A selective JNK3-targeting miniprotein confers neuroprotection in a mouse model of ischemic stroke

Abstract Ischemic stroke remains a leading cause of death and long-term disability, and current reperfusion therapies do not prevent progressive neuronal loss and neurological decline. Thus, therapeutic strategies targeting intracellular pathways underlying post-ischemic neurodegeneration remain a critical unmet clinical need. The neuron-enriched kinase c-Jun N-terminal kinase 3 (JNK3) is a key mediator of ischemia-induced neuronal death. Still, efforts to therapeutically inhibit this pathway have been limited by poor isoform selectivity and toxicity. Here, we tested whether specific modulation of pathological JNK3 signaling could provide sustained neuroprotection after stroke. Starting from structural information on the interaction between JNK3 and its selective scaffold protein β-arrestin2 (BA2), we designed a BA2-derived miniprotein to attenuate ischemia-induced JNK3 signaling. This strategy led to the development of SIMBA2, a brain-penetrant miniprotein that selectively modulates JNK3 signaling and reduces phosphorylation of the pro-apoptotic effector c-Jun. In a mouse model of transient middle cerebral artery occlusion, SIMBA2 administered 3 h post-ischemia reduced infarct volume, cerebral edema, and cytotoxic damage, while improving functional recovery and preserving neuronal and synaptic integrity. These effects persisted for at least 7 days, consistent with sustained neuroprotection. Circulating JNK3 blood levels increased after ischemia but were reduced in SIMBA2-treated mice, consistent with reduced neuronal injury. Notably, this reduction of plasma JNK3 correlated with neuroprotection. Together, these findings identify SIMBA2 as a selective JNK3-targeting miniprotein that confers sustained neuroprotection after ischemic stroke, highlighting its potential as a neuroprotective therapy that may complement reperfusion treatments. Moreover, plasma JNK3 levels may represent a peripheral biomarker associated with ischemic neuronal injury.