REVIEW |
Correspondence to: Paul C. Letourneau, Dept. of Neuroscience, University of Minnesota, 6-145 Jackson Hall, 321 Church St SE, Minneapolis, MN 55455. E-mail: letour@lenti.med.umn.edu
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Summary |
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Nerve growth factor (NGF) and semaphorin3A (Sema3A) are guidance cues found in pathways and targets of developing dorsal root ganglia (DRG) neurons. DRG growth cone motility is regulated by cytoplasmic signaling triggered by these molecules. We investigated interactions of NGF and Sema3A in modulating growth cone behaviors of axons extended from E7 chick embryo DRGs. Axons extending in collagen matrices were repelled by Sema3A released from transfected HEK293 cells. However, if an NGF-coated bead was placed adjacent to Sema3A-producing cells, axons converged at the NGF bead. Growth cones of DRGs raised in 10-9 M NGF were more resistant to Sema3A-induced collapse than when DRGs were raised in 10-11 M NGF. After overnight culture in 10-11 M NGF, 1-hr treatment with 10-9 M NGF also increased growth cone resistance to Sema3A. Pharmacological studies indicated that the activities of ROCK and PKG participate in the cytoskeletal alterations that lead to Sema3A-induced growth cone collapse, whereas PKA activity is required for NGF-mediated reduction of Sema3A-induced growth cone collapse. These results support the idea that growth cone responses to a guidance cue can be modulated by interactions involving coincident signaling by other guidance cues.
(J Histochem Cytochem 51:435444, 2003)
Key Words: neurotrophin, semaphorin3A, protein kinase A, protein kinase G, ROCK, growth cone
DEVELOPING AXONS are guided to their targets by physical and molecular cues encountered by growth cones in their local environment (
Sensory neurons of dorsal root ganglia (DRG) extend peripheral processes to skin, muscle, and other organs, and DRG central processes make synapses in the spinal cord. The neurotrophin NGF and the semaphorin Sema3A regulate the in vitro motility of DRG growth cones and regulate in vivo axon morphogenesis, as shown by experimentation and by analyses of mice with mutations for NGF, Sema3A, and the neuropilin-1 Sema3A receptor (
NGF promotes differentiation, survival and morphogenesis of trkA-expressing sensory neurons (
We investigated interactions of NGF and Sema3A signaling in regulating chick DRG growth cones. Elevated levels of NGF reduced the collapse of DRG growth cones by Sema3A. Pharmacological studies indicated opposite roles for protein kinases PKA and PKG in mediating signaling by these molecules. Inhibition of the RhoA effector ROCK also reduced Sema3A-induced growth cone collapse. Our results support the idea that the response of growth cones to a single guidance cue is not invariant but depends on interactions with signaling triggered from other cues and on the activities of other second messenger pathways (
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Materials and Methods |
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NGF was obtained from R & D Systems (Minneapolis MN). 8-Bromo-cyclic AMP, 8-bromo-cyclic GMP, ODQ, KT5720, KT5823, Sp-cAMP, Y27632, and HA1077 were purchased from Biomol Research Laboratories (Plymouth Meeting, PA). PKI and YC-1 were purchased from Calbiochem (La Jolla, CA). Purified Sema3A and HEK293 cells transfected to produce and secrete Sema3A were generously provided by Drs. Yuling Luo and Sheldon Ng of Exelixis (South San Francisco, CA). Drugs were prepared in water or in DMSO and were aliquotted.
DRG Cultures
Culture dishes were treated overnight with 10 µg/ml laminin. Explants of E7 chick DRGs were cultured overnight in a warmed, humidified incubator in 2 ml F12 medium (Gibco/BRL; Gaithersburg, MD) buffered with 10 mM HEPES and with supplements (5 µg/ml transferrin, 40 µg/ml sodium pyruvate, 5 µg/ml phosphocreatine, 5 µg/ml progesterone, 5 µg/ml Na selenite) and NGF. DRG explants were experimentally treated with neurotrophins and drugs in several ways.
