Synergistic effect of GDNF and NGF on axonal branching and elongation in vitro
Introduction
Peripheral nerves regenerate spontaneously after injury, because of a permissive environment and the activation of the intrinsic growth capacity of neurons (Chen et al., 2007). Functional recovery after nerve injury requires effective axonal regeneration and reinnervation of the target organs (Gillingwater et al., 2004, Kingham and Terenghi, 2006). However, neuronal survival is a prerequisite for axonal regeneration following injury. Neurotrophic factors (NTFs) are known to play a crucial role both in the neuronal survival and axonal regeneration (Frostick et al., 1998, Huang and Reichardt, 2001, Sofroniew et al., 2001). Important NTFs in this context are: (i) neurotrophins, (ii) glial cell line-derived neurotrophic factor family ligands (GFLs), (iii) neuropoietic cytokines, and (iv) insulin-like growth factor (IGF-I). Members of the neurotrophin family include nerve growth factor (NGF), brain-derived neurotrophic growth factor (BDNF), and neurotrophin-3, -4, -5 (NT-3, NT-4, NT-5), whereas GFLs encompass the glial cell line-derived neurotrophic factor (GDNF), artemin (ARTN), neurturin (NRTN), and persephin (PSPN).
Many studies have demonstrated the neuroprotective activities of these factors in vitro and in vivo. Target-derived GDNF and NGF possess neuroprotective activities for the development and maintenance of distinct sets of central and peripheral neurons (Sofroniew et al., 2001, Sariola and Saarma, 2003). Actually, GDNF is a potent survival factor for the motor neurons of peripheral nerves (Henderson et al., 1994) and also protective for sensory neurons (Matheson et al., 1997, Leclere et al., 1998). Mice lacking the tyrosine kinase receptor Ret, or its ligand GDNF, or the GDNF family co-receptor α1 (GFRα1), die soon after birth, whereas mice lacking other GFLs or co-receptors are viable and fertile (Taraviras et al., 1999). NGF administered exogenously has been shown to protect the sensory neurons from axotomy-induced cell death (Yip et al., 1984, Otto et al., 1987, Rich et al., 1987, Melville et al., 1989).
Despite the important knowledge on the physiological role of NTFs in neural development and repair, little is known on the impact of regional-temporal NTF concentrations on axonal outgrowth. Knowledge of biologically adequate NTF concentration–time profiles may be critical for promoting repair and functional recovery after nerve injury. As a first step to acquire this knowledge, we developed an in vitro bioassay using chicken embryonic dorsal root ganglion (DRG) explants. Chicken embryonic DRGs are readily available and exhibit growth similarities with other animal neural systems (Stoeckli et al., 1989, Stoeckli et al., 1991, Kuhn et al., 1991). The present study demonstrates that the axonal growth of embryonic DRG-explants is distinctively affected by GDNF, NGF, and combinations of the two factors in terms of neurite length, branching and growth rate. GDNF and NGF also enhanced the expression of their respective functional receptors Ret and TrkA on the DRG neurons.
Section snippets
Materials
Fertilized white chicken eggs were obtained from Eier Hungerbühler Company (Flawil, Switzerland). Human recombinant glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) were kindly supplied by Amgen (Thousand Oaks, CA, USA) and Genentech (South San Francisco, CA, USA), respectively. Except specified otherwise, all reagents for cell culture were purchased from Invitrogen (Basel, Switzerland).
Preparation of chicken embryonic DRG-explants
Fertilized chicken eggs were incubated at 39 ± 0.2 °C under 100% relative
Results
Treatment of the DRG-explants with GDNF (Fig. 1) or NGF (Fig. 2) induced important and readily visible axonal outgrowth as compared with control explants. GDNF promoted primarily axonal elongation with little axonal branching (Fig. 1), whereas NGF induced extensive axonal branching with less axonal elongation (Fig. 2).
