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 Freund's 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 Freund's 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.

RISK-1: a novel MAPK homologue in axoplasm that is activated and retrogradely transported after nerve injury.

Sensory neurons (SNs) of Aplysia are widely used to study the molecular correlates of learning. Among these is the activation of an Aplysia (ap) MAPK that phosphorylates the transcription factor apC/EBPbeta. Because crushing the axons of the SNs induces changes similar to learning, we tested the hypothesis that apMAPK is a point of convergence on the pathways for learning and injury. One event in common is long-term hyperexcitability (LTH), and LTH was induced in the SNs after intrasomatic injection of active vertebrate extracellular signal-regulated kinase 1 (ERK1; as an apMAPK surrogate). Nerve crush activated an axoplasmic kinase at the site of injury that phosphorylated apC/EBPbeta. Surprisingly, this was not apMAPK, but a kinase that was recognized by antibodies to vertebrate ERKs and to doubly phosphorylated, activated ERKs. The activated kinase was transported to the cell body and nucleus and its arrival was concurrent with an injury-induced increase in apC/EBPbeta mRNA and protein. We call this retrogradely transported kinase RISK-1. RISK-1 initiated the binding of apC/EBPbeta to the ERE enhancer site in vitro and an increase in ERE-binding was detected in injured neurons containing active RISK-1. Thus, Aplysia neurons contain two MAPK homologues, one of which is a late acting retrogradely transported injury signal.

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.

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.

Inflammation causes a long-term hyperexcitability in the nociceptive sensory neurons of Aplysia.

Nerve injury, tissue damage, and inflammation all cause hyperalgesia. A factor contributing to this increased sensitivity is a long-term (>24 hr) hyperexcitability (LTH) in the sensory neurons that mediate the responses. Using the cluster of nociceptive sensory neurons in Aplysia californica as a model, we are examining how inflammation induces LTH. A general inflammatory response was induced by inserting a gauze pad into the animal Within 4 days, the gauze is enmeshed in an amorphous material that contains hemocytes, which comprise a cellular immune system. Concurrently, LTH appears in both ipsilateral and contralateral sensory neurons. The LTH is manifest as increased action potential discharge to a normalized stimulus. Immunocytochemistry revealed that hemocytes have antigens recognized by antibodies to TGFbeta1, IL-6, and 5HT. When a localized inflammation was elicited on a nerve, hemocytes containing the TGFbeta1 antigen were present near axons within the nerve and those containing the IL-6 were on the surface. Western blots of hemocytes, or of gauze that had induced a foreign body response, contained a 28-kD polypeptide recognized by the anti-TGFbeta1 antibody. Exposure of the nervous system to recombinant human TGFbeta1 elicited increased firing of the nociceptive neurons and a decrease in threshold. The TGFbeta1 also caused an activation of protein kinase C (PKC) in axons but did not affect a kinase that is activated in axons after injury. Our findings, in conjunction with previous results, indicate that a TGFbeta1-homolog can modulate the activity of neurons that respond to noxious stimuli. This system could also contribute to interactions between the immune and nervous systems via regulation of PKC.

Activation of protein kinase A contributes to the expression but not the induction of long-term hyperexcitability caused by axotomy of Aplysia sensory neurons.

Nociceptive sensory neurons (SNs) in Aplysia provide useful models to study both memory and adaptive responses to nerve injury. Induction of long-term memory in many species, including Aplysia, is thought to depend on activation of cAMP-dependent protein kinase (PKA). Because Aplysia SNs display similar alterations in models of memory and after nerve injury, a plausible hypothesis is that axotomy triggers memory-like modifications by activating PKA in damaged axons. The present study disproves this hypothesis. SN axotomy was produced by (1) dissociation of somata from the ganglion [which is shown to induce long-term hyperexcitability (LTH)], (2) transection of neurites of dissociated SNs growing in vitro, or (3) peripheral nerve crush. Application of the competitive PKA inhibitor Rp-8-CPT-cAMPS at the time of axotomy failed to alter the induction of LTH by each form of axotomy, although the inhibitor antagonized hyperexcitability produced by 5-HT application. Strong activation of PKA in the nerve by coapplication of a membrane-permeant analog of cAMP and a phosphodiesterase inhibitor was not sufficient to induce LTH of either the SN somata or axons. Furthermore, nerve crush failed to activate axonal PKA or stimulate its retrograde transport. Therefore, PKA activation plays little if any role in the induction of LTH by axotomy. However, the expression of LTH was reduced by intracellular injection of the highly specific PKA inhibitor PKI several days after nerve crush. This suggests that long-lasting activation of PKA in or near the soma contributes to the maintenance of long-term modifications produced by nerve injury.

