Objective. To assess the effects of human intervertebral disc aggrecan on nerve growth and guidance, using in vitro techniques.Methods. Aggrecan extracted from human lumbar intervertebral discs was incorporated into tissue culture substrata for the culture of the human neuronal cell line, SH-SY5Y, or explants of chick dorsal root ganglia. The effects on nerve growth of different concentrations of aggrecan extracted from the anulus fibrosus and nucleus pulposus, and of these aggrecan preparations following enzymic deglycosylation, were compared.Results. Disc aggrecan inhibited the growth of neurites from SH-SY5Y cells and induced growth cone turning of chick sensory neurites in a concentrationdependent manner. Aggrecan isolated from the anulus fibrosus was more inhibitory than that isolated from the nucleus pulposus, but enzymic pretreatments to reduce the glycosylation of both types of disc aggrecan partially abrogated their inhibitory effects.Conclusion. Nerve growth into degenerate intervertebral discs has been linked with the development of low back pain, but little is known about factors affecting disc innervation. The finding that disc aggrecan inhibits nerve growth in vitro, and that this inhibitory activity depends on aggrecan glycosylation, has important implications for our understanding of mechanisms that may regulate disc innervation in health and disease.

Proteoglycans influence aging and plasticity in the nervous system. Particularly prominent are the chondroitin sulfate proteoglycans (CSPGs), which are generally inhibitory to neurite outgrowth. During development, CSPGs facilitate normal guidance, but following nervous system injury and in diseases of aging (e.g., Alzheimer's disease), they block successful regeneration, and are associated with axon devoid regions and degenerating nerve cells. Whereas previous studies used non-nervous system sources of CSPGs, this study analyzed the morphology and behavior of sensory (dorsal root ganglia) neurons, and a human nerve cell model (SH-SY5Y neuroblastoma cells) as they contacted nervous system-derived CSPGs, using a variety of microscopy techniques. The results of these qualitative analyses show that growth cones of both nerve cell types contact CSPGs via actin-based filopodia, sample the CSPGs repeatedly without collapse, and alter their trajectory to avoid nervous system-derived CSPGs. Turning and branching are correlated with increased filopodial sampling, and are common to both neurons and Schwann cells. We show that CSPG expression by rat CNS astrocytes in culture is correlated with sensory neuron avoidance. Further, we show for the first time the ultrastructure of sensory growth cones at a CSPG-laminin border and reveal details of growth cone and neurite organization at this choice point. This type of detailed analysis of the response of growth cones to nervous system-derived CSPGs may lead to an understanding of CSPG function following injury and in diseases of aging, where CSPGs are likely to contribute to aberrant neurite outgrowth, failed or reduced synaptic connectivity, and/or ineffective plasticity.

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Rit, by sequence homology, is a member of the Ras subfamily of small guanine triphosphatases (GTPases). In PC6 cells, Rit signals through pathways both common to and different from those activated by Ras to promote cell survival and neurite outgrowth. However, the specific morphological changes induced by Rit in human cells are not known. Here, we show in a human neuronal model that Rit increases neurite outgrowth and branching through MEK-dependent and MEK-independent signaling mechanisms, respectively. Adenoviral expression of wild-type or constitutively active Rit increased neurite initiation,elongation and branching on endogenous matrix or a purified laminin-1 substratum of SH-SY5Y cells as assessed using image analysis. This outgrowth was morphologically distinct from that promoted by constitutively active Ras or Raf (evidenced by increased branching and elongation). Constitutively active Rit increased phosphorylation of ERK 1/2, but not Akt, and the MEK inhibitor PD 098059 blocked constitutively active Rit-induced neurite initiation but not elongation or branching. These results suggest that Rit plays a key role in human neuronal development and regeneration through activating both known and as yet undefined signaling pathways.

Many lines of evidence indicate the importance of the Rho family guanine nucleotide triphosphatases (GTPases) in directing axon extension and guidance. The signaling networks that involve these proteins regulate actin cytoskeletal dynamics in navigating neuronal growth cones. However, the intricate patterns that regulate Rho GTPase activation and signaling are not yet fully defined. Activity and subcellular localization of the Rho GTPases are regulated by post-translational modification. The addition of a geranylgeranyl group to the carboxy (C-) terminus targets Rho GTPases to the plasma membrane and promotes their activation by facilitating interaction with guanine nucleotide exchange factors and allowing sequestering by association with guanine dissociation inhibitors. However, it is unclear how these modifications affect neurite extension or how subcellular localization alters signaling from the classical Rho GTPases (RhoA, Rac1, and Cdc42). Here, we review recent data addressing this issue and propose that Rho GTPase geranylgeranylation regulates outgrowth.