Measurement of the average axonal length confirmed the visual observations. After 3 days of incubation, the axons of the treated DRG-explants were between 0.5 and 2.2 mm longer
Discussion
Neurotrophic factors (NTFs) play a crucial role in regulating the development and function of different sets of neurons of the mammalian nervous system (Lewin and Barde, 1996, Huang and Reichardt, 2001, Tucker et al., 2001, Whitmarsh and Davis, 2001, Price et al., 2005, Zweifel et al., 2005). NGF and GDNF, for example, have been shown to promote neuronal survival and regeneration both in vitro and in vivo (Trupp et al., 1995, Barras et al., 2002, Fine et al., 2002, Boyd and Gordon, 2003, Price
Acknowledgements
We thank Genentech Inc. (South San Francisco, CA, USA) for kind provision of NGF and Amgen Inc. (Thousand Oaks, CA, USA) for kind provision of GDNF. We further acknowledge the enabling financial support by GEBERT RÜF FOUNDATION (Basel, Switzerland).
References (50)
- et al.
A rapid method for semi-quantitative analysis of neurite outgrowth from chick DRG explants using image analysis
J. Neurosci. Methods
(1999) - et al.
Glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor sustain the axonal regeneration of chronically axotomized motoneurons in vivo
Exp. Neurol.
(2003) - et al.
Investigating the synergistic effect of combined neurotrophic factor concentration gradients to guide axonal growth
Neuroscience
(2003) Axonal regeneration of sensory nerves is delayed by continuous intrathecal infusion of nerve growth factor
Neuroscience
(1997)- et al.
Intrathecal administration of nerve growth factor delays GAP 43 expression and early phase regeneration of adult rat peripheral nerve
Brain Res.
(2002) - et al.
Expression pattern of GDNF, c-ret, and GFRalphas suggests novel roles for GDNF ligands during early organogenesis in the chick embryo
Dev. Biol.
(2000) - et al.
Glial cell line-derived neurotrophic factor and nerve growth factor receptor mRNAs are expressed in distinct subgroups of dorsal root ganglion neurons and are differentially regulated by peripheral axotomy in the rat
Neurosci. Lett.
(1998) - et al.
Overlapping and additive effects of neurotrophins and CNTF on cultured human spinal cord neurons
Exp. Neurol.
(1994) - et al.
Preservation of sensory cells by placing stumps of transected nerve in an impermeable tube
Exp. Neurol.
(1989) - et al.
Pharmacological effects of nerve growth factor and fibroblast growth factor applied to the transectioned sciatic nerve on neuron death in adult rat dorsal root ganglia
Neurosci. Lett.
(1987)
Quantitation of neurite growth parameters in explant cultures using a new image processing program
J. Neurosci. Methods
The initial period of peripheral nerve regeneration and the importance of the local environment for the conditioning lesion effect
Brain Res.
NGF utilizes c-Ret via a novel GFL-independent, inter-RTK signaling mechanism to maintain the trophic status of mature sympathetic neurons
Neuron
Nerve growth factor enhances neurite arborization of adult sensory neurons; a study in single-cell culture
Brain Res.
The GDNF family: signalling, biological functions and therapeutic value
Nat. Rev. Neurosci.
Glial cell line-derived neurotrophic factor released by synthetic guidance channels promotes facial nerve regeneration in the rat
J. Neurosci. Res.
Gelatin-tricalcium phosphate membranes immobilized with NGF, BDNF, or IGF-1 for peripheral nerve repair: an in vitro and in vivo study
J. Biomed. Mater. Res. A
Peripheral regeneration
Annu. Rev. Neurosci.
Endogenous NGF and nerve impulses regulate the collateral sprouting of sensory axons in the skin of the adult rat
J. Neurosci.
Structure and expression of the chicken beta nerve growth factor gene
EMBO J.
Brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor are required simultaneously for survival of dopaminergic primary sensory neurons in vivo
J. Neurosci.
GDNF and NGF released by synthetic guidance channels support sciatic nerve regeneration across a long gap
Eur. J. Neurosci.
Schwann cells, neurotrophic factors, and peripheral nerve regeneration
Microsurgery
Growth responses of different subpopulations of adult sensory neurons to neurotrophic factors in vitro
Eur. J. Neurosci.
Myo-GDNF increases non-functional polyinnervation of reinnervated mouse muscle
Neuroreport
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