Neurotrophic activity of proNGF in vivo

Abstract

Ectopic expression of nerve growth factor (NGF) in transgenic mice results in the directional growth of sympathetic and/or sensory fibers. For instance, mice that over-express NGF under the control of the glial fibrillary acidic protein (GFAP) promoter exhibit robust axonal sprouting into the cerebellum, with no apparent loss of neurons in peripheral ganglia. Given the disagreement in the literature over whether pro-NGF exerts neurotrophic or apoptotic effects, we assessed the relative levels of proNGF and mature NGF in the cerebella of these transgenic mice. Blinded western blot analyses revealed that proNGF was the major species in both transgenic and wild type mice, with very low levels of mature NGF expression. While transgenic mice displayed significantly higher levels of cerebellar proNGF protein as compared to wild type mice, both strains possessed comparable levels of mature NGF. These data reveal that the ectopic expression of NGF in the cerebellum results in an increase in proNGF rather than mature NGF levels. Together with the robust axonal growth and lack of neuronal death in the ganglia in these animals, our results are clearly consistent with proNGF exhibiting neurotrophic activity in vivo.

Introduction

Nerve growth factor (NGF) is responsible for the development, differentiation, maintenance and repair of neurons (Bibel and Barde, 2000, Ebadi et al., 1997, Huang and Reichardt, 2001, Tucker et al., 2001). In mouse, translation produces a preproNGF protein from two major transcripts (Edwards et al., 1986, Fahnestock, 1991, Selby et al., 1987). The signal sequence is cleaved in the endoplasmic reticulum, producing 32 kDa and 25 kDa proNGF molecules. Neurotrophins in the CNS are most commonly secreted as the pro form (Bruno and Cuello, 2006, Fahnestock et al., 2004a, Lee et al., 2001, Lou et al., 2005, Seidah et al., 1996). Cleavage of proNGF may occur intracellularly by proteases such as γ-NGF, a kallikrein (Berger and Shooter, 1977, Edwards et al., 1988), or by furin or other proprotein convertases (Seidah et al., 1996). ProNGF may be cleaved extracellularly by plasmin to the 13 kDa mature NGF or degraded by matrix metalloproteinases (Bruno and Cuello, 2006, Lee et al., 2001).

NGF binds two distinct receptors. One is a high-affinity, membrane bound, receptor tyrosine kinase known as TrkA. Activation of TrkA induces several signaling cascades including the phosphatidylinositol-3-kinase Akt pathway (Crowder and Freeman, 1998), which mediates sympathetic neuron survival and axonal growth (Atwal et al., 2003). Another pathway mediated by TrkA is the Ras-mitogen activated protein kinase (MAPK) pathway, which also mediates cellular survival, differentiation and neurite outgrowth (Kaplan and Miller, 2000). The second receptor is the common neurotrophin receptor p75NTR, a member of the tumor necrosis family receptor family. This receptor can associate with a variety of transmembrane proteins which can influence diverse functions, including survival/cell death and inhibition of axonal outgrowth (Bandtlow and Dechant, 2004, Barker, 2004, Dhanoa et al., 2006, Hannila and Kawaja, 1999, Hannilla et al., 2004, Walsh et al., 1999a).

In the brain, NGF is found primarily as proNGF (Fahnestock et al., 2001). There is general disagreement in the literature over whether proNGF is neurotrophic or apoptotic (Fahnestock et al., 2004a). In vitro, proNGF binds to TrkA and activates the Ras-MAPK pathway (Fahnestock et al., 2004b). In addition, proNGF binds to p75NTR with high affinity (Lee et al., 2001). ProNGF has been reported as both neurotrophic and apoptotic for oligodendrocytes (Althaus and Kloppner, 2006, Beattie et al., 2002), superior cervical ganglion neurons and PC12 cells (Fahnestock et al., 2004b, Lee et al., 2001). In vivo, the apoptotic action of proNGF has been demonstrated, but only in injured neural systems expressing increased p75NTR and/or reduced or absent TrkA (Harrington et al., 2004, Kumar et al., 2006, Volosin et al., 2006). While previous reports (Kawaja et al., 1997, Kawaja and Crutcher, 1997, Walsh et al., 1999b) have shown that the ectopic expression of the NGF gene under the control of the glial fibrillary acidic protein promoter (GFAP) induces robust sympathetic and sensory sprouting into NGF-rich target tissues (e.g., the cerebellum), there remains a paucity of information as to the relative abundance of proNGF or mature NGF in these transgenic tissues. To address this issue, we now provide direct evidence that, while mature NGF is present in the cerebellum of these transgenic mice, levels of proNGF are significantly elevated, strongly suggesting that the directional ingrowth of sympathetic and sensory axons is induced by the neurotrophic activity of proNGF.