All collapse assays were performed similarly. Purified Sema3A or conditioned medium from Sema3A-trasfected 293 cells was added for 30 min, followed by fixation with 0.5% glutaraldehyde in PBS for 30 min. Fixed DRGs were viewed by phase-contrast optics with a x20 objective, and the morphology of randomly selected axon endings was scored as either a normal growth cone with lamellipodia and filopodia or a collapsed growth cone (a tapered axon terminal without lamellipodia or less than 3 filopodia;
Conditioned Media from Sema3A-transfected 293 Cells
HEK 293 cells stably transfected to express human Sema3A were prepared as described in
Immunocytochemistry
DRG explants from E7 chick embryos were cultured on laminin-coated coverslips for 24 hr as described above. After 24 hr of culture, the cultures were fixed with 4% paraformaldehyde (PF) in PBS by adding warm fixative directly to the culture medium for 15 min, followed by immunocytochemistry. After rinsing off the fix, cultures were quenched with 0.1 M glycine in PBS for 15 min, and the cells were blocked and permeabilized with 0.1% Triton X-100 in PBS with 1% fish gelatin for 30 min. The fixed cells were incubated at 1:100 dilutions of polyclonal antibodies against the catalytic subunit of the -isoform of PKA or PKGI
(both from Stressgen Biotechnologies, San Diego, CA) for 1 hr at room temperature (RT). Staining for tubulin was done with a 1:100 dilution of a monoclonal antibody against ß-tubulin (ßIII; Covance, Princeton, NJ). RhoA was localized with a monoclonal antibody (Santa Cruz Biothechnology) One percent fish gelatin was incubated together with the primary antibody. After rinsing in PBS, the samples were incubated with secondary rhodamine-conjugated goat anti-rabbit and fluorescein-conjugated goat anti-mouse antibodies (Jackson Laboratories; West Grove, PA), each diluted 1:400 in PBS with 1% fish gelatin for 1 hr at RT. F-actin was labeled with rhodamine-conjugated phalloidin (Molecular Probes; Eugene, OR).
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Results |
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To first examine how elongating axons integrate simultaneous signaling from multiple guidance cues, we cultured explants of E7 chick DRGs for 2448 hr in collagen gels adjacent either to cell aggregates of HEK293 cells transfected to produce and release Sema3A or to glass beads that release NGF from their surfaces. Fig 1 shows that many more DRG axons extended from the side of a DRG explant that was away from Sema3A-producing HEK cells (Fig 1, upper panels). In the presence of non-transfected HEK cell aggregates, axon outgrowth was equal from all sides of DRG explants (not shown). When DRG explants were adjacent to an NGF-coated glass bead, axon growth toward the bead was denser than elsewhere around the explant, although many axons extended beyond the NGF beads (Fig 1, middle panels). When DRG explants were cultured in collagen gels with both an NGF bead and Sema3A-transfected HEK cell aggregate on the same side of the explant, a different pattern of axon outgrowth was observed. Axons extended from the side of the explant facing the bead and the cell aggregate but, in a unique pattern, the axons converged on the NGF bead, very different from the profuse axon growth beyond the NGF bead in the absence of the Sema3A-secreting HEK cells (Fig 1, lower panels). These in vitro results show that both Sema3A and NGF influence axon growth in a manner reminiscent of the axon outgrowth from DRGs through peripheral tissues towards their targets.
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We continued investigating growth cone integration of signaling by Sema3A and NGF with more rapid and efficient in vitro assays of growth cone collapse. Exposure of DRG growth cones to global application of Sema3A causes rapid withdrawal of filopodial and lamellipodial protrusions and collapse of growth cones and distal axons (
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Elevated NGF Concentrations Can Reduce Sema3A-induced Growth Cone Collapse
Explants of E7 chick DRGs were cultured overnight in media containing 10-11 M, 10-10 M, or 10-9 M NGF. When DRG explants were cultured overnight with 10-9 M or 10-10 M NGF, growth cone collapse in response to a standard amount of Sema3A was significantly less than when DRGs were cultured in 10-11 M NGF (Fig 3). We next investigated whether a briefer exposure to high NGF concentrations would reduce the collapse response to Sema3A. Explants were cultured for 24 hr with 10-11 M NGF, and then the neurotrophin concentration of the medium was elevated to 10-9 M NGF for 1 hr before adding Sema3A for 30 min. One hour of exposure to 10-9 M NGF was sufficient to reduce the collapse response of DRG growth cones to Sema3A (Fig 4). Therefore, elevated concentrations of NGF can act within 1 hr to reduce the Sema3A-induced collapse of growth cones of DRG neurons raised in 10-11 M NGF.
