous system connections that are essential for cognition, coordination, strength, sensation, and mobility. Dysfunction of these brain and nerve functions is associated with Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Amyotrophic lateral sclerosis, and motor neuron disease. In addition to these, 50% of people living with HIV develop a spectrum of cognitive, motor, and/or mood problems collectively referred to as HIV-Associated Neurocognitive Disorders (HAND) despite the widespread use of a combination of antiretroviral therapies. Neuroinflammation and neurotransmitter systems have a pathological correlation and play a critical role in developing neurodegenerative diseases. Each of these diseases has a unique pattern of dysregulation of the neurotransmitter system, which has been attributed to different forms of cell-specific neuronal loss. In this review, we will focus on a discussion of the regulation of dopaminergic and cholinergic systems, which are more commonly disturbed in neurodegenerative disorders. Additionally, we will provide evidence for the hypothesis that disturbances in neurotransmission contribute to the neuronal loss observed in neurodegenerative disorders. Further, we will highlight the critical role of dopamine as a mediator of neuronal injury and loss in the context of NeuroHIV. This review will highlight the need to further investigate neurotransmission systems for their role in the etiology of neurodegenerative disorders.

The disruption of dopamine (DA) neurotransmission by the HIV-1 transactivator of transcription (Tat) during HIV-1 infection has been linked to the development of neurocognitive disorders, even under combined antiretroviral therapy treatment. We have demonstrated that Southern Research Institute (SRI) 32742, a novel allosteric modulator of DA transporter (DAT), attenuates cocaine- and Tat-binding to DAT, alleviates Tat-induced cognitive deficits and potentiation of cocaine reward in inducible Tat transgenic mice. The current study determined the in vitro pharmacological profile of SRI-32743 and its optimized second-generation analogs and their effects as allosteric modulators. Through structure-activity relationship studies of SRI-32743, 170 compounds were synthesized and evaluated for their ability to modulate DAT function. We identified 21 analogs as atypical competitors of DAT (maximum attributable drug effect, ≤60%). Four compounds, SRI-46564, SRI-47056, SRI-46286, and SRI-47867, displayed IC₅₀ values for [³H]DA uptake inhibition from 9.33 ± 0.50 to 0.96 ± 0.05 μM and from 3.96 ± 1.36 to 1.29 ± 0.19 for DAT binding, respectively. The 4 analogs also displayed high potency at 2 different concentrations (0.5 nM and 0.05 nM) to attenuate Tat-induced inhibition of [³H]DA uptake and cocaine-mediated dissociation of [³H]WIN35,428 binding in Chinese hamster ovary cells expressing human DAT, suggesting that the effects occur through an allosteric mechanism. In further ex vivo studies using fast scan cyclic voltammetry, we demonstrated that the analogs do not disrupt the baseline phasic-like DA release. These findings provide a new insight into the potential for development of novel therapeutic agents to attenuate DAT-Tat interactions to normalize DA neurotransmission in NeuroHIV. SIGNIFICANCE STATEMENT: The allosteric inhibition of the dopamine (DA) transporter by the HIV-1 transactivator of transcription (Tat) disrupts DA homeostasis, leading to HIV-associated neurocognitive disorders. Analogs of Southern Research Institute 32743, a novel allosteric modulator of the Tat-DA transporter (DAT) interaction, were evaluated in the current study and characterized as atypical ligands of DA uptake. Four novel lead compounds demonstrated high potency to attenuate Tat-induced inhibition of human DAT-mediated DA uptake in an allosteric modulatory manner with no effects on the dynamics of DA uptake-release in DAT.

Fast scan cyclic voltammetry (FSCV) allows for real -time analysis of phasic neurotransmitter levels. Tryptophan (TRP) is an aromatic amino acid responsible for facilitating electron transfer kinetics in oxidoreductase enzymes. Previous work with TRP-modified electrodes showed increased sensitivity for cyclic voltammetry detection of dopamine (DA) when used with slower scan rates (0.05 V/s). Here, we outline an in vitro proof of concept for TRP-modified electrodes in FSCV detection of DA, and decreased sensitivity for ascorbic acid (AA). TRP-modified electrodes had a limit of detection (LOD) for DA of 2.480 ± 0.343 nM compared to 8.348 ± 0.405 nM for an uncoated electrode. Selectivity for DA/ascorbic acid (AA) was 1.107 ± 0.3643 for uncoated and 15.57 ± 4.184 for TRP-modified electrodes. Additionally, these TRP-modified electrodes demonstrated reproducibility when exposed to extended cycling. TRP-modified electrodes will provide an effective modification to increase sensitivity for DA.

