William D. Snider

NGF-Induced Axon Growth Is Mediated by Localized Inactivation of GSK-3β and Functions of the Microtubule Plus End Binding Protein APC

Little is known about how nerve growth factor (NGF) signaling controls the regulated assembly of microtubules that underlies axon growth. Here we demonstrate that a tightly regulated and localized activation of phosphatidylinositol 3-kinase (PI3K) at the growth cone is essential for rapid axon growth induced by NGF. This spatially activated PI3K signaling is conveyed downstream through a localized inactivation of glycogen synthase kinase 3beta (GSK-3beta). These two spatially coupled kinases control axon growth via regulation of a microtubule plus end binding protein, adenomatous polyposis coli (APC). Our results demonstrate that NGF signals are transduced to the axon cytoskeleton via activation of a conserved cell polarity signaling pathway.

Development of Sensory Neurons in the Absence of NGF/TrkA Signaling In Vivo

The neurotrophin survival dependence of peripheral neurons in vitro is regulated by the proapoptotic BCL-2 homolog BAX. To study peripheral neuron development in the absence of neurotrophin signaling, we have generated mice that are double null for BAX and nerve growth factor (NGF), and BAX and the NGF receptor TrkA. All dorsal root ganglion (DRG) neurons that normally die in the absence of NGF/TrkA signaling survive if BAX is also eliminated. These neurons extend axons through the dorsal roots and collateral branches into the dorsal horn. In contrast, superficial cutaneous innervation is absent. Furthermore, rescued sensory neurons fail to express biochemical markers characteristic of the nociceptive phenotype. These findings establish that NGF/TrkA signaling regulates peripheral target field innervation and is required for the full phenotypic differentiation of sensory neurons.

Raf and akt mediate distinct aspects of sensory axon growth.

Nerve growth factor (NGF) induces dramatic axon growth from responsive embryonic peripheral neurons. However, the roles of the various NGF-triggered signaling cascades in determining specific axon morphological features remain unknown. Here, we transfected activated and inhibitory mutants of Trk effectors into sensory neurons lacking the proapoptotic protein Bax. This allowed axon growth to be studied in the absence of NGF, enabling us to observe the contributions of individual signaling mediators. While Ras was both necessary and sufficient for NGF-stimulated axon growth, the Ras effectors Raf and Akt induced distinct morphologies. Activated Raf-1 caused axon lengthening comparable to NGF, while active Akt increased axon caliber and branching. Our results suggest that the different Trk effector pathways mediate distinct morphological aspects of developing neurons.

GSK-3 is a master regulator of neural progenitor homeostasis

The development of the brain requires the exquisite coordination of progenitor proliferation and differentiation to achieve complex circuit assembly. It has been suggested that glycogen synthase kinase 3 (GSK-3) acts as an integrating molecule for multiple proliferation and differentiation signals because of its essential role in the RTK, Wnt and Shh signaling pathways. We created conditional mutations that deleted both the α and β forms of GSK-3 in mouse neural progenitors. GSK-3 deletion resulted in massive hyperproliferation of neural progenitors along the entire neuraxis. Generation of both intermediate neural progenitors and postmitotic neurons was markedly suppressed. These effects were associated with the dysregulation of β-catenin, Sonic Hedgehog, Notch and fibroblast growth factor signaling. Our results indicate that GSK-3 signaling is an essential mediator of homeostatic controls that regulate neural progenitors during mammalian brain development.

Neurotrophic factors and axonal growth.

Neuronal morphological differentiation is regulated by numerous polypeptide growth factors (neurotrophic factors). Recently, significant progress has been achieved in clarifying the roles of neurotrophins as well as glial cell line-derived neurotrophic factor family members in peripheral axon elongation during development. Additionally, advances have been made in defining the signal transduction mechanisms employed by these factors in mediating axon morphological responses. Several studies addressed the role of neurotrophic factors in regenerative axon growth and suggest that signaling mechanisms in addition to those triggered by receptor tyrosine kinases may be required for successful peripheral nervous system regeneration. Finally, recent investigations demonstrate that neurotrophic factors can enhance axon growth after spinal cord injuries.