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PKA Activity Is Involved in NGF Modulation of Sema3A-induced Growth Cone Collapse
It has been proposed that the cAMP-regulated protein kinase A (PKA) and the cGMP-regulated protein kinase G (PKG) modulate growth cone responses to many extrinsic guidance molecules (
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We tested the effects of activation of PKA by adding the cAMP analogues 8-bromo cAMP or Sp-cAMP (Fig 6A). Either PKA activator alone significantly reduced the growth cone response to Sema3A. Furthermore, when both PKA activation and elevation of NGF preceded the addition of Sema3A, the reduction in collapse response to Sema3A was even greater. Next we investigated the effect of inhibition of PKA activity on NGF modulation of growth cone responses to Sema3A. Two inhibitors of PKA activity were used (Fig 6B). When PKA was inhibited with KT5720 before addition of Sema3A (
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In the above experiments using blockers of PKA activity, NGF was elevated to 10-9 M for 60 min before adding the drugs. This sequence of treatment would allow 60 min of signaling by elevated NGF before addition of a PKA inhibitor. In the next experiments we added the PKA inhibitor PKI either before elevating NGF to block all signaling that might involve PKA activity or after elevation of NGF. We found that the ability of 10-9 M NGF to reduce the 69% Sema3A collapse response to 40% collapse was diminished to the same extent whether PKI was added before (58% collapsed) or after (62% collapsed) elevating NGF to 10-9 M.
PKG Activity Is Involved in Sema3A-induced Growth Cone Collapse
The cGMP-regulated kinase PKG has been implicated in mediating growth cone responses to Sema3A. Therefore, we investigated the effects of manipulations that may affect PKG activity on chick DRG growth cone responses to Sema3A. First, we examined the effects of a PKG inhibitor, the drug KT5823 (
To examine the effects of activating PKG, we added the cGMP analogue 8-bromo cGMP before addition of Sema3A (Fig 7B). We found that 8-bromo cGMP alone induced a significant amount of growth cone collapse. This collapse was not attenuated by elevation of NGF to 10-9 M. When DRG explants were exposed to 8-bromo cGMP and then to Sema3A, growth collapse was significantly higher than when explants were treated with Sema3A alone. Elevation of NGF to 10-9 M did not reduce this extensive growth cone collapse. Therefore, PKG activity does appear to be involved in growth cone collapse. Another means to activate PKG is to stimulate quanylate cyclase activity with the drug YC-1 (
Inhibition of ROCK Reduces Sema3A-induced Growth Cone Collapse
RhoA GTPase and its effector, ROCK, have been implicated in cellular contractility (
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Discussion |
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Axon pathfinding is controlled by extracellular proteins that act through ligandreceptor signaling to regulate growth cone behaviors (
Our studies showed that the collapse response of sensory growth cones to Sema3A was reduced by elevated NGF. This is like our previous finding that BDNF protects retinal growth cones from nitric oxide-induced collapse (
Our evidence suggests that NGF triggers signaling that reduces the effectiveness of Sema3A signaling to induce growth cone collapse. Several pathways activated by neurotrophins may modulate Sema3A-induced collapse (
Our results indicated that cGMP-activated protein kinase G (PKG) is involved in Sema3A-induced growth cone collapse. Inhibiting PKG or guanylate cyclase reduced the response to Sema3A, whereas activation of PKG or guanylyl cyclase promoted collapse. That elevated NGF did not reduce growth cone collapse induced by PKG activation to the same extent as Sema3A-induced collapse suggests that PKG acts downstream or independently of the influence of NGF signaling. Although cGMP is implicated in growth cone guidance (
Rho family GTPases regulate axon morphogenesis and guidance (
As stated above, these studies investigated the idea that coincident signaling by Sema3A and neurotrophins occurs as DRG growth cones traverse tissues and within their targets. Fig 10 summarizes how interactions between signaling pathways may regulate growth cone behaviors. Several regions of chick and mouse embryos contain high Sema3A levels, the ventral spinal cord and epidermis (chicks) of their targets and the dermamyotome in the peripheral pathway. These regions are not entered by NGF-dependent axons. However, Sema3A is also broadly expressed at low levels in mesenchyme (
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Footnotes |
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1 Presented as part of the Cytoskeletal Dynamics and Path Finding of Neuronal Growth Cones Symposium, 6th Joint Meeting of the Histochemical Society and the Japan Society of Histochemistry and Cytochemistry, University of Washington, Seattle, WA, July 1821, 2002.
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Acknowledgments |
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Supported by National Institutes of Health grant HD19950 (PCL), National Science Foundation grant IBN-0080932 (PCL), a grant from the NIH/Fogarty International Center to V.D. Dontchev while in the Letourneau laboratory, and a Minnesota Medical Foundation grant.
Florence Roche and Eric Veien provided valuable technical assistance, and Dr Gianluca Gallo provided valuable comments on the manuscript.
Received for publication September 19, 2002; accepted January 14, 2003.
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