A high-affinity, bivalent PDZ domain inhibitor complexes PICK1 to alleviate neuropathic pain

Maladaptive plasticity involving increased expression of AMPA‐type glutamate receptors is involved in several pathologies, including neuropathic pain, but direct inhibition of AMPARs is associated with side effects. As an alternative, we developed a cell‐permeable, high‐affinity (~2 nM) peptide inhibitor, Tat‐P$_4$‐(C5)$_2$, of the PDZ domain protein PICK1 to interfere with increased AMPAR expression. The affinity is obtained partly from the Tat peptide and partly from the bivalency of the PDZ motif, engaging PDZ domains from two separate PICK1 dimers to form a tetrameric complex. Bivalent Tat‐P$_4$‐(C5)$_2$ disrupts PICK1 interaction with membrane proteins on supported cell membrane sheets and reduce the interaction of AMPARs with PICK1 and AMPA‐receptor surface expression in vivo. Moreover, Tat‐P$_4$‐(C5)$_2$ administration reduces spinal cord transmission and alleviates mechanical hyperalgesia in the spared nerve injury model of neuropathic pain. Taken together, our data reveal Tat‐P$_4$‐(C5)$_2$ as a novel promising lead for neuropathic pain treatment and expand the therapeutic potential of bivalent inhibitors to non‐tandem protein–protein interaction domains

Serine 77 in the PDZ domain of PICK1 is a protein kinase Cα phosphorylation site regulated by lipid membrane binding

PICK1 (protein interacting with C-kinase 1) contains an N-terminal protein binding PDZ domain and a C-terminal lipid binding BAR domain. PICK1 plays a key role in several physiological processes including synaptic plasticity. However, little is known about cellular mechanisms governing the activity of PICK1 itself. Here we show that PICK1 is a substrate in vitro both for PKCα (Protein kinase Cα), as previously shown, and for CaMKIIα (Ca(2+)-calmodulin dependent protein kinase IIα). By mutation of predicted phosphorylation sites, we identify Ser77 in the PDZ domain as a major phosphorylation site for PKCα. Mutation of Ser77 reduced PKCα mediated phosphorylation ~50% whereas no reduction was observed upon mutation of seven other predicted sites. Addition of lipid vesicles increased phosphorylation of Ser77 ~10-fold, indicating that lipid binding is critical for optimal phosphorylation. Binding of PKCα to the PICK1 PDZ domain was not required for phosphorylation but a PDZ domain peptide ligand reduced overall phosphorylation ~30%. The phosphomimic S77D reduced cytosolic clustering of eYFP-PICK1 in COS7 cells and thereby conceivably its lipid binding and/or polymerization capacity. We propose that PICK1 is phosphorylated at Ser77 by PKCα preferentially when bound to membrane vesicles and that this phosphorylation in turn modulates its cellular distribution

Loss of Synapse Repressor MDGA1 Enhances Perisomatic Inhibition, Confers Resistance to Network Excitation, and Impairs Cognitive Function

Synaptopathies contributing to neurodevelopmental disorders are linked to mutations in synaptic organizing molecules, including postsynaptic neuroligins, presynaptic neurexins, and MDGAs, which regulate their interaction. The role of MDGA1 in suppressing inhibitory versus excitatory synapses is controversial based on in vitro studies. We show that genetic deletion of MDGA1 in vivo elevates hippocampal CA1 inhibitory, but not excitatory, synapse density and transmission. Furthermore, MDGA1 is selectively expressed by pyramidal neurons and regulates perisomatic, but not distal dendritic, inhibitory synapses. Mdga1−/− hippocampal networks demonstrate muted responses to neural excitation, and Mdga1−/− mice are resistant to induced seizures. Mdga1−/− mice further demonstrate compromised hippocampal long-term potentiation, consistent with observed deficits in spatial and context-dependent learning and memory. These results suggest that mutations in MDGA1 may contribute to cognitive deficits through altered synaptic transmission and plasticity by loss of suppression of inhibitory synapse development in a subcellular domain- and cell-type-selective manner