Regulation of AQP4 Expression and Investigation of the Underlying Mechanisms by HIV-1 Tat Through the NMDAR/cAMP/PKA Signaling Pathway in Astrocytes

Chuo  Li; Ran  Duan; Congcong  Fu

doi:10.18502/ijaai.v25i4.21746

Chuo Li Department of Neurology, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
Ran Duan The Second Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou, China
Congcong Fu Department of Neurology, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China

DOI: https://doi.org/10.18502/ijaai.v25i4.21746

Keywords: AQP4; HAND; NMDAR/cAMP/PKA signaling pathway; Tat protein

Abstract

Human immunodeficiency virus (HIV)-associated neurocognitive disorder (HAND) remains a major neurological complication in people living with HIV despite effective antiretroviral therapy. Neurotoxicity caused by viral proteins, particularly the HIV-1 transactivator of transcription (Tat), contributes significantly to HAND. Although N-methyl-D-aspartate receptors (NMDARs) in astrocytes are known to regulate aquaporin-4 (AQP4) the mechanisms by which Tat influences NMDAR signaling and AQP4 expression remain unclear. This study investigated how HIV-1 Tat regulates AQP4 expression in astrocytes through the NMDAR/CaMKII/AC/cAMP/PKA signaling pathway and how secondary Ca²⁺ dynamics modulate this process.

Astrocytic Ca²⁺ influx was measured using the Fluo-3 AM probe. Western blotting quantified AQP4, NR1, NR2A/B, CaMKII, p-CaMKII, PKA, and PKG expression. Real-time quantitative polymerase chain reaction (RT-qPCR) assessed mRNA levels of AQP4 and NMDAR-related genes. Enzyme-linked immunosorbent assay (ELISA) evaluated nitric oxide synthase activity, adenylate cyclase activity, and intracellular cAMP levels. Pharmacologic inhibitors-MK-801 (NMDAR blocker), H89 (PKA inhibitor), and KT5823 (protein kinase G [PKG] inhibitor)-were applied to investigate pathway interactions.

HIV-1 Tat induced robust activation of NMDAR, resulting in increased Ca²⁺ influx and sequential activation of the CaMKII/AC/cAMP/PKA pathway, ultimately elevating AQP4. After prolonged Tat exposure (approximately 36 hours), a secondary surge in Ca²⁺ activated PKG, which acts as a protective negative feedback mechanism to inhibit excessive NMDAR activity, thereby stabilizing Ca²⁺ influx and preventing abnormal overexpression of AQP4. Cotreatment with MK-801, H89, or KT5823 suppressed Tat-induced Ca²⁺ influx and attenuated AQP4 upregulation, although persistent Tat exposure gradually restored Ca²⁺ elevations through compensatory mechanisms.

HIV-1 Tat dynamically regulates AQP4 expression in astrocytes via the NMDAR/CaMKII/AC/cAMP/PKA pathway, with PKG-mediated feedback contributing to later stabilization. These findings highlight AQP4 as a potential therapeutic target for HAND.