The neurotrophin family, comprising nerve growth factor (NGF) and related proteins, regulates survival, differentiation and axon growth in populations of peripheral and central neurons (Lewin and Barde 1996; Huang and Reichardt 2001; Ernsberger 2009). These actions occur via receptor tyrosine kinases (Trks), and p75 that modulates tyrosine kinase receptor (Trk) receptor binding (Barbacid 1994; Kaplan and Miller 2000). Neurotrophins also promote axon regeneration in injured neurons, so understanding their mechanism in adult systems may suggest therapeutic targets for repair (Chen et al. 2007).
Neurotrophins are synthesised as precursors (proneurotrophins) that are cleaved by proteases to the mature form (Seidah et al. 1996; Lee et al. 2001). Proneurotrophins are important signalling molecules in their own right, with many studies showing that proNGF binds to p75 and the Vps10p domain protein, sortilin, to induce apoptosis (Lee et al. 2001; Nykjaer et al. 2004). The theme emerging is that proNGF acts as a death-inducing ligand under pathological conditions, including Alzheimer’s disease and spinal cord injury (Fahnestock et al. 2001; Harrington et al. 2004; Nykjaer et al. 2004; Volosin et al. 2006; Domeniconi et al. 2007; Jansen et al. 2007; Yune et al. 2007; Al-Shawi et al. 2008). This has led to the hypothesis that mature and proneurotrophins exert opposing effects using distinct (i.e., Trk vs. p75/sortilin) signalling pathways (Teng et al. 2010). However, there are also reports of neurotrophic proNGF actions, potentially mediated by TrkA (Fahnestock et al. 2004; Al-Shawi et al. 2008; Masoudi et al. 2009). It is not known what determines the type of action (neurotrophic vs. apoptotic) of proNGF, although this may be influenced by the ratio of TrkA to p75/sortilin expressed at the cell surface (Fahnestock et al. 2004; Al-Shawi et al. 2008; Clewes et al. 2008; Masoudi et al. 2009). It has also been reported that proNGF does not bind TrkA (Nykjaer et al. 2004; Boutilier et al. 2008), or activates this receptor more weakly than NGF (Clewes et al. 2008). To reconcile these observations, based on cell line experiments it has been proposed that TrkA-dependent neurotrophic effects of proNGF require p75-mediated endocytosis and cleavage to mature NGF (Boutilier et al. 2008). The site and mechanism of this conversion has not been identified. To fully integrate the available mechanistic data it is important to recognise that it has been obtained from diverse cell types, including embryonic sympathetic, sensory and central neurons, as well as cell lines; these vary in the expression of each receptor type and their physiological functions and growth behaviours, so they may also differ in their mechanisms of proNGF signalling.
ProNGF is a significant form of NGF in adult sensory neurons, peripheral target tissues and the spinal cord (Reinshagen et al. 2000; Bierl et al. 2005; Arnett et al. 2007; Buttigieg et al. 2007). Many sensory neurons in dorsal root ganglia (DRG) express one or more of the receptors, TrkA, p75 and sortilin (Wright and Snider 1995; Arnett et al. 2007), raising the possibility of complex effects induced by proNGF. Our goal is to determine the mechanism of proNGF trophic actions in mature sensory neurons, as embryonic systems are more difficult to extrapolate to injury, regeneration and pain. In this study we found that neurotrophic responses to proNGF are dependent on TrkA, p75 and sortilin. Our results support a model in which sortilin- and p75-dependent cleavage of proNGF occurs in peri-somatic glia, to produce NGF that activates TrkA in the adjacent neuronal soma. The proximity and activity of glial cells may determine whether proNGF has pro-apoptotic or trophic actions in adult sensory neurons.
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Whereas many studies have investigated the role of proNGF as a death-inducing ligand, its potential to drive neurotrophic events is less well understood, especially in adult neuronal systems. We used growth assays, pharmacological and immunocytochemical approaches to investigate the site and mechanism of proNGF-induced neurite growth in sensory neurons of adult mice. Here, NGF has been strongly implicated in diverse responses to injury, including axon regeneration (Segal 2003; Chen et al. 2007) and, in the cases of nociceptors, sensitisation (Pezet and McMahon 2006).
