Molecular glue neferine induces BAP31 homodimerisation to disrupt endoplasmic reticulum–mitochondria crosstalk for anti‐neuroinflammation in ischaemic stroke

BACKGROUND AND PURPOSE: B-cell receptor-associated protein 31 (BAP31), an endoplasmic reticulum (ER)-resident transmembrane protein, has emerged as a critical regulator of immune cell activation, yet its role in neuroinflammation remains unexplored. Here, we uncovered the natural compound neferine (Nef) as a pharmacological modulator of BAP31 that suppressed microglial activation. EXPERIMENTAL APPROACH: Using thermal protein profiling (TPP), we identified BAP31 as the primary target of Nef. Biochemical and structural analyses were employed to characterise Nef-BAP31 interactions. We evaluated ER stress and mitochondrial energy metabolism homeostasis using techniques such as STER super-resolution technology, flow cytometry, western blot, etc. In vivo validation utilised two models: lipopolysaccharide (LPS)-induced endotoxaemia and middle cerebral artery occlusion (MCAO) for ischaemic stroke, combining behavioural tests, cytokine profiling and histopathological assessments. KEY RESULTS: Nef functioned as a 'molecular glue' by binding to BAP31's coiled-coil CC2 domain to induce stable dimerisation. We revealed that dimerised BAP31 triggered ER membrane remodelling, which disrupted ER-mitochondria contact sites and preserved mitochondrial energy metabolism homeostasis, thereby blocking inflammatory cytokine release. In vivo, Nef attenuated neuroinflammation in endotoxaemia mice and further conferred neuroprotection against ischaemic stroke in a MCAO model by inhibiting microglia-driven neuronal injury. CONCLUSIONS AND IMPLICATIONS: In summary, our work reveals that BAP31 is a master regulator of ER-mitochondria communication during microglial activation and introduces a paradigm-shifting 'molecular glue' strategy for targeting ER-resident proteins. Additionally, these findings redefine the pharmacological landscape for modulating organelle interactions in microglia involved in neuroinflammatory diseases.