Mechanism of tcirg1b deficiency in disrupting central nervous system homeostasis of zebrafish

Microglial phagocytosis is crucial for maintaining central nervous system (CNS) homeostasis, a process that depends on normal lysosomal acidification and is precisely regulated by vacuolar-type ATPase (V-ATPase). While mutations in the V-ATPase a3 subunit (encoded by the tcirg1b gene in zebrafish) are a major cause of human malignant osteopetrosis, the subunit's function in the CNS remains unknown. To investigate the role of the V-ATPase a3 subunit in the zebrafish CNS, we generated a tcirg1b knockout model. Although mutant zebrafish displayed no early neuronal defects, adult brains exhibited significant pathological alterations and behavioral abnormalities. Previous data showed that loss of tcirg1b resulted in enlarged microglia, suggesting potential functional alterations. In this study, transcriptome sequencing analysis of zebrafish macrophages revealed that phagosome formation and intracellular pH regulation pathway genes were significantly down-regulated. Functional analysis confirmed that V-ATPase a3 subunit deficiency impairs lysosomal acidification and digestive function in microglia, leading to the accumulation of apoptotic cell debris and TMR-dextran. Notably, specific restoration of tcirg1b expression in microglia successfully rescued the behavioral phenotypes of mutants, suggesting that the regulation of CNS homeostasis by the V-ATPase a3 subunit is primarily mediated through microglia. In summary, this study provides the first in vivo evidence that tcirg1b deficiency disrupts microglial function, thereby indirectly leading to an imbalance in CNS homeostasis. Our findings reveal a key role for the V-ATPase a3 subunit in regulating neural homeostasis and offer a new theoretical framework for studying the mechanisms of neurological diseases.