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J H Skene

Publications and source records attributed to J H Skene.

At least 19 recordsLinked to original sources
PubMed2001

Spinal axon regeneration evoked by replacing two growth cone proteins in adult neurons.

In contrast to peripheral nerves, damaged axons in the mammalian brain and spinal cord rarely regenerate. Peripheral nerve injury stimulates neuronal expression of many genes that are not generally induced by CNS lesions, but it is not known which of these genes are required for regeneration. Here we show that co-expressing two major growth cone proteins, GAP-43 and CAP-23, can elicit long axon extension by adult dorsal root ganglion (DRG) neurons in vitro. Moreover, this expression triggers a 60-fold increase in regeneration of DRG axons in adult mice after spinal cord injury in vivo. Replacing key growth cone components, therefore, could be an effective way to stimulate regeneration of CNS axons.

Animals↗
PubMed2001

GAP-43 promoter elements in transgenic zebrafish reveal a difference in signals for axon growth during CNS development and regeneration.

A pivotal event in neural development is the point at which differentiating neurons become competent to extend long axons. Initiation of axon growth is equally critical for regeneration. Yet we have a limited understanding of the signaling pathways that regulate the capacity for axon growth during either development or regeneration. Expression of a number of genes encoding growth associated proteins (GAPs) accompanies both developmental and regenerative axon growth and has led to the suggestion that the same signaling pathways regulate both modes of axon growth. We have tested this possibility by asking whether a promoter fragment from a well characterized GAP gene, GAP-43, is sufficient to activate expression in both developing and regenerating neurons. We generated stable lines of transgenic zebrafish that express green fluorescent protein (GFP) under regulation of a 1 kb fragment of the rat GAP-43 gene, a fragment that contains a number of evolutionarily conserved elements. Analysis of GFP expression in these lines confirms that the rat 1 kb region can direct growth-associated expression of the transgene in differentiating neurons that extend long axons. Furthermore, this region supports developmental down-regulation of transgene expression which, like the endogenous gene, coincides with neuronal maturation. Strikingly, these same sequences are insufficient for directing expression in regenerating neurons. This finding suggests that signaling pathways regulating axon growth during development and regeneration are not the same. While these results do not exclude the possibility that pathways involved in developmental axon growth are also active in regenerative growth, they do indicate that signaling pathway(s) controlling activation of the GAP-43 gene after CNS injury differ in at least one key component from the signals controlling essential features of developmental axon growth.

Animals↗
PubMed1999

A shift in protein S-palmitoylation, with persistence of growth-associated substrates, marks a critical period for synaptic plasticity in developing brain.

In the mammalian cortex, the initial formation of synaptic connections is followed by a prolonged period during which synaptic circuits are functional, but retain an elevated capacity for activity-dependent remodeling and functional plasticity. During this period, synaptic terminals appear fully mature, morphologically and physiologically. We show here, however, that synaptic terminals during this period are distinguished by their simultaneous accumulation of multiple growth-associated proteins at levels characteristic of axonal growth cones, and proteins involved in synaptic transmitter release at levels characteristic of adult synapses. We show further that newly formed synapses undergo a switch in the dynamic S-palmitoylation of proteins early in the critical period, which includes a large and specific decrease in the palmitoylation of GAP-43 and other major substrates characteristic of growth cones. Previous studies have shown that a similar reduction in ongoing palmitoylation of growth cone proteins is sufficient to stop advancing axons in vitro, suggesting that a developmental switch in protein S-palmitoylation serves to disengage the molecular machinery for axon extension in the absence of local triggers for remodeling during the critical period. Only much later does a decline in the availability of major growth cone components mark the molecular maturation of cortical synapses at the close of the critical period.

Aging↗
PubMed1999

Rapid arrest of axon elongation by brefeldin A: a role for the small GTP-binding protein ARF in neuronal growth cones.

Members of the ADP-ribosylation factor (ARF) family of small guanosine triphosphate-binding proteins play an essential role in membrane trafficking which subserves constitutive protein transport along exocytic and endocytic pathways within eukaryotic cell bodies. In growing neurons, membrane trafficking within motile growth cones distant from the cell body underlies the rapid plasmalemmal expansion which subserves axon elongation. We report here that ARF is a constituent of axonal growth cones, and that application of brefeldin A to neurons in culture produces a rapid arrest of axon extension that can be ascribed to inhibition of ARF function in growth cones. Our findings demonstrate a role for ARF in growth cones that is coupled tightly to the rapid growth of neuronal processes characteristic of developmental and regenerative axon elongation, and indicate that ARF participates not only in constitutive membrane traffic within the cell body, but also in membrane dynamics within growing axon endings.

