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

Izhak Michaelevski,Katalin F Medzihradszky,Aenoch Lynn,Alma L Burlingame,Mike Fainzilber

doi:10.1074/mcp.m900369-mcp200

Abstract

Investigations of the molecular mechanisms underlying responses to nerve injury have highlighted the importance of axonal transport systems. To obtain a comprehensive view of the protein ensembles associated with axonal transport in injured axons, we analyzed the protein compositions of axoplasm concentrated at ligatures following crush injury of rat sciatic nerve. LC-MS/MS analyses of iTRAQ-labeled peptides from axoplasm distal and proximal to the ligation sites revealed protein ensembles transported in both anterograde and retrograde directions. Variability of replicates did not allow straightforward assignment of proteins to functional transport categories; hence, we performed principal component analysis and factor analysis with subsequent clustering to determine the most prominent injury-related transported proteins. This strategy circumvented experimental variability and allowed the extraction of biologically meaningful information from the quantitative neuroproteomics experiments. 299 proteins were highlighted by principal component analysis and factor analysis, 145 of which correlate with retrograde and 154 of which correlate with anterograde transport after injury. The analyses reveal extensive changes in both anterograde and retrograde transport proteomes in injured peripheral axons and emphasize the importance of RNA binding and translational machineries in the axonal response to injury.

Highlights

Investigations of the molecular mechanisms underlying responses to nerve injury have highlighted the importance of axonal transport systems
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 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]

Summary

Introduction

Investigations of the molecular mechanisms underlying responses to nerve injury have highlighted the importance of axonal transport systems. Comparison of all three experiments by factor scores (Fig. 5A and supplemental Fig. S3) revealed that 30 proteins belonging to this category were common to all biological replicates of the injury experiment, whereas a less stringent cutoff taking proteins found in two of the three replicates resulted in a total of 144 proteins correlated positively (71 proteins) or negatively (73 proteins) to the retrograde transport ensemble (Fig. 5B; supplemental Tables S1, S3, and S5; and supplemental supporting spectra).

Results

Conclusion