Role of the NLRP3/caspase-1/GSDMD signaling pathway in dopaminergic neurons during Japanese encephalitis virus infection

Abstract

Background: Japanese encephalitis virus (JEV) is a highly neurotropic virus that can enter the central nervous system after high levels of peripheral replication, causing severe neuroinflammation. JEV targets dopaminergic neuron-rich areas such as the thalamus, midbrain, and striatum, causing pyroptosis. Pyroptosis, which is a novel mode of programmed cell death, plays an important role in infection by other flaviviruses, but whether it plays a role in JEV-infected neurons has not been confirmed. Methods: In this study, the damaging effect of JEV infection on dopaminergic neurons was examined in vivo and in vitro through HE staining, immunohistochemical staining, double immunofluorescence labeling, determination of cytotoxicity, qPCR and western blotting for testing pyroptosis. Results: Mice infected with JEV showed glial cell activation and proliferation, neuronal degeneration and necrosis, perivascular cuffing, and neuronophilia in brain tissue, and a large number of microglia were significantly activated around the virus-positive signal, as shown by immunofluorescence staining. JEV specifically targeted dopaminergic neurons, activated the nucleotide oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3)/cysteinyl aspartate specific proteinase-1 (caspase-1)/gasdermin D (GSDMD) signaling pathway and induced an inflammatory response. In vitro infection of mouse midbrain dopaminergic neuron cells revealed that JEV significantly replicated in MN9D cells. Infected cells were significantly lesioned at 24 hpi, and the number of lesioned cells increased significantly with time. JEV infection resulted in a gradual increase in LDH release, indicating damage to the cytosol. JEV clearly colocalized with MN9D cells, and apoptosis occurred via an NLRP3/caspase-1/GSDMD-dependent pathway. Conclusions: These results show that inhibiting pyroptosis can delay motor impairment caused by JEV to some extent and that protecting dopaminergic neurons from viral infection may be an effective strategy for the treatment of JEV with late effects.