Richard T. Ambron

Priming events and retrograde injury signals. A new perspective on the cellular and molecular biology of nerve regeneration.

Successful axon regeneration requires that signals from the site of injury reach the nucleus to elicit changes in transcription. In spite of their obvious importance, relatively few of these signals have been identified. Recent work on regeneration in the marine molluskAplysia californica has provided several insights into the molecular events that occur in neurons after axon injury. Based on these findings, we propose a model in which axon regeneration is viewed as the culmination of a series of temporally distinct but overlapping phases. Within each phase, specific signals enter the nucleus to prime the cell for the arrival of subsequent signals. The first phase begins with the arrival of injury-induced action potentials, which act via calcium and cAMP to turn on genes used in the early stages of repair. In the next phase, MAP-kinases and other intrinsic constituents activated at the injury site are retrogradely transported through the axon to the nucleus, informaing the nucleus of the severity of the axonal injury, reinforcing the earlier events, and triggering additional changes. The third phase is characterized by the arrival of signals that originate from extrinsic growth factors and cytokines released by cells at the site of injury. In the last phase, signals from target-derived growth factors arrive in the cell soma to stop growth. Because many of these events appear to be universal, this framework may be useful in studies of nerve repair in both invertebrates and vertebrates.

Activity-dependent transcription regulation of PSD-95 by neuregulin-1 and Eos

Neuregulin-1 (Nrg-1) contains an intracellular domain (Nrg-ICD) that translocates into the nucleus, where it may regulate gene expression upon neuronal depolarization. However, the identity of its target promoters and the mechanisms by which it regulates transcription have been elusive. Here we report that, in the mouse cochlea, synaptic activity increases the level of nuclear Nrg-ICD and upregulates postsynaptic density protein-95 (PSD-95), a scaffolding protein that is enriched in post-synaptic structures. Nrg-ICD enhances the transcriptional activity of the PSD-95 promoter by binding to a zinc-finger transcription factor, Eos. The Nrg-ICD–Eos complex induces endogenous PSD-95 expression in vivo through a signaling pathway that is mostly independent of γ-secretase regulation. This upregulation of PSD-95 expression by the Nrg-ICD–Eos complex provides a molecular basis for activity-dependent synaptic plasticity.

Long-term alterations induced by injury and by 5=HT in Aplysia sensory neurons: convergent pathways and common signals?

Bodily injury in Aplysia, as in mammals, produces long-lasting memory traces at various neural loci. One consequence of injury, damage to peripheral axons, produces long-term hyperexcitability, synaptic facilitation, and growth in Aplysia sensory neurons. Similar effects are induced in these cells by repeated exposure to 5-HT that is released during aversive learning. An interesting question is to what extent cellular pathways that mediate the effects of axonal injury and 5-HT overlap. One current focus is on identifying cytoplasmic signals that initiate persistent sensory alterations that contribute to both long-term sensitization and memory of injury.

A signal sequence mediates the retrograde transport of proteins from the axon periphery to the cell body and then into the nucleus

The presynaptic terminal and axon of neurons can undergo structural changes in response to environmental signals. Since these changes require protein synthesis in the cell body, the needs of the periphery must somehow be communicated to the cell soma. To look for such a mechanism, we used artificial protein constructs with properties expected of a signal that is transported from the axon to the nucleus. One construct consisted of the nuclear import signal peptide (sp) of the SV40 large T antigen, coupled to human serum albumin (HSA) and rhodamine (r). When injected into the axoplasm of Aplysia californica neurons in vitro, the rHSA-sp was transported in the retrograde direction through the axon to the cell body and then into the nucleus. Little, if any, moved in the anterograde direction toward growth cones. The retrograde movement of injected rHSA-sp was rapid (greater than 25 mm/d) and depended upon intact microtubules. The sp portion of rHSA-sp provided access to both the retrograde transport system and the nuclear import apparatus. Thus, rHSA was not transported at all, but accumulated in organelles near the injection site. Also, rHSA-sp containing an sp with a Lys to Thr substitution, which is known to reduce nuclear import markedly, was transported only poorly. To look for endogenous molecules that use this system, we affinity-purified a rabbit polyclonal antibody to the signal sequence. The antibody recognized an 83 kDa polypeptide on Western blots of Aplysia nervous tissue. These data indicate that Aplysia neurons contain the machinery to convey macromolecules from the axon periphery to the nucleus.

Positive injury signals induce growth and prolong survival in Aplysia neurons.

