Vps34 knockdown attenuated AlNPs-induced neurodevelopmental toxicity in zebrafish larvae and adults by inhibiting mitophagy

Nano-alumina (AlNPs) are susceptible to inducing neurotoxicity, mainly through excessive autophagy/mitophagy. Vacuolar protein sorting 34 (Vps34) is a target for modulating autophagy. This study aimed to investigate whether Vps34 knockdown could reduce AlNPs' neurodevelopmental toxicity. Zebrafish embryos were exposed to 100 mg/L of 13 nm AlNPs and 2.5 mmol/L of 3-methyladenine (3MA) until 144 h post-fertilization (hpf) to assess their development, neurobehavior, oxidative stress, and the expression of autophagy-related gene. Vps34 was knocked down using 500 µL of morpholino oligonucleotide (MO) at 1 mM. Additionally, embryos were treated with control, negative control, Vps34 knockdown, AlNPs (100 mg/L), and AlNPs + Vps34 knockdown. Evaluations included developmental parameters, locomotor activity, oxidative stress, autophagy/mitophagy gene expression, and neuronal cell counts at 144 hpf. Long-term effects were assessed up to 90 dpf, including locomotor activity, mitophagy-related genes expression, and ultrastructural analysis. The findings showed that 3MA effectively counteracted AlNPs-induced developmental stunting at 24 hpf, neurobehavioral impairments, oxidative damage, and upregulation of autophagy-related Beclin1, Vps34, and LC3II genes in larvae. AlNPs significantly elevated Vps34 gene expression in zebrafish. Additionally, Vps34 knockdown alleviated AlNPs-induced developmental retardation at 24 hpf, locomotor deficits, oxidative damage, neuronal loss, and abnormal expression of autophagy/mitophagy-related genes and P62 protein levels. Vps34 knockdown ameliorated AlNPs' early developmental retardation and neurobehavioral deficits in zebrafish larvae and adults by reducing mitophagy. These findings suggest that Vps34 could be a promising therapeutic target for attenuating the neurotoxic effects of AlNPs.