Cry2 deficiency leads to cognitive impairment through the microbiota-gut-brain axis mediated S1P/NLRP3/IL-1β pathway in mice

Alzheimer’s disease (AD) is characterized by extracellular Aβ deposition and tau hyperphosphorylation, leading to synaptic dysfunction and cognitive decline. Mounting evidence indicates that circadian rhythm disorders are associated with increased AD risks. Growing evidence implicates the microbiota-gut-brain axis and its metabolites as critical modulators of both circadian physiology and AD pathology. However, the molecular mechanism through which circadian disturbance modulates gut-brain communication to influence AD pathogenesis remains poorly understood. Core circadian clock gene expression was assessed across four AD human brain datasets, and found Cry2 to be the only gene consistently downregulated. To investigate its functional role in vivo, we established a mouse model with hippocampal-specific Cry2 knockdown. Cognitive performance, gut microbiota composition, and metabolic alterations were evaluated using the Morris water maze, 16 S rRNA sequencing, and untargeted metabolomics, respectively. Intestinal barrier integrity, blood-brain barrier function, and neuroinflammatory signaling were examined through immunohistochemistry, immunofluorescence, and Western blotting. The contribution of microbiota disturbance was tested using fecal microbiota transplantation (FMT). The involvement of sphingolipid signaling was further assessed through FMT, pharmacological modulation with the S1PR agonist FTY720, NLRP3 knockout mice, and microglial assays. We found that the expression of Cry2 consistently decreased in the AD group in four AD-related datasets. Then, knockdown of Cry2 in the hippocampus (shCry2) caused dysbiosis of gut microbiota, intestinal barrier impairment, cognitive dysfunction and tau pathology in mice. Intriguingly, along with the disturbance in intestinal sphingolipid metabolism pathways, activation of the S1P/NLRP3/IL-1β pathway was found in the brain of shCry2 mice. Transplantation of “shCry2 microbiota” mimicked the pathological and behavioral changes induced by hippocampal Cry2 deficiency. Administration of S1PR agonist FTY720 significantly improved cognitive impairment and decreased the expression of NLRP3 in shCry2 mice, and knockdown of Cry2 in NLRP3−/− mice alleviated tau pathology and cognitive impairment. FTY720 and S1PR1 antagonist W146 dose-dependently modulated the expression of NLRP3 in BV2 cells. Overexpressing Cry2 in the hippocampus significantly alleviated the tau pathology and cognitive decline in APP/PS1 mice. Hippocampal Cry2 deficiency leads to cognitive impairment through the gut-brain axis mediated S1P/NLRP3/IL-1β pathway and might provide a potential therapeutic target for AD.