Tehran University of Medical Sciences Iranian Journal of Allergy, Asthma and Immunology 1735-1502 0 0 2026 04 16 1 15 Chuo Li Department of Neurology, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China AND Institute of Infectious Diseases, Guangzhou Eighth People’s Hospital, Guangzhou Medical University, Guangzhou, China AND Guangzhou Key Laboratory of Clinical Pathogen Research for Infectious Diseases, 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 AND Institute of Infectious Diseases, Guangzhou Eighth People’s Hospital, Guangzhou Medical University, Guangzhou, China AND Guangzhou Key Laboratory of Clinical Pathogen Research for Infectious Diseases, Guangzhou, China 2025 11 26 2025 12 24 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.  https://ijaai.tums.ac.ir/index.php/ijaai/article/view/4683 https://ijaai.tums.ac.ir/index.php/ijaai/article/download/4683/2320