![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER
1 The Ludwig Institute for Cancer Research, Stockholm Branch, S-171 77 Stockholm, Sweden , 2 Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden , 3 X-Ceptor Pharmaceuticals Inc., San Diego, California 92121, USA , 4 Department of Cell and Molecular Biology, Karolinska Institutet, S-171 77 Stockholm, Sweden
 |
Abstract |
|---|
 Top Abstract ResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
[Keywords: Nuclear receptor; orphan receptor; retinoid X receptor; dopamine neurons; neuronal survival]
Received June 23, 2003; revised version accepted October 28, 2003.
; Aranda and Pascual 2001). Several NRs, including, for example, the retinoic acid receptor (RAR) and thyroid hormone receptor, require heterodimerization with the common partner the retinoid X receptor, or RXR (NR2B1-3), for efficient DNA binding and function in response to their cognate ligands (Mangelsdorf et al. 1990; Mangelsdorf and Evans 1995). RXR can also bind ligands such as 9-cis-retinoic acid (9cis RA) and fatty acids including the brain-enriched docosahexanoic acid (DHA; Mangelsdorf and Evans 1995; Mata de Urquiza et al. 2000). However, given its essential function as a heterodimerization partner/cofactor, it has remained possible that RXR's only role might be to provide silent support to other members of the NR family, and definitive evidence for a function as a true "signaling" NR in vivo has so far been lacking.
The possibility that RXR may be activated by endogenous ligands also raises questions concerning the identity of relevant RXR partners in such signaling events. RXR is unable to respond to cognate RXR ligands in several so-called nonpermissive heterodimers (Mangelsdorf and Evans 1995; Germain et al. 2002). However, ligand activation can occur in "permissive" heterodimeric complexes with other NRs (Mangelsdorf and Evans 1995). The orphan NR Nurr1 (NR4A2) forms heterodimers with RXR that are robustly activated by RXR ligands (Law et al. 1992; Forman et al. 1995; Perlmann and Jansson 1995), but can also bind to specific DNA-binding sites either as monomers or homodimers and function as a constitutively active transcription factor (Wilson et al. 1993; Philips et al. 1997). Interestingly, Nurr1 lacks a cavity for ligand binding as revealed from the recently solved X-ray crystal structure of the Nurr1 ligand binding domain (LBD; Wang et al. 2003). Nurr1 is thus distinct from other known RXR partners, which are all capable of binding cognate ligands.
Nurr1 is almost exclusively confined to the central nervous system (CNS) and is essential for the development of midbrain dopamine (DA) neurons (Zetterström et al. 1997; Castillo et al. 1998; Saucedo-Cardenas et al. 1998; Le et al. 1999a; Wallén et al. 1999). In addition, analyses of Nurr1 heterozygous mutant mice show that DA neurons from these animals are more vulnerable to toxic stress compared with wild-type animals, indicating that Nurr1 has a role in neuroprotection of mature DA cells (Le et al. 1999b; Eells et al. 2002). Indeed, recent identification of mutations in the human Nurr1 gene in familial cases of Parkinson's disease have provided clinically relevant evidence for such a role (Le et al. 2003).
Nurr1 is not only expressed in developing and mature DA neurons but is also localized to several additional brain areas including the hippocampus and cerebral cortex (Zetterström et al. 1996a,b). In addition, Nurr1 and its highly homologous family members NGFI-B (NR4A1) and Nor1 (NR4A3) can be rapidly induced by various stimuli, including hypoxic/ischemic stress and kainic acid-induced excitotoxicity (Law et al. 1992; Neumann-Haefelin et al. 1994; Lin et al. 1996; Crispino et al. 1998; Honkaniemi and Sharp 1999; Johansson et al. 2000). It seems likely, therefore, that Nurr1 functions in neuroprotection and/or other neuronal processes are not limited to dopaminergic cells.
