Two-fold elevation of endogenous GDNF levels in mice improves motor coordination without causing side-effects

Glial cell line-derived neurotrophic factor (GDNF) promotes the survival of dopaminergic neurons in vitro and in vivo. For this reason, GDNF is currently in clinical trials for the treatment of Parkinson’s disease (PD). However, how endogenous GDNF influences dopamine system function and animal behavior is not fully understood. We recently generated GDNF hypermorphic mice that express increased levels of endogenous GDNF from the native locus, resulting in augmented function of the nigrostriatal dopamine system. Specifically, Gdnf wt/hyper mice have a mild increase in striatal and midbrain dopamine levels, increased dopamine transporter activity, and 15% increased numbers of midbrain dopamine neurons and striatal dopaminergic varicosities. Since changes in the dopamine system are implicated in several neuropsychiatric diseases, including schizophrenia, attention deficit hyperactivity disorder (ADHD) and depression, and ectopic GDNF delivery associates with side-effects in PD models and clinical trials, we further investigated Gdnf wt/hyper mice using 20 behavioral tests. Despite increased dopamine levels, dopamine release and dopamine transporter activity, there were no differences in psychiatric disease related phenotypes. However, compared to controls, male Gdnf wt/hyper mice performed better in tests measuring motor function. Therefore, a modest elevation of endogenous GDNF levels improves motor function but does not induce adverse behavioral outcomes.Peer reviewe

Chronic 2-Fold Elevation of Endogenous GDNF Levels Is Safe and Enhances Motor and Dopaminergic Function in Aged Mice

Glial cell line-derived neurotrophic factor (GDNF) supports function and survival of dopamine neurons that degenerate in Parkinson's disease (PD). Ectopic delivery of GDNF in clinical trials to treat PD is safe but lacks significant therapeutic effect. In pre-clinical models, ectopic GDNF is effective but causes adverse effects, including downregulation of tyrosine hydroxylase, only a transient boost in dopamine metabolism, aberrant neuronal sprouting, and hyperactivity. Hindering development of GDNF mimetic increased signaling via GDNF receptor RET by activating mutations results in cancer. Safe and effective mode of action must be defined first in animal models to develop successful GDNF-based therapies. Previously we showed that about a 2-fold increase in endogenous GDNF expression is safe and results in increased motor and dopaminergic function and protection in a PD model in young animals. Recently, similar results were reported using a novel Gdnf mRNA-targeting strategy. Next, it is important to establish the safety of a long-term increase in endogenous GDNF expression. We report behavioral, dopamine system, and cancer analysis of five cohorts of aged mice with a 2-fold increase in endogenous GDNF. We found a sustained increase in dopamine levels, improvement in motor learning, and no side effects or cancer. These results support the rationale for further development of endogenous GDNF-based treatments and GDNF mimetic.Peer reviewe

Elevated expression of endogenous glial cell line-derived neurotrophic factor impairs spatial memory performance and raises inhibitory tone in the hippocampus

Parvalbumin-positive interneurons (PV+) are a key component of inhibitory networks in the brain and are known to modulate memory and learning by shaping network activity. The mechanisms of PV+ neuron generation and maintenance are not fully understood, yet current evidence suggests that signalling via the glial cell line-derived neurotrophic factor (GDNF) receptor GFR alpha 1 positively modulates the migration and differentiation of PV+ interneurons in the cortex. Whether GDNF also regulates PV+ cells in the hippocampus is currently unknown. In this study, we utilized a Gdnf "hypermorph" mouse model where GDNF is overexpressed from the native gene locus, providing greatly increased spatial and temporal specificity of protein expression over established models of ectopic expression. Gdnf(wt/hyper) mice demonstrated impairments in long-term memory performance in the Morris water maze test and an increase in inhibitory tone in the hippocampus measured electrophysiologically in acute brain slice preparations. Increased PV+ cell number was confirmed immunohistochemically in the hippocampus and in discrete cortical areas and an increase in epileptic seizure threshold was observed in vivo. The data consolidate prior evidence for the actions of GDNF as a regulator of PV+ cell development in the cortex and demonstrate functional effects upon network excitability via modulation of functional GABAergic signalling and under epileptic challenge.Peer reviewe

GDNF Overexpression from the Native Locus Reveals its Role in the Nigrostriatal Dopaminergic System Function

Peer reviewe

GDNF is not required for catecholaminergic neuron survival in vivo.

Glial cell line-derived neurotrophic factor (GDNF) has been tested in clinical trials to treat Parkinson’s disease with promising but variable results. Improvement of therapeutic effectiveness requires solid understanding of the physiological role of GDNF in the maintenance of the adult brain catecholamine system. However, existing data on this issue is contradictory. Here we show with three complementary approaches that, independent of the time of reduction, Gdnf is not required for maintenance of catecholaminergic neurons in adult mice

Correction: GDNF Overexpression from the Native Locus Reveals its Role in the Nigrostriatal Dopaminergic System Function.

