Semaphorin 3A-induced Growth Cone Collapse Research Articles

Role of calpain-mediated p53 truncation in semaphorin 3A-induced axonal growth regulation

Neurite outgrowth represents a critical stage in the correct development of neuronal circuitries, and is dependent on the complex regulation of actin filament and microtubule dynamics by intrinsic as well as extrinsic signals. Previous studies have implicated the tumor suppressor factor, p53, in the regulation of axonal outgrowth through a nontranscriptional effect involving local regulation of the Rho kinase signaling pathway that controls these dynamics. In the present study, we first showed that semaphorin 3A-induced growth cone collapse in cultured hippocampal neurons was associated with the partial truncation of phosphorylated p53, and that both effects were prevented by calpain inhibition with either m-calpain-specific siRNA or inhibitors. We further determined that semaphorin 3A-mediated calpain activation and growth cone collapse were associated with m-calpain phosphorylation and prevented by inhibition of MAPK, ERK, or p38. In vitro studies confirmed that p53 and especially phosphorylated p53 were partially truncated by calpain. Thus, our results indicate that semaphorin 3A-mediated growth cone collapse is mediated in part by m-calpain activation, possibly through MAPK-mediated phosphorylation, and the resulting truncation of phosphorylated p53, leading to Rho kinase activation and cytoskeletal reorganization. They provide a pathway by which extrinsic signals regulate axonal growth through activation of m-calpain and p53 truncation.

S1.5 Sympathetic and parasympathetic adult rat pelvic ganglion neurons utilize distinct cyclic nucleotide signaling pathways to mediate Semaphorin 3A-induced growth cone collapse

After nerve injury, appropriate target reconnection is required for successful functional regeneration, but this can be limited by aberrant reinnervation of inappropriate targets. These processes may recapitulate developmental events during which both attractive and repulsive guidance factors determine axonal targeting. Semaphorins constitute a major family of guidance molecules that typically signal to repel axons from inappropriate targets. Semaphorin 3A (Sema3A) has been shown to guide many sensory projections, and has been linked to motor and autonomic nerve pathfinding in development.

Alpha2-chimaerin, cyclin-dependent Kinase 5/p35, and its target collapsin response mediator protein-2 are essential components in semaphorin 3A-induced growth-cone collapse.

Neurite outgrowth is influenced by positive and negative signals that include the semaphorins, an important family of axonal outgrowth inhibitors. Here we report that the Rac GTPase activating protein (GAP)alpha2-chimaerin is involved in Semaphorin 3A (Sema 3A) signaling. In dorsal root ganglion neurons, Sema 3A-induced growth cone collapse was inhibited by alpha2-chimaerin mutated to eliminate GAP activity or interaction with phosphotyrosine. Activation of alpha2-chimaerin by phorbol ester caused growth cone collapse. Active alpha2-chimaerin interacts with collapsin response mediator protein-2 (CRMP-2) and cyclin-dependent kinase (Cdk) 5/p35 kinase through its SH2 and GAP domains, respectively. Cdk5 phosphorylates CRMP-2 at serine 522, possibly facilitating phosphorylation of serine 518 and threonine 514 by glycogen synthase kinase 3beta (GSK3beta), a kinase previously implicated in Sema 3A signaling. Phosphorylation of CRMP-2 serine 522 was essential for Sema 3A-induced growth cone collapse, which is dependent on Cdk5 but not Rho kinase activity. alpha2-chimaerin, like CRMP-2, can associate with the Sema 3A receptor. These results indicate that active alpha2-chimaerin Rac GAP, Cdk5/p35, and its substrate CRMP-2, are implicated in the dynamics of growth cone guidance initiated through Sema 3A signaling.

Xanthofulvin, a Novel Semaphorin Inhibitor Produced by a Strain of Penicillium.

A new semaphorin inhibitor xanthofulvin was isolated from the cultured broth of a fungus Penicillium sp. SPF-3059 along with a known compound vinaxanthone by solvent extraction and bioassay-guided fractionation. The tautomeric structure of xanthofulvin was determined by spectroscopic analyses. The two compounds exhibited significant semaphorin inhibitory activity with IC50 values of 0.09 and 0.1μg/ml, respectively, in semaphorin3A-induced growth cone collapse assay using cultured chick dorsal root ganglia neurons.

