Activation of Retinoid X Receptor increases dopamine cell survival in models for Parkinson's disease
© Friling et al; licensee BioMed Central Ltd. 2009
Received: 1 July 2009
Accepted: 11 December 2009
Published: 11 December 2009
Parkinson's disease (PD) is caused by degeneration of dopamine (DA) neurons in the ventral midbrain (vMB) and results in severely disturbed regulation of movement. The disease inflicts considerable suffering for the affected and their families. Today, the opportunities for pharmacological treatment are meager and new technologies are needed. Previous studies have indicated that activation of the nuclear receptor Retinoid X Receptor (RXR) provides trophic support for DA neurons. Detailed investigations of these neurotrophic effects have been hampered by the lack of readily available DA neurons in vitro. The aim of this study was to further describe the potential neurotrophic actions of RXR ligands and, for this and future purposes, develop a suitable in vitro-platform using mouse embryonic stem cells (mESCs).
We studied the potential neurotrophic effects of the RXR ligand LG100268 (LG268) and the RXR-Nurr1 ligand XCT0139508 (XCT) in neuronal cultures derived from rat primary vMB and mESCs. RXR ligands protect DA neurons from stress, such as that induced by the PD-modeling toxin 6-hydroxy dopamine (6-OHDA) and hypoxia, but not from stress induced by oxidative hydrogen peroxide (H2O2) or the excitotoxic agent kainic acid (KA). The neurotrophic effect is selective for DA neurons. DA neurons from rat primary vMB and mESCs behaved similarly, but the mESC-derived cultures contained a much higher fraction of DA cells and thus provided more accessible experimental conditions.
RXR ligands rescue DA neurons from degeneration caused by the PD simulating 6-OHDA as well as hypoxia. Thus, RXR is a novel promising target for PD research. mESC-derived DA cells provide a valid and accessible in vitro-platform for studying PD inducing toxins and potential trophic agents.
PD is caused by progressive degeneration of dopaminergic neurons in the substantia nigra of the vMB [1–3]. The resulting lack of the neurotransmitter DA leads to decreased signaling within the nigro-striatal pathway and produces disturbed regulation of movement with tremor, bradykinesia and rigidity . As of today, there is no cure for PD.
Nuclear hormone receptors (NRs) are emerging as interesting factors in PD research. Most NRs are regulated by small, lipophilic ligands that easily enter the cell nucleus to control transcription. RXR (NR2B1-3) is activated by the synthetic ligand LG268 . Interestingly, this activation has been shown to rescue DA neurons from degeneration in survival assays based on primary cultures . Increased survival was dependent on activation of the heterodimer between RXR and the orphan NR, Nurr1 (NR4A2), as substantiated by several findings. Survival was selective for Nurr1-expressing neurons in vMB as well as cortex and the effects were abolished in cortical cultures from Nurr1 knock-out mice. Furthermore, the ligand XCT, which is selective for the Nurr1-RXR heterodimer, also increased vMB DA neuron survival. Nurr1 is essential for vMB DA neuron development , regulates genes essential for DA synthesis and storage [8–10], and has been indicated to have a role in neuroprotection of mature DA cells in several studies [11, 12]. Indeed, human Nurr1-mutations have been associated with familial PD , providing clinically relevant evidence for such a role.
The origin of DA cell degeneration in PD is largely unknown, but it is suggested to be caused by agents causing oxidative damage and energy depletion in the brain [14–16]. The hydroxylated DA analogue 6-OHDA is commonly used to model nigral degeneration in experimental animals as well as in vitro, where it causes DA cell death and neurotransmitter depletion . In vivo, its uptake is selective for DA cells through the DA transporter, and when inside the neuron, 6-OHDA produces oxidative stress [18, 19] as well as mitochondrial inhibition . Several other stressors can also be used to induce neurodegeneration and stress, for example hypoxic environments , the oxidative agent H2O2 [22, 23], and the excitotoxic glutamate analogue KA [24–26].