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.

An NF-kappaB-like transcription factor in axoplasm is rapidly inactivated after nerve injury in Aplysia.

We found a protein in Aplysia neurons that has many characteristics of the transcription factor NF-kappaB. Thus, the protein recognized a radiolabeled probe containing the kappaB sequence from the human interferon-beta gene enhancer element (PRDII), and the binding was not affected by PRDIV, an ATF-2 enhancer sequence from the same gene. Binding was efficiently inhibited, however, by nonradioactive oligonucleotides containing H2, the kappaB site from the major histocompatibility complex I gene promotor. In addition, recombinant mammalian IkappaB-alpha, which associates specifically with the P65 subunit of NF-kappaB, inhibited the binding to the PRDII probe in a dose-dependent manner. The nuclear form of the Aplysia protein was constitutively active. Axoplasm, however, contained the constitutively active form as well as a latent form. The latter was activated by treatment with deoxycholate under the same conditions as mammalian NF-kappaB. Based on these findings, we believe the protein to be a homolog of NF-kappaB. To investigate the role of apNF-kappaB in the axon, we crushed the peripheral nerves to the body wall. Surprisingly, there was a rapid loss of apNF-kappaB binding at the crush site and, within 15 min, as far as 2.5 cm along the axon. In contrast, exposing either the intact animal or the nervous system in situ to levels of 5-HT that induce synaptic facilitation did not affect apNF-kappaB activity.

Intrinsic injury signals enhance growth, survival, and excitability of Aplysia neurons.

Neurons undergo extensive changes in growth and electrophysiological properties in response to axon injury. Efforts to understand the molecular mechanisms that initiate these changes have focused almost exclusively on the role of extrinsic signals, primarily neurotrophic factors released from target and glial cells. The objective of the present investigation was to determine whether the response to axonal injury also involves intrinsic axoplasmic signals. Aplysia neurons were removed from their ganglia and placed in vitro on a substratum permissive for growth, but in the absence of glia and soluble growth factors. Under these conditions, neurites emerged and grew for approximately 4 d. Once growth had ceased, the neurites were transected. In all, 46 of 50 cells regenerated, either by resorbing the remaining neurites and elaborating a new neuritic arbor or by merely replacing the neurites that had been severed. Cut cells also exhibited enhanced excitability and, paradoxically, prolonged survival, when compared with uninjured neurons. These findings indicate that axons contain intrinsic molecular signals that are directly activated by injury to trigger changes underlying regeneration and compensatory plasticity.

Targeting of glycoprotein I (gE) of varicella-zoster virus to the trans-Golgi network by an AYRV sequence and an acidic amino acid-rich patch in the cytosolic domain of the molecule.

