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.
Highlights
From the ‡Molecular NeuroPathobiology Laboratory, Sobell Department of Motor Neuroscience and Movement Disorders, UCL Institute of Neurology, University College London, London WC1N 3BG, UK; §Bioinformatics and Biostatistics Group, The Francis Crick Institute, London WC2A 3LY, UK; ¶Protein Analysis and Proteomics Group, The Francis Crick Institute, South Mimms EN6 3LD, UK
Rab5 is exchanged with Rab7, a late endosome (LE)/lysosome marker, in a process that is required for fast axonal retrograde transport of these organelles to the soma (30 min; Fig. 1A) [6]
Once they reached the soma, HCT and NT-receptor complexes are sorted to different fates: activated NT receptors are mainly sent to degradation [32], whereas HCT remains in nonacidic compartments [31, 33] and is transcytosed to adjacent interneurons (60 min; Fig. 1A)
Summary
Motor neurons (MNs) are characterized by very long axons whose nerve terminals can be as far as one meter away from the soma in humans These long distances require the development of specialized mechanisms to ensure effective long-range communication between axonal and somatodendritic compartments. Retrograde transport relies on cytoplasmic dynein for the movement of mitochondria, autophagosomes, lysosomes, and aging proteins targeted for degradation and/or recycling from axon terminals to the soma Other cargoes such as neurotrophins (NTs) and their receptors are transported in a retrograde fashion. Spatio-Temporal Characterization of Signaling Endosomes internalized in endocytic carriers and transported along the axon to the soma, where they exert their trophic responses [3, 4] These specialized organelles, the so-called signaling endosomes, are key players in this process. Further links between motor neuron disease and mutations of the dynein/dynactin complex have been reported [2, 11, 12]
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