Axonal transcription factors signal retrogradely in lesioned peripheral nerve.

Retrograde axonal injury signalling stimulates cell body responses in lesioned peripheral neurons. The involvement of importins in retrograde transport suggests that transcription factors (TFs) might be directly involved in axonal injury signalling. Here, we show that multiple TFs are found in axons and associate with dynein in axoplasm from injured nerve. Biochemical and functional validation for one TF family establishes that axonal STAT3 is locally translated and activated upon injury, and is transported retrogradely with dynein and importin α5 to modulate survival of peripheral sensory neurons after injury. Hence, retrograde transport of TFs from axonal lesion sites provides a direct link between axon and nucleus.

Signaling to transcription networks in the neuronal retrograde injury response.

Retrograde signaling from axon to soma activates intrinsic regeneration mechanisms in lesioned peripheral sensory neurons; however, the links between axonal injury signaling and the cell body response are not well understood. Here, we used phosphoproteomics and microarrays to implicate approximately 900 phosphoproteins in retrograde injury signaling in rat sciatic nerve axons in vivo and approximately 4500 transcripts in the in vivo response to injury in the dorsal root ganglia. Computational analyses of these data sets identified approximately 400 redundant axonal signaling networks connected to 39 transcription factors implicated in the sensory neuron response to axonal injury. Experimental perturbation of individual overrepresented signaling hub proteins, including Abl, AKT, p38, and protein kinase C, affected neurite outgrowth in sensory neurons. Paradoxically, however, combined perturbation of Abl together with other hub proteins had a reduced effect relative to perturbation of individual proteins. Our data indicate that nerve injury responses are controlled by multiple regulatory components, and suggest that network redundancies provide robustness to the injury response.

A human neuron injury model for molecular studies of axonal regeneration.

The enhancement of regeneration of damaged axons in both the peripheral and central nervous systems is a widely pursued goal in clinical medicine. Although some of the molecular mechanisms involved in the intrinsic neurite regeneration program have been elucidated, much additional study is required for development of new therapeutics. The majority of studies in the field of axonal regeneration have utilized animal models due to obvious limitations of the accessibility of human neural tissues. Here we describe the use of human embryonic stem cell (hESC)-derived neurons as a novel model for studying neuronal responses to axonal injury. Neurons were generated using PA6 induction and neurites injured in vitro using trituration or laser microdissection. Lesioned neurons re-extended neurites with distinct growth cones. Expression of proteins associated with regeneration were observed in this human in vitro system, including appearance of importin beta1 in processes after neuritomy. Laser-transected hESC-derived neuronal cultures were analyzed for their transcriptional response to injury using Affymetrix expression microarrays. Profound changes in gene expression were observed over a time course of 2 to 24 hours after lesion. The expression of several genes reported to be involved in axonal injury responses in animal models changed following injury of hESC-derived neurons. Thus, hESC-derived neurons may be a useful in vitro model system for mechanistic studies on human axonal injury and regeneration.

Localized regulation of axonal RanGTPase controls retrograde injury signaling in peripheral nerve.

Peripheral sensory neurons respond to axon injury by activating an importin-dependent retrograde signaling mechanism. How is this mechanism regulated? Here, we show that Ran GTPase and its associated effectors RanBP1 and RanGAP regulate the formation of importin signaling complexes in injured axons. A gradient of nuclear RanGTP versus cytoplasmic RanGDP is thought to be fundamental for the organization of eukaryotic cells. Surprisingly, we find RanGTP in sciatic nerve axoplasm, distant from neuronal cell bodies and nuclei, and in association with dynein and importin-alpha. Following injury, localized translation of RanBP1 stimulates RanGTP dissociation from importins and subsequent hydrolysis, thereby allowing binding of newly synthesized importin-beta to importin-alpha and dynein. Perturbation of RanGTP hydrolysis or RanBP1 blockade at axonal injury sites reduces the neuronal conditioning lesion response. Thus, neurons employ localized mechanisms of Ran regulation to control retrograde injury signaling in peripheral nerve.

Vimentin-dependent spatial translocation of an activated MAP kinase in injured nerve.

How are phosphorylated kinases transported over long intracellular distances, such as in the case of axon to cell body signaling after nerve injury? Here, we show that the MAP kinases Erk1 and Erk2 are phosphorylated in sciatic nerve axoplasm upon nerve injury, concomitantly with the production of soluble forms of the intermediate filament vimentin by local translation and calpain cleavage in axoplasm. Vimentin binds phosphorylated Erks (pErk), thus linking pErk to the dynein retrograde motor via direct binding of vimentin to importin beta. Injury-induced Elk1 activation and neuronal regeneration are inhibited or delayed in dorsal root ganglion neurons from vimentin null mice, and in rats treated with a MEK inhibitor or with a peptide that prevents pErk-vimentin binding. Thus, soluble vimentin enables spatial translocation of pErk by importins and dynein in lesioned nerve.

The neuroprotective peptide NAP inhibits the aggregation of the beta-amyloid peptide.

Alzheimer's disease (AD) is characterized by brain plaques containing the beta-amyloid peptide (Abeta). One approach for treating AD is by blocking Abeta aggregation. Activity-dependent neuroprotective protein contains a peptide, NAP that protects neurons in culture against Abeta toxicity. Here, NAP was shown to inhibit Abeta aggregation using: (1) fluorimetry; (2) electron microscopy; (3) high-throughput screening of Abeta deposition onto a synthetic template (synthaloid); and (4) Congo Red staining of neurons. Further assays showed biotin-NAP binding to Abeta. These results suggest that part of the neuroprotective mechanism exerted by NAP is through modulation of toxic protein folding in the extracellular milieu.