Axonal Transport Proteomics Reveals Mobilization of Translation Machinery to the Lesion Site in Injured Sciatic Nerve

Abstract

Peripheral nerve injuries elicit a cascade of axonal responses that are required for a successful regenerative response. The lesioned axons must signal retrogradely to their cell bodies to activate intrinsic neurite outgrowth mechanisms (1-3) and then overcome physical barriers and inhibitory cues in the extracellular environment to achieve functional regeneration (4, 5). The first indications of a breach in axonal integrity upon injury are most likely abnormal generation of action potentials and/or waves of calcium propagating from the lesion site toward the intact portions of the cell (6, 7). At a later stage, signals carried by motor-driven transport systems start to affect the cell body. This phase includes both an interruption of the normal supply of retrogradely transported molecules such as trophic factor signals (8) and arrival of new signals elicited at the injury site (3, 9). The latter interact with a variety of dynein-associated carriers including importins (10, 11) and kinase family scaffolds (12, 13).In mammalian neurons, the distances between axonal lesion sites and the nucleus can reach many centimeters (up to 1 m in humans); hence, retrograde signaling events within the first few hours after injury must be independent of new transcription in the cell body. Proteolysis, local protein synthesis, and post-translational signaling modifications such as phosphorylation have all been implicated in generation of the retrograde signaling ensemble (1). This prominent role for post-transcriptional processes suggests that comprehensive characterization of retrograde signaling will require proteomics approaches. In a previous study we used two-dimensional PAGE and mass spectrometry to analyze retrogradely concentrated axoplasm from injured mollusc nerve, identifying a vesicular ensemble blocked by the lesion and an up-regulated ensemble highly enriched in calpain cleavage products of an intermediate filament (14,15). Follow-up studies in rodent sciatic nerve showed that the mammalian intermediate filament vimentin is produced by local translation of axonal mRNA upon axonal injury and then undergoes calpain-mediated proteolysis, generating a cleavage product that interacts with importins bound to dynein and enables protected retrograde transport of phosphorylated forms of the mitogen-activated protein kinases Erk1 and Erk2 (16,17). Here, we extend our efforts to determine the components of the retrograde injury signaling ensemble in lesioned nerve by using LC-MS/MS coupled with iTRAQ TM1 labeling to directly analyze mammalian axoplasm samples after nerve injury. The analyses reveal extensive changes in both anterograde and Research

Mammalian Target of Rapamycin (mTOR) Activation Increases Axonal Growth Capacity of Injured Peripheral Nerves

“…Protein synthesis plays a critical role in both injury signaling (16,(35)(36)(37) and the formation of new growth cones during regeneration (38). Several proteins translated in peripheral neurons after injury, including vimentin and importin ␤, have been shown to be critical for regeneration (16).…”

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Proteomic Analysis of Dynein-Interacting Proteins in Amyotrophic Lateral Sclerosis Synaptosomes Reveals Alterations in the RNA-Binding Protein Staufen1

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