# Japanese encephalitis virus activates the NLRP3/caspase-1/GSDMD signaling pathway in dopaminergic neurons

## TL;DR

This study shows that Japanese encephalitis virus harms dopaminergic neurons by triggering a specific inflammatory cell death pathway, suggesting new targets for treatment.

## Contribution

The study provides evidence that JEV activates the NLRP3/caspase-1/GSDMD pathway in dopaminergic neurons, contributing to neuroinflammation.

## Key findings

- JEV targets dopaminergic neurons and activates the NLRP3 inflammasome, causing neuroinflammation.
- Inhibiting NLRP3 reduces viral load and clinical symptoms but does not prevent glial-mediated inflammation.
- Sustained glial activation may drive inflammation independently of NLRP3, suggesting combined targeting is needed.

## Abstract

Japanese encephalitis virus (JEV) preferentially targets brain regions rich in dopaminergic neurons, including the thalamus, midbrain, and striatum, leading to severe neuroinflammation. However, the underlying pathological mechanisms remain unclear. This study hypothesizes that JEV-induced pyroptosis exacerbates neuroinflammation by activating the NLRP3/caspase-1/GSDMD signaling pathway. In vivo experiments using JEV-infected mice revealed virus-specific targeting of dopaminergic neurons, concurrent activation of the NLRP3 inflammasome, and induction of inflammatory responses. In vitro studies with mouse midbrain dopaminergic cells showed significant viral replication, progressive cell membrane damage, and colocalization of JEV with pyroptotic markers. The pharmacological inhibition of NLRP3, while suppressing viral load and NLRP3 inflammasome activation, fails to delay the onset of neuroinflammation, potentially due to the activation of glial cells. This suggests that glial cell-mediated inflammatory pathways independent of NLRP3 may drive neuroinflammation despite NLRP3 inhibition. The unimpeded activation of glia could sustain pro-inflammatory responses, highlighting the need to target glial activation alongside NLRP3 to effectively mitigate neuroinflammation.

Japanese encephalitis virus infection can induce severe neurological symptoms, including tremors, slow movements, and motor impairments, indicating severe infection of dopaminergic neurons. These symptoms primarily occur in the later stages of infection, suggesting that sustained inflammatory responses may be the primary cause of neuronal damage. Our research team has previously confirmed significant upregulation of genes related to NLRP3, caspase-1, GSDMD, and other molecules after infection, indicating their involvement in neuroinflammation. Pyroptosis, as a mechanism of inflammatory cell death, has been shown to participate in many viral infections, but there is no specific evidence supporting its exact pathogenic mechanism in JEV-infected neurons. To determine the role of pyroptosis in JEV infection, we conducted cell infection and animal infection experiments. After confirming the crucial role of NLRP3, corresponding inhibitors were used in regression experiments, confirming that inhibiting the NLRP3-related signaling pathway effectively alleviates the clinical symptoms induced by the virus.