Nerve growth factor (NGF) and insulin-like growth factor-1 (IGF-1) play an important role in promoting axonal growth from dorsal root ganglion (DRG) neurons. Adult DRG neurons exhibit neurotrophin-independent survival, providing an excellent system with which to study trophic factor effects on neurite growth in the absence of significant survival effects. Using young adult rat DRG neurons we have demonstrated a synergistic effect of NGF plus IGF (N + I), compared with either factor alone, in promoting neurite growth. Not only does the presence of NGF and IGF-1 enhance neurite initiation, it also significantly augments the extent of neurite branching and elongation. We have also examined potential mechanism(s) underlying this synergistic effect. Immunoblotting experiments of classical growth factor intermediary signalling pathways (PI 3-K-Akt-GSK-3 and Ras-Raf-MAPK) were performed using phospho-specific antibodies to assess activation state. We found that activation of Akt and MAPK correlated with neurite elongation and branching. However, using pharmacological inhibitors, we observed that a PI 3-K pathway involving both Akt and GSK-3 appeared to be more important for neurite extension and branching than MAPK-dependent signalling. In fact, inhibition of activation of MAPK with U0126 resulted in increased neuritic branching, possibly as a result of the concomitant increase observed in phospho-Akt. Furthermore, inhibition of GSK3 (which is negatively regulated by phosphorylation on S9/S21) also resulted in increased growth. Our data point to signalling convergence upon the PI 3-K-Akt-GSK-3 pathway that underlies the NGF plus IGF synergism. In addition, to our knowledge, this is the first report in primary neurons that inhibition of GSK3 results in an enhanced neurite growth. Keywords: adult sensory neurons, glycogen synthase kinase, insulin-like growth factor-1, mitogen activated protein kinase, nerve growth factor, phosphoinositide 3-kinase.

Neurite growth is influenced by many factors, including the availability of trophic support as well as the extracellular environment. In this study, we have investigated whether attachment to a permissive culture substrate such as laminin is sufficient to promote neurite outgrowth from dorsal root ganglion neurons in the absence of added nerve growth factor (NGF) and whether this attachment can enhance the response of these neurons to NGF. Adult dorsal root ganglia neurons plated on surfaces coated with a thin film of laminin exhibited increased neurite outgrowth. This effect was integrin-dependent as it was attenuated by treatment with RGD (arginine-glycine-aspartate) peptides and by a beta1-integrin blocking antibody. The addition of NGF resulted in a significant increase in the integrin-dependent outgrowth. We have correlated this increase in growth with increased expression of integrin subunits and activation of known downstream signaling intermediates such as focal adhesion kinase, Src, and Akt. We have also examined pathway cooperation through the use of an Src-specific inhibitor, PP2, and a beta1-integrin blocking antibody, beta1i, by observing downstream signaling intermediates in both integrin and growth factor signaling pathways. These results are among the first to detail the importance of interactions between neurotrophin- and integrin-activated signaling in adult primary neurons.

No abstract

Neurons in the adult rat dorsal root ganglion (DRG) can be classified into at least three separate subpopulations based on morphologic and phenotypic differences. In this study we have focused on the growth response of these specific subpopulations in vitro with respect to laminin (LN) and growth factor receptor activation. Using a cell selection approach we show that LN-induced neurite growth occurs in the absence of added trophic factors only in heavy-chain neurofilament-positive and calcitonin gene-related peptide-positive DRG neurons [nerve growth factor (NGF)-responsive population]. In contrast, LN alone is not sufficient to stimulate significant neurite growth from lectin Griffonia simplicifolia IB4-positive neurons (IB4+ve), although it is still required to elicit a growth response from these cells in the presence of glial-derived neurotrophic factor (GDNF, e.g. neurite growth occurred only when cells were plated on LN in the presence of GDNF). By using chemical inhibitors we demonstrate that only the phosphatidylinositol 3 kinase (PI 3-K)/Akt pathway is required for neurite growth from the NGF-responsive cell population. However, both the PI 3-K/Akt and MEK/mitogen-activated protein kinase signaling pathways are required for neurite growth from the IB4+ve cell population. Thus, we have identified specific signaling events and environmental requirements associated with neurite growth for different subpopulations of adult DRG neurons, pointing to potential therapeutic targets while identifying an inability for any one treatment alone to repair peripheral nerve damage.

We have investigated the role of stress-activated signaling pathways and the small heat shock protein, Hsp27, in protecting PC12 cells from heat shock and nerve growth factor (NGF) withdrawal-induced apoptosis. PC12 cells and a stable cell line overexpressing Hsp27 (HSPC cells) were subjected to heat shock. This resulted in the rapid activation of Akt followed by p38 mitogen-activated protein kinase (MAPK) signaling, with phosphorylation and intracellular translocation of Hsp27 also detectable. Hsp27 was found to form an immunoprecipitable complex with Akt and p38 MAPK in both nonstimulated and heat shocked cells, although after heat shock there was a gradual dissociation of Akt and p38 from the Hsp27. Cells were differentiated with NGF and then subjected to NGF withdrawal, a treatment which results in substantial cell death over 24-72 h. Hsp27 was shown to be protective against this treatment, since HSPC cells which overexpress Hsp27 showed significantly less cell death than the parental PC12 cells. In addition, we observed that phosphorylation of Akt was maintained in HSPC cells subjected to heat shock and NGF withdrawal compared with the parental cells. Taken together, our results suggest that Hsp27 may protect Akt from dephosphorylation and may also act in stabilizing Akt.