Here we provide evidence showing that RXR is active in Nurr1-RXR heterodimers in the developing CNS in vivo. Moreover, regions in which Nurr1-RXR heterodimers are active contain endogenous RXR ligand activities. Finally, in experiments using neuronal primary cultures, we reveal that RXR ligands increase the number of surviving DA cells via a mechanism that requires ligand binding to RXR in Nurr1-RXR heterodimers. Thus, these findings provide evidence for active RXR signaling in vivo, demonstrate a functional role for Nurr1 as a ligand-independent partner of RXR, and suggest a role of RXR ligands in neuronal cell survival.
|
Results |
|---|
|
TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
We have previously used transgenic mice to assess activation of NR LBDs in vivo as a strategy to facilitate characterization of ligand distribution and NR function (Solomin et al. 1998; Mata de Urquiza et al. 1999). To analyze the activity of the Nurr1 LBD in vivo, a DNA sequence encoding a fusion protein of the Nurr1 LBD fused to the DNA-binding domain of the yeast transcription factor Gal4 was cloned into a transgenic vector also containing a LacZ reporter with upstream Gal4-binding sites. In transgenic mice, this approach allows analysis by X-gal staining of sites in vivo where Gal4-Nurr1 is active and thereby inducing the LacZ reporter gene. X-Gal staining of transgenic embryonic day 11.5 (E11.5) embryos revealed robust LacZ expression in several regions of the CNS including the cerebral cortex, medulla oblongata, spinal cord, and in the ventral midbrain (VMB) where DA neurons develop (Fig. 1A; data not shown). Moreover, staining was also seen in the proximal parts of the developing limbs (data not shown). Coronal sections through the midbrain revealed LacZ staining in both the ventricular and mantle zones of the VMB. Staining correlated with that of Nurr1 mRNA expression and TH immunoreactivity (IR) in the mantle zone, suggesting that Gal4-Nurr1 was active in developing DA neurons (Fig. 1A). Within the ventricular zone, staining was localized to a domain expressing Aldh1a1, an aldehydrogenase previously shown to be expressed in proliferating DA progenitor cells as well as in maturing postmitotic DA neurons (Fig. 1A; Wallén et al. 1999).
|
). This mutation does not disrupt other functions such as DNA binding or the ability of the Nurr1 LBD to promote transcriptional activation in certain cell lines (Aarnisalo et al. 2002). The mutation was introduced in Gal4-Nurr1 to generate the dimerization-deficient derivative Gal4-Nurr1dim. Strikingly, in Gal4-Nurr1dim transgenic embryos, the CNS was not stained by X-gal, although staining similar to that observed in Gal4-Nurr1 embryos was detected in the limbs (Fig. 1B; data not shown). Nine out of 19 (47%) Gal4-Nurr1 and 0 out of 17 (0%) Gal4-Nurr1dim embryos showed X-gal staining in the CNS. Because all these embryos represent independent transgenic integration events, the data clearly demonstrate that in E11.5 CNS, activation of Gal4-Nurr1 is entirely dependent on dimerization with RXR. The results suggest that CNS activation of Gal4-Nurr1 might depend on ligand-mediated activation of the heterodimerization partner RXR. Indeed, reporter gene analysis in cell transfection experiments demonstrated that Gal4-Nurr1 functions as a sensor of RXR ligand activation (Fig. 1C). In contrast, Gal4-Nurr1dim was entirely inactive even when cells were treated with high doses of RXR ligands (Fig. 1C). In conclusion, our data suggest that Gal4-Nurr1 can be activated via RXR in vivo and that endogenous RXR ligands are present in several embryonic regions including the developing VMB.
In vitro reporter gene assays were used to analyze if embryonic VMB tissue explants, added to transfected human chorion carcinoma JEG-3 cells, contain and release RXR ligand activity. Consistent with data in transgenic embryos, VMB tissue activated Gal4-Nurr1 but failed to activate Gal4-Nurr1dim (Fig. 2A). Dorsal midbrain tissue did not activate Gal4-Nurr1, consistent with the absence of X-gal staining in this region. Importantly, the RXR-specific antagonist LG849 (Sockanathan and Jessell 1998; Solomin et al. 1998), added together with VMB tissue, blocked activation of the reporter gene, demonstrating that Gal4-Nurr1 activation was mediated via heterodimerization with ligand-bound RXR (Fig. 2B). Equal amounts of VMB tissue from stages E13.5-E15.5 were incubated with the Gal4-Nurr1-transfected reporter cells. A significant increase in activity was observed with increasing age (Fig. 2C), suggestive of an age-dependent accumulation of RXR-specific ligand activity.
