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Unlabelled: Phosphorylation of serine/threonine residues preceding a proline regulates the fate of its targets through postphosphorylation conformational changes catalyzed by the peptidyl-prolyl cis-/trans isomerase Pin1. By flipping the substrate between two different functional conformations, this enzyme exerts a fine-tuning of phosphorylation signals. Pin1 has been detected in dendritic spines and shafts where it regulates protein synthesis required to sustain the late phase of long-term potentiation (LTP). Here, we demonstrate that Pin1 residing in postsynaptic structures can interact with postsynaptic density protein-95 (PSD-95), a key scaffold protein that anchors NMDA receptors (NMDARs) in PSD via GluN2-type receptor subunits. Pin1 recruitment by PSD-95 occurs at specific serine-threonine/proline consensus motifs localized in the linker region connecting PDZ2 to PDZ3 domains. Upon binding, Pin1 triggers structural changes in PSD-95, thus negatively affecting its ability to interact with NMDARs. In electrophysiological experiments, larger NMDA-mediated synaptic currents, evoked in CA1 principal cells by Schaffer collateral stimulation, were detected in hippocampal slices obtained from Pin1(-/-) mice compared with controls. Similar results were obtained in cultured hippocampal cells expressing a PSD-95 mutant unable to undergo prolyl-isomerization, thus indicating that the action of Pin1 on PSD-95 is critical for this effect. In addition, an enhancement in spine density and size was detected in CA1 principal cells of Pin1(-/-) or in Thy-1GFP mice treated with the pharmacological inhibitor of Pin1 catalytic activity PiB.Our data indicate that Pin1 controls synaptic content of NMDARs via PSD-95 prolyl-isomerization and the expression of dendritic spines, both required for LTP maintenance., Significance Statement: PSD-95, a membrane-associated guanylate kinase, is the major scaffolding protein at excitatory postsynaptic densities and a potent regulator of synaptic strength and plasticity. The activity of PSD-95 is tightly controlled by several post-translational mechanisms including proline-directed phosphorylation. This signaling cascade regulates the fate of its targets through postphosphorylation conformational modifications catalyzed by the peptidyl-prolyl cis-/trans isomerase Pin1. Here, we uncover a new role of Pin1 in glutamatergic signaling. By interacting with PSD-95, Pin1 dampens PSD-95 ability to complex with NMDARs, thus negatively affecting NMDAR signaling and spine morphology. Our findings further emphasize the emerging role of Pin1 as a key modulator of synaptic transmission., (Copyright © 2016 the authors 0270-6474/16/365437-11$15.00/0.)