5‑HT₄ Receptors Constitutively Promote the Non-Amyloidogenic Pathway of APP Cleavage and Interact with ADAM10

In addition to the amyloidogenic pathway, amyloid precursor protein (APP) can be cleaved by α-secretases, producing soluble and neuroprotective APP alpha (sAPPα) (nonamyloidogenic pathway) and thus preventing the generation of pathogenic amyloid-β. However, the mechanisms regulating APP cleavage by α-secretases remain poorly understood. Here, we showed that expression of serotonin type 4 receptors (5-HT₄Rs) constitutively (without agonist stimulation) induced APP cleavage by the α-secretase ADAM10 and the release of neuroprotective sAPPα in HEK-293 cells and cortical neurons. This effect was independent of cAMP production. Interestingly, we demonstrated that 5-HT₄ receptors physically interacted with the mature form of ADAM10. Stimulation of 5-HT₄ receptors by an agonist further increased sAPPα secretion, and this effect was mediated by cAMP/Epac signaling. These findings describe a new mechanism whereby a GPCR constitutively stimulates the cleavage of APP by α-secretase and promotes the nonamyloidogenic pathway of APP processing

Structure-Based Design of PDZ Ligands as Inhibitors of 5‑HT_2A Receptor/PSD-95 PDZ1 Domain Interaction Possessing Anti-hyperalgesic Activity

Disrupting the interaction between the PDZ protein PSD-95 and the C-terminal domain of the 5-HT_2A serotonin receptor has been shown to reduce hyperalgesia in a rodent model of neuropathic pain. Here, we designed and synthesized PDZ ligands capable of binding to the first PDZ domain (PDZ1) of the PSD-95 protein and evaluated their biological activity <i>in vitro</i> and <i>in vivo</i>. A series of substituted indoles was identified by docking simulations, and six novel analogues were synthesized. Three analogues displayed strong interactions with the first PDZ domain (PDZ1) of PDZ-95 in ¹H–¹⁵N heteronuclear single-quantum coherence (HSQC) experiments and two of them were able to inhibit the interaction between PSD-95 and the 5-HT_2A receptor <i>in vitro</i>. We identified compound <b>8b</b> as the analogue able to significantly suppress mechanical hyperalgesia in an experimental model of traumatic neuropathic pain in the rat. This effect was suppressed by the coadministration of the 5-HT_2A receptor antagonist M100907, consistent with an inhibitory effect upon 5-HT_2A receptor/PSD-95 interaction. Finally, we determined an NMR-restraint driven model structure for the PSD95 PDZ1/<b>8b</b> complex, which confirms that indole <b>8b</b> binds to the putative PDZ-ligand binding site