ADP-Ribosylation Factors↗
PubMed1998

The activity of a highly promiscuous AP-1 element can be confined to neurons by a tissue-selective repressive element.

Tissue-specific gene transcription can be determined by the use of either positive-acting or negative-acting DNA regulatory elements. We have analyzed a promoter from the growth-associated protein 43 (GAP-43) gene and found that it uses both of these mechanisms to achieve its high degree of neuron-specific activity. Two novel transcription factor binding sites, designated Cx1 and Cx2, drive promoter activity in neurons from developing cerebral cortex but not in several other cell types. The promoter also contains an activator protein 1 (AP-1) site that contributes to activity in neurons. The AP-1 site can drive promoter activity in a wide range of non-neuronal cells that express little or no endogenous GAP-43, but only in the absence of a tissue-specific repressive element located downstream of the GAP-43 TATA box. These findings suggest that the GAP-43 repressive element plays an important role in allowing AP-1 signaling pathways to modulate activity of the GAP-43 gene in neurons, without also causing inappropriate activation by AP-1 transcription factors in other cell types.

Amphibians↗
PubMed1997

Identification of a novel repressive element that contributes to neuron-specific gene expression.

Multiple signaling pathways are thought to control the selective expression of genes over the course of neuronal differentiation. One approach to elucidating these pathways is to identify specific cis-acting elements that serve as the final targets for these signaling pathways in neural-specific genes. We now identify a novel repressive element from the growth-associated protein 43 (GAP-43) gene that can contribute to neuron-specific gene expression by inhibiting transcription in a wide range of non-neuronal cell types. This repressive element is located downstream of the GAP-43 TATA box and is highly position-dependent. When transferred to viral promoters this element preferentially inhibits transcription in non-neuronal cells. Electrophoretic mobility shift assays show that the repressive element comprises at least two protein recognition sites. One of these is a novel sequence motif that we designate the SNOG element, because it occurs downstream of the TATA boxes of the synaptosomal-associated protein of 25 kDa and neuronal nitric oxide synthase genes, as well as the GAP-43 gene. The GAP-43 repressive element is distinct in sequence and position dependence from the repressor element 1/neuron-restrictive silencer element previously described in other neural genes and therefore is a likely target for a distinct set of signaling pathways involved in the control of neuronal differentiation.

Animals↗
PubMed1997

A transcription-dependent switch controls competence of adult neurons for distinct modes of axon growth.

Although maturing neurons undergo a precipitous decline in the expression of genes associated with developmental axon growth, structural changes in axon arbors occur in the adult nervous system under both normal and pathological conditions. Furthermore, some neurons support extensive regrowth of long axons after nerve injury. Analysis of adult dorsal root ganglion (DRG) neurons in culture now shows that competence for distinct types of axon growth depends on different patterns of gene expression. In the absence of ongoing transcription, newly isolated neurons can extend compact, highly branched arbors during the first day in culture. Neurons subjected to peripheral axon injury 2-7 d before plating support a distinct mode of growth characterized by rapid extension of long, sparsely branched axons. A transition from "arborizing" to "elongating" growth occurs in naive adult neurons after approximately 24 hr in culture but requires a discrete period of new transcription after removal of the ganglia from the intact animal. Thus, peripheral axotomy-by nerve crush or during removal of DRGs--induces a transcription-dependent change that alters the type of axon growth that can be executed by these adult neurons. This transition appears to be triggered, in large part, by interruption of retrogradely transported signals, because blocking axonal transport in vivo can elicit competence for elongating growth in many DRG neurons. In contrast to peripheral axotomy, interruption of the centrally projecting axons of DRG neurons in vivo leads to subsequent growth in vitro that is intermediate between "arborizing" and "elongating" growth. This suggests that the transition between these two modes of growth is a multistep process and that individual steps may be regulated separately. These observations together suggest that structural remodeling in the adult nervous system need not involve the same molecular apparatus as long axon growth during development and regeneration.

Afferent Pathways↗
PubMed1994

Novel inhibitory action of tunicamycin homologues suggests a role for dynamic protein fatty acylation in growth cone-mediated neurite extension.

