Hippocampal Caspase-4 non-canonical inflammasome activation is associated with microglia-synapse crosstalk disruption and drives depression-like behaviors.

BACKGROUND: The neuroinflammation hypothesis has reshaped our understanding of depression; however, most studies have focused on the canonical NLRP3 inflammasome, leaving the role of the non-canonical inflammasome, particularly Caspase-4, largely unexplored. This study aimed to investigate whether hippocampal non-canonical inflammasome activation drives microglial neuroinflammation and impairs synaptic plasticity to precipitate depression-like behavior. METHODS: Male C57BL/6J mice were subjected to chronic unpredictable mild stress (CUMS) for 4 weeks, followed by behavioral testing tail suspension test (TST), forced swimming test (FST), sucrose preference test (SPT), novelty-suppressed feeding test (NSF), and open field test (OFT) and hippocampal transcriptomic profiling. Because the murine genome lacks a Caspase-4 ortholog (its functional counterpart being Caspase-11), human Caspase-4 (hCasp4) was ectopically overexpressed in the mouse hippocampus via adeno-associated virus (AAV9-CMV-hCasp4-eGFP) stereotaxic injection, with AAV9-CMV-eGFP as the control. Reverse-transcription quantitative PCR (RT-qPCR) and western blot were used to detect Caspase-4 expression. Immunofluorescence was performed to detect NLRP3, nuclear factor-κB (NF-κB) p65, guanylate-binding protein 1 (GBP1), ionized calcium-binding adapter molecule 1 (IBA-1), Ca2+/calmodulin-dependent protein kinase II (CaMKII), postsynaptic density protein 95 (PSD95), and Synapsin1, with semi-quantitative and co-localization analyses using ImageJ. RESULTS: CUMS induced depression-like behaviors and significantly upregulated hippocampal Caspase-4 expression, accompanied by with significant enrichment of the NOD-like receptor signaling pathway and activation of the NLRP3 inflammasome enrichment. Ectopic hippocampal expression of human Caspase-4 was sufficient to reduce sucrose preference and increase immobility time in the TST, without affecting locomotor activity. Mechanistically, Caspase-4 activation elevated overexpression was accompanied by elevated GBP1 and increased promoted IBA-1+ microglial immunoreactivity activation, enhanced NLRP3 expression and NF-κB p65 nuclear immunoreactivity, and ultimately suppressed hippocampal CaMKII expression while decreasing together with decreased PSD95 and Synapsin1 levels and their spatial co-localization area. These molecular associations are correlative; loss-of-function, rescue, and functional synaptic assays were not performed. CONCLUSIONS AND RELEVANCE: Hippocampal Caspase-4-associated non-canonical inflammasome activation via Caspase-4 serves as a critical upstream driver of depression-like behavior by orchestrating GBP1-dependent microglial inflammation and disrupting CaMKII-dependent synaptic plasticity signaling may contribute to depression-like behavior through a putative pathway involving GBP1-related microglial inflammation and downregulation of CaMKII and synaptic proteins. Because the evidence is partly correlative and the model relies on ectopic expression of human Caspase-4 in mice, these findings should be regarded as hypothesis-generating. Targeting the Caspase-4 non-canonical inflammasome warrants further investigation as a potential, may offer a novel immunotherapeutic strategy for depression.