["The Journal of Physiology, EarlyView. ", "\nAbstract figure legend This study demonstrates that cholinergic neuron‐specific heat shock protein 60 (HSP60) is crucial in modulating lipopolysaccharide (LPS)‐induced neuroinflammation and depressive‐like behaviours in mice. HSP60 knockout mice exhibited mitigated weight loss and alleviated depressive‐like behaviours, with behavioural tests confirming these effects. Additionally, HSP60 deficiency reversed LPS‐induced pro‐inflammatory cytokine elevation and neuroinflammation markers in the hippocampus, underscoring its therapeutic potential.\n\n\n\n\n\n\n\n\n\nAbstract\nDepression is a significant global health issue characterized by complex underlying mechanisms. This study aimed to investigate the role of cholinergic neuron‐specific heat shock protein 60 (HSP60) in modulating lipopolysaccharide (LPS)‐induced neuroinflammation and depressive‐like behaviours in mice.\nCholinergic neuron‐specific HSP60 knockout mice were generated by crossing Hsp60‐flox mice with Chat‐cre mice. Genotyping confirmed the successful creation of the knockout. The effects of LPS on weight loss, behavioural changes, cytokine levels, neuroinflammation markers and signalling pathway proteins were assessed. Behavioural assessments included the tail suspension test and sucrose preference test.\nHSP60 knockout mice exhibited mitigated weight loss in response to LPS. Behavioural tests indicated that HSP60 deficiency alleviated LPS‐mediated depressive‐like behaviours without affecting locomotor activity. In the hippocampus, LPS treatment significantly altered cytokine levels, increasing pro‐inflammatory cytokines at the same time as decreasing anti‐inflammatory cytokines. HSP60 knockout mice partially reversed these effects, showing increased anti‐inflammatory and decreased pro‐inflammatory cytokines. LPS‐induced upregulation of neuroinflammation markers such as glial fibrillary acidic protein, NLRP3 (i.e. NOD‐, LRR‐ and pyrin domain‐containing protein 3) and p‐IKKα/β [i.e. the phosphorylated forms of the catalytic subunits of the IκB kinase (IKK) complex, specifically IKKα (CHUK) and IKKβ] was significantly reduced in HSP60 knockout mice. Additionally, LPS‐induced elevation of phosphorylated eukaryotic translation initiation factor 2α levels in the hippocampus was attenuated by HSP60 deficiency, without affecting other signalling pathway proteins.\nThese findings suggest that HSP60 in cholinergic neurons plays a critical role in regulating LPS‐induced neuroinflammation and depressive‐like behaviours. Targeting HSP60 in cholinergic neurons may provide a therapeutic approach for mitigating neuroinflammation and associated depressive symptoms.\n\n\n\n\n\n\n\n\n\nKey points\n\nSpecific knockout of HSP60 in cholinergic neurons could protect against LPS‐induced physiological and behavioral impairments, including mitigated weight loss, improved depressive‐like behaviors, and unaffected locomotor activity.\nCholinergic neuron‐specific HSP60 deletion could attenuate neuroinflammation by reversing LPS‐induced cytokine imbalance, suppressing key inflammatory markers (GFAP, NLRP3, cGAS, p‐IKKα/β), and crucially, by preserving hippocampal acetylcholine levels, a key neurotransmitter with established anti‐inflammatory properties.\nHSP60 deficiency selectively reduces the LPS‐induced phosphorylation of the eukaryotic translation initiation factor 2α (p‐eIF2α) in the hippocampus, indicating a targeted modulation of the cellular stress and protein synthesis regulation pathway, without altering other major signaling molecules.\n\n\n"]