RESUMEN
Lowe Syndrome (LS) is a rare X-linked disorder characterized by renal dysfunction, cataracts, and several central nervous system (CNS) anomalies. The mechanisms underlying the neurological dysfunction in LS remain unclear, albeit they share some phenotypic characteristics similar to the deficiency or dysfunction of the Reelin signaling, a relevant pathway with roles in CNS development and neuronal functions. In this study, we investigated the role of OCRL1, an inositol polyphosphate 5-phosphatase encoded by the OCRL gene, mutated in LS, focusing on its impact on endosomal trafficking and receptor recycling in human neuronal cells. Specifically, we tested the effects of OCRL1 deficiency in the trafficking and signaling of ApoER2/LRP8, a receptor for the ligand Reelin. We found that loss of OCRL1 impairs ApoER2 intracellular trafficking, leading to reduced receptor expression and decreased levels at the plasma membrane. Additionally, human neurons deficient in OCRL1 showed impairments in ApoER2/Reelin-induced responses. Our findings highlight the critical role of OCRL1 in regulating ApoER2 endosomal recycling and its impact on the ApoER2/Reelin signaling pathway, providing insights into potential mechanisms underlying the neurological manifestations of LS.
Asunto(s)
Moléculas de Adhesión Celular Neuronal , Endosomas , Proteínas de la Matriz Extracelular , Proteínas Relacionadas con Receptor de LDL , Proteínas del Tejido Nervioso , Neuronas , Monoéster Fosfórico Hidrolasas , Transporte de Proteínas , Proteína Reelina , Serina Endopeptidasas , Humanos , Monoéster Fosfórico Hidrolasas/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/deficiencia , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/deficiencia , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/deficiencia , Moléculas de Adhesión Celular Neuronal/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/deficiencia , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/deficiencia , Endosomas/metabolismo , Neuronas/metabolismo , Proteínas Relacionadas con Receptor de LDL/metabolismo , Proteínas Relacionadas con Receptor de LDL/genética , Transducción de Señal , Síndrome Oculocerebrorrenal/genética , Síndrome Oculocerebrorrenal/metabolismoRESUMEN
Adaptor protein complex 4 (AP-4) is a heterotetrameric complex that promotes export of selected cargo proteins from the trans-Golgi network. Mutations in each of the AP-4 subunits cause a complicated form of Hereditary Spastic Paraplegia (HSP). Herein, we report that ApoER2, a receptor in the Reelin signaling pathway, is a cargo of the AP-4 complex. We identify the motif ISSF/Y within the ApoER2 cytosolic domain as necessary for interaction with the canonical signal-binding pocket of the µ4 (AP4M1) subunit of AP-4. AP4E1- knock-out (KO) HeLa cells and hippocampal neurons from Ap4e1-KO mice display increased co-localization of ApoER2 with Golgi markers. Furthermore, hippocampal neurons from Ap4e1-KO mice and AP4M1-KO human iPSC-derived cortical i3Neurons exhibit reduced ApoER2 protein expression. Analyses of biosynthetic transport of ApoER2 reveal differential post-Golgi trafficking of the receptor, with lower axonal distribution in KO compared to wild-type neurons, indicating a role of AP-4 and the ISSF/Y motif in the axonal localization of ApoER2. Finally, analyses of Reelin signaling in mouse hippocampal and human cortical KO neurons show that AP4 deficiency causes no changes in Reelin-dependent activation of the AKT pathway and only mild changes in Reelin-induced dendritic arborization, but reduces Reelin-induced ERK phosphorylation, CREB activation, and Golgi deployment. This work thus establishes ApoER2 as a novel cargo of the AP-4 complex, suggesting that defects in the trafficking of this receptor and in the Reelin signaling pathway could contribute to the pathogenesis of HSP caused by mutations in AP-4 subunits.