We showed that in isolated mature sensory neurons, proNGF has neurotrophic actions that require sortilin, TrkA and p75. These three receptors are expressed in partially overlapping populations of adult DRG neurons, as shown in the present study and by Arnett et al. (2007). The major finding of our study is that the neurotrophic action of proNGF required sortilin but the locus of growth did not correlate spatially with neuronal sortilin expression. Instead, we identified that many glial cells were sortilin-immunoreactive and commonly associated with the soma and proximal neurites of those neurons that demonstrated basal growth (i.e., grew processes overnight in the absence of added neurotrophins). Therefore, our results suggest that glial sortilin mediates these neurotrophic effects of proNGF. This was further supported by our growth cone collapse studies that highlighted the distinct actions of NGF and proNGF – i.e., the poor response to proNGF in this assay correlates well with the distant location of sortilin-positive glia in relation to growth cones. The model of proNGF action we propose based on our results involves sortilin- and p75-dependent conversion of proNGF to NGF at the level of peri-somatic glia (satellite cells), providing a local source of NGF to the adjacent neuronal soma. Therefore, the activity of satellite glia may have significant implications for whether a neuron undergoes a regenerative or apoptoptic event in the presence of proNGF. Peri-somatic glial expression of p75 is up-regulated by injury (Zhou et al. 1996; Hu and McLachlan 2003; Obata et al. 2006), which could also potentially influence the participation of sortilin in cleavage of proNGF.
A critical observation made early in our study was that in contrast to NGF, proNGF was unable to stimulate a robust neurite initiation response. By revealing this distinguishing feature of proNGF signalling we were able to infer that the neurotrophic action (elongation and branching of neurites) of proNGF quantified here had occurred in neurons that were already undergoing growth in the absence of added neurotrophins (basal growth). By carefully assessing this population of neurons, we were able to directly correlate the neurotrophin action of proNGF with neurons having closely associated sortilin-positive glia. We did not specifically quantify p75-positive glia in our cultures, however, many showed low to moderate levels of granular expression, and previous studies in sensory ganglia have reported that glial expression of p75 is up-regulated by injury (Zhou et al. 1996; Hu and McLachlan 2003; Obata et al. 2006). Although not revealed by our study, our proposed model does not exclude an effect of proNGF on neurite initiation. However, we could only assess trophic factor-induced neurite initiation in neurons that had not already grown neurites (i.e. did not contribute to basal growth). Only a very small proportion of these were both TrkA-positive and associated with sortilin-positive peri-somatic glia, i.e. the cellular machinery to elicit a growth response through our proposed glial mechanism. In contrast, neurons with the appropriate characteristics to be targeted by locally cleaved proNGF were the same population that underwent basal growth of neurites, so any potential for additional growth of this type could not be assessed.
We used furin-resistant proNGF (Lee et al. 2001) to reduce the possibility of intracellular cleavage and conversion to NGF to potentially be released into the culture medium. Our western blotting analysis failed to detect NGF in media from proNGF-treated DRG cultures. Moreover, if high levels of NGF were produced and secreted into the medium, proNGF should have mimicked all of the actions of NGF. However, there was little effect of proNGF on neurite initiation or growth cone formation, indicating that significant levels of NGF were not released into the culture medium. Rather, our model predicted that production of NGF from proNGF occurred in a highly localised manner, such that only the soma adjacent to sortilin- and p75-expressing glia were able to be stimulated by newly produced NGF. It is possible that this locally produced NGF is secreted in a very targeted manner so is difficult to measure in culture media using conventional western blotting, so alternative approaches will be needed to probe this potential mechanism further.
The model suggested by our results may resolve some of the previous complexities of the literature that identified an ability of proNGF to stimulate TrkA phosphorylation and MAPK activity (Fahnestock et al. 2004; Masoudi et al. 2009) but having a low affinity for TrkA (Nykjaer et al. 2004) (contrasting with its high affinity for p75 and sortilin to form a pro-apoptotic signalling complex). Such discrepancies may have partly arisen from the diverse experimental systems studied but, irrespective, these different types of experimental outcomes have delayed the broad acceptance of proNGF as a genuine neurotrophic molecule. To at least partly resolve this, Boutilier et al. (2008) provided evidence that the neurotrophic actions of proNGF are indirect and involve conversion to NGF, which may be subsequently secreted to activate TrkA. They showed in PC12 cells (i.e., cultures that do not include glial cells) that activation of TrkA and downstream Erk and Akt required endocytosis and intracellular cleavage of proNGF by the protease furin. In addition, TrkA activation by proNGF has a delayed time course compared with NGF, and evidence from immunoprecipitation experiments indicates that proNGF does not bind to TrkA. Our results from adult ganglion cultures extend this model by proposing that a site of cleavage may be the population of glial cells close to neuronal somata and proximal neurites.