Previous studies suggested that varicella-zoster virus (VZV) envelope glycoproteins (gps) are selectively transported to the trans-Golgi network (TGN) and that the cytosolic domain of gpI (gE) targets it to the TGN. To identify targeting signals in the gpI cytosolic domain, intracellular protein trafficking was studied in transfected cells expressing chimeric proteins in which a full-length or mutated gpI cytosolic domain was fused to the gpI transmembrane domain and interleukin-2 receptor (tac) ectodomain. Expressed protein was visualized with antibodies to tac. A targeting sequence (AYRV) and a second, acidic amino acid-rich region of the gpI cytosolic domain (putative signal patch) were each sufficient to cause expressed protein to colocalize with TGN markers. This targeting was lost when the tyrosine of the AYRV sequence was replaced with glycine or lysine, when arginine was replaced with glutamic acid, or when valine was substituted with lysine. In contrast, tyrosine could be replaced by phenylalanine and valine could be substituted with leucine. Mutation of alanine to aspartic acid or deletion of alanine abolished TGN targeting. Exposure of transfected cells to antibodies to the tac ectodomain revealed that the TCN targeting of expressed tac-gpI chimeric proteins occurred as a result of selective retrieval from the plasmalemma. These data suggest that the AYRV sequence and a second signaling patch in the cytosolic domain of gpI are responsible for its targeting to the TGN. The observations also support the hypothesis that the TGN plays a critical role in the envelopment of VZV.

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 mollusk Aplysia 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, informing 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.

Envelopment of varicella-zoster virus: targeting of viral glycoproteins to the trans-Golgi network.

Previous studies suggested that varicella-zoster virus derives its final envelope from the trans-Golgi network (TGN) and that envelope glycoproteins (gps) are transported to the TGN independently of nucleocapsids. We tested the hypothesis that gpI is targeted to the TGN as a result of a signal sequence or patch encoded in its cytosolic domain. cDNAs encoding gpI wild type (wt) and a truncated mutant gpI(trc) lacking transmembrane and cytosolic domains were cloned by using the PCR. Cells transfected with cDNA encoding gpI(wt) or gpI(trc) synthesized and N glycosylated the proteins. gpI(wt) accumulated in the TGN, some reached the plasmalemma, but none was secreted. In contrast, gpI(trc) was retained and probably degraded in the endoplasmic reticulum; none was found on cell surfaces, but some was secreted. The distribution of gpI(trc) was not affected by deletion of potential glycosylation sites. To locate a potential gpI-targeting sequence, cells were transfected with cDNA encoding chimeric proteins in which the ectodomain of a plasmalemmal marker, the interleukin-2 receptor (tac), was fused to different domains of gpI. A chimeric protein in which tac was fused with the transmembrane and cytoplasmic domains of gpI was targeted to the TGN. In contrast, a chimeric protein in which tac was fused only with the gpI transmembrane domain passed through the TGN and concentrated in endosomes. We conclude that gpI is targeted to the TGN as a result of a targeting sequence or patch in its cytosolic domain.

Axoplasm enriched in a protein mobilized by nerve injury induces memory-like alterations in Aplysia neurons.

Axon regeneration after injury and long-term alterations associated with learning both require protein synthesis in the neuronal cell body, but the signals that initiate these changes are largely unknown. Direct evidence that axonal injury activates molecular signals in the axon was obtained by injecting axoplasm from crushed or uncrushed nerves into somata of sensory neurons with uncrushed axons. Those injected with crush axoplasm behaved as if their axons had been crushed, exhibiting increases in both repetitive firing and spike duration, and a decrease in spike afterhyperpolarization 1 d after injection. Because similar changes occur in the same cells after learning, these data suggest that some of the long-lasting adaptive changes that occur after injury and learning may be induced by common axoplasmic signals. Since the signals in axoplasm must be conveyed to the cell soma, we have begun to test the hypothesis that at least some of these signals are proteins containing a nuclear localization signal (NLS). Axoplasmic proteins at the crush site and those that accumulated at a ligation proximal to the crush were probed with an antibody to an amino acid sequence (sp) containing a NLS that provides access to the retrograde transport/nuclear import pathway. One protein, sp97, displayed properties expected of an axonal injury signal: it responded to injury by undergoing an anterograde-to-retrograde change in movement and, when the ligation was omitted, it was transported to the cell bodies of the injured neurons.

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.

Intracellular transport of newly synthesized varicella-zoster virus: final envelopment in the trans-Golgi network.

