Song Xin Ying Rachel, Jansen Margo Iris, Marzagalli Rubina, Musumeci Giuseppe, D'Agata Velia, Castorina Alessandro
Laboratory of Cellular and Molecular Neuroscience, School of Life Sciences, Faculty of Science, University of Technology Sydney, Sydney, NSW 2007, Australia.
Department of Biomedical and Biotechnological Sciences, Section of Anatomy, Histology and Movement Sciences, University of Catania, 95100 Catania, Italy.
Cells. 2025 May 23;14(11):769. doi: 10.3390/cells14110769.
Microglial cells, the resident immune cells of the central nervous system (CNS), are essential for maintaining CNS homeostasis. Dysregulation of microglial function is implicated in the pathogenesis of various neurodegenerative diseases. Vasoactive intestinal polypeptide receptors 1 and 2 (VPAC1 and VPAC2) are G-protein-coupled receptors (GPCRs) expressed by microglia, with their primary ligands being pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP). However, the specific roles of VPAC-type receptors in microglial regulation remain poorly understood. In this study, we generated VPAC1 and VPAC2 BV2 microglial cell lines using CRISPR-Cas9 gene editing and conducted a series of biological and molecular assays to elucidate the functions of these receptors. Our findings demonstrated that both mutant cell lines exhibited a polarized phenotype and increased migratory activity. VPAC1 cells showed enhanced survivability and baseline activation of the unfolded protein response (UPR), a protective mechanism triggered by endoplasmic reticulum (ER) stress, whereas this response appeared impaired in VPAC2 cells. In contrast, under lipopolysaccharide (LPS)-induced inflammatory conditions, UPR activation was impaired in VPAC1 cells but restored in VPAC2 cells, resulting in improved survival of VPAC2 cells, whereas VPAC1 cells exhibited reduced resilience. Overall, our findings suggest that VPAC1 and VPAC2 receptors play distinct yet complementary roles in BV2 microglia. VPAC2 is critical for regulating survival, ER stress responses, and polarization under basal conditions, while VPAC1 is essential for adaptive responses to inflammatory stimuli such as LPS. These insights advance our understanding of microglial receptor signaling and may inform therapeutic strategies targeting microglial dysfunction in neurodegenerative diseases.
小胶质细胞是中枢神经系统（CNS）中的常驻免疫细胞，对于维持CNS的稳态至关重要。小胶质细胞功能失调与多种神经退行性疾病的发病机制有关。血管活性肠肽受体1和2（VPAC1和VPAC2）是小胶质细胞表达的G蛋白偶联受体（GPCR），其主要配体是垂体腺苷酸环化酶激活多肽（PACAP）和血管活性肠肽（VIP）。然而，VPAC型受体在小胶质细胞调节中的具体作用仍知之甚少。在本研究中，我们使用CRISPR-Cas9基因编辑技术构建了VPAC1和VPAC2基因敲除的BV2小胶质细胞系，并进行了一系列生物学和分子分析以阐明这些受体的功能。我们的研究结果表明，两种突变细胞系均表现出极化表型并增强了迁移活性。VPAC1基因敲除细胞显示出增强的生存能力和未折叠蛋白反应（UPR）的基线激活，UPR是一种由内质网（ER）应激触发的保护机制，而这种反应在VPAC2基因敲除细胞中似乎受损。相反，在脂多糖（LPS）诱导的炎症条件下，VPAC1基因敲除细胞中的UPR激活受损，但在VPAC2基因敲除细胞中恢复，从而导致VPAC2基因敲除细胞的存活率提高，而VPAC1基因敲除细胞的恢复力降低。总体而言，我们的研究结果表明，VPAC1和VPAC2受体在BV2小胶质细胞中发挥着不同但互补的作用。VPAC2对于在基础条件下调节存活、内质网应激反应和极化至关重要，而VPAC1对于对LPS等炎症刺激的适应性反应至关重要。这些见解推进了我们对小胶质细胞受体信号传导的理解，并可能为针对神经退行性疾病中小胶质细胞功能障碍的治疗策略提供参考。