In neuronal growth cones, the advancing tips of elongating axons and dendrites, specific protein substrates appear to undergo cycles of posttranslational modification by covalent attachment and removal of long-chain fatty acids. We show here that ongoing fatty acylation can be inhibited selectively by long-chain homologues of the antibiotic tunicamycin, a known inhibitor of N-linked glycosylation. Tunicamycin directly inhibits transfer of palmitate to protein in a cell-free system, indicating that tunicamycin inhibition of protein palmitoylation reflects an action of the drug separate from its previously established effects on glycosylation. Tunicamycin treatment of differentiated PC12 cells or dissociated rat sensory neurons, under conditions in which protein palmitoylation is inhibited, produces a prompt cessation of neurite elongation and induces a collapse of neuronal growth cones. These growth cone responses are rapidly reversed by washout of the antibiotic, even in the absence of protein synthesis, or by addition of serum. Two additional lines of evidence suggest that the effects of tunicamycin on growth cones arise from its ability to inhibit protein long-chain acylation, rather than its previously established effects on protein glycosylation and synthesis. (a) The abilities of different tunicamycin homologues to induce growth cone collapse very systematically with the length of the fatty acyl side-chain of tunicamycin, in a manner predicted and observed for the inhibition of protein palmitoylation. Homologues with fatty acyl moieties shorter than palmitic acid (16 hydrocarbons), including potent inhibitors of glycosylation, are poor inhibitors of growth cone function. (b) The tunicamycin-induced impairment of growth cone function can be reversed by the addition of excess exogenous fatty acid, which reverses the inhibition of protein palmitoylation but has no effect on the inhibition of protein glycosylation. These results suggest an important role for dynamic protein acylation in growth cone-mediated extension of neuronal processes.

Acylation↗
PubMed1994

Neural selective activation and temporal regulation of a mammalian GAP-43 promoter in zebrafish.

Neurons throughout the vertebrate nervous system selectively activate the gene for a growth cone component, GAP-43, during embryonic development, and then decrease its expression abruptly as they form synapses. Distal interruption of mature axons in the central nervous system (CNS) of fish and amphibians, but not in the mammalian CNS reverses the developmental down-regulation of GAP-43 expression. To explore functional conservation and divergence of cis-acting elements that regulate expression of the GAP-43 gene, we studied activation, in transgenic zebrafish embryos, of mammalian GAP-43 genomic sequences fused to a marker gene. The DNA fragments containing the GAP-43 promoter, including a short fragment of 386 base pairs, were preferentially activated in the embryonic fish nervous system at times when extensive neuronal differentiation and neurite outgrowth take place. After 2 days of development, expression of the mammalian transgenes was specifically downregulated in the fish spinal cord but increased in more rostral regions of the CNS. This expression pattern was well correlated with the regulation of the endogenous fish GAP-43 gene revealed by in situ hybridization. Elements of the mammalian gene located a substantial distance upstream of the minimal promoter directed additional expression of the marker gene in a specific set of non-neural cells in zebrafish embryos. Our results indicate that cis-acting elements of the GAP-43 gene, and signaling pathways controlling these elements during embryonic development, have been functionally conserved in vertebrate evolution.

Animals↗
PubMed1993

Neuronal growth cone collapse and inhibition of protein fatty acylation by nitric oxide.

Nitric oxide, a free-radical gas produced endogenously by several mammalian cell types, has been implicated as a diffusible intercellular messenger subserving use-dependent modification of synaptic efficacy in the mature central nervous system. It has been suggested on theoretical grounds that nitric oxide might play an analogous role during the establishment of ordered connections by developing neurons. We report here that nitric oxide rapidly and reversibly inhibits growth of neurites of rat dorsal root ganglion neurons in vitro. In addition, we show that exposure to nitric oxide inhibits thioester-linked long-chain fatty acylation of neuronal proteins, possibly through a direct modification of substrate cysteine thiols. Our results demonstrate a potential role for nitric oxide in the regulation of process outgrowth and remodelling during neuronal development, which may be effected at least in part through modulation of dynamic protein fatty acylation in neuronal growth cones.

Acylation↗
PubMed1993

Injury-associated induction of GAP-43 expression displays axon branch specificity in rat dorsal root ganglion neurons.

