title: Analysis of Signaling Endosome Composition and Dynamics Using SILAC in Embryonic Stem Cell-Derived Neurons.
abstract: Neurons require efficient transport mechanisms such as fast axonal transport to ensure neuronal homeostasis and survival. Neurotrophins and their receptors are conveyed via fast axonal retrograde transport of signaling endosomes to the soma, where they elicit transcriptional responses. Despite the essential roles of signaling endosomes in neuronal differentiation and survival, little is known about their molecular identity, dynamics, and regulation. Gaining a better mechanistic understanding of these organelles and their kinetics is crucial, given the growing evidence linking vesicular trafficking deficits to neurodegeneration. Here, we exploited an affinity purification strategy using the binding fragment of tetanus neurotoxin (HCT) conjugated to monocrystalline iron oxide nanoparticles (MIONs), which in motor neurons, is transported in the same carriers as neurotrophins and their receptors. To quantitatively assess the molecular composition of HCT-containing signaling endosomes, we have developed a protocol for triple Stable Isotope Labeling with Amino acids in Cell culture (SILAC) in embryonic stem cell-derived motor neurons. After HCT internalization, retrograde carriers were magnetically isolated at different time points and subjected to mass-spectrometry and Gene Ontology analyses. This purification strategy is highly specific, as confirmed by the presence of essential regulators of fast axonal transport in the make-up of these organelles. Our results indicate that signaling endosomes undergo a rapid maturation with the acquisition of late endosome markers following a specific time-dependent kinetics. Strikingly, signaling endosomes are specifically enriched in proteins known to be involved in neurodegenerative diseases and neuroinfection. Moreover, we highlighted the presence of novel components, whose precise temporal recruitment on signaling endosomes might be essential for proper sorting and/or transport of these organelles. This study provides the first quantitative proteomic analysis of signaling endosomes isolated from motor neurons and allows the assembly of a functional map of these axonal carriers involved in long-range neuronal signaling.
authors.affiliation: From the ‡Molecular NeuroPathobiology Laboratory, Sobell Department of Motor Neuroscience and Movement Disorders, UCL Institute of Neurology, University College London, London WC1N 3BG, UK;
authors.forename: Solène
authors.initials: S
authors.lastname: Debaisieux
authors.affiliation: ¶Protein Analysis and Proteomics Group, The Francis Crick Institute, South Mimms EN6 3LD, UK.
authors.forename: Vesela
authors.initials: V
authors.lastname: Encheva
authors.affiliation: §Bioinformatics and Biostatistics Group, The Francis Crick Institute, London WC2A 3LY, UK;
authors.forename: Probir
authors.initials: P
authors.lastname: Chakravarty
authors.affiliation: ¶Protein Analysis and Proteomics Group, The Francis Crick Institute, South Mimms EN6 3LD, UK.
authors.forename: Ambrosius P
authors.initials: AP
authors.lastname: Snijders
authors.affiliation: From the ‡Molecular NeuroPathobiology Laboratory, Sobell Department of Motor Neuroscience and Movement Disorders, UCL Institute of Neurology, University College London, London WC1N 3BG, UK; giampietro.schiavo@ucl.ac.uk.
authors.forename: Giampietro
authors.initials: G
authors.lastname: Schiavo
mesh_terms: D000818:Animals; D001370:Axonal Transport / Q000187:drug effects / Q000235:genetics; D001369:Axons / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D004705:Endocytosis / Q000235:genetics; D011992:Endosomes / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D005290:Ferric Compounds / Q000008:administration & dosage / Q000737:chemistry; D006801:Humans; D007553:Isotope Labeling; D053768:Metal Nanoparticles / Q000008:administration & dosage / Q000737:chemistry; D008666:Metalloendopeptidases / Q000008:administration & dosage / Q000737:chemistry; D051379:Mice; D009046:Motor Neurons / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D009414:Nerve Growth Factors / Q000235:genetics / Q000378:metabolism*; D019636:Neurodegenerative Diseases / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D040901:Proteomics; D015398:Signal Transduction; D013744:Tetanus Toxin / Q000008:administration & dosage / Q000737:chemistry
chemical_list: D005290:Ferric Compounds; D009414:Nerve Growth Factors; D013744:Tetanus Toxin; C035979:tetanospasmin; C000499:ferric oxide; D008666:Metalloendopeptidases