Development of neurocognitive disorder in HIV infected patients has been linked to dysregulation of dopamine by HIV-1 transactivator of transcription (Tat) protein, a negative allosteric modulator of dopamine transporter (DAT). Using Fast-Scan Cyclic Voltammetry, the present study determined the effects of in vivo Tat expression on dopamine release in the caudate putamen of inducible Tat transgenic (iTat-tg) mice and the impact of a novel DAT allosteric modulator, SRI-32743, on the Tat effect. We found that 7- or 14-day doxycycline (Dox)-induced Tat expression in iTat-tg mice resulted in a two-fold increase in phasic but not tonic stimulated baseline dopamine release relative to saline control mice. To determine whether the Tat-induced increase in dopamine release is mediated by DAT regulation, we examined the effect of an in vitro applied DAT inhibitor, nomifensine, on the dopamine release. Nomifensine (1 nM-10 µM) concentration-dependently enhanced phasic stimulated dopamine release in saline- and Dox-treated iTat-tg mice, while the magnitude of the nomifensine-mediated dopamine release was unchanged between saline and Dox treatment groups. A single systemic administration of SRI-32743 prior to animal sacrifice reversed the increased dopamine release in the baseline of phasic dopamine release and nomifensine-augmented dopamine levels in Dox-treated iTat-tg mice, while SRI-32743 alone did not alter baseline of dopamine release. These findings suggest that Tat expression-induced increase in extracellular dopamine levels by not only inhibiting DAT-mediated dopamine transport but also stimulating dopamine release. Thus, DAT allosteric modulators may serve as a potential therapeutic intervention for HIV infection-dysregulated dopamine system observed in HIV-1 positive individuals. Significance Statement HIV infection-induced dysregulation of dopaminergic system has been implicated in the development of neurocognitive impairments observed in HIV positive patients. Understanding the mechanisms underlying HIV-1 Tat protein-induced alteration of extracellular dopamine levels will provide insights into the development of molecules that can attenuate Tat interaction with targets in the dopaminergic system. Here, we determined whether Tat alters dopamine release and how the novel DAT allosteric modulator, SRI-32743, impacts dopamine neurotransmission to attenuate Tat-induced effects on extracellular dopamine dynamics.

Dysregulation of dopaminergic transmission induced by the HIV-1 transactivator of transcription (Tat) has been implicated as a central factor in the development of HIV-1 associated neurocognitive disorders (HAND). We have demonstrated that the tyrosine470 residue of the human dopamine transporter (hDAT) plays a critical role in Tat-hDAT interaction. Based on the computational modeling predictions, the present study sought to examine the mutational effects of the tyrosine467 residue of the human norepinephrine transporter (hNET), a corresponding residue of the hDAT tyrosine470, on Tat-induced inhibition of reuptake of dopamine through the hNET. Mutations of the hNET tyrosine467 to a histidine (Y467H) or a phenylalanine (Y467F) displayed similar kinetic properties of reuptake of [³H]dopamine and [³H]norepinephrine in PC12 cells expressing wild-type hNET and its mutants. Compared to wild-type hNET, neither of Y467H or Y467F altered B_max and K_d values of [³H]WIN35,428 binding, whereas Y467H but not Y467F decreased the B_max of [³H]nisoxetine binding without changes in K_d. Y467H also increased the affinity of nisoxetine for inhibiting [³H]dopamine uptake relative to wild-type hNET. Recombinant Tat_1-86 (140 nM) induced a significant reduction of [³H]dopamine uptake in wild-type hNET, which was attenuated in both Y467H and Y467F. Compared to wild-type hNET, neither Y467H or Y467F altered [³H]dopamine efflux in CHO cells expressing WT hNET and mutants, whereas Y467F but not Y467H decreased [³H]MPP⁺ efflux. These results demonstrate tyrosine467 as a functional recognition residue in the hNET for Tat-induced inhibition of dopamine transport and provide a novel insight into the molecular basis for developing selective compounds that target Tat-NET interactions in the context of HAND.