小胶质细胞作为中枢神经系统的固有免疫细胞，可通过吞噬作用有序地清除坏死神经元、细胞碎片及病原体并维持整个神经系统的稳定。吞噬作用是细胞内一系列蛋白协同调控的动态过程，包括捕获货物形成吞噬体、吞噬体成熟以及溶酶体降解等步骤。其中，吞噬体成熟过程依赖V-ATPase为溶酶体持续提供酸化，而V-ATPase的质子转运效率则主要取决于a亚基。在哺乳动物中，V-ATPase a亚基有4种亚型(a1、a2、a3和a4)，斑马鱼(Danio rerio)仅有前3种(a1、a2和a3)，且它们所靶向的细胞类型和亚细胞器定位具有显著差异。据报道，V-ATPase a3亚基主要定位于破骨细胞的溶酶体并辅助酸化从而促进骨吸收，本实验室前期的研究结果显示V-ATPase a3亚基同样定位于斑马鱼小胶质细胞的溶酶体，但V-ATPase a3亚基在小胶质细胞发育过程中的作用及其对吞噬体成熟的调控机制尚不明确。本研究利用V-ATPase a3亚基缺陷(tcirg1b^−/−)的斑马鱼模型，通过整体原位杂交、免疫荧光、免疫共沉淀和凋亡实验等探究了V-ATPase a3亚基调控小胶质细胞吞噬体成熟的分子机制。结果显示，V-ATPase a3亚基在斑马鱼发育早期即开始表达，a3亚基缺陷会导致小胶质细胞吞噬体异常堆积，细胞胀大且活跃度降低，呈现“消化不良”表型。通过外源性标记晚期吞噬体并进行免疫荧光染色，进一步验证了a3亚基缺陷会引起小胶质细胞的晚期吞噬体和溶酶体融合障碍。随后，免疫共沉淀实验结果表明V-ATPase a3亚基可与晚期吞噬体标记物Rab7蛋白相结合，而rab7敲低实验可重现小胶质细胞“消化不良”表型，以上结果综合提示V-ATPase a3亚基可能通过结合Rab7并促进小胶质细胞的吞噬体顺利降解。综上所述，本研究阐明了V-ATPase a3亚基对小胶质细胞发育的影响以及其调控吞噬体和溶酶体膜融合的分子机制，为进一步理解吞噬体成熟过程提供了数据支持和理论依据。

Microglia, as the resident immune cells of the central nervous system (CNS), maintain neural homeostasis by orchestrating the clearance of necrotic neurons, cellular debris, and pathogens through phagocytosis. This intracellular process involves a dynamic cascade of protein-mediated events, including cargo recognition and phagosome formation, phagosome maturation, and lysosomal degradation. Phagosome maturation critically relies on V-ATPase-driven acidification, with proton transport efficiency predominantly determined by its a subunit. In mammals, the V-ATPase a subunit has four isoforms (a1, a2, a3 and a4), whereas zebrafish (Danio rerio) possess only the three isoforms (a1, a2 and a3), with marked differences observed in their targeted cell types and suborganellar localization patterns. Notably, the V-ATPase a3 subunit is primarily localized on osteoclast lysosomes, facilitating bone resorption by acidification. The preliminary studies have revealed its conserved lysosomal localization in zebrafish microglia; however, its functional role in microglial development and regulatory mechanisms underlying phagosome maturation remain elusive. To address this knowledge gap, we employed a V-ATPase a3 subunit-deficient (tcirg1b^−/−) zebrafish model, integrating whole-mount in situ hybridization, immunofluorescence, co-immunoprecipitation (Co-IP), and apoptosis assays. Key findings include: The a3 subunit is expressed during early zebrafish development; a3 subunit deficiency caused abnormal phagosome accumulation, microglial swelling and decreased activity, showing an “indigestion-like” phenotype; By exogenous labeling of late phagosomes and immunofluorescence staining, it was confirmed that the defect of V-ATPase a3 subunit impaired late phagosome-lysosome fusion in microglia; Co-IP demonstrated direct binding between the a3 subunit and Rab7. Rab7 knockdown phenocopied the “indigestion” morphology, suggesting a cooperative role in phagosome degradation. These results demonstrate that the V-ATPase a3 subunit regulates microglial phagosome maturation by mediating Rab7-dependent phagolysosomal fusion. In conclusion, this study not only elucidates the molecular mechanism underlying microglial phagocytic capacity but also provides novel insights into the evolutionarily conserved roles of V-ATPase isoforms in cellular clearance pathways.