Peripheral nerve injury results in the increased synthesis and axonal transport of the growth-associated protein GAP-43 in dorsal root ganglion (DRG) neurons, coincident with regenerative growth of the injured peripheral axon branches. To determine whether the injury-associated signalling mechanism which leads to GAP-43 induction also operates through the central branches of DRG axons, we used immunocytochemistry to compare the expression of GAP-43 in adult rat DRG neurons 2 weeks after dorsal root crush lesions (central axotomy) or peripheral nerve crush lesions (peripheral axotomy). In uninjured ganglia, a subpopulation of smaller DRG neurons expresses moderate levels of GAP-43, whereas larger neurons generally do not. At 2 weeks following peripheral axotomy, virtually all axotomized neurons, large and small, express high levels of GAP-43. At 2 weeks following dorsal root lesions, no increase in GAP-43 expression is detected. Thus, the injury-associated up-regulation of GAP-43 expression in DRG neurons is triggered by a mechanism that is responsive to injury of only the peripheral, and not the central, axon branches. These findings support the hypothesis that GAP-43 induction in DRG neurons is caused by disconnection from peripheral target tissue, not by axon injury per se.

Animals↗
PubMed1992

The 25 kDa synaptosomal-associated protein SNAP-25 is the major methionine-rich polypeptide in rapid axonal transport and a major substrate for palmitoylation in adult CNS.

A conspicuous correlate of the developmental transformation of axonal growth cones to synaptic terminals is a marked increase in synthesis and axonal transport of a methionine-rich, acidic polypeptide of approximately 25 kDa. This polypeptide, designated "super protein" (SuP), is the most prominent species among methionine-labeled proteins conveyed by rapid axonal transport in mature CNS and PNS neurons of warm- and cold-blooded vertebrates. We show here that SuP is identical to SNAP-25, a highly conserved synaptic protein of known primary structure, by immunoprecipitation with anti-SNAP-25 antiserum of SuP labeled with 35S-methionine and transported by retinal ganglion cells of rat and cat. In addition, we show that SNAP-25/SuP is the most prominent species among retinal polypeptides that incorporate 3H-palmitate in vivo, that it is fatty acylated through a hydroxylamine-labile, thioester bond, and that palmitoylated SNAP-25/SuP is axonally transported. Thus, SNAP-25/SuP is a rapidly transported constituent of the presynaptic apparatus and a major neuronal substrate for long-chain fatty acylation.

Animals↗
PubMed1992

Multiple elements may be used for regulation of the GAP-43 gene in different cell-types.

Recent evidence suggests that GAP-43 expression is not restricted to the nervous system, but may also occur outside the neural cell lineage. Two distinct patterns of GAP-43 regulation can therefore be distinguished. The first is the regulation of GAP-43 expression in multiple cell-types, and the second is the gene's temporal modulation within one specific cell-type. The latter type is well documented for neurons, where GAP-43 regulation is regulated in a fashion that is dependent on axon integrity. Results from partial analysis of the GAP-43 promoter/enhancer region indicate that at least some of these aspects of GAP-43 gene regulation may be accounted for by distinct cis-acting elements. For example, the expression of a rat GAP-43 promoter fusion gene in epidermal cells of transgenic zebrafish is dependent on an enhancer element, that is clearly distinct from the minimal neural-specific promoter. Characterization of specific GAP-43 regulatory elements responsible for particular aspects of its regulation may provide insight to signal pathways also utilized by other genes during development. Ultimately, a better understanding of the molecular events during development could help to define more precisely the complex sequences necessary for the establishment of an intact organism.

Animals↗
PubMed1992

A neural-specific GAP-43 core promoter located between unusual DNA elements that interact to regulate its activity.

In an effort to identify cis-acting elements that respond to signals controlling different stages of neural differentiation, we have analyzed the promoter and surrounding regulatory sequences of the rat GAP-43 gene. Expression of this gene is both neural specific and, within neurons, strongly modulated by signals related to axon integrity. Expression analysis in cell lines and primary rat cortical cultures demonstrates that neural-selective gene expression can be directed by a 386 base pair GAP-43 promoter fragment that contains canonical TATA and CCAAT box consensus sequences. A short region of homology with other neural-specific genes, identified upstream of the core promoter, is not essential for selective expression in neuronal cells. Within cortical cell cultures, expression is strongly modulated by two interacting elements on either side of the promoter, each of which contains a sequence with the potential to adopt an unusual DNA conformation. While each of these flanking elements reduces expression when added alone to the core promoter, each counteracts the negative influence of the other when both elements are present.