Primary neuronal cultures provide data of high biological relevance. However, their use is diminished by low yields and high technical demands. Recently, we developed a platform for in vitro studies using DA cells derived from mESCs. Overexpression of the homeobox domain containing transcription factor Lmx1a under the control of the neuroprogenitor specific Nestin enhancer (NesE) induces formation of high numbers of bona fide vMB DA neurons in culture . These neurons express all relevant neurotransmitters, show proper electrophysiological characteristics as well as physiological levels of DA and metabolites. Moreover, they survive and regenerate when grafted into 6-OHDA lesioned rat brains .
Here we have used DA neurons derived from primary vMB cultures as well as mESCs to further establish the neurotrophic role of RXR activity. We can show that RXR ligands selectively protect DA neurons from stress caused by 6-OHDA and hypoxia, but not from KA and H2O2. The protective effects are only seen in Nurr1-expressing DA cells. To conclude, RXR ligands and mESC-derived DA cells represent promising platforms in the search for novel PD therapies.
Results and discussion
Since primary neurons are retrieved directly from developing brain tissue, artifacts are small, assuring data of high biological relevance. Primary neuronal cultures were prepared from rat E14.5 vMB. The initial extensive neurodegeneration declined significantly within approximately 24 hours of plating and after three days in vitro (DIV) the cell death was negligible, whereby the cultures could be maintained for weeks (also see ). After three DIV, neurons have clearly visible neurites and a mature neuronal morphology. Approximately 2-3% of the neurons in the vMB cultures was dopaminergic and could be identified by immunostaining against the rate-limiting enzyme in DA synthesis, tyrosine hydroxylase (TH). To investigate the specificity of the RXR effects on neuronal survival, several stressors were used.
RXR ligands rescue primary vMB DA neurons from 6-OHDA-induced degeneration
RXR ligand rescues primary vMB DA neurons from degeneration caused by hypoxia, but not from KA and H2O2
RXR ligands rescue mESC-derived DA neurons from 6-OHDA-induced degeneration
PD inflicts considerable suffering for the affected and their families, and it constitutes a great cost for the society. Today, the opportunities for pharmacological treatment are meager and mainly consist of substitution with the DA precursor Levodopa. However its medical potential decreases severely over time . The development of neuroprotective or neuroregenerative therapies would provide great benefits. As such, glial cell line-derived neurotrophic factor (GDNF) has been tested in several clinical trials against PD [31–35]. Unfortunately, lack of efficacy and potential hazardous side effects has lessened the initial enthusiasm, and future development of GDNF-based drug therapies seems uncertain. Thus, there is a high need for alternative strategies to treat PD.
NR ligands are small and lipophilic, and they easily pass the blood-brain-barrier, providing excellent targets for the pharmaceutical industry. Indeed, the characterization of novel NR-mediated signaling pathways during the last decade has resulted in development of novel drugs used in the treatment of metabolic disease and cancer. Specifically, RXR ligands have limited toxicity in humans  and selective RXR ligands would minimize the risk of adverse effects. Our findings that RXR activity leads to neuroprotection  provide novel possible approaches to PD research. The neuroprotective effect is mediated, at least partly, by the Nurr1-RXR dimer. Here, we further show that RXR ligands provide neurotrophic support after stress induced by the PD modeling toxin 6-OHDA. The effects are selective for the Nurr1-expressing DA cells and do not affect total neuronal number in the cultures. Interestingly, neuroprotective effects are also seen after hypoxia-induced neurodegeneration. No protective effects are seen after excitotoxic or pure oxidative stress induced by KA and H2O2, respectively. Thus, the effects of ligand activation is not generally neuroprotective, but rather likely to affect individual cell death pathways.
Ligands activating RXR and the RXR-Nurr1 heterodimer selectively protect DA neurons from stress induced by the PD-modeling toxin 6-OHDA and hypoxia. Thus, the regulation of RXR activity holds promises to contribute to a novel, alternative strategy in PD treatment. Furthermore, mESC-derived cultures offer accessible platforms of DA neurons and may provide novel means to conduct valid in vitro-based PD research.
