Methamphetamine and HIV-1-induced neurotoxicity: role of trace amine associated receptor 1 cAMP signaling in astrocytes

Abstract

Methamphetamine (METH) is abused by about 5% of the United States population with approximately 10-15% of human immunodeficiency virus-1 (HIV-1) patients reporting its use. METH abuse accelerates the onset and severity of HIV-associated neurocognitive disorders (HAND) and astrocyte-induced neurotoxicity. METH activates G-protein coupled receptors such as trace amine associated receptor 1 (TAAR1) increasing intracellular cyclic adenosine monophosphate (cAMP) levels in presynaptic cells of monoaminergic systems. In the present study, we investigated the effects of METH and HIV-1 on primary human astrocyte TAAR1 expression, function and glutamate clearance. Our results demonstrate combined conditions increased TAAR1 mRNA levels 7-fold and increased intracellular cAMP levels. METH and beta-phenylethylamine (β-PEA), known TAAR1 agonists, increased intracellular cAMP levels in astrocytes. Further, TAAR1 knockdown significantly reduced intracellular cAMP levels in response to METH/β-PEA, indicating signaling through astrocyte TAAR1. METH±HIV-1 decreased excitatory amino acid transporter-2 (EAAT-2) mRNA and significantly decreased glutamate clearance. RNA interference for TAAR1 prevented METH-mediated decreases in EAAT-2. TAAR1 knockdown significantly increased glutamate clearance, which was further heightened significantly by METH. Moreover, TAAR1 overexpression significantly decreased EAAT-2 levels and glutamate clearance that were further reduced by METH. Taken together, our data show that METH treatment activated TAAR1 leading to intracellular cAMP in human astrocytes and modulated glutamate clearance abilities. Furthermore, molecular alterations in astrocyte TAAR1 levels correspond to changes in astrocyte EAAT-2 levels and function. To our knowledge this is the first report implicating astrocyte TAAR1 as a novel receptor for METH during combined injury in the context of HAND.

Keywords: Astrocytes; Human immunodeficiency virus-1; Methamphetamine; Trace amine associated receptor 1; cAMP.

Figure 1. METH and HIV-1 modulate excitotoxicity and astrocyte viability

Astrocytes were treated with METH (500 μM) and/or HIV-1 (p24 10 ng/mL). RNA was isolated at 8 hr and assayed for EAAT-2 levels by real-time PCR. EAAT-2 levels were significantly lower in astrocytes treated with METH (*p<0.05), HIV-1 (***p<0.001) and METH + HIV-1 (A, ***p<0.001, n=5). Glutamate clearance was measured at 4 hr and 10 hr post-glutamate addition and was significantly decreased with METH, HIV-1 and in combined conditions at 10 hr (B, ***p<0.001, n=3). No significant differences in GFAP mRNA levels were observed (C). To determine astrocyte viability, metabolic activity (D), cytotoxicity (E) and apoptosis (F) were measured following 24 hr activation with METH and/or HIV-1. Transient treatment of METH and/or HIV-1 did not significantly increase MTT, LDH activity or apoptosis (D–F, n=3). Multiple astrocyte donors (n) were tested, each analyzed in a minimum of triplicate determinations.

Figure 2. Astrocyte TAAR1 expression and localization is regulated by METH and HIV-1

Human astrocytes were treated with METH (500 μM) and/or HIV-1 (p24 10 ng/mL) for 8 hr and RNA was isolated and assayed for TAAR1. TAAR1 mRNA levels were significantly increased with METH + HIV-1 cotreatments (A, ***p<0.001, n=3, independent astrocyte donors). METH-and/or HIV-1-induced intracellular cAMP was measured at 2 min and 10 min post-activation (B). METH alone significantly induced intracellular cAMP at both time points (**p<0.01). HIV-1 alone and in combination with METH significantly induced intracellular cAMP at 10 min post-activation (**p<0.01). Astrocytes were fixed and immunostained for GFAP (red) and TAAR1 (green). TAAR1 expression was localized in both cytoplasm and nucleus (C, D, E and F). To further confirm nuclear localization, confocal microscopy was performed (C1, D1, E1 and F1). Activation with HIV-1 alone (E1) and in combination with METH (F1) increased nuclear TAAR1 localization.

Figure 3. Forskolin, METH and β-PEA induce astrocyte intracellular cAMP signaling

Astrocyte intracellular cAMP signaling was induced with forskolin (A) as a positive control, METH (B) and β-PEA (C), known TAAR1 agonists. Representative donor EC₅₀ values for forskolin (10.96 μM), METH (32.86 μM), and β-PEA (11.04 μM) are shown. Intracellular cAMP induction assays were determined for multiple biological donors (n), and cumulative data is illustrated in panel D.

Figure 4. RNA interference downregulates astrocyte TAAR1 protein levels and intracellular cAMP in response to METH and β-PEA

Human astrocytes were MOCK-, siCON- and siTAAR1-transfected and plated for 48 hr followed by 24 hr of METH (500 μM) treatment. Cells were fixed and probed for TAAR1 (green) and GFAP (red). MOCK- (A) and siCON- (C) transfected astrocytes showed TAAR1 protein expression with and without METH treatment at 24 hr. METH (500 μM) treated MOCK- and siCON-transfected astrocytes demonstrated similar levels of TAAR1 protein (B and D). As expected siTAAR1-transfected astrocytes demonstrated reduced TAAR1 expression (E and F). Intracellular cAMP levels were quantified in MOCK-, siCON- and siTAAR1-transfected astrocytes. siTAAR1-transfected astrocytes showed significantly reduced METH and β-PEA-induced intracellular cAMP (G, ***p<0.001, n=4, independent astrocyte donors).

Figure 5. METH-induced TAAR1 activation regulates EAAT-2 mRNA levels and function

Primary human astrocytes were MOCK-, siCON-, siTAAR1-, CON-GFP- or TAAR1-GFP-transfected and treated with METH (500 μM). Two independent astrocyte donors were tested with a minimum of triplicate determinations in each experiment. Data was analyzed as fold change to internal control and presented as cumulative data for both donors. RNA was collected at 8 hr and glutamate clearance was measured at 24 hr post-treatment. (A) EAAT-2 mRNA levels significantly decreased in MOCK- and siCON-transfected astrocytes treated with METH (*p<0.05, **p<0.01). Changes in METH-mediated EAAT-2 levels were blocked by siTAAR1 transfection. (B) Glutamate clearance was performed in parallel. METH treatment significantly decreased glutamate clearance in MOCK- and siCON-transfected astrocytes while siTAAR1 transfection significantly increased glutamate clearance compared to METH treated controls (***p<0.001). (C & D) Both TAAR1-GFP-transfection and METH treatment significantly lowered levels of EAAT-2 mRNA expression in astrocytes and in parallel resulted in an exacerbated reduction in glutamate clearance abilities (***p<0.001).