Xanthofulvin, a novel semaphorin inhibitor produced by a strain of Penicillium.

A new semaphorin inhibitor xanthofulvin was isolated from the cultured broth of a fungus Penicillium sp. SPF-3059 along with a known compound vinaxanthone by solvent extraction and bioassay-guided fractionation. The tautomeric structure of xanthofulvin was determined by spectroscopic analyses. The two compounds exhibited significant semaphorin inhibitory activity with IC50 values of 0.09 and 0.1 microg/ml, respectively, in semaphorin3A-induced growth cone collapse assay using cultured chick dorsal root ganglia neurons.

CGMP-mediated signaling via cGKIalpha is required for the guidance and connectivity of sensory axons.

Previous in vitro studies using cGMP or cAMP revealed a cross-talk between signaling mechanisms activated by axonal guidance receptors. However, the molecular elements modulated by cyclic nucleotides in growth cones are not well understood. cGMP is a second messenger with several distinct targets including cGMP-dependent protein kinase I (cGKI). Our studies indicated that the α isoform of cGKI is predominantly expressed by sensory axons during developmental stages, whereas most spinal cord neurons are negative for cGKI. Analysis of the trajectories of axons within the spinal cord showed a longitudinal guidance defect of sensory axons within the developing dorsal root entry zone in the absence of cGKI. Consequently, in cGKI-deficient mice, fewer axons grow within the dorsal funiculus of the spinal cord, and lamina-specific innervation, especially by nociceptive sensory neurons, is strongly reduced as deduced from anti-trkA staining. These axon guidance defects in cGKI-deficient mice lead to a substantial impairment in nociceptive flexion reflexes, shown using electrophysiology. In vitro studies revealed that activation of cGKI in embryonic dorsal root ganglia counteracts semaphorin 3A–induced growth cone collapse. Our studies therefore reveal that cGMP signaling is important for axonal growth in vivo and in vitro.

Rac1-Mediated Endocytosis during Ephrin-A2- and Semaphorin 3A-Induced Growth Cone Collapse

Negative guidance molecules are important for guiding the growth of axons and ultimately for determining the wiring pattern in the developing nervous system. In tissue culture, growth cones at the tips of growing axons collapse in response to negative guidance molecules, such as ephrin-A2 and semaphorin 3A. The small GTPase Rac1 is involved in growth cone collapse, but the nature of its role is not clear. Rac1 activity assays showed that Rac1 is transiently inactivated after treatment with ephrin-A2. Ephrin-induced growth cone collapse, however, correlated with resumption of Rac1 activity. We demonstrate that Rac1 is required for endocytosis of the growth cone plasma membrane and reorganization of F-actin but not for the depolymerization of F-actin during growth cone collapse in response to ephrin-A2 and semaphorin 3A. Rac1, however, does not regulate constitutive endocytosis in growth cones. These findings show that in response to negative guidance molecules, the function of Rac1 changes from promoting actin polymerization associated with axon growth to driving endocytosis of the plasma membrane, resulting in growth cone collapse. Furthermore, Rac1 antisense injected into the embryonic chick eyein vivocaused the retinotectal projection to develop without normal topography in a manner consistent with Rac1 having an obligatory role in mediating ephrin signaling.

Rac1-mediated endocytosis during ephrin-A2- and semaphorin 3A-induced growth cone collapse.

