Neurotrophin-induced upregulation of p75NTR via a protein kinase C-delta-dependent mechanism.

Neurotrophins exert their biological effects via p75NTR and Trk receptors. Functional interplay between these two receptors has been widely explored with respect to p75NTR enhancing the activation and signalling of Trk, but few studies address the bidirectional aspects. We have previously demonstrated that the expression of p75NTR can be differentially modulated by different Trk receptor mutations. Here we investigate the mechanism of Nerve Growth Factor (NGF)-induced upregulation of p75NTR expression. We utilize pharmacological inhibition to investigate the role of various TrkA-associated signalling intermediates in this regulatory cascade. Notably, the inhibition of phospholipase C-gamma (PLC-gamma) using U73122, prevented the NGF-induced upregulation of p75NTR protein and mRNA. The inhibition of protein kinase C-delta (PKC-delta) activation by rottlerin, a selective PKC-delta inhibitor, and by small interfering RNA (siRNA) directed against PKC-delta also inhibited this NGF-induced upregulation. Finally, we also show that in cerebellar granule neurons, BDNF acting via TrkB increases p75NTR expression in a PKC-delta dependent manner. These results indicate the importance of Trk-dependent PLC-gamma and PKC-delta activation for downstream regulation of p75NTR protein expression in response to neurotrophin stimulation, a process that has implications to the survival and growth of the developing nervous system.

Src and FAK are key early signalling intermediates required for neurite growth in NGF-responsive adult DRG neurons.

Axonal regeneration is influenced by factors in the extracellular environment, including neurotrophins, such as NGF, and adhesion molecules, such as laminin. The provision of both NGF and a permissive substrate to cultured adult NGF-responsive DRG neurons results in enhanced levels of neurite growth not achievable by either factor alone. In this study, we have investigated the early signalling events that contribute to NGF and laminin-induced neurite growth. Adult NGF-responsive DRG neurons were plated on poly-d-lysine for 2 h then stimulated with NGF, laminin, or laminin plus NGF for 10 min, 1 h, or 6 h. Signalling pathways were subsequently analysed using Western blotting and pharmacological inhibition of specific signalling components. While activation of the various signalling intermediates (Src, FAK, Akt, MAPK) could be detected as early as 10 min-1 h after stimulation, significant neurite growth was observed mainly at the 6 h time point. The results of the time course experiments showed differential activation of the signalling intermediates. Src was activated by all treatments (NGF, laminin and the combination) at the earliest time point analysed, 10 min. NGF stimulation also resulted in detectable activation of FAK, Akt and MAPK by 10 min. However, laminin stimulation alone did not result in detectable activation of FAK, Akt or MAPK until the 1 h time point. Inhibition of either Src or FAK activity attenuated both laminin and/or NGF-induced PI 3-K/Akt and MEK/MAPK signalling pathways, as well as neurite growth. Downstream inhibition of Akt by Akt knockdown also blocked observed neurite growth, while inhibition of MEK/MAPK had no significant effect. Together, these results demonstrate that signalling underlying neurite growth can be detected within minutes of stimulation and provide a mechanism for the observed enhancement of neurite growth when both NGF and the permissive substrate, laminin, are provided.

Laminin and growth factor receptor activation stimulates differential growth responses in subpopulations of adult DRG neurons.

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.

Heat shock protein 27 is involved in neurite extension and branching of dorsal root ganglion neurons in vitro.

Alteration of the cytoskeleton in response to growth factors and extracellular matrix proteins is necessary for neurite growth. The cytoskeletal components, such as actin and tubulin, can be modified through interaction with other cellular proteins, including the small heat shock protein Hsp27. Our previous work suggested that Hsp27 influences neurite growth, potentially via its phosphorylation state interactions with actin. To investigate further the role of Hsp27 in neurite outgrowth of adult dorsal root ganglion (DRG) neurons, we have both down-regulated endogenous Hsp27 and expressed exogenous Hsp27. Down-regulation of Hsp27 with Hsp27 siRNA resulted in a decrease of neuritic tree length and complexity. In contrast, expression of exogenous Hsp27 in these neurons resulted in an increase in neuritic tree length and branching. Collectively, these results demonstrate that Hsp27 may play a role in neuritic growth via modulation of the actin cytoskeleton.

A method to assess multiple aspects of the motile behaviour of adherent PC12 cells on applied biological substrates.

Cellular migration is central to a wide range of biological and pathological processes in vivo. In vitro cell migration assays can be used to obtain invaluable information relating to the mechanism of cell movement, but current available methods can be limiting. Here we describe a novel motility assay that allows the simultaneous investigation of both quantitative and qualitative aspects of a population of motile cells as they move across a variety of substrates. By plating cells in a confluent monolayer on a coverslip, the monolayer can then be inverted to migrate over a larger substrate-coated coverslip, which can subsequently be reliably quantified, and subjected to immunocytochemistry and confocal imaging. This assay can be used to assess multiple aspects of motility, including distance, quantity, morphology, polarization and component colocalization. To demonstrate the utility of this assay, it was applied to the study of a stimulator of PC12 cell migration, nerve growth factor (NGF), and how this migration is influenced by the extracellular substrate, laminin. Furthermore, since mutations to the NGF receptor, TrkA, have been noted to alter the behaviour of PC12 cells in response to NGF, a PC12 subline that expresses a mutated TrkA receptor was utilized to illustrate that a Y785F mutation in the cytoplasmic tail of TrkA results in increased migration in response to the stimulus compared to the control PC12s.

Stress-induced heat shock protein 27 expression and its role in dorsal root ganglion neuronal survival.