It is not known exactly how sortilin and p75 are involved in the trophic response, e.g., whether they are required for internalisation of proNGF or if they bind proNGF for extracellular presentation to a local source of secreted MMPs (see below). Given the technical challenges involved in separately manipulating glial and neuronal function in this unavoidably mixed short-term culture system, new approaches will be required to ascertain exactly how glial are involved. Nevertheless, highlighting this potential target of neurotrophic factor signalling provides a new way to investigate the function of proNGF in injury or disease states. This complements the report that showed mature pig oligodendrocytes can cleave proNGF (Althaus and Kloppner 2006). Although our results are consistent with glial involvement in proNGF actions, we also initially considered neurons as a site of sortilin-dependent proNGF cleavage. However, very few of the sortilin-positive neurons undergo basal growth during the culture period so do not closely match the properties of proNGF-responsive neurons.
We were able to prevent the neurotrophic response to proNGF by pre-treatment with the broad spectrum MMP inhibitor, batimastat. Therefore, it is possible that glial-derived MMPs are involved with proNGF cleavage in our cultures (although the lack of action of doxycycline suggests additional contributing mechanisms). This contrasts with PC12 cells where proNGF cleavage depends on endocytosis and intracellular cleavage of proNGF by furins, and in cerebellar granule cells proBDNF conversion is mediated by a combination of intracellular furins and MMPs (Boutilier et al. 2008). MMPs are widely implicated in tissue remodelling and neural plasticity after injury (Yong et al. 2001; Rivera et al. 2010). In vivo, the activity of plasmin and MMPs is tightly regulated by tissue plasminogen activator and tissue inhibitors of MMPs (TIMPs) respectively. These are expressed in DRG satellite glia, where they are regulated by injury (Yamanaka et al. 2004, 2005; Kozai et al. 2007; Huang et al. 2011). It would be of great interest to explore further the large family of MMPs and their endogenous regulators in the context of proNGF cleavage, both in vitro and in vivo.
The properties of our culture system, focused on peptidergic TrkA-expressing sensory neurons, allowed us to specifically visualise the proNGF neurotrophic response on neurite length and branching in peptidergic neurons undergoing basal growth (neurite initiation). However, sortilin was also expressed by some non-peptidergic DRG neurons and their associated glia, so proNGF may also have TrkA-independent actions, such as promoting cell death. Although we did not specifically investigate degenerative events in our cultures, we noted that in our control cultures some neurons had the appearance of unhealthy or dying neurons and that these neurons typically expressed high levels of sortilin (data not shown). Arnett et al. (2007) also noted that sortilin-positive DRG neurons were lost after sciatic nerve injury and proposed that these were more susceptible to injury- induced death. We did not see any reduction in the proportion of neurons initiating neurites after proNGF treatment, which one would have expected if proNGF had a significant negative effect on neuronal health in this early period. However, investigating a potential action of proNGF on neuronal death is an interesting question that would be best examined after longer treatments; our culture protocol (short-term cultures, low neuronal density) was designed to optimise analysis of neurites.
In conclusion, proNGF is able to stimulate neurotrophic responses in adult sensory neurons and we propose that these responses are mediated by NGF produced locally by perisomatic glia in a sortilin-, p75- and MMP-dependent manner. Further understanding of the signalling events underlying the neurotrophic response and how these components are regulated in vivo may provide new targets for enhancing regeneration.
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This work was supported by the National Health and Medical Research Council of Australia (Senior Research Fellowships # 570877 and 632903 to J.R.K), the New South Wales (NSW) Office for Science and Medical Research “Spinal Cord Injury and Other Neurological Conditions” Program Grant (J.R.K), National Institutes of Health (NS 30687 to B.L.H.) and Australian Postgraduate Award (to A.K). Current affiliation of A.K.: Department of Physiology, Development & Neuroscience, Anatomy Building, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK. AK, MN, AA and JK participated in conducting the experimental work. All authors contributed to conception and design of the study, analysis or interpretation of the data and writing the manuscript. The authors have no conflicts of interest to declare.