Retrograde injury signaling in lesioned nerve

The cell body of a lesioned neuron must receive accurate and timely information on the site and extent of axonal damage, in order to mount an appropriate response. Specific mechanisms must therefore exist to transmit such information along the length of the axon from the lesion site to the cell body. Three distinct types of signals have been postulated to underlie this process, starting with injury-induced discharge of axon potentials, and continuing with two distinct types of retrogradely transported macromolecular signals. The latter include, on the one hand, an interruption of the normal supply of retrogradely transported trophic factors from the target; and on the other hand activated proteins emanating from the injury site. Over the past five years we have combined proteomics and cell biology approaches to characterize the mechanisms by which positive injury signals traffic retrogradely in injured nerve. We demonstrated that the importin/karyopherin alpha and beta families underlie this process. We found importins in axons at significant distances from the cell body and demonstrated that importin beta protein is increased after nerve lesion by local translation of axonal mRNA. This leads to formation of a high-affinity nuclear localization signal (NLS) binding complex that traffics retrogradely with the motor protein dynein. Trituration of synthetic NLS peptide at the injury site of axotomized dorsal root ganglion (DRG) neurons delays their regenerative outgrowth, and NLS introduction to sciatic nerve concomitantly with a crush injury suppresses the conditioning lesion induced transition from arborizing to elongating growth in L4/L5 DRG neurons. These data suggest a model whereby lesion-induced upregulation of axonal importin beta enables retrograde transport of signals that modulate the regeneration of injured neurons.

Schematic model for formation of the importin-targeted retrograde injury-signaling complex

Importin-alpha protein is constitutively associated with the retrograde motor dynein (D) in axons, whereas importin-beta is normally present only as mRNA (upper panel). Upon lesion, local translation of importin-beta to protein induces formation of the importin heterodimer, thus creating a high affinity NLS-binding site associated with dynein. Concomitant modification of NLS-bearing signaling proteins in axoplasm creates a signaling cargo that binds to the complex, thus accessing the retrograde transport pathway (middle and lower panels).

Our current efforts in this project are focused on the following three questions:

Retrograde signaling by neurotrophic factors

The NGF family of neurotrophins has crucial roles in development and maintenance of the nervous system. Trk receptors are receptor tyrosine kinases with specificity for individual neurotrophins, while the p75 neurotrophin receptor (p75) binds all known neurotrophins, as well as other ligands. In addition to activating independent signaling pathways, p75 can function as part of a receptor complex with the trks or with other receptors. A large number of intracellular molecules have been found to interact with p75, thus this receptor integrates multiple extracellular signals with a range of intracellular signaling pathways, leading to diverse biological consequences. Although retrograde signaling from trk receptors has been intensively studied in recent years, p75 retrograde signaling in neurons has remained enigmatic. We have shown that p75 is internalized to the recycling endosome upon neurotrophin binding, but at much slower kinetics than trk-neurotrophin complexes in the same cells. Nonetheless p75 is internalized together with its ligand, and intracellular interactors from the MAGE gene family are recruited to the complex, thus creating a potential signaling endosome or proteolytically cleaved signaling complex. How might this entity connect with the retrograde transport machinery? Strikingly, many of the intracellular p75 interactors contain nuclear localization signals (NLS). Thus, if p75 signaling somehow also induces local formation of an importins complex, the signaling endosome/complex may be transported in a similar manner to the injury-signaling mechanism described above. Current efforts are geared to examination of this hypothesis.

A hypothetical model for transport of p75 signaling endosomes or complexes

Internalization of p75 with ligand to the recycling endosome (upper) or cleavage of p75 to release the intracellular domain (lower) may be followed in both cases by recruitment of intracellular interactors with nuclear localization signals. These might then serve as adaptors to link the p75 signalling complexes to an importin/dynein retrograde transport ensemble on the microtubules.

Differentiation/Survival Signaling in neurons or in tumors of neural origin

Over the past decade we have carried out a number of protein-protein interaction screens to study neurotrophic signaling networks. A yeast RRS screen using the p75 intracellular domain as bait pulled out necdin, a member of the MAGE gene family known primarily as a facilitator of cell cycle exit and neuronal differentiation. The necdin gene is located in the chromosomal region affected in Prader-Willi syndrome (PWS), and necdin-deficient mice display phenotypes mimicking some aspects of PWS. Necdin has been shown to be present in different subcellular compartments ranging from membrane receptor association to cytoplasmic and nuclear localization, raising the question whether its functional importance is as a signal transduction protein or an effector of gene expression, or both. Additional RRS screens with necdin as bait suggest that it is involved in two interaction networks – one primarily nuclear and one including cytoplasmic and plasma membrane interactors. Current efforts are focusing on the mode of translocation of necdin between cytoplasmic and nuclear compartments, and the role of necdin in each of these compartments for differentiation and survival. We are also studying a new necdin interactor that we call Karet, which is involved in TrkA-dependent cell death in transfected cells. TrkA-dependent cell death is an atypical phenomenon that has been described in very few reports in the literature, primarily in pediatric tumor cells of neural origin, underscoring the interest in elucidating the functions and mechanisms of action of karet.