|
The tissue activity was further characterized after partial purification. Activity from 
80 E15.5 mouse midbrains and cortices was recovered after hexane extraction and tested in a reporter gene assay (Fig. 2E). The most active fraction from reversed phase high performance liquid chromatography (HPLC; fraction #4) was reconstituted and analyzed by negative ion mass spectrometry (Fig. 2F). The previously identified RXR ligand DHA (Mata de Urquiza et al. 2000) was the main constituent in this fraction (Fig. 2F). No detectable levels of 9cis RA were observed. Additional fractions showed limited activity and contained other fatty acid derivatives but no detectable levels of 9cisRA (data not shown). Because the active fraction is not purified to homogeneity, the active substance cannot be conclusively identified from these data. However, the experiments provide additional clues regarding its chemical properties and indicate that it is distinct from any known retinoid derivatives.
RXR ligands increase the number of surviving cultured DA neurons
The accumulation of RXR ligand with increasing embryonic age and their abundance in the postnatal brain suggest a possible role in maturing and postnatal neurons (Fig. 2C; Mata de Urquiza et al. 2000). To assess the consequences of exogenously administered RXR ligands on VMB neuronal cells, we used primary rat cell cultures from E14.5-E15.5 VMB, a stage when DA cell fate commitment is already determined (Hynes and Rosenthal 1999). Mesencephalic TH-positive neurons degenerate progressively when maintained in a serum-free culture medium, and these cultures are therefore used to assay for survival-promoting factors (see, e.g., Hyman et al. 1991; Lin et al. 1993; Branton and Clarke 1999). DA cells constituted 2%-5% of total cells in the VMB cultures and expressed the characteristic marker genes Nurr1, TH, and Aldh1a1 (Fig. 3A; data not shown). By treating cells with the synthetic RXR-specific agonist LG100268 (hereafter referred to as LG268; Boehm et al. 1995; Repa et al. 2000), a dose-dependent increase in the number of surviving TH-positive neurons was observed, reaching 100% with the most effective concentrations 0.03 and 0.1 µM (Fig. 3B). A similar concentration-dependent increase in the number of TH-positive neurons was also seen using a different RXR agonist (SR11237; Fig. 3B; data not shown; Lehmann et al. 1992). The observed increase in the number of surviving DA neurons could be due to an RXR-ligand-induced effect on neuronal proliferation rather than survival. However, the increase in TH-positive neurons was not correlated with an increased proliferation as determined by BrdU incorporation in either the absence or presence of LG268 (data not shown). Therefore, we conclude that it was the progressive degeneration of DA neurons that was negatively influenced by RXR ligands.
|
), in potentiating the increase in DA cell number, whereas LG268 (0.1 µM) was almost twice as potent (Fig. 3C). The concentration of GDNF used in this experiment (60 ng/mL) was shown to generate optimal survival in parallel dose-response experiments (data not shown). The use of an RXR-specific antagonist, LG1208 (Canan Koch et al. 1996, 1999), together with LG268 blocked the response, thereby verifying that the effect was due to RXR activation (Fig. 3D). To verify the specificity of LG268 and LG1208 in neuronal cells, primary neuronal cultures were transfected with vectors encoding Gal4-Nurr1 and Gal4-Nurr1dim together with a Gal4-responsive luciferase reporter vector. As shown in Figure 3E, LG268 activates Gal4-Nurr1, but not Gal4-Nurr1dim. As predicted, LG1208 blocks this response. Because Gal4-Nurr1 or Gal4-Nurr1dim was transfected without expression vector encoding RXR, these results show that endogenous RXR is available for heterodimerization with transgenic Gal4-Nurr1. In conclusion, these results demonstrate the potential of Nurr1-RXR heterodimers to transduce signaling by RXR ligands in neuronal cells.
