A Brain‐Penetrant Nanobody Reveals GSK3β‐Driven Proline‐Directed Phosphorylation as a Master Regulator of Ischemic Neurodegeneration

ABSTRACT Serine/threonine‐proline (S/T‐P) phosphorylation is a fundamental mechanism maintaining cellular homeostasis. Although glycogen synthase kinase 3β (GSK3β) is a key regulator in ischemic stroke, the contribution of its proline‐directed kinase activity to cellular dysfunction and disease progression remains unclear. Here, we developed Nb.29E9, a nanobody that selectively targets the proline‐directed kinase domain of GSK3β. Under ischemic conditions, Nb.29E9 inhibited S/T‐P phosphorylation of key substrates, including RNA‐binding motif protein 38 (RBM38), HIF1α, and p53, thereby enhancing neuronal and microglial viability while reducing oxidative stress and neuroinflammation. Phosphoproteomic analysis revealed broad reprogramming of S/T‐P phosphorylation networks. In mice after ischemic injury, Nb.29E9 delivered via a brain‐penetrant, MMP‐9‐responsive nanoparticle reduced infarct volume, restored neurovascular integrity, and improved motor function. Mechanistically, Nb.29E9 corrected pathological hyperphosphorylation of SMAD2/3‐Thr8 (TGFβ signaling), calcium/calmodulin‐dependent protein kinase kinase 2 (CAMKK2)‐Ser495 (AMPK pathway), and AKT1 substrate 1 (AKT1S1)‐Ser183 (mTORC1 regulation). These findings demonstrate that GSK3β’s intrinsic proline‐directed kinase activity drives ischemic neurodegeneration, establishing its pathogenic role in vivo.