Asunto(s)
Complejo 4 de Proteína Adaptadora , Proteínas Relacionadas con Receptor de LDL , Paraplejía Espástica Hereditaria , Animales , Humanos , Ratones , Complejo 4 de Proteína Adaptadora/genética , Complejo 4 de Proteína Adaptadora/metabolismo , Células HeLa , Proteínas Relacionadas con Receptor de LDL/genética , Proteínas Relacionadas con Receptor de LDL/metabolismo , Receptores de Superficie Celular , Paraplejía Espástica Hereditaria/genética , Paraplejía Espástica Hereditaria/metabolismoRESUMEN
Adaptor protein complex 4 (AP-4) is a heterotetrameric complex that promotes protein export from the trans -Golgi network. Mutations in each of the AP-4 subunits cause a complicated form of Hereditary Spastic Paraplegia (HSP). Herein, we report that ApoER2, a receptor in the Reelin signaling pathway, is a cargo of the AP-4 complex. We identify the motif ISSF/Y within the ApoER2 cytosolic domain as necessary for interaction with the canonical signal-binding pocket of the µ4 (AP4M1) subunit of AP-4. AP4E1 -knock-out (KO) HeLa cells and hippocampal neurons from Ap4e1 -KO mice display increased Golgi localization of ApoER2. Furthermore, hippocampal neurons from Ap4e1 -KO mice and AP4M1 -KO human iPSC-derived cortical i3Neurons exhibit reduced ApoER2 protein expression. Analyses of biosynthetic transport of ApoER2 reveal differential post-Golgi trafficking of the receptor, with lower axonal distribution in KO compared to wild-type neurons, indicating a role of AP-4 and the ISSF/Y motif in the axonal localization of ApoER2. Finally, analyses of Reelin signaling in mouse hippocampal and human cortical KO neurons show that AP4 deficiency causes no changes in Reelin-dependent activation of the AKT pathway and only mild changes in Reelin-induced dendritic arborization, but reduces Reelin-induced ERK phosphorylation, CREB activation, and Golgi deployment. Altogether, this work establishes ApoER2 as a novel cargo of the AP-4 complex, suggesting that defects in the trafficking of this receptor and in the Reelin signaling pathway could contribute to the pathogenesis of HSP caused by mutations in AP-4 subunits.
RESUMEN
Megalin/LRP2 is the primary multiligand receptor for the re-absorption of low molecular weight proteins in the proximal renal tubule. Its function is significantly dependent on its endosomal trafficking. Megalin recycling from endosomal compartments is altered in an X-linked disease called Lowe Syndrome (LS), caused by mutations in the gene encoding for the phosphatidylinositol 5-phosphatase OCRL1. LS patients show increased low-molecular-weight proteins with reduced levels of megalin ectodomain in the urine and accumulation of the receptor in endosomal compartments of the proximal tubule cells. To gain insight into the deregulation of megalin in the LS condition, we silenced OCRL1 in different cell lines to evaluate megalin expression finding that it is post-transcriptionally regulated. As an indication of megalin proteolysis, we detect the ectodomain of the receptor in the culture media. Remarkably, in OCRL1 silenced cells, megalin ectodomain secretion appeared significantly reduced, according to the observation in the urine of LS patients. Besides, the silencing of APPL1, a Rab5 effector associated with OCRL1 in endocytic vesicles, also reduced the presence of megalin's ectodomain in the culture media. In both silencing conditions, megalin cell surface levels were significantly decreased. Considering that GSK3ß-mediated megalin phosphorylation reduces receptor recycling, we determined that the endosomal distribution of megalin depends on its phosphorylation status and OCRL1 function. As a physiologic regulator of GSK3ß, we focused on insulin signaling that reduces kinase activity. Accordingly, megalin phosphorylation was significantly reduced by insulin in wild-type cells. Moreover, even though in cells with low activity of OCRL1 the insulin response was reduced, the phosphorylation of megalin was significantly decreased and the receptor at the cell surface increased, suggesting a protective role of insulin in a LS cellular model.
RESUMEN
Coupling of protein synthesis with protein delivery to distinct subcellular domains is essential for maintaining cellular homeostasis, and defects thereof have consistently been shown to be associated with several diseases. This function is particularly challenging for neurons given their polarized nature and differential protein requirements in synaptic boutons, dendrites, axons, and soma. Long-range trafficking is greatly enhanced in neurons by discrete mini-organelles resembling the Golgi complex (GC) referred to as Golgi outposts (GOPs) which play an essential role in the development of dendritic arborization. In this context, the morphology of the GC is highly plastic, and the polarized distribution of this organelle is necessary for neuronal migration and polarized growth. Furthermore, synaptic components are readily trafficked and modified at GOP suggesting a function for this organelle in synaptic plasticity. However, little is known about GOPs properties and biogenesis and the role of GOP dysregulation in pathology. In this review, we discuss current literature supporting a role for GC dynamics in prevalent neurological disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and epilepsy, and examine the association of these disorders with the wide-ranging effects of GC function on common cellular pathways regulating neuronal excitability, polarity, migration, and organellar stress. First, we discuss the role of Golgins and Golgi-associated proteins in the regulation of GC morphology and dynamics. Then, we consider abnormal GC arrangements observed in neurological disorders and associations with common neuronal defects therein. Finally, we consider the cell signaling pathways involved in the modulation of GC dynamics and argue for a master regulatory role for Reelin signaling, a well-known regulator of neuronal polarity and migration. Determining the cellular pathways involved in shaping the Golgi network will have a direct and profound impact on our current understanding of neurodevelopment and neuropathology and aid the development of novel therapeutic strategies for improved patient care and prognosis.