Animals↗
PubMed1991

Fate of GAP-43 in ascending spinal axons of DRG neurons after peripheral nerve injury: delayed accumulation and correlation with regenerative potential.

Proteins characteristic of growing axons often fail to be induced or transported along axons that have been interrupted far from their cell bodies in the adult mammalian CNS. Here, we inquire whether long axons in the mammalian CNS can support efficient axonal transport and deposition of one such protein, GAP-43, when the protein is induced in neuron cell bodies. We have used immunocytochemistry to follow the fate of GAP-43 in dorsal column axons ascending the rat spinal cord from dorsal column axons ascending the rat spinal cord from dorsal root ganglion (DRG) neurons, after synthesis of the protein is induced in these cells by peripheral nerve injury. Sciatic nerve lesions do lead to an accumulation of GAP-43 in dorsal column axons derived from the lumbar DRG. However, in distal segments of these CNS axons, accumulation of GAP-43 is apparent only after a delay of 1-2 weeks, in contrast to its rapid accumulation in axon segments within the PNS environment, suggesting that deposition and stabilization of GAP-43 can be limited by local, posttranslational regulation. GAP-43 immunoreactivity subsides to control levels within 8 weeks after crush lesions that permit peripheral axon regeneration, but remains robust 8 weeks after resection lesions that prevent peripheral regeneration. Accumulation of GAP-43 in cervical dorsal column axons after peripheral nerve injury is closely correlated with the ability of these axons to respond to local cues capable of eliciting axon growth (Richardson and Verge, 1986).

Animals↗
PubMed1990

GAP-43 as a 'calmodulin sponge' and some implications for calcium signalling in axon terminals.

In most neurons, the maturation of axonal growth cones to become stable synaptic terminals is accompanied by a dramatic decline in the abundance of a major growth cone component, GAP-43. Accumulation of GAP-43 persists, however, in a minority of mature synaptic terminals. What properties of axons and their terminals are affected by these changes in GAP-43 expression? Storm and colleagues first noted that the membrane- and calmodulin-binding properties of GAP-43 (a.k.a. P-57 or neuromodulin) could allow it to sequester a large fraction of calmodulin to the submembranous regions, and to release free calmodulin in response to protein kinase C activation. Analysis of evolutionarily conserved sequences in GAP-43 indicates that these properties are central to the biological effects of the protein. If GAP-43 is presumed to inactivate bound calmodulin, the network of GAP-43 in an axon terminal could be considered a regulatable calmodulin buffer, or 'calmodulin sponge', absorbing free calmodulin and releasing it in response to activation of protein kinase C. Such a calmodulin sponge has properties that could be useful in modulating the responses of membrane and cytoskeletal assembly events to calcium signals in growth cones, and in mediating long-term potentiation of neurotransmitter release from some pre-synaptic terminals.

Animals↗
PubMed1990

Changes in the distribution of GAP-43 during the development of neuronal polarity.

GAP-43, a neuron specific growth-associated protein, is selectively distributed to the axonal domain in developing neurons; it is absent from dendrites and their growth cones. Using immunofluorescence microscopy, we have further examined the distribution of GAP-43 during the development of hippocampal neurons in culture, in order to determine when this polarized distribution arises. Cultured hippocampal neurons initially extend several short processes which have the potential to become either axons or dendrites. At this stage, before the morphological expression of polarity, GAP-43 is concentrated in the growth cones of these processes but is distributed more or less equally among them. Polarity becomes established when one of these processes elongates to become the axon. At the earliest stage when the emerging axon can be identified, GAP-43 is preferentially concentrated in its growth cone. During the next few days, as the remaining processes take on dendritic properties, they lose their residual GAP-43 immunoreactivity. Throughout development, GAP-43 remains highly concentrated in the axonal growth cone, but the concentration of GAP-43 in the axon shaft increases, beginning near the growth cone and progressing proximally until GAP-43 is uniformly distributed along the entire axon. At all stages of development, GAP-43 is also concentrated in the region of the Golgi apparatus. These results suggest that the selective sorting of at least one membrane protein into the axon coincides with the morphological expression of polarity. These results also raise the possibility that GAP-43 may play an important role in the early phases of axonal outgrowth, by which the functional polarity of neurons is established.

Animals↗