The maturation and envelopment of varicella-zoster virus (VZV) was studied in infected human embryonic lung fibroblasts. Transmission electron microscopy confirmed that nucleocapsids acquire an envelope from the inner nuclear membrane as they enter the perinuclear-cisterna-rough endoplasmic reticulum (RER). Tegument is not detectable in these virions; moreover, in contrast to the mature VZV envelope, the envelope of VZV in the RER is not radioautographically labeled in pulse-chase experiments with [3H]mannose, and it lacks gpI immunoreactivity and complex oligosaccharides. This primary envelope fuses with the RER membrane (detected in cells incubated at 20 degrees C), thereby releasing nucleocapsids to the cytosol. Viral glycoproteins, traced by transmission electron microscopy radioautography in pulse-chase experiments with [3H]mannose, are transported to the trans-Golgi network (TGN) by a pathway that runs from the RER through an intermediate compartment and the Golgi stack. At later chase intervals, [3H]mannose labeling becomes associated with enveloped virions in post-Golgi locations (prelysosomes and plasma membrane). Nucleocapsids appear to be enveloped by wrapping in specialized cisternae, identified as the TGN with specific markers. Tegument-like material adheres to the cytosolic face of the concave surface of TGN sacs; nucleocapsids adhere to this protein, which is thus trapped between the nucleocapsid and the TGN-derived membrane that wraps around it. Experiments with brefeldin A suggest that tegument may bind to the cytosolic tails of viral glycoproteins. Fusion and fission convert the TGN-derived wrapping sacs into an inner enveloped virion and an outer transport vesicle that carries newly enveloped virions to cytoplasmic vacuoles. These vacuoles are acidic and were identified as prelysosomes. It is postulated that secreted virions are partially degraded by their exposure to the prelysosomal internal milieu and rendered noninfectious. This process explains the cell-associated nature of VZV in vitro; however, the mechanism by which the virus escapes diversion from the secretory pathway to the lysosomal pathway in vivo remains to be determined.

Endogenous axoplasmic proteins and proteins containing nuclear localization signal sequences use the retrograde axonal transport/nuclear import pathway in Aplysia neurons.

When the nuclear localization signal peptide (sp) of the SV 40 large T antigen was coupled to human serum albumin (HSA), rhodaminated (r), and microinjected into axons of Aplysia neurons in vitro, the rHSA-sp was conveyed through the axon to the cell body and then into the nucleus (Ambron et al., 1992). But since rHSA-sp is an artificial construct, we needed to determine whether naturally occurring nuclear proteins use this pathway. We therefore injected calf thymus histone H-1 and Xenopus oocyte nucleoplasmin into axons. By 3 hr both were retrogradely transported and targeted into the nucleus, though histone H-1 less efficiently than rHSA-sp or nucleoplasmin. In contrast, neither rHSA, nor rHSA conjugated to a peptide with a random distribution of basic amino acids, was transported or imported. To see if proteins that use the pathway remain intact, we coupled sp to HRP. When injected into varicosities, the HRP-sp was transported/imported to the nucleus, where it was enzymatically active. A key issue was to determine whether endogenous proteins use this pathway. Consequently, axoplasm was extruded from Aplysia nerves and the proteins were fractionated by size. SDS-PAGE and Western blots showed that two fractions contained proteins that were recognized by an affinity-purified antibody to sp: fraction 3 included sp83, and fraction 4 contained sp75. In addition, these two proteins were found in nuclei isolated from neurons. To assess transport, the total proteins in the fractions were rhodaminated and injected into varicosities. Fraction 3, but not fraction 4, contained protein that was transported through the axon to the nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

An 83 kDa O-GlcNAc-glycoprotein is found in the axoplasm and nucleus of Aplysia neurons.