Injury to a peripheral nerve initiates changes that can lead to regeneration of the damaged axons. How information about a distant injury is communicated to the cell body is not clear. Using the nervous system of Aplysia californica, we tested the idea that some of this information is conveyed via positive injury signals-axoplasmic proteins that are activated at the injury site and transported to the cell soma. We collected these proteins by crushing pedal nerves and then placing a ligation proximal to the ligation. The contralateral nerves were ligated as controls. Twenty h later, axoplasm was extruded from the nerve segment just distal to the ligation on the crushed nerves (cr/lig) and on the control nerves (lig). The total proteins were rhodaminated and injected into the cytoplasm of neurons in vitro to look for nuclear import. Punctate fluorescence was detected in the nucleus of all seven neurons injected with the cr/lig axoplasm. Only two of five neurons injected with lig axoplasm had any fluorescence. Equal amounts of cr/lig and lig axoplasm were then injected directly into the cell bodies of neurons maintained in vitro. The cells injected with cr/lig axoplasm exhibited renewed growth and significantly longer survival: 25.9 +/- 2.1 days (mean +/- SEM: n = 22) relative to the cells injected with lig axoplasm (15.3 +/- 1.2 days; n = 14) and to those that were not injected (12.2 +/- 1.7 days; n = 24). Fractionation of the cr/lig axoplasm indicated that different factors are responsible for growth and survival.

A nuclear localization signal targets proteins to the retrograde transport system, thereby evading uptake into organelles in aplysia axons

The turnover of soluble proteins in axons and terminals is effected by replacing used proteins with newly synthesized constituents from the cell body. To investigate this complex process, which is especially important during nerve regeneration, we microinjected proteins into varicosities on axons of Aplysia neurons in vitro. When human serum albumin (HSA) coupled to rhodamine (r) was injected, it initially filled the varicosity; within seconds, however, it began to accumulate in packets and by 15 min was punctate. A similar pattern was observed after injecting soluble proteins from extruded axoplasm. In contrast, when we injected rHSA covalently attached to the SV-40 nuclear localization sequence (sp), the distribution was never punctate and the rHSA-sp was retrogradely transported from the varicosity to the cell body and into the nucleus. Electron microscopy of varicosities injected with HSA-gold showed that >90% of the particles were inside vacuoles and multivesicular bodies. These organelles probably function as storage rather than degradatory sites since they did not contain acid phosphatase. In contrast, when HSAsp-colloidal gold was injected, only 25% of the particles were in organelles. Thus, HSA and resident axonal proteins can be removed from axoplasm by uptake into organelles. The presence of a nuclear localization sequence (the sp) may avoid uptake by providing access to the retrograde transport/nuclear import pathway.

Activation and retrograde transport of protein kinase G in rat nociceptive neurons after nerve injury and inflammation

Nerve injury elicits both universal and limited responses. Among the former is regenerative growth, which occurs in most peripheral neurons, and among the latter is the long-term hyperexcitability that appears selectively in nociceptive sensory neurons. Since positive injury signals communicate information from the site of an injury to the cell body, we hypothesize that a nerve injury activates both universal and limited positive injury signals. Studies in Aplysia indicate that protein kinase G is a limited signal that is responsible for the induction of long-term hyperexcitability. Given that long-term hyperexcitability contributes to chronic pain after axotomy in rodent neuropathic pain models, we investigated its underlying basis in the rat peripheral nervous system. Using biochemical assays, Western blots, and immunocytochemistry we found that the Type 1alpha protein kinase G is the predominant isoform in the rat periphery. It is present primarily in axons and cell bodies of nociceptive neurons, including populations that are isolectin B4-positive, isolectin B4-negative, and those that express transient receptor potential vanilloid receptor-1. Surprisingly, protein kinase G is not present in the facial nerve, which overwhelmingly contains axons of motor neurons. Crushing the sciatic nerve or a cutaneous sensory nerve activates protein kinase G in axons and results in its retrograde transport to the neuronal somata in the DRG. Preventing the activation of protein kinase G by injecting Rp-8-pCPT-cGMPS into the crush site abolished the transport. The protein kinase A inhibitor Rp-8-pCPT-cAMPS had no effect. Extracellular signal-related kinases 42/44 are also activated and transported after nerve crush, but in both motor and sensory axons. Chronic pain has been linked to long-term hyperexcitability following a nerve inflammation in several rodent models. We therefore injected complete Freunds adjuvant into the hindpaw to induce an inflammation and found that protein kinase G was activated in the sural nerve and transported to the DRG. In contrast, the extracellular signal-related kinases in the sensory axons were not activated by the complete Freunds adjuvant. These studies support the idea that the extracellular signal-related kinases are universal positive axonal signals and that protein kinase G is a limited positive axonal signal. They also establish the association between protein kinase G, the induction of long-term hyperexcitability, and chronic pain in rodents.

