In clathrin-mediated membrane trafficking, adaptin proteins (AP1, AP2, AP3) play a pivotal role by binding to clathrin, cargo, and accessory proteins, thereby serving as essential contributors to both vesicle formation and sorting (Shin et al., 2021). The APs assemble as heterotetramers, comprising a small (σ), medium (μ), and two large (γ, α, δ, ϵ or ζ and β1–5) subunits. AP1 is specifically involved in clathrin-mediated transport between the trans-Golgi network and the endosome, while the AP2 adaptor assumes a central role in clathrin-mediated endocytosis (CME). Since knocking out any subunit of AP complex results in its inactivation, the knockout of a specific subunit is a common strategy for functional studies. The evidence indicates that a deficiency in the AP1 subunit Ap1g1 causes inner ear, retina, thyroid, testes, and neurodevelopmental disorders in mice, and human AP1G1 variants lead to neurodevelopmental deficiency (Johnson et al., 2016; Usmani et al., 2021). Deficiency in AP1M1 results in a typical NPC-like phenotype characterized by an intracellular accumulation of cholesterol (Poirier et al., 2013). Mutations in AP1S3 cause pustular psoriasis, an immune disease in skin tissue (Setta-Kaffetzi et al., 2014). AP2A1 is reported to be involved in the synaptic vesicle cycle pathway and related neurodegenerative diseases (Wang et al., 2017), and AP2A2 improves β-adrenergic signaling in adipose tissue, increasing lipolysis in adipocytes (Montgomery et al., 2019).

AAGAB (alpha and gamma adaptin binding protein), identified in a genome-scale CRISPR screen of CME, is crucial for the formation of AP1 and AP2 complexes (Owen et al., 2004). AAGAB mutation abolishes AP1 and AP2 assembly, leading to the degradation of AP1 and AP2 subunits and disruption of CME, thereby disrupting AP1- and AP2-mediated cargo trafficking simultaneously. This disruption reflects the combinatorial effects of AP1 and AP2 deficiency (Gulbranson et al., 2019; Wan et al., 2021). The human genetic disease associated with AAGAB gene mutation is punctate palmoplantar keratoderma type 1 (PPPK1). PPPK1 is a rare autosomal dominant disorder characterized by hyperkeratotic papules on the palms and soles, and AAGAB dimerization mediated by its C-terminal domain (CTD) is critical for AAGAB stability, and is missing in mutant proteins found in patients with this skin disease (Pohler et al., 2012; Giehl et al., 2016; Wan et al., 2021; Tian et al., 2023). However, due to the lack of aagab deficient animal model, the function of aagab gene during embryonic development remains unclear.

In this study, we constructed a loss-of-function mutation of aagab in zebrafish. aagab mutants exhibited swimming defects and died during 4-10 days post fertilization (dpf). To explain these phenotypes, we focused on the function of aagab in the brain, where gene expression is high and ubiquitous, and clathrin-mediated endocytosis is essential for synaptic vesicle formation and recycling, influencing synaptic transmission and neuronal communication (Koenig and Ikeda, 1989). Although the gross morphology of neurons remained normal in mutants, neural activity significantly decreased at 7 dpf. The attenuated synaptic vesicle (SV) retrieval also down-regulated the neurotransmitter release of tectal neurons. Therefore, Aagab plays an important role in the nervous system of zebrafish, regulating neural activity and swimming behavior. Additionally, our study also reveals that the amino acids of the missense mutations in patients (V147, E288) and the highly conserved sequence at the C-terminus are all indispensable for the survival of the mutants. Therefore, our study sheds light on the biological function of Aagab in the nervous system, providing insights into the underlying pathology and potential treatments for AAGAB mutation-associated genetic disease.