Negative guidance molecules are important for guiding the growth of axons and ultimately for determining the wiring pattern in the developing nervous system. In tissue culture, growth cones at the tips of growing axons collapse in response to negative guidance molecules, such as ephrin-A2 and semaphorin 3A. The small GTPase Rac1 is involved in growth cone collapse, but the nature of its role is not clear. Rac1 activity assays showed that Rac1 is transiently inactivated after treatment with ephrin-A2. Ephrin-induced growth cone collapse, however, correlated with resumption of Rac1 activity. We demonstrate that Rac1 is required for endocytosis of the growth cone plasma membrane and reorganization of F-actin but not for the depolymerization of F-actin during growth cone collapse in response to ephrin-A2 and semaphorin 3A. Rac1, however, does not regulate constitutive endocytosis in growth cones. These findings show that in response to negative guidance molecules, the function of Rac1 changes from promoting actin polymerization associated with axon growth to driving endocytosis of the plasma membrane, resulting in growth cone collapse. Furthermore, Rac1 antisense injected into the embryonic chick eye in vivo caused the retinotectal projection to develop without normal topography in a manner consistent with Rac1 having an obligatory role in mediating ephrin signaling.

Semaphorin Signaling to Actin

Modulation of the actin cytoskeleton underlies the protrusion and retraction responses of migrating neuronal growth cones. Semaphorins (Semas) are transmembrane proteins that elicit growth cone collapse, but the signaling events that affect actin dynamics when Semas engage their cell surface receptors, called plexins, remain unclear. Aizawa et al. report that Sema 3 may alter actin through the action of cofilin, an actin-binding protein that stimulates actin filament depolymerization. When dorsal root ganglion (DRG) neurons were treated with Sema 3A, sequential phoshorylation and then dephosphorylation of cofilin at the growth cone correlated with growth cone collapse. The authors suggest that serine phosphorylation of cofilin stabilizes peripheral actin filaments in the growth cone, but subsequent dephosphorylation by a phosphatase, that has yet to be identified, then reactivates cofilin, resulting in the rapid actin depolymerization that is characteristic of semaphorins. Inhibition of LIM-kinase in DRG neurons indicates that LIM-kinase is necessary, but not sufficient, for this growth cone collapse response. How plexins activate LIM-kinase remains to be determined. Interestingly, LIM-kinase activity is known to be regulated by Rac, the small GTP-binding protein that has also been implicated in gowth cone collapse by semaphorins. Driessens et al. report that activation of plexin-B1 that is overexpressed in fibroblasts results in activation of the small GTP-binding protein Rho in a Rac -dependent manner, causing actin stress fiber formation. The authors determined that activated Rac binds to the cytoplasmic domain of plexin-B1, but the significance of this interaction is not yet clear as plexin that lacks the Rac-binding region is still functional in control of the cytoskeleton. H. Aizawa, S. Wakatsuki, A. Ishii, K. Moriyama, Y. Sasaki, K. Ohashi, Y. Sekine-Aizawa, A. Sehara-Fujisawa, K. Mizuno, Y. Goshima, I. Yahara, Phosphorylation of cofilin by LIM-kinase is necessary for semaphorin 3A-induced growth cone collapse. Nature Neurosci. 4 , 367-373 (2001). [Online Journal] M. H. E. Driessens, H. Hu, C. D. Nobes, A. Self, I. Jordens, C. S. Goodman, A. Hall, Plexin-B semaphorin receptors interact directly with active Rac and regulate the actin cytoskeleton by activating Rho. Curr. Biol. 11 , 339-344 (2001). [Online Journal]

Phosphorylation of cofilin by LIM-kinase is necessary for semaphorin 3A-induced growth cone collapse.

Semaphorin 3A is a chemorepulsive axonal guidance molecule that depolymerizes the actin cytoskeleton and collapses growth cones of dorsal root ganglia neurons. Here we investigate the role of LIM-kinase 1, which phosphorylates an actin-depolymerizing protein, cofilin, in semaphorin 3A-induced growth cone collapse. Semaphorin 3A induced phosphorylation and dephosphorylation of cofilin at growth cones sequentially. A synthetic cell-permeable peptide containing a cofilin phosphorylation site inhibited LIM-kinase in vitro and in vivo, and essentially suppressed semaphorin 3A-induced growth cone collapse. A dominant-negative LIM kinase, which could not be activated by PAK or ROCK, suppressed the collapsing activity of semaphorin 3A. Phosphorylation of cofilin by LIM-kinase may be a critical signaling event in growth cone collapse by semaphorin 3A.