Glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor sustain the axonal regeneration of chronically axotomized motoneurons in vivo

Abstract

In contrast to injuries in the central nervous system, injured peripheral neurons will regenerate their axons. However, axotomized motoneurons progressively lose their ability to regenerate their axons, following peripheral nerve injury often resulting in very poor recovery of motor function. A decline in neurotrophic support may be partially responsible for this effect. The initial upregulation of glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) by Schwann cells of the distal nerve stump after nerve injury has led to the speculation that they are important for motor axonal regeneration. However, few experiments directly measure the effects of exogenous BDNF or GDNF on motor axonal regeneration. This study provided the first direct and quantitative evidence that long-term continuous treatment with exogenous GDNF significantly increased the number of motoneurons which regenerate their axons, completely reversing the negative effects of chronic axotomy. The beneficial effect of GDNF was not dose-dependent. A combination of exogenous GDNF and BDNF on motor axonal regeneration was significantly greater than either factor alone, and this effect was most pronounced following long-term continuous treatment. The ability of GDNF, either alone or in combination with BDNF, to increase the number of motoneurons that regenerated their axons correlated well with an increase in axon sprouting within the distal nerve stump. Thus long-term continuous treatment with neurotrophic factors, such as GDNF and BDNF, can be used as a viable treatment to sustain motor axon regeneration.

Introduction

One of the major contributing factors to the poor functional recovery observed following peripheral nerve injury is the progressive decline in the capacity of motoneurons to regenerate their axons, particularly during the prolonged period in which motoneurons remain axotomized prior to reinnervating denervated muscle targets Fu and Gordon 1995, Fu and Gordon 1997, Funakoshi et al 1993. Neurotrophic factors that are expressed after nerve injury are important for neuronal survival, and may also be important for axonal regeneration Meyer et al 1992, Sendtner et al 1992, Koliatsos et al 1993, Kobayashi et al 1997, Novikov et al 1997, Naveilhan et al 1997). However, neurotrophic factor expression after injury is generally transient, and the failure to maintain the expression of neurotrophic factors over an extended period of time may partially account for the poor axonal regeneration of chronically axotomized motoneurons. Thus exogenous application of these neurotrophic factors may be able to sustain motor axonal regeneration after peripheral nerve injury.

We have recently examined the role of brain-derived neurotrophic factor (BDNF) and its receptors in motor axonal regeneration Boyd and Gordon 2001, Boyd and Gordon 2002. BDNF mediates its effects via two classes of receptors: a member of the tropomyosin receptor kinase (trk) family of receptors, trkB, and a member of the tumor necrosis factor family of receptors, the p75 neurotrophin receptor (p75NTR; Friedman and Greene, 1999; Yano and Chao, 2000). Although low doses of exogenous BDNF completely reverse the negative effects of chronic axotomy, progressively higher doses potently inhibit axonal regeneration (Boyd and Gordon, 2002). The facilitatory and inhibitory effects of BDNF are attributed to its binding to trkB and p75NTR, respectively. A differential role for trkB and p75NTR in motor axonal regeneration is supported by several lines of experimental evidence. First, the inhibitory effects of high-dose BDNF can be eliminated by preventing BDNF binding to p75NTR with function blocking antibodies (Boyd and Gordon, 2002). Second, there is decreased axonal regeneration in trkB heterozygous mutant mice, and increased axonal regeneration in p75NTR hypomorphic mice (Boyd and Gordon, 2001). The strong dose-dependent effects of exogenous BDNF may limit its effectiveness as a therapeutic strategy to promote motor axonal regeneration.

Glial cell line-derived neurotrophic factor (GDNF) is another candidate trophic factor for motoneurons during axonal regeneration. GDNF belongs to a subfamily within the transforming growth factor-β superfamily, together with neurturin, persephin, and artemin Lin et al 1993, Kotzbauer et al 1996, Creedon et al 1997. In contrast to BDNF, which binds to two distinct classes of receptors, GDNF mediates its effects via a single receptor complex composed of a ligand-binding glycosylphosphatidyl inositol membrane-linked receptor subunit, termed GDNF-family receptor α-1 (GFRα-1), and a signal-transducing tyrosine kinase subunit, Ret (reviewed in Saarma and Sariola, 1999). The expression of GDNF in the denervated distal nerve stump and its receptors in axotomized motoneurons after injury are consistent with a functional role for GDNF in motor axonal regeneration. Specifically, the GDNF receptor subunits are upregulated in axotomized motoneurons Tsujino et al 1999, Tuszynski et al 1996, Burazin and Gundlach 1998, Naveilhan et al 1997. GDNF is rapidly upregulated in denervated Schwann cells after sciatic nerve injury Naveilhan et al 1997, Hoke et al 2002, and is retrogradely transported by motoneurons (Yan et al., 1995).

There is considerable evidence demonstrating that GDNF is a potent survival factor for axotomized neonatal and adult motoneurons Henderson et al 1994, Yan et al 1995, Oppenheim et al 1995, Li et al 1995, Vejsada et al 1998, Yuan et al 2000. In addition, overexpression of GDNF in developing muscle causes hyperinnervation of motor endplates during development (Nguyen et al., 1998) and adenoviral gene transfection of axotomized neonatal motoneurons with GDNF increases the numbers of myelinated axons in the distal nerve stump of the facial nerve after a crush injury. This increased number of myelinated axons is associated with significantly improved whisker function compared to nontransfected controls (Baumgartner and Shine, 1998). However, a direct role for GDNF in increasing the number of axotomized motoneurons that regenerate their axons remains to be established. In the present study, we will test whether exogenous GDNF, either alone or in combination with exogenous BDNF, will mediate only positive effects on motor axonal regeneration under conditions of both acute and chronic axotomy.