Surprisingly, when added alone, the endogenous RXR ligand 9cis RA did not affect the number of surviving DA cells. Given that 9cis RA also activates RAR, this suggested that activation of RAR might negatively influence the ability of RXR ligands to increase DA cell number (Fig. 4A). Two observations corroborated this hypothesis. First, when 9cis RA signaling via RAR was blocked by an RAR-specific antagonist (Ro41-5253; Apfel et al. 1992), a robust increase of surviving DA cells was observed (Fig. 4A). Second, an RAR-specific agonist TTNPB [(E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tatramethyl-2-naphtalenyl1) propen-1-yl)]benzoic acid] (Sporn et al. 1984) abolished the increase in surviving DA cells when added together with LG268 (Fig. 4B).
|
Nurr1 is essential for RXR-ligand-dependent neuroprotection
DA neurons constitute only a minority of total neurons in VMB cultures. To evaluate whether RXR ligands selectively stimulate survival of DA neurons, cells were stained for a general neuronal marker (NeuN) to visualize the entire neuronal population. Notably, RXR-selective ligands did not promote a significant increase in total neuronal number, demonstrating that the increased survival is not general in all neuronal cell types in these cultures (Fig. 5A,B).
|
|
Although the data described above indicate that Nurr1 is essential for the ability of RXR ligands to promote neuronal survival, it remained possible that the requirement for Nurr1 is indirect. A pharmacological approach was used to address this possibility. Screening of a chemical library resulted in the identification of an aminopyrimidine derivative (XCT0135908; Fig. 7A) that is highly selective in activation of heterodimers formed between Gal4-Nurr1 and cotransfected RXR in African green monkey CV-1 cells (Fig. 7A). Notably, at the concentrations used in the experiments (1 µM), this compound failed to activate other Gal4-NR derivatives cotransfected with RXR and did not activate RXR alone (Fig. 7A; data not shown). Moreover, activation of Gal4-Nurr1/RXR was blocked by the addition of an RXR antagonist, indicating that the RXR subunit in these heterodimers is activated by XCT0135908 (data not shown). In line with the results presented above, addition of the Nurr1/RXR-selective compound XCT0135908 to primary cultures from rat VMB increased the number of surviving DA cells whereas nondopaminergic neurons were unaffected (Fig. 7B; data not shown). In transfection experiments in primary neuronal cultures, XCT0135908 activated Gal4-Nurr1 but not Gal4-Nurr1dim (Fig. 7C). As predicted, activation of Gal4-Nurr1 was blocked by the RXR antagonist LG1208. Together these data provide additional strong evidence substantiating that Nurr1 is an essential heterodimer partner in RXR-ligand-induced neuronal survival.
|
|
Discussion |
|---|
|
TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
Existence of endogenous RXR ligands
RXR was initially defined as a retinoid receptor based on its ability to become activated and bind 9cis RA (Heyman et al. 1992; Levin et al. 1992). In addition, 9cis RA can also activate RARs. Such dual activation potential would suggest that 9cis RA is essential in vitamin A-dependent processes in vivo. Indeed, ligand activation of both subunits in RAR-RXR heterodimers often results in strongly synergistic effects (see, e.g., Roy et al. 1995; Botling et al. 1997; Lu et al. 1997), and previous data have indicated RXR activation in the embryonic spinal cord, likely as a result of 9cis RA ligand binding (Solomin et al. 1998). Moreover, mutations introduced in the ligand-dependent activation function 2 in RXR
by gene targeting in mice also suggested an active ligand-binding role of RXR in retinoid-mediated signaling (Mascrez et al. 1998). However, despite these data, the signaling status of RXR in vivo has been a matter of debate because 9cis RA has been difficult to detect in tissues, and specific isomerases converting atRA into 9cis RA have not been identified (Horton and Maden 1995; Ulven et al. 2001). Thus, 9cis RA may be important at sites containing very high levels of all-trans RA, for example, in the developing embryonic spinal cord, where sufficient amounts of 9cis RA can be derived from nonenzymatic atRA isomerization.
