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

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.