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Abstract
Glial cell line-derived neurotrophic factor (GDNF) family signaling has
been implicated in development, survival and function of many different cell
types, including neurons in the central nervous system. The family consists of
four ligands [GDNF, neurturin (NTN), artemin and persephin] which bind to four
co-receptors (GFRα1-4) and recruit the tyrosine kinase receptor RET into the
signaling complex. In vivo, the ligands bind to the co-receptors with preferential
affinity, e.g., GDNF to GFRα1 and NTN to GFRα2, whereas in vitro, cross-talk
between the ligands and co-receptors has been demonstrated. RET exists as
two main alternatively spliced isoforms, RET9 and RET51. These isoforms differ
in their carboxyl termini and have been implicated with distinct functions in the
enteric and central nervous systems. Clinically, GDNF and NTN have been
examined as potential therapeutic agents for treatment of neurodegenerative
disorders, particularly Parkinson’s disease (PD), as these proteins were found
to provide essential trophic support to dopaminergic (DA) neurons, one of the
main neuronal populations affected by this pathology. To progress development
of such therapies further, a greater understanding of GDNF family signaling is
necessary, which was the aim of the current work.
Two regions of the brain were studied -- the olfactory system, a unique
region of the nervous system that supports lifelong neurogenesis; and the
substantia nigra (SN), the site of DA neuron degeneration in PD. Expression of
GDNF, NTN, GFRα1 and GFRα2 was previously characterized in the olfactory
system (Maroldt et al., 2005; Kaplinovsky and Cunningham, 2011). The current
work specifically focused on expression of the RET isoforms in this system.
Double-labeling fluorescent immunohistochemistry in vivo and in an olfactory
cell culture model illustrated RET9 to be the predominantly expressed isoform
in a pattern similar to the previously reported expression for GDNF and GFRα2.
RET51 was generally expressed in a subset of RET9-positive cells and in vivo
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restricted to the same region as NTN. Treatment of the olfactory cultures with
GDNF initiated a strong neurogenerative response, potentially by acting on the
GFRα2/RET9 signaling complex. NTN exposure promoted olfactory progenitor
proliferation, again potentially via the GFRα2/RET9 signaling complex, as well as
differentiation towards a nestin-positive non-neuronal lineage, speculated to be
sustentacular or olfactory ensheathing cells. The differentiating affect may have
been propagated by GFRα1/RET51 as these proteins were expressed in the
same cells in culture.
In the adult rat and human SN, RET9 was again found to be the predominantly
expressed isoform, localized to all DA neurons, even in PD pathology. RET51
was expressed in a subset of RET9-positive DA neurons in the rat. In human
tissue, this isoform was virtually absent from healthy SN whereas in PD, a
strong up-regulation was noted in DA neurons as well as s100B-positive glia.
The results of this study demonstrate several important features regarding GDNF
family signaling mechanisms. First, RET9 was established as the main functional
isoform and RET51 as a secondary receptor likely important for a specialized
role in a subset of the RET9-positive cells. Second, GDNF may promote neural
differentiation by activating the GFRα2/RET9 signaling complex whereas NTN
activation of this complex yields proliferative mechanisms. NTN acting on GFRα1/
RET51, however, may cause differentiation towards a supporting cell lineage.
Third, RET51 signaling may be involved in the glial response associated with
PD pathology. The current study provided evidence for distinct functional roles
between the ligands GDNF and NTN, and illustrated the signaling complexes
which may be involved in promoting these functions. These findings may facilitate
future therapy development for PD as well as other neurodegenerative disorders.