More recent findings have indicated the existence of endogenous nonretinoid RXR-selective ligands. Phytanic acid is a chlorophyll metabolite activating RXR, but is probably only accumulating at sufficiently high levels to allow activation of RXR in patients with certain metabolic disorders (Kitareewan et al. 1996; Lemotte et al. 1996). The polyunsaturated fatty acid DHA was recently identified as a brain-derived RXR ligand (Mata de Urquiza et al. 2000). Our more recent studies have identified additional RXR-activating fatty acid derivatives in the brain, but also in other tissues (A. Mata de Urquiza and T. Perlmann, unpubl.). These fatty acids are mostly associated with membrane phospholipids, but they can be released in free form and are in several cases highly abundant. Thus, although the exact identity of physiologically relevant endogenous RXR ligands remains to be established, we can conclude that RXR-selective ligands exist in tissues and, as indicated in this study, RXR is apparently ligand activated in the embryonic CNS. As demonstrated by partial purification, one of the active components of E15.5 midbrain tissue was indeed identified as DHA (Fig. 2F).
Nurr1 as a silent partner in Nurr1-RXR heterodimers
This study defines a new role of Nurr1 in RXR-ligand-mediated signaling in neurons. Evidence for the involvement of Nurr1 in RXR-ligand-dependent signaling is derived from several observations: (1) Nurr1-expressing cells, but not other neurons in the VMB, cortex, and hippocampus respond to RXR ligand treatment. (2) Cells cultured from the cortex of Nurr1-gene-targeted mice fail to respond to RXR ligand treatment. (3) A synthetic compound (XCT0135908) selectively activating Nurr1-RXR heterodimers stimulated DA neuron survival. (4) Nurr1-RXR heterodimers can be activated in vivo, apparently as a result of RXR ligand binding, as demonstrated in our transgenic mouse experiments. Together, these observations provide compelling evidence showing that Nurr1-RXR heterodimers are responsible for transducing the neuroprotective effect.
Nurr1 can also bind to DNA either as monomers or homodimers, and it seems likely that both RXR-dependent and -independent activities are essential for Nurr1 functions in vivo. For example, although previous data indicated that Nurr1 functions independently of RXR at early stages of DA cell development (Castro et al. 2001), the RXR-dependent survival-promoting effect presented here may be more relevant at later stages of brain maturation and in the postnatal brain. Such effects will remain undetected in Nurr1-null-gene-targeted mice, as these die immediately after birth (Zetterström et al. 1997; Castillo et al. 1998; Saucedo-Cardenas et al. 1998).
Nurr1 belongs to an evolutionarily conserved subgroup of NRs that can bind DNA as monomers, homodimers, and heterodimers with RXR. Despite intense efforts, neither synthetic nor natural compounds that can modulate the activity of Nurr1 in an apparent ligand-dependent manner have been identified. The recently described X-ray crystal structure of the LBDs of Nurr1 and its Drosophila homolog DHR38 have explained the reason for these difficulties (Baker et al. 2003; Wang et al. 2003). Accordingly, although the Nurr1 LBD largely resembles LBDs of other NRs, it lacks a cavity for ligand binding, thus defining Nurr1 as a ligand-independent orphan NR. This remarkable property distinguishes Nurr1 (and most likely NGFI-B) from other RXR heterodimer partners, which all have identified ligands. We speculate that an important ligand-independent function of Nurr1 is to function as a silent partner of RXR and thereby indirectly promote ligand-mediated signaling in vivo. Curiously, in such a mechanism Nurr1 resembles the function of RXR in other heterodimers.