Essential Roles for GSK-3s and GSK-3-Primed Substrates in Neurotrophin-Induced and Hippocampal Axon Growth

Glycogen synthase kinase-3beta (GSK-3beta) is thought to mediate morphological responses to a variety of extracellular signals. Surprisingly, we found no gross morphological deficits in nervous system development in GSK-3beta null mice. We therefore designed an shRNA that targeted both GSK-3 isoforms. Strong knockdown of both GSK-3alpha and beta markedly reduced axon growth in dissociated cultures and slice preparations. We then assessed the role of different GSK-3 substrates in regulating axon morphology. Elimination of activity toward primed substrates only using the GSK-3 R96A mutant was associated with a defect in axon polarity (axon branching) compared to an overall reduction in axon growth induced by a kinase-dead mutant. Consistent with this finding, moderate reduction of GSK-3 activity by pharmacological inhibitors induced axon branching and was associated primarily with effects on primed substrates. Our results suggest that GSK-3 is a downstream convergent point for many axon growth regulatory pathways and that differential regulation of primed versus all GSK-3 substrates is associated with a specific morphological outcome.

Peripheral NT3 Signaling Is Required for ETS Protein Expression and Central Patterning of Proprioceptive Sensory Afferents

To study the role of NT3 in directing axonal projections of proprioceptive dorsal root ganglion (DRG) neurons, NT3(-/-) mice were crossed with mice carrying a targeted deletion of the proapoptotic gene Bax. In Bax(-/-)/NT3(-/-) mice, NT3-dependent neurons survived and expressed the proprioceptive neuronal marker parvalbumin. Initial extension and collateralization of proprioceptive axons into the spinal cord occurred normally, but proprioceptive axons extended only as far as the intermediate spinal cord. This projection defect is similar to the defect in mice lacking the ETS transcription factor ER81. Few if any DRG neurons from Bax(-/-)/NT3(-/-) mice expressed ER81 protein. Expression of a NT3 transgene in muscle restored DRG ER81 expression in NT3(-/-) mice. Finally, addition of NT3 to DRG explant cultures resulted in induction of ER81 protein. Our data indicate that NT3 mediates the formation of proprioceptive afferent-motor neuron connections via regulation of ER81.

Intracellular control of developmental and regenerative axon growth

Axon growth is a highly regulated process that requires stimulating signals from extracellular factors. The extracellular signals are then transduced to regulate coordinately gene expression and local axon assembly. Growth factors, especially neurotrophins that act via receptor tyrosine kinases, have been heavily studied as extracellular factors that stimulate axon growth. Downstream of receptor tyrosine kinases, recent studies have suggested that phosphatidylinositol-3 kinase (PI3K) regulates local assembly of axonal cytoskeleton, especially microtubules, via glycogen synthase kinase 3β (GSK-3β) and multiple microtubule binding proteins. The role of extracellular signal regulated kinase (ERK) signalling in regulation of local axon assembly is less clear, but may involve the regulation of local protein translation. Gene expression during axon growth is regulated by transcription factors, among which cyclic AMP response element binding protein and nuclear factors of activated T-cells (NFATs) are known to be required for neurotrophin (NT)-induced axon extension. In addition to growth factors, extracellular matrix molecules and neuronal activity contribute importantly to control axon growth. Increasingly, evidence suggests that these influences act to enhance growth via coordinating with growth factor signalling. Finally, evidence is emerging that developmental versus regenerative axon growth may be mediated by distinct signalling pathways, both at the level of gene transcription and at the level of local axon assembly.

Signaling the Pathway to Regeneration

Robust axon regeneration occurs after peripheral nerve injury through coordinated activation of a genetic program and local intracellular signaling cascades. Although regeneration-associated genes are being identified with increasing frequency, most aspects of regeneration-associated intracellular signaling remain poorly understood. Two independent studies now report that upregulation of cAMP is a component of the PNS regeneration program that can be exploited to enhance axon regeneration through the normally inhibitory CNS environment.

Specific Functions for ERK/MAPK Signaling during PNS Development

We have established functions of the stimulus-dependent MAPKs, ERK1/2 and ERK5, in DRG, motor neuron, and Schwann cell development. Surprisingly, many aspects of early DRG and motor neuron development were found to be ERK1/2 independent, and Erk5 deletion had no obvious effect on embryonic PNS. In contrast, Erk1/2 deletion in developing neural crest resulted in peripheral nerves that were devoid of Schwann cell progenitors, and deletion of Erk1/2 in Schwann cell precursors caused disrupted differentiation and marked hypomyelination of axons. The Schwann cell phenotypes are similar to those reported in neuregulin-1 and ErbB mutant mice, and neuregulin effects could not be elicited in glial precursors lacking Erk1/2. ERK/MAPK regulation of myelination was specific to Schwann cells, as deletion in oligodendrocyte precursors did not impair myelin formation, but reduced precursor proliferation. Our data suggest a tight linkage between developmental functions of ERK/MAPK signaling and biological actions of specific RTK-activating factors.