Neurons require efficient transport mechanisms such as fast axonal Alisertib transport to ensure neuronal homeostasis and survival. 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 Alisertib 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. Intracellular communication is essential to maintain neuronal homeostasis differentiation and survival. Motor neurons (MNs)1 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. Fast axonal transport represents the backbone of these trafficking mechanisms and is responsible for the Rabbit polyclonal to ZC3H12A. shuttling of several types of organelles proteins and RNAs on microtubule tracks (1 2 Anterograde transport is mainly driven by kinesin motors and ensures delivery of Alisertib newly synthesized proteins synaptic vesicle precursors lipids and organelles to synapses and distal parts of dendrites. In contrast 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 Alisertib the soma. Other cargoes such as neurotrophins (NTs) and their receptors are transported in a retrograde fashion. Upon binding to their receptors NTs are 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. On their delimiting membrane signaling endosomes host several factors required for NT signaling (2) and for their sorting and transport such as the small GTPases Rab5 and Rab7 (5 6 (Fig. 1Schematics summarizing current understanding on the trafficking of NTs HCT and their receptors in MNs. At the neuromuscular junction (NMJ) HCT binds its plasma membrane receptors … Increasing evidence show that deficits in axonal transport lead to neurodevelopmental and neurodegenerative diseases. Mutations in molecular motors their regulators and cytoskeletal components have been associated with motor neuron disease Alzheimer’s disease and Huntington’s disease to cite a few (7 8 Moreover mutations in Rab proteins and their regulating factors cause motor and sensory pathologies such as Charcot-Marie-Tooth type 2B (CMT2B) disease which is caused by mutations in Rab7 (9) and a familial form of amyotrophic lateral sclerosis (ALS) which is linked to mutations in the Rab5 regulator alsin (10). Further links between motor neuron disease and mutations of the dynein/dynactin complex have also been reported (2 11 12 Although several studies focused on unraveling the signaling properties the role of actin and microtubules-dependent motors and the sequential.

Neurons require efficient transport mechanisms such as fast axonal Alisertib transport

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