Regulation of AQP4 expression and investigation of the underlying mechanisms by HIV-1 Tat through the NMDAR/cAMP/PKA signaling pathway in astrocytes

Abstract

Introduction: The neurotoxicity caused by human immunodeficiency virus (HIV) proteins is a significant factor contributing to the development of HIV-associated neurocognitive disorder (HAND), and currently, effective treatment options for HAND are still lacking. The transactivator of transcription (Tat) protein, a crucial regulatory protein encoded by HIV-1, has not been extensively studied in relation to N-methyl-D-aspartate receptor (NMDAR) in astrocytes. However, studies in traumatic brain injury (TBI) models have confirmed that the expression of aquaporin-4 (AQP4), a water channel protein in astrocytes, is regulated by the NMDA signaling pathway. Animal models have shown that HIV-associated neurocognitive impairment is initially characterized by reduced AQP4 levels and subsequent cortical degeneration, suggesting a pivotal role for AQP4 in the progression of HAND. Reports have indicated that the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway contributes to AQP4 expression. Nevertheless, the mechanisms by which the HIV-1 Tat protein regulates AQP4 expression and its potential for ameliorating HIV-related neurocognitive impairment remain to be elucidated. Objectives This study aims to explore the regulatory mechanism by which the HIV-1 Tat protein modulates the expression of aquaporin-4 (AQP4) through the NMDAR/cAMP/PKA signaling pathway. Methods The intracellular calcium ion concentration in astrocytes was measured using the Fluo-3, AM fluorescent probe. Protein expression levels of AQP4, N-methyl-D-aspartate receptor subunit 1 (NR1), N-methyl-D-aspartate receptor subunit 2A (NR2A), N-methyl-D-aspartate receptor subunit 2B (NR2B), Calmodulin-dependent protein kinase II (CaMKII), phosphorylated calcium/calmodulin-dependent protein kinase II (p-CaMKII), PKA, and protein kinase G (PKG) were detected using Western blotting (WB). Real-time quantitative PCR (RT-qPCR) was employed to determine the mRNA transcription levels of AQP4, NR1, NR2A, NR2B, and CaMKII. Enzyme-linked immunosorbent assay (ELISA) was utilized to assess the activity levels of nitric oxide synthase (NOS) and adenylate cyclase (AC), as well as the cAMP content in the cells. Results In astrocytes, HIV-1 Tat activates NMDAR, leading to an enhanced Ca²⁺ influx and subsequent activation of the CaMKⅡ/AC/cAMP/PKA pathway, inducing an upregulation of AQP4 expression. However, after 36h of HIV-1 Tat induction, the secondary enhanced Ca²⁺ influx within the cells activates the PKG pathway, which inhibits NMDAR. This results in a decrease in Ca²⁺ influx, the expression of factors related to the NMDAR/CaMKⅡ/AC/cAMP/PKA pathway tends to stabilize, and the expression of AQP4 also tends to stabilize. When NMDAR antagonist MK-801 was added simultaneously with HIV-1 Tat induction, there was a decrease in Ca²⁺ influx and a reduction in the expression of AQP4 compared to the HIV-1 Tat-induced group. Despite the inhibition of the NMDAR pathway, the continuous induction of HIV-1 Tat resulted in an upward trend of Ca²⁺ influx. The increased Ca²⁺ influx and the addition of MK-801 led to the suppression of the NMDAR/CaMKⅡ/AC/cAMP/PKA pathway, reaching the peak expression of AQP4 at 24h. After 24h, the secondary enhanced Ca2⁺ influx further inhibited the NMDAR through the PKG pathway, resulting in a peak Ca²⁺ influx at 36h. Subsequently, the Ca²⁺ influx stabilized, and the expression of AQP4 tended to remain steady. Adding PKA inhibitor H89 simultaneously with HIV-1 Tat induction resulted in decreased Ca²⁺ influx and reduced expression of AQP4 compared to the HIV-1 Tat-induced group. Due to the persistent induction of HIV-1 Tat, Ca²⁺ influx continued to show an upward trend. The enhanced Ca²⁺ influx, along with the addition of H89, suppressed the NMDAR/CaMK Ⅱ/AC/cAMP/PKA pathway, and the expression of AQP4 reached its peak at 36h. After 36h, the secondary enhanced Ca²⁺ influx within the cells further inhibited the NMDAR through the PKG pathway, resulting in a stabilization of Ca²⁺ influx at 48h, and the expression of AQP4 also tended to stabilize. When PKG inhibitor KT5823 was added simultaneously with HIV-1 Tat induction, there was a decrease in Ca²⁺ influx and reduced expression of AQP4 compared to the HIV-1 Tat-induced group. However, due to the persistent induction of HIV-1 Tat, Ca²⁺ influx continued to show an upward trend. The enhanced Ca²⁺ influx led to the suppression of the NMDAR/CaMK Ⅱ/AC/cAMP/PKA pathway, resulting in the peak expression of AQP4 at 48h. After 48h, the secondary enhanced Ca²⁺ influx within the cells further inhibited the NMDAR through the PKG pathway, leading to a stabilization of Ca²⁺ influx, and the expression of AQP4 also tended to stabilize. Conclusion This study elucidates the participation of Tat protein in the pathological and physiological processes of HAND, by modulating the expression of AQP4. These findings provide a novel therapeutic target for the treatment of HAND.