Abstract
Degeneration of nigrostriatal dopaminergic system is the principal lesion in Parkinson’s disease. Because glial cell line-derived neurotrophic factor (GDNF) promotes survival of dopamine neurons in vitro and in vivo, intracranial delivery of GDNF has been attempted for Parkinson’s disease treatment but with variable success. For improving GDNF-based therapies, knowledge on physiological role of endogenous GDNF at the sites of its expression is important. However, due to limitations of existing genetic model systems, such knowledge is scarce. Here, we report that prevention of transcription of Gdnf 3’UTR in Gdnf endogenous locus yields GDNF hypermorphic mice with increased, but spatially unchanged GDNF expression, enabling analysis of postnatal GDNF function. We found that increased level of GDNF in the central nervous system increases the number of adult dopamine neurons in the substantia nigra pars compacta and the number of dopaminergic terminals in the dorsal striatum. At the functional level, GDNF levels increased striatal tissue dopamine levels and augmented striatal dopamine release and re-uptake. In a proteasome inhibitor lactacystin-induced model of Parkinson’s disease GDNF hypermorphic mice were protected from the reduction in striatal dopamine and failure of dopaminergic system function. Importantly, adverse phenotypic effects associated with spatially unregulated GDNF applications were not observed. Enhanced GDNF levels up-regulated striatal dopamine transporter activity by at least five fold resulting in enhanced susceptibility to 6-OHDA, a toxin transported into dopamine neurons by DAT. Further, we report how GDNF levels regulate kidney development and identify microRNAs miR-9, miR-96, miR-133, and miR-146a as negative regulators of GDNF expression via interaction with Gdnf 3’UTR in vitro. Our results reveal the role of GDNF in nigrostriatal dopamine system postnatal development and adult function, and highlight the importance of correct spatial expression of GDNF. Furthermore, our results suggest that 3’UTR targeting may constitute a useful tool in analyzing gene function.
Highlights
Applied glial cell line-derived neurotrophic factor (GDNF) promotes the survival, function, and neurite growth of nigrostriatal dopamine (DA) neurons both in vitro and in vivo [1,2]
Using a reporter gene assay, we found an 8-fold increase in luciferase expression from the construct containing Firefly-puΔtk proceeded by Gdnf 3’UTR in a cell line derived from human embryonic kidney cells (HEK293) and a 2-fold increase in a cell line derived from human brain cells (U87) (S1D Fig)
Increased GDNF levels increase the nigrostriatal dopaminergic system in adult mice To assess whether DA system was changed in adult animals, we studied Gdnfwt/hyper mice at 2.5–4 months of age and noted that the DA levels in the striatum of Gdnfwt/hyper mice were increased by 25% compared to wild-type littermates (Fig 4D)
Summary
Applied glial cell line-derived neurotrophic factor (GDNF) promotes the survival, function, and neurite growth of nigrostriatal dopamine (DA) neurons both in vitro and in vivo [1,2]. The classic motor deficit in Parkinson’s disease is characterized by a gradual loss of nigrostriatal DA neurons, leading to a reduction in striatal dopamine levels, resting tremor, rigidity, and an inability to initiate voluntary movement [3]. It has been reported that 50% reduction in GDNF levels in adult Gdnf conditional knockout mice has profound consequences on midbrain dopamine neuron survival upon aging [9]. Based on current evidence it is possible that GDNF either has no physiological role in the brain DA system, that GDNF reduction or deletion in the brain is compensated by another mechanism, or that GDNF regulates the DA system at the functional level, rather than at the level of supporting the survival of the DA cell bodies in the midbrain. GDNF is known to be essential for initiating kidney development [7], our understanding of the role of endogenous GDNF in kidney maturation has remained limited
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