Abstract
Methamphetamine (METH) use, referred to as methamphetamine use disorder (MUD), results in neurocognitive decline, a characteristic shared with HIV-associated neurocognitive disorders (HAND). MUD exacerbates HAND partly through glutamate dysregulation. Astrocyte excitatory amino acid transporter (EAAT)-2 is responsible for >90% of glutamate uptake from the synaptic environment and is significantly decreased with METH and HIV-1. Our previous work demonstrated astrocyte trace amine associated receptor (TAAR) 1 to be involved in EAAT-2 regulation. Astrocyte EAAT-2 is regulated at the transcriptional level by cAMP responsive element binding (CREB) protein and NF-κB, transcription factors activated by cAMP, calcium and IL-1β. Second messengers, cAMP and calcium, are triggered by TAAR1 activation, which is upregulated by IL-1β METH-mediated increases in these second messengers and signal transduction pathways have not been shown to directly decrease astrocyte EAAT-2. We propose CREB activation serves as a master regulator of EAAT-2 transcription, downstream of METH-induced TAAR1 activation. To investigate the temporal order of events culminating in CREB activation, genetically encoded calcium indicators, GCaMP6s, were used to visualize METH-induced calcium signaling in primary human astrocytes. RNA interference and pharmacological inhibitors targeting or blocking cAMP-dependent protein kinase A and calcium/calmodulin kinase II confirmed METH-induced regulation of EAAT-2 and resultant glutamate clearance. Furthermore, we investigated METH-mediated CREB phosphorylation at both serine 133 and 142, the co-activator and co-repressor forms, respectively. Overall, this work revealed METH-induced differential CREB phosphorylation is a critical regulator for EAAT-2 function and may thus serve as a mechanistic target for the attenuation of METH-induced excitotoxicity in the context of HAND.
Highlights
Methamphetamine use disorder (MUD) is correlated to heightened transmission of human immunodeficiency virus (HIV) (1–3) and increases the severity and onset of HIVassociated neurocognitive disorders (HAND) (4, 5)
We have previously demonstrated astrocyte trace amine associated receptor 1 (TAAR1) overexpression resulted in a significant decrease in astrocyte excitatory amino acid transporter-2 (EAAT-2) and glutamate clearance (7)
Since METH abuse during central nervous system (CNS) inflammation and human immunodeficiency virus-1 (HIV-1) poses greater threat due to their potential to increase TAAR1 levels/activity and crosstalk, we investigated whether interleukin 1 beta (IL-1β) and HIV-1 downregulated excitatory amino acid transporter (EAAT)-2 and affected TAAR1 levels and function (Figure 1)
Summary
Methamphetamine use disorder (MUD) is correlated to heightened transmission of human immunodeficiency virus (HIV) (1–3) and increases the severity and onset of HIVassociated neurocognitive disorders (HAND) (4, 5). We identified astrocyte trace amine associated receptor (TAAR) 1 is activated by METH, leading to increased intracellular cAMP and regulation of astrocyte EAAT-2 (7). We investigate signal transduction cascades, downstream of TAAR1 regulation and activation, to elucidate the mechanisms of astrocyte EAAT-2 downregulation. Previous studies reveal that HIV-1 proteins, including negative regulatory factor (Nef), transactivator of transcription (tat), and glycoprotein 120 (gp120), activate NFκB signaling pathways in astrocytes (33, 34). Our previous data demonstrating METH increases intracellular cAMP via TAAR1 activation in astrocytes and subsequently regulates EAAT-2 and glutamate clearance levels, sets a strong basis for further investigations of METHinduced TAAR1 signaling in the transcriptional regulation of astrocyte EAAT-2. Our data revealed that differential CREB phosphorylation results in EAAT-2 regulation, indicating that tipping the balance of METH-induced signaling to favor cAMP/PKA/pCREBSer[133] serves as a promising countermeasure in reversing METH-induced EAAT-2 downregulation
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