G protein-coupled receptors (GPCRs) are considered to function primarily at the plasma membrane, where they interact with extracellular ligands and couple to G proteins that transmit intracellular signals. Consequently, therapeutic drugs are designed to target GPCRs at the plasma membrane. Activated GPCRs undergo clathrin-dependent endocytosis. Whether GPCRs in endosomes control pathophysiological processes in vivo and are therapeutic targets remains uncertain. We investigated the contribution of endosomal signaling of the calcitonin receptor-like receptor (CLR) to pain transmission. Calcitonin gene-related peptide (CGRP) stimulated CLR endocytosis and activated protein kinase C (PKC) in the cytosol and extracellular signal regulated kinase (ERK) in the cytosol and nucleus. Inhibitors of clathrin and dynamin prevented CLR endocytosis and activation of cytosolic PKC and nuclear ERK, which derive from endosomal CLR. A cholestanol-conjugated antagonist, CGRP, accumulated in CLR-containing endosomes and selectively inhibited CLR signaling in endosomes. CGRP caused sustained excitation of neurons in slices of rat spinal cord. Inhibitors of dynamin, ERK, and PKC suppressed persistent neuronal excitation. CGRP-cholestanol, but not unconjugated CGRP, prevented sustained neuronal excitation. When injected intrathecally to mice, CGRP-cholestanol inhibited nociceptive responses to intraplantar injection of capsaicin, formalin, or complete Freund's adjuvant more effectively than unconjugated CGRP Our results show that CLR signals from endosomes to control pain transmission and identify CLR in endosomes as a therapeutic target for pain. Thus, GPCRs function not only at the plasma membrane but also in endosomes to control complex processes in vivo. Endosomal GPCRs are a drug target that deserve further attention.
Membrane trafficking pathways are essential for the viability and growth of cells, and play a major role in the interaction of cells with their environment. In this At a Glance article and accompanying poster, we outline the major cellular trafficking pathways and discuss how defects in the function of the molecular machinery that mediates this transport lead to various diseases in humans. We also briefly discuss possible therapeutic approaches that may be used in the future treatment of trafficking-based disorders.
Vaz, McDermott et al. identify variants in PCYT2, which encodes a key gene in phospholipid biosynthesis, in five individuals with a new complex hereditary spastic paraplegia. Functional studies in fibroblasts and a zebrafish model confirm the pathogenic nature of the variants, while lipidomic analysis reveals potential treatment strategies and plasma biomarkers.
Muscle degeneration is the most prevalent cause for frailty and dependency in inherited diseases and ageing, affecting hundreds of millions of people. Elucidation of pathophysiological mechanisms, as well as effective treatments for muscle diseases represents an important goal in improving human health. Here, we show that phosphatidylethanolamine cytidyltransferase (PCYT2/ECT), the critical enzyme of the Kennedy branch of phosphatidylethanolamine (PE) synthesis pathway, has an essential role in muscle health and lifespan. Human genetic deficiency in PCYT2 causes a severe disease with failure to thrive and progressive weakness. Pcyt2 mutant zebrafish recapitulate patient phenotypes, indicating that the role of PCYT2/PE in muscle is evolutionary conserved. Muscle specific Pcyt2 knockout mice exhibited failure to thrive, impaired muscle development, progressive muscle weakness, muscle loss and accelerated ageing. Interestingly, from several organs tested, this pathology is muscle specific. Mechanistically, in muscle deficiency of PCYT2 triggers dramatic alterations of physicochemical properties of the myofiber membrane lipid bilayer, compromising membrane stability and durability under strain. We also show that PCYT2 activity declines in the aging muscles of humans and mice, and Pcyt2 gene-therapy in aged mice improved muscle strength. AAV-based delivery of PCYT2 also rescued muscle weakness in Pcyt2 knock-out mice, offering a feasible novel therapeutic avenue for rare disease patients and to alleviate muscle aging. Thus, PCYT2 plays a fundamental, specific, and conserved role in vertebrate muscle health, linking PCYT2 and PCYT2 synthesized PE lipids to severe muscle dystrophy, exercise intolerance and aging.
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