Glycoproteins containing O-linked N-acetylglucosamine (O-GlcNAc) are present in axons of Aplysia neurons (Gabel et al., 1989) and among transcription factors and other proteins in the nucleus of eukaryotic cells (Jackson and Tjian, 1988). A recently discovered pathway in neurons transports proteins through the axon and then into the nucleus (Ambron et al., 1992). If any of the axonal O-GlcNAc glycoproteins use this pathway, then the axon and the nucleus will have these glycoproteins in common. We addressed this issue by using galactosyltransferase and UDP-3H-galactose to label and identify the glycoproteins in three regions of Aplysia neurons: axoplasm, extruded from nerves; nuclei, isolated by manual dissection of single neurons; and cytoplasm, obtained after removal of nuclei. At least 21 glycoproteins were labeled by this procedure; several, at 200, 180, 83, 76, and 66 kDa, from the nucleus and axoplasm comigrated after SDS-PAGE. Radiolabeled galactosyl-N-acetylglucosaminitol was released from the glycoproteins by base/borohydride, thereby verifying the presence of O-GlcNAc. Comparison of the 83 kDa glycoprotein from the nucleus and axoplasm revealed that both were soluble, had multiple O-GlcNAcs, and were bound to WGA. Thus, the 83 kDa constituent is a good candidate to use the axonal transport/nuclear import pathway.

The morphological localization and biochemical characterization of a synapsin I-like antigen in the nervous system of Aplysia californica.

Synapsins are a well-characterized class of phosphoproteins found at synapses in the mammalian nervous system. One member of this family, synapsin I, has been extensively studied and shown to associate in a phosphorylation-dependent manner with both small synaptic vesicles and cytoskeletal elements. Though the characteristics of synapsin I suggest an important function in synaptic transmission, its definitive role is still in question. In an effort to find a model system in which to test directly the function of synapsin I, we have looked in the nervous system of the marine mollusc Aplysia californica for synapsin I-like antigens (SILA). Light microscope immunocytochemical studies using polyclonal and monoclonal antibodies to bovine brain synapsin I demonstrate Aplysia SILA in neuronal somata, in the neuropil, and at some identified synapses. Though SILA were exclusively associated with neuronal structures in Aplysia, the pattern of staining suggested that they are not present at all synaptic terminals. This interpretation was corroborated by ultrastructural studies in which SILA were present at some synaptic terminals but absent, or in low abundance, in adjacent terminals. In axons, SILA were associated with vesicles of 120-150 nm diameter, as well as with filamentous structures. Biochemical studies identified small amounts of SILA of 40 and 50 kD molecular weight that are recognized by several antibodies to mammalian synapsin I, and are acid extractable, collagenase-sensitive phosphoproteins; these are criteria used to define synapsin I homologues in other species. Our studies indicate that SILA are present in neurons in Aplysia californica but suggested that they represent only a small percentage of the total protein within the nervous system.

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.

Cytochrome oxidase activity as a measure of synaptogenesis by multifunctional neuron L7 of Aplysia.

Neuron L7 of the marine mollusc, Aplysia californica, is unique in that it innervates five different target tissues in the animal. We show that when L7 is grown in vitro with two of these targets, that is, muscle cells isolated from the auricle or the gill vein, newly formed L7 neurites contact the muscle cells. Chemical synapses are formed since intracellular stimulation of L7 elicits contraction of individual muscle cells. Interestingly, auricle muscles are also innervated by neuron RBhe and co-cultures of RBhe and auricle muscle cells also exhibit synapse formation. To explore the molecular basis for synaptogenesis between L7 and its targets, it would be useful to quantify the extent of synapse formation in vitro, that is, to determine how many muscle cells can be innervated by a single L7. We show that this can be attained by staining for cytochrome oxidase activity. Cultures of auricle and gill vein muscles were exposed to the appropriate neurotransmitter in order to elicit contraction. The cells were then fixed and stained. In both cases, only cells that contracted were stained and electron microscopy showed reaction product associated with the cristae of mitochondria. When this procedure was applied to cultures of L7 and muscle cells, 38 +/- 2.8% (S.E.M.; n = 7) of the cells on the neurites were stained and therefore responded to L7 stimulation. Thus, part of the L7-RBhe circuit can be assembled in vitro and the extent of synaptogenesis can be accurately quantitated.