Nurr1 in neuroprotection
The results presented here demonstrate that Nurr1 has a survival-promoting function in Nurr1-expressing neurons. Previous studies have provided suggestive evidence for a neuroprotective role of Nurr1 in mature DA neurons. Compared with DA neurons derived from wild-type animals, these cells are more vulnerable in Nurr1-gene-targeted heterozygous mice (Le et al. 1999b; Eells et al. 2002; Le et al. 2003). In addition, recent genetic analyses in familial Parkinson's patients underscored the importance of Nurr1 for the maintenance of dopaminergic cells in the human brain (Le et al. 2003). It is important to emphasize, however, that the results presented here implicate Nurr1 and RXR in a more versatile neuroprotective function, as not only midbrain DA cells, but also Nurr1-expressing neurons in the cortex and hippocampus respond to RXR ligand treatment. Given the ubiquitous expression of RXR (Mangelsdorf et al. 1992; Dolle et al. 1994; Zetterström et al. 1999), Nurr1 availability may thus determine the responsiveness of neurons to RXR-ligand-promoted survival. Nurr1 is encoded by an immediate early gene that is rapidly induced by various stressful insults including ischemia, and it is interesting to speculate that such up-regulation in response to stressful insults reflects a cytoprotective mechanism mediated by Nurr1-RXR heterodimers (Law et al. 1992; Crispino et al. 1998; Honkaniemi and Sharp 1999).
Endogenous RXR ligands in neuroprotection
Are endogenous RXR ligands neuroprotective in vivo? The apparent accumulation of RXR ligand activity during development and in the postnatal brain seems consistent with a functional role in maintenance of developing and mature neurons. Ligands that are biologically relevant in such a neuronal survival pathway should be selective for RXR, because RAR ligands, including the RAR/RXR ligand 9cis RA, inhibit RXR-ligand-mediated neuronal survival (Fig. 4). Notably, although their exact biochemical nature remains to be determined, the endogenous ligand activities defined here fulfill this criterion because they are specific for RXR and pharmacologically distinct from 9cis RA. It should be noted, however, that definitive evidence for a role of RXR and endogenous RXR ligands in the adult brain remains to be provided.
A previously identified ligand that may contribute to activation of Nurr1-RXR heterodimers in vivo is the polyunsaturated fatty acid DHA. Of note, DHA has in previous studies been shown to be neuroprotective (Glozman et al. 1998; Lauritzen et al. 2000; Politi et al. 2001), and we demonstrate here that DHA has a robust ability to increase the survival of DA neurons. An RXR antagonist blocked this effect, supporting the hypothesis that DHA can be neuroprotective via activation of RXR. DHA is mainly associated with membrane phospholipids. However, it can be released as free acid, for example, upon stressful insults such as ischemia (Neuringer et al. 1988; Baker and Chang 1992). Because Nurr1 is encoded by an immediate early gene, it is intriguing to speculate that both Nurr1 and endogenous RXR ligands are made available under situations requiring engagement by acutely induced neuroprotective mechanisms.
RXR as a target for treatment of neurodegenerative disease
NRs have major potential as drug targets. However, whereas several metabolic disorders are clearly amenable to NR ligand treatment (eg., via activation of PPAR
), much less progress has been made in understanding NR functions of potential relevance in neurological disorders. The results presented here suggest that RXR ligand administration may provide a novel approach in treatment of neurodegenerative disease. The high levels of Nurr1 expression in DA neurons and in cortical and hippocampal neurons in response to hypoxic stress (Honkaniemi and Sharp 1996) suggest that both Parkinson's disease and stroke may be relevant disorders in further studies toward this goal. RXR's versatile role as a common heterodimer partner might increase the risk for unwanted side effects and is a concern in any pharmacological approach involving RXR agonists. The unique pharmacological properties of the Nurr1-RXR-specific agonist XCT0135908 are therefore intriguing and warrant further in vitro and in vivo studies in models of neurodegenerative disease.
|
Materials and methods |
|---|
|
TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
NMRI mouse embryo midbrains were dissected (dissection microscope SMZ-2T; Nikon) and either placed directly on transfected JEG3 cells or fine-dissected into separate ventral and dorsal pieces and placed on the cells. The tissue was weighed to get equal amounts of tissue on the cells. For generation of tissue-conditioned medium, tissue was excised, cut into small pieces, and placed in serum-free minimal essential medium (MEM; GIBCO). Incubation in cell culture incubator was overnight, followed by centrifugation and removal of tissue. This conditioned medium was placed on transfected cells.