[This corrects the article DOI: 10.1371/journal.pgen.1005710.]

Identification of <i>Gdnf</i>-regulating miRNAs.

<p><b>(A)</b> Putative miR binding sites cluster within the conserved areas of the <i>Gdnf</i> 3’UTR. The miRNAs underlined with red bars were co-immunoprecipitated with <i>Gdnf</i> mRNA in a genome-wide screen of a mouse brain tissue [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005710#pgen.1005710.ref034" target="_blank">34</a>]. Predicted putative RNA-binding protein (RBP) sites are indicated with triangles. Source: Blast, TragetScan, AREsite, and <a href="http://servers.binf.ku.dk/antar/" target="_blank">http://servers.binf.ku.dk/antar/</a>; Hs, human; Mm, mouse. <b>(B)</b> Luciferase expression from a Ren-<i>Gdnf</i> 3’UTR construct after co-transfection with the indicated pre-miRNAs in HEK293 cells. scr1 and scr2 are scrambled pre-miRNA controls; N = 3 experiments/miRNA with 3–5 biological repeats/miRNA/experiment. <b>(C)</b> Predicted binding sites (ddG<-3) for miR-9 (green), miR-96 (blue), miR-133a/b (orange), and miR-146a (red) based on “predict microRNA targets” analysis (<a href="http://genie.weizmann.ac.il/" target="_blank">http://genie.weizmann.ac.il/</a>). Boxes indicate the mutated miRNA-binding sites in each mutant. Note that miR-9/96/133m contains overlapping sites for miR-9, miR-96, and miR-133, all of which were mutated in this construct. <b>(D)</b> Luciferase assay of miR-9, miR-96, miR-133a, and miR-146a mutants (mutated sequences indicated with boxes in panel C; scr1 is a scrambled pre-miRNA control; N = 2 experiments/miRNA with 3 biological repeats/miRNA/experiment. <b>(E-F)</b> Expression of endogenous <i>GDNF</i> mRNA (E) and GDNF protein (F) is inhibited in U87 cells by co-transfection with the indicated pre-miRNAs; N = 3–4 experiments with 2–3 biological replicates/experiment. <b>(G)</b> Adenoviral transduction of Cre—but not GFP—in homozygous “floxed” Dicer-1^F/F primary cortical neurons increases endogenous GDNF protein levels; N = 2 experiments with 3–4 mice/group. <b>(H)</b> AAV-based constructs encoding shRNAs against miR-9, miR-96, and miR-146a, as well as siRNA against Dicer, increase endogenous <i>GDNF</i> expression in HEK293 cells; N = 5 experiments/construct (except for the shRNA against miR-9, where N = 2 experiments/construct) with 2 biological repeats per experiment. Ad, adenovirus; m, mutant.</p

<i>Gdnf</i> expression is increased in cells that normally express <i>Gdnf</i>.

<p><b>(A)</b><i>In situ</i> hybridization showing <i>Gdnf</i> mRNA expression in thalamic nuclei at P7.5 (white arrowheads). <b>(B)</b><i>In situ</i> hybridization showing <i>Gdnf</i> mRNA expression in Clarke’s column in the thoracic part of the spinal cord at P12.5 (white arrowheads). <b>(C)</b><i>Gdnf</i> mRNA-positive cells (black dots) in the whole striatum (upper panel); a magnified view of <i>Gdnf</i> mRNA-positive cells (arrowheads) is shown in the lower panel. <b>(D)</b> Representative images of <i>Pvalb (PV</i>, blue) and <i>Gdnf</i> (red) mRNA in the striatum of 3-month-old mice detected using RNAscope. <b>(E)</b> Summary of <i>PV</i>-positive only, <i>Gdnf</i>-positive only, and double-positive cells in striatal slices obtained from <i>Gdnf</i>^<i>wt/wt</i> and <i>Gdnf</i>^{<i>wt/hyper</i>} mice; N = 5 animals/group. <b>(F)</b> QPCR analysis of <i>Gdnf</i> mRNA levels in the indicated brain regions in P7.5 mice; N = 6–8 mice/group. <b>(G)</b> QPCR analysis of <i>Gdnf</i> mRNA levels in the indicated brain regions of adult mice; N = 4–8 mice/group. <b>(H)</b> ELISA analysis of GDNF protein levels in the dorsal striatum of adult mice; N = 6–8 mice/group. Scale bars: A, 150 μm; B, 200 μm; C (upper panels), 500 μm; C (lower panels), 50 μm; D, 10 μm. Abbreviations: dSTR, dorsal striatum; OB, olfactory bulb; Hyp, hypothalamus; PFC, prefrontal cortex; vSTR, ventral striatum; Hip, hippocampus; vMB, ventral midbrain; CB, cerebellum; RN, dorsal raphe nucleus; SC, spinal cord; SN, substantia nigra; VTA, ventral tegmental area; PV, parvalbumin; m, months; P, postnatal day.</p