Richard T. Ambron, Ph.D.
Signals that mediate changes in transcription following nerve injury
The failure of nerves to regenerate after injury results in tactile deficits and motor paralysis and can cause the formation of painful neuromas. At present, surgery is the method of choice in nerve repair, but complete restoration of function is rare. The successful regeneration of an axon after injury requires alterations in the pattern of protein synthesis in the cell body and an important unresolved issue is the identity of the signals that inform the cell soma and nucleus that the axon has been damaged. Identifying these signals would be important in the devising of strategies to promote nerve regeneration after injury. Reasoning that neurons have a mechanism to communicate between the distant presynaptic terminal and the nucleus we hypothesized that, because these signals would have to enter the nucleus, they would have a nuclear localization sequence (NLS). Consequently, we synthesized a peptide containing a known NLS, coupled it to an inert protein, and injected the construct into axons of neurons in vitro. All of the construct was transported retrogradely along the axon to the cell body and then into the nucleus. We used the nervous system of the marine mollusc Aplysia for these studies since it contains large neurons with defined functions.
The discovery of the retrograde transport/nuclear import pathway prompted us to ask whether events at the site of axon injury lead to the formation of proteins that utilize this pathway. We were particularly interested in signals that would induce the hyperexcitability that occurs in sensory neurons after injury since this is a cause of chronic neuropathic pain. We had already shown that axoplasm extruded from injured axons could induce hyperexcitability when micro-injected into non-injured sensory neurons. To identify the protein responsible, we screened axoplasm for proteins that can enter the nucleus and identified a homologue of the transcription factor NF-kB and several protein kinases. To our surprise, all of the NF-kB activity rapidly disappeared after nerve crush, implying that the normal function of this factor is to repress one or more transcriptional programs for regeneration. One of the kinases that is activated and retrogradely transported after axon injury is a SAPK that enters the nucleus to phosphorylate and activate members of the AP-1 family of transcription factors. We have shown that AP-1 activity increases after axotomy. Also activated at the injury site is a kinase immunologically related to ERK1. This is significant because when recombinant ERK1 was injected into sensory neurons it induced hyperexcitability. We call this kinase IAK-1 and recently found that it can phosphorylate the acute phase transcription factor C/EBP.
Based on these studies, our working hypothesis is that injury to an axon causes the rapid degradation of NF-kB, thereby de-repressing the programs for growth. Concurrently, a SAPK and IAK-1 are activated and use the retrograde transport nuclear import pathway. In the nucleus, these kinases modify transcriptional programs that contribute to survival, growth, and the changes in excitability underlying axon regeneration. How this accomplished is now under investigation.
Selected publicationsSchmied R, Huang C-C, Zhang X-P, Ambron D, Ambron RT (1993) Endogenous axoplasmic proteins and proteins containing nuclear localization signal sequences use the retrograde axonal transport/nuclear import pathway in Aplysia neurons. J Neurosci., 13:4064-7
Ambron RT, Walters ET (1996) Priming events and retrograde injury signals: a new perspective on the cellular and molecular biology of nerve regeneration. Mol. Neurobiol. , 13:61-79.
Ambron RT, Dulin MF, Zhang X-P, Schmied R, Walters ET (1995) Axoplasm enriched in a protein mobilized by nerve injury induces memory-like alterations in Aplysia neurons. J Neurosci., 15:3440-3446.
Povelones M, Tran K, Thanos D, Ambron RT (1997) An Nf-kB-like transcription factor in axoplasm is rapidly inactivated after nerve injury in Aplysia. J Neurosci. , 17:4915-4920
Walters ET, Ambron RT (1995) Long-term alterations induced by injury and by 5-HT in Aplysia sensory neurons: convergent pathways and common signals? Trends Neurosci 18:137-142.