Investigating the neurodevelopmental toxicity of graphene oxides using 3D human brain organoids and zebrafish models: emphasis on GABAergic neuron alterations at single-cell resolution.

Exposure to graphene oxides (GOs) may induce neurotoxic effects, but the mechanisms and extent of these effects remain unclear, partly due to limitations in traditional neurotoxicity models. In this study, we utilized 3D human brain organoids and zebrafish larvae to investigate GO-induced neurodevelopmental toxicity. In 3D human brain organoids, GOs significantly reduced cellular viability in a dose-dependent manner, with bulk RNA-sequencing indicating minimal impact on neuron-related pathways. However, single-cell RNA-sequencing revealed significant alterations in glutamatergic and GABAergic neurons, particularly in gene ontology terms related to synaptic function and neuronal development. Further analysis showed that GO exposure affected the localization, morphology, and electrophysiological activity of GABAergic neurons. Additionally, key genes involved in neurodegeneration (ko05022) and protein processing in endoplasmic reticulum (ko04141) were dysregulated in GABAergic neurons. In vivo studies using zebrafish larvae demonstrated altered locomotor behaviors, including hyperactivity during dark cycles and reduced swimming distances during light cycles, alongside a loss of GABAergic neurons in specific brain regions. These findings highlight the potential neurodevelopmental toxicity of GOs, especially through their impact on GABAergic neurons, and underscore the value of combining 3D human brain organoids and zebrafish models with single-cell resolution techniques to better understand the neurotoxic effects of nanomaterials.