# Oxidative stress-driven disease-associated microglia (DAM)-like polarization in human microglia (HMC3) cells exposed to small-size silver nanoparticles in a transwell co-culture system with neurons (cholinergic differentiated SH-SY5Y) cells in vitro

## TL;DR

Small silver nanoparticles may cause harmful inflammation in brain cells, potentially contributing to neurodegenerative diseases.

## Contribution

Demonstrates that small silver nanoparticles induce a disease-associated microglia (DAM)-like response in human microglia-neuron co-cultures via oxidative stress and inflammation.

## Key findings

- Small AgNPs caused dose- and time-dependent cytotoxicity in human microglia and neurons.
- AgNP exposure activated TLR4/NF-κB pathways, leading to upregulation of DAM-related proteins like ICAM1 and ApoE.
- Neuronal dysfunction was indicated by reduced SYN1/SNAP-25 and increased oxidative stress markers in SH-SY5Y cells.

## Abstract

Silver nanoparticles (AgNPs) are well-established pro-oxidative and pro-inflammatory factors, with the strongest effects observed in smaller NPs. This is particularly important due to the increasing accumulation of AgNPs in the environment and the human food chain in recent years. This accumulation has been correlated with an increased number of neurodegenerative diseases, in which microglia-driven inflammation plays a pivotal role. Importantly, the disease-associated microglia (DAM) phenotype has gained special attention. Therefore, this study aimed to determine the role of small-size AgNPs in inducing a DAM-like phenotype in a transwell co-culture model of human microglia (HMC3) and neurons (differentiated SH-SY5Y) in vitro. Our results demonstrated time- and dose-dependent cytotoxicity of small-size AgNPs in both tested cell models. Moreover, in the transwell co-culture system of HMC3 and differentiated SH-SY5Y cells, exposure to AgNPs led to ROS-dependent inflammation, associated with TLR4 and NF-κB activation, subsequently upregulating DAM-related proteins (e.g., ICAM1, ITGAX, ApoE, and B2M). A time-dependent increase in •NO levels was also observed. Notably, reduced SYN1 and SNAP-25 expression, along with increased CAT, SOD, TRAF6, and TLR4 expression in differentiated SH-SY5Y cells, indicates ROS- and inflammation-mediated neuronal dysfunction. These findings indicate that the use of small-size AgNPs should be reconsidered due to their potential neurotoxic effects, but further investigation is necessary in future, using in vivo models.

The online version contains supplementary material available at 10.1007/s00204-025-04183-0.