Heat shock protein 27 (Hsp27), a molecular chaperone ubiquitously expressed in many cell types, has been shown to play a role in protecting neurons from cellular stresses. Unlike adult DRG neurons in vitro, neonatal DRG neurons require NGF for survival; withdrawal of NGF results in apoptosis of a majority of neonatal neurons. We hypothesized that Hsp27 contributes to the neurotrophin-independent survival of adult DRG neurons. Constitutive Hsp27 expression is higher in adult DRG neurons compared to neonates, although both upregulate Hsp27 expression after heat shock (HS). We found that increasing endogenous Hsp27 by HS in neonatal neurons was able to inhibit NGF withdrawal-induced apoptosis. Heat shock of adult and neonatal neurons also resulted in Akt activation, which could be a mechanism for the increased survival. Hsp27 siRNA treatment of adult neurons effected a decreased expression of Hsp27, which correlated with increased apoptosis in these neurons. Downregulation of Hsp27 via siRNA also blocked the HS-induced rescue of neonatal neurons after NGF withdrawal. These results indicate that physiologically induced upregulation of Hsp27 is sufficient to provide some degree of neuronal protection. Further, this induction appears to be regulated by the transcriptional activation of HSF1 as shown by HSF1 nuclear translocation and by EMSA analyses of HSF1 binding to nuclear protein.

A procedure for selecting and culturing subpopulations of neurons from rat dorsal root ganglia using magnetic beads.

Current protocols for preparing primary sensory neuron cultures are inadequate when studying individual subpopulations of dorsal root ganglion (DRG) neurons. The DRG is made up of a heterogeneous population of cells, making it difficult to study treatment effects on any given population in mass cultures. Thus, we describe a procedure using magnetic beads from Dynal to select and plate viable populations of neurons based on expression of specific cell surface markers. We show that, by the use of the lectin IB4, we can select a highly enriched viable subpopulation of GDNF-responsive DRG neurons, leaving a viable population of non-selected IB4-ve, Trk+ve neurons. Key factors for successful cultures are (i) quick and careful dissection of DRGs from 4- to 5-week-old Sprague-Dawley rats, (ii) adequate removal of debris and non-neuronal contamination and (iii) gentle handling of bead-bound cells during selection.

Integrin activation and neurotrophin signaling cooperate to enhance neurite outgrowth in sensory neurons.

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.

Hsp27 and axonal growth in adult sensory neurons in vitro.

BACKGROUND: Neurite growth can be elicited by growth factors and interactions with extracellular matrix molecules like laminin. Among the targets of the signalling pathways activated by these stimuli are cytoskeletal elements, such as actin, tubulin and neurofilaments. The cytoskeleton can also be modulated by other proteins, such as the small heat shock protein Hsp27. Hsp27 interacts with actin and tubulin in non-neuronal cells and while it has been suggested to play a role in the response of some neurons to injury, there have been no direct studies of its contribution to axonal regeneration. RESULTS: We have investigated neurite initiation and process extension using cultures of adult dorsal root ganglion (DRG) sensory neurons and a laminin stimulation paradigm. Employing confocal microscopy and biochemical analyses we have examined localization of Hsp27 at early and later stages of neurite growth. Our results show that Hsp27 is colocalized with actin and tubulin in lamellopodia, filopodia, focal contacts and mature neurites and growth cones. Disruption of the actin cytoskeleton with cytochalasin D results in aberrant neurite initiation and extension, effects which may be attributable to alterations in actin polymerization states. Inhibition of Hsp27 phosphorylation in our cultures results in an atypical growth pattern that may be attributable to an effect of pHsp27 on the stability of the actin cytoskeleton. CONCLUSION: We observed colocalization of the phosphorylated and non-phosphorylated forms of Hsp27 with actin and tubulin in both very early and later stages of neurite growth from cultured adult DRG neurons. The colocalization of Hsp27 and pHsp27 with actin in lamellopodia and focal contacts at early stages of neurite growth, and in processes, branch points and growth cones at later stages, suggests that Hsp27 may play a role in neuritogenesis and subsequent neurite extension, and potentially in the patterning of this growth. Hsp27 has been reported to play a key role in modulating actin cytoskeletal dynamics as an actin-capping protein in non-neuronal cells. Our results suggest that this may also be the case in neurons and support a role for Hsp27 in neurite outgrowth via its phosphorylation state-dependent interactions with actin.

The synergistic effects of NGF and IGF-1 on neurite growth in adult sensory neurons: convergence on the PI 3-kinase signaling pathway.

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.

Factors contributing to neurotrophin-independent survival of adult sensory neurons.

Dorsal root ganglion (DRG) sensory neurons become less dependent upon neurotrophins for their survival as they mature. DRG neurons from young adult rats were dissociated and cultured in vitro in serum-free defined medium. We show that adult DRG sensory neurons are able to survive for at least 2 weeks in culture in the absence of nerve growth factor (NGF). We then investigated potential mechanisms contributing to this apparent neurotrophin-independent survival in these neurons through the use of inhibitors of cellular signaling pathways. The phosphoinositide kinase-3 (PI 3-K) inhibitor LY294002, and a protein kinase C (PKC) inhibitor, chelerythrine resulted in significant decreases in neuronal survival. Neither the mitogen activated protein kinase kinase (MEK) inhibitor U0126 nor two other PKC inhibitors (bisindolylmaleimide and rottlerin) had any significant effect on survival. Our results point to the importance of PI 3-K and PKC signaling in the neurotrophin-independent survival of adult DRG neurons.