Primary vMB Cultures
All experiments were performed in accordance with guidelines from the Swedish National Board for Laboratory Animals. Primary vMB cultures were obtained as previously described . Briefly, vMB from rat embryos at stage E14.5 were dissected, mechanically dissociated and plated on poly-D-lysine coated 12 or 24 well plates in serum free medium (N2) consisting of a 1:1 mixture of MEM (Gibco, UK) with 15 mM Hepes buffer (Gibco, UK) and Ham's F12 medium (Gibco, UK). The mixture was supplemented with 6 mg/ml glucose, 1 mg/ml bovine serum albumine, 5 μg/ml insulin, 100 μg/ml transferrin, 60 μM putrescine, 20 nM progesterone, 30 nM selenium and 1 mM glutamine (Sigma). Cultures were incubated for three to five DIV before treatment. Cultures were kept in 37°C, 5% CO2 and 99% humidity unless otherwise stated.
DA cells derived from Lmx1a-expressing mESCs
DA cells were derived from E14.1 mESCs as previously described [27, 28]. Briefly, mESCs were propagated in feeder free conditions in DMEM (Invitrogen) supplemented with 2000 U/ml LIF (Chemicon), 9% KSR, 3% FBS, 0.1 mM non-essential amino acids, 1 mM pyruvate (Invitrogen) and 0.1 μM β2-mercaptoethanol (Sigma). For generation of stable ESC lines, 2 × 10^6 cells were nucleofected with 7 μg linearized NesE-mLmx1a-PGK-neo vector according to protocol (mouse ESC nucleofector kit, Amaxa biosystems Gmbh, Koeln, Germany), selected with G418, replated on gelatinized dishes and induced to differentiate in N2B27 differentiation medium  supplemented with 20 ng/ml bFGF, 100 ng/ml FGF8 and 70 nM Shh. Cells were incubated for 18 days before treatment. Cultures were kept in 37°C, 5% CO2 and 99% humidity throughout all experiments.
Ligands and stressors
Experiments were performed in triplicates. The ligands (stock solutions in DMSO; LG268 and XCT, kindly provided by Dr. Mark Leibowitz at Ligand Pharmaceuticals and Dr. Peter Ordentlich at X-ceptor pharmaceuticals, respectively), were diluted to working dilutions in culture medium and added to the cultures for two to three hours prior to neurodegenerative stressors. Final concentrations were 0.1 and 1 μM for LG268 and XCT, respectively. The DA analogue 6-OHDA was given to the vMB primary neuron cultures at 5-15 μM for 20 minutes before medium and ligand replacement. Hypoxic stress was induced by placing cultures in a modular incubator chamber (Billups-Rothenberg Inc., Del Mar, CA) at 37°C filled with 5% CO2 and 0-1% O2 (balanced with N2) from one to 36 hours. The excitotoxic glutamate analogue KA and the oxidative agent H2O2 were added over night, in the range of 100 to 500 μM and 80 to 110 μM, respectively. To stress mESC-derived DA cells, 150 to 500 μM 6-OHDA in 0.1% ascorbic acid were added three hours before medium and ligand exchange. The cultures were left for another 36 hours in the incubator.
Paraformaldehyde fixed cultures were incubated overnight with TH (1:1000, Pel-Freez, Arkansas), TuJ1 (1:1000, Babco) antiserum in PBS containing 5% fetal calf serum and 0.3% triton X-100. Following rinses, cultures were incubated with FiTC- and Cy3 conjugated secondary antibodies (Jackson, ImmunoResearch, West Grove, PA) for direct detection or with biotinylated secondary antibodies followed by detection of immuno-staining using the ABC immunoperoxidase kit from Vector (Buringame, CA).
Microscopical analysis and image collection
Analysis, imaging and cell counting were performed on Eclipse E1000M and Eclipse TE300 microscopes (both Nikon) coupled to the Spot2 camera (Diagnostic Instruments, Sterling Heights, MI). Scoring was performed by cell counting, and counts were made blind to avoid observation bias. Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test when appropriate.
one-way analysis of variance
days in vitro
Fibroblast growth factor
glial cell line-derived neurotrophic factor
mouse embryonic stem cells
non-significant NRs: Nuclear hormone receptors
Rexinoid × receptor
We thank Prof. Thomas Perlmann at LICR for facilities and valuable scientific discussions. We also thank Dr. Mark Leibowitz at Ligand Pharmaceuticals and Dr. Peter Ordentlich at X-ceptor Pharmaceuticals for providing the RXR ligands used in this study. This work was funded by The Swedish Research Council support to Strategic Center for Developmental Biology and Regenerative Medicine (DBRM).
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