Pathways that elicit long-term changes in gene expression in nociceptive neurons following nerve injury: contributions to neuropathic pain

Abstract Chronic neuropathic pain following nerve injury or inflammation is mediated by transcription-dependent changes in neurons that comprise the nociceptive pathway. Among these changes is often a long-term hyperexcitiability (LTH) in primary nociceptors that persists long after the lesion has healed. LTH is manifest by a reduction in threshold and an increased tendency to fire action potentials. This increased excitability activates higher order neurons in the pathway, leading to the perception of pain. Efforts to ameliorate chronic pain would therefore benefit if we understood how LTH is induced, but studies toward this goal are impeded by the complexity and heterogeneity of vertebrate nervous systems. Fortunately, LTH is an evolutionarily conserved mechanism that underlies defensive behaviors across phyla, including invertebrates. Thus, the same electrophysiological changes that underlie LTH in vertebrate nociceptive neurons are seen in their counterparts in the experimentally favorable mollusk Aplysia californica. Nociceptive neurons of Aplysia are readily accessible and large enough to approach using a variety of cell and molecular approaches not possible in higher organisms. Studies of the molecular cascades activated by injury to Aplysia peripheral nerves has focused on a group of positive injury signals that are retrogradely transported from the injury site in the axon to the cell nucleus where they regulate gene transcription. One of these, protein kinase G, is activated by nitric oxide synthetase and its activation in axons is required for the induction of LTH after injury. This pathway, and the transcriptional events that it activates, are targets for therapeutic intervention for chronic pain.

Engineering Novel Spinal Circuits to Promote Recovery after Spinal Injury

We have developed an innovative way to establish a functional bridge around a spinal lesion. We disconnected the T13 nerve from its muscle targets, leaving the proximal end intact. The cut end was inserted either into an intact spinal cord, to assess regeneration of T13 axons into the cord and synapse formation with spinal neurons, or caudal to a hemisection at L2/3, to assess restoration of function below the injury. Four to 28 weeks later, anterograde tracers indicated that axons from the inserted T13 nerve regenerated into the ventral horn, the intermediate zone, and dorsal horn base, both in intact and hemisected animals. Antibodies to cholinergic markers showed that many regenerating axons were from T13 motoneurons. Electrical stimulation of the T13 nerve proximal to the insertion site 4 weeks or more after insertion into the intact cord evoked local field potentials in the intermediate zone and ventral horn, which is where T13 axons terminated. Stimulation of T13 in 71% of the animals (8 hemisected, 7 intact) evoked contraction of the back or leg muscles, depending on the level of insertion. Animals in which T13 was inserted caudal to hemisection had significantly less spasticity and muscle wasting and greater mobility at the hip, knee, ankle, and digits in the ipsilateral hindlimb than did animals with a hemisection only. Thus, T13 motor axons form novel synapses with lumbosacral motor circuits. Because the T13 motor neurons retain their connections to the brain, these novel circuits might restore voluntary control to muscles paralyzed below a spinal lesion.

Direct interactions between immunocytes and neurons after axotomy in Aplysia.

Axon growth during development and after injury has processes in common, but also differs in that regeneration requires the participation of cells of the immune system. To investigate how neuron-immunocyte interactions might influence regeneration, we developed an in vitro model whereby neurons and hemocytes from Aplysia californica were cocultured. The hemocytes, which behave like vertebrate macrophages, migrated randomly throughout the dish. When a neuron was encountered, some hemocytes exhibited an avoidance response, whereas others formed stable contacts. Hemocytes did not distinguish between neurons from different animals. Stable contacts occurred on neurites and growth cones, but not the cell soma, and were benign in that the hemocytes did not impede neurite growth. When hemocytes attached to the cell body, it presaged the destruction of the neuron. Destruction was a dynamic process that was initiated when groups of one to three hemocytes adhered to various regions of the cell soma. Each group was then joined by other hemocytes. They did not contact the neuron, but interconnected the initial groups, forming a network around the neuron. The network then contracted to dismember the cell. Once a neuron was destroyed, hemocytes removed the debris by phagocytosis. Both damaged neurons and those without apparent damage were targets for destruction. Severing neurites with a needle resulted in the destruction of only one of six cells. Our studies suggest that hemocytes, and by extrapolation, vertebrate macrophages, exhibit highly complex interactions with neurons that can exert a variety of influences on the course of nerve regeneration.