Tissue culture and transfections
Human choriocarcinoma JEG3 cells were transfected in triplicates with 100 ng of receptor CMX-Gal4-Nurr1, CMX-Gal4-Nurr1dim (point mutation in LBD I-box P560A), ±100 ng of CMX-RXR and 100 ng of Gal4-binding luciferase reporter MH100-tk-luc using the calcium phosphate method (Perlmann et al. 1993). In the experiment with conditioned media and titration of 9cis RA, CMX-Gal4-RAR was also used. In all experiments, CMX-
-Gal (200 ng) was used as an internal control. Calcium precipitate was removed by rinses 6 h posttransfection, and relevant substances were added to cells. After 24 h, cells were harvested and assayed for luciferase and reference -galactosidase activity on a luminometer/photometer reader (Lucy-1; Anthos).
CV-1 cells were cultured in DMEM (Sigma) with 10% charcoal stripped FCS (HyClone), 50 µg/mL Gentamycin (GIBCO, Invitrogen), and plated at 10,000 cells/well in DMEM with 5% charcoal stripped FCS (HyClone) and 50 µg/mL Gentamycin into 96-well plates 1 d previous to transfecting. Transfections were carried out using FuGENE6 reagent (Roche). Briefly, 70 ng of DNA was transfected per well with 0.25 µL of FuGENE6 reagent. For heterodimer selectivity assays, 20 ng of MH100x4-tk-luciferase, 20 ng of the appropriate CMX-GAL4-LBD construct, 20 ng of CMX-RXR-LBD, and 10 ng of CMX--Gal, were transfected. The compound was added 5 h after transfection at the concentrations indicated in the figure, and the transfection was assayed 18 h later unless otherwise indicated. Cells were lysed, and the luciferase activity was measured using an LJL Analyst plate reader and normalized to -galactosidase activity. All experiments were carried out a minimum of three times.
Partial purification of tissue factor from brain-conditioned medium
Embryonal conditioned medium was prepared using forebrain tissue from 80 mouse embryos (E15.5) as described above. Medium was extracted using hexane as described (Mata de Urquiza et al. 2000), reconstituted in a minimum of HPLC-mobile phase (methanol:isopropanol:water, 80:10:10 [v/v], with 0.5% acetic acid) and fractionated using an isocratic gradient (0.2 mL/min) on a reverse-phase C18-column (ACE, 250 x 2.1 mm, Advanced Chromatography Technologies). Fractions were reconstituted in ethanol and tested for activity by adding small aliquots to HEK293 cells transfected with effector and reporter plasmids (Mata de Urquiza et al. 2000). Active fractions were analyzed by negative ion electrospray mass spectrometry using a Quattro Micro triple-quadrupole mass spectrometer (Micromass).
Gal4-transgenic mice
The bicistronic UAS-hsp-gRAR/lacZ construct (Mata De Urquiza et al. 1999) was cleaved with XhoI and NheI to replace part of the Gal4 (g) and entire RAR-LBD cDNA with the corresponding Gal4 sequence and LBD cDNA from CMX-gNurr1 and CMX-gNurr1dim LBDs, respectively. Constructs purified from vector sequences (Not1 cleavage) were microinjected into pro-nuclei of fertilized eggs from matings of C57BL/6xCBA hybrid mice. For the Gal4-Nurr1 wild type, seven founder lines were established and mated to wild-type mice for generation of off-spring for embryonic analyses. In addition, transient transgenic embryos were generated. For Gal4-Nurr1dim, transient transgenic embryos only were used. In total, 19 Gal4-Nurr1 and 17 Gal4-Nurr1dim mouse lines were obtained. Embryos were dissected at E11.5, fixed for 30 min in 0.2% glutaraldehyde, followed by immersion in X-gal staining solution (2 mM MgCl2, 0.02% NP-40, 0.01% Na-deoxycholate, 5 mM K4Fe[CN]6, 5 mM K3Fe[CN]6, and 1 mg/mL X-gal [5-bromo-4-chloro-3-indoyl--D-galactopyranoside]) overnight at 37°C. Embryos were rinsed in PBS and postfixed in 4% paraformaldehyde. Amnion-derived DNA was used for genotyping.
