RESUMO
Dendritic filopodia are dynamic structures thought to be the precursors of spines during synapse development. Morphological maturation to spines is associated with the stabilization and strengthening of synapses, and can be altered in various neurological disorders. Telencephalin (TLN/intercellular adhesion molecule-5 (ICAM5)) localizes to dendritic filopodia, where it facilitates their formation/maintenance, thereby slowing spine morphogenesis. As spines are largely devoid of TLN, its exclusion from the filopodia surface appears to be required in this maturation process. Using HeLa cells and primary hippocampal neurons, we demonstrate that surface removal of TLN involves internalization events mediated by the small GTPase ADP-ribosylation factor 6 (ARF6), and its activator EFA6A. This endocytosis of TLN affects filopodia-to-spine transition, and requires Rac1-mediated dephosphorylation/release of actin-binding ERM proteins from TLN. At the somato-dendritic surface, TLN and EFA6A are confined to distinct, flotillin-positive membrane subdomains. The co-distribution of TLN with this lipid raft marker also persists during its endosomal targeting to CD63-positive late endosomes. This suggests a specific microenvironment facilitating ARF6-mediated mobilization of TLN that contributes to promotion of dendritic spine development.
Assuntos
Fatores de Ribosilação do ADP/fisiologia , Moléculas de Adesão Celular/metabolismo , Dendritos/fisiologia , Espinhas Dendríticas/metabolismo , Endossomos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Pseudópodes/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/química , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Sequência de Aminoácidos , Animais , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Microambiente Celular/genética , Microambiente Celular/fisiologia , Dendritos/genética , Dendritos/metabolismo , Espinhas Dendríticas/genética , Espinhas Dendríticas/fisiologia , Células HeLa , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Cultura Primária de Células , Transporte Proteico/genética , Pseudópodes/genética , Pseudópodes/fisiologia , Homologia de Sequência de AminoácidosRESUMO
Amyloid ß (Aß) peptides, the primary constituents of senile plaques and a hallmark in Alzheimer's disease pathology, are generated through the sequential cleavage of amyloid precursor protein (APP) by ß-site APP cleaving enzyme 1 (BACE1) and γ-secretase. The early endosome is thought to represent a major compartment for APP processing; however, the mechanisms of how BACE1 encounters APP are largely unknown. In contrast to APP internalization, which is clathrin-dependent, we demonstrate that BACE1 is sorted to early endosomes via a route controlled by the small GTPase ADP ribosylation factor 6 (ARF6). Altering ARF6 levels or its activity affects endosomal sorting of BACE1, and consequently results in altered APP processing and Aß production. Furthermore, sorting of newly internalized BACE1 from ARF6-positive towards RAB GTPase 5 (RAB5)-positive early endosomes depends on its carboxyterminal short acidic cluster-dileucine motif. This ARF6-mediated sorting of BACE1 is confined to the somatodendritic compartment of polarized neurons in agreement with Aß peptides being primarily secreted from here. These results demonstrate a spatial separation between APP and BACE1 during surface-to-endosome transport, suggesting subcellular trafficking as a regulatory mechanism for this proteolytic processing step. It thereby provides a novel avenue to interfere with Aß production through a selective modulation of the distinct endosomal transport routes used by BACE1 or APP.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Endossomos/enzimologia , Processamento de Proteína Pós-Traducional , Fator 6 de Ribosilação do ADP , Motivos de Aminoácidos , Secretases da Proteína Precursora do Amiloide/química , Animais , Antígenos CD59/metabolismo , Compartimento Celular , Polaridade Celular , Dendritos/metabolismo , Endocitose , Células HeLa , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Leucina/metabolismo , Camundongos , Modelos Biológicos , Transporte Proteico , Ratos , Receptores da Transferrina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
The gamma-secretase complex, consisting of presenilin, nicastrin, presenilin enhancer-2 (PEN-2), and anterior pharynx defective-1 (APH-1) cleaves type I integral membrane proteins like amyloid precursor protein and Notch in a process of regulated intramembrane proteolysis. The regulatory mechanisms governing the multistep assembly of this "proteasome of the membrane" are unknown. We characterize a new interaction partner of nicastrin, the retrieval receptor Rer1p. Rer1p binds preferentially immature nicastrin via polar residues within its transmembrane domain that are also critical for interaction with APH-1. Absence of APH-1 substantially increased binding of nicastrin to Rer1p, demonstrating the competitive nature of these interactions. Moreover, Rer1p expression levels control the formation of gamma-secretase subcomplexes and, concomitantly, total cellular gamma-secretase activity. We identify Rer1p as a novel limiting factor that negatively regulates gamma-secretase complex assembly by competing with APH-1 during active recycling between the endoplasmic reticulum (ER) and Golgi. We conclude that total cellular gamma-secretase activity is restrained by a secondary ER control system that provides a potential therapeutic value.