Nurr1-gene-targeted mice
Generation and genotyping of Nurr1 mutant mice was described previously (Zetterström et al. 1997).
Histology
Coronal cryosections of Gal4-Nurr1 embryos were prepared at 14 µm thickness.
In situ hybridization histochemistry
For Nurr1 mRNA analysis, the protocol previously described was used (Wallén et al. 1999).
Immunofluorescent histochemistry
TH (1:200; Pel-Freez) and Aldh1a1 (1:400; generous gift from R. Lindahl, University of South Dakota, Vermillion) primary antiserum was diluted in PBS with 0.3% Triton X-100 and 0.5% fetal calf serum and incubated on paraformaldehyde-fixed sections overnight at 4°C, followed by rinses in PBS and immunodetection by Cy3-conjugated IgG (Jackson ImmunoResearch).
Primary cultures
VMB, hippocampus, and cortex from rat (B&K) and mouse (Zetterström et al. 1997) embryos at stage E14.5-E15.5 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) with 15 mM HEPES buffer (GIBCO) and Ham's F12 medium (GIBCO). The mixture was supplemented with 6 mg/mL glucose, 1 mg/mL bovine serum albumin, 5 µg/mL insulin, 100 µg/mL transferrin, 60 µM putrescine, 20 nM progesterone, 30 nM selenium, and 1 mM glutamine (Sigma). Ligands included stock solutions in DMSO; 9cis RA (Sigma); LG100268, LG100849, and LG1001208 (kindly provided by Mark Leibowitz at Ligand Pharmaceuticals); (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid (TTNPB), DHA (Sigma), GDNF (Sigma-Aldrich), SR11237 (Roche), and Ro41-5253 (kindly provided by Louise Foley at Hoffman-LaRoche). These were diluted to working concentrations in N2 and added to cells in triplicates. Cells were incubated 3-5 d. Paraformaldehyde-fixed cultures were incubated overnight with TH (1:1000; Pel-Freez), NeuN (1:200; Chemicon), or Nurr1 (1:10.000; Santa Cruz Biotechnology) antiserum in PBS containing 0.5% (TH and NeuN) or 10% (Nurr1) fetal calf serum and 0.3% Triton X-100. Following rinses, cultures were incubated with biotinylated secondary antibody followed by detection of immunostaining using the ABC immunoperoxidase kit from Vector. Cortical primary neurons were transfected using the Nucleofector technology and the Rat Neuron Nucleofector kit (Amaxa biosystems Gmbh) according to the manufacturer's protocol. A total of 1.5 µg of CMX-Gal4-Nurr1 or CMX-Gal4-Nurr1dim, ±1.5 µg of CMX-RXR and 1 µg of MH100-tk-luc and 0.5 µg of CMX--Gal were added per 6 x 106 cells; 1 x 106 cells were plated per 2-cm2 well. Ligands were added within 4 h after plating. Cells were lysed after 18-20 h and assayed for luciferase and reference -galactosidase activity as described above.
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). To obtain unbiased results, counts were made by two persons. Statistical analyses were by Student's t-test.
|
Acknowledgments |
|---|
|
TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
|
Footnotes |
|---|
5 These authors contributed equally to this work. 
6 Corresponding author.
E-MAIL Thomas.Perlmann{at}licr.ki.se; FAX 46-8-33-28-12.
|
References |
|---|
|
TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
Apfel, C., Bauer, M., Crettaz, M., Forni, L., Kamber, M., Kaufman, F., LeMotte, P., Pirson, W., and Klaus, M. 1992. A retinoic acid receptor antagonist selectively counteracts retinoic acid effects. Proc. Natl. Acad. Sci. 89: 7129-7133.
Aranda, A. and Pascual, A. 2001. Nuclear hormone receptors and gene expression. Physiol. Rev. 81: 1269-1304.
Baker, R.R. and Chang, H.Y. 1992. The hydrolysis of natural phosphatidylethanolamines by phospholipase A2 from rat serum: A degree of selectivity is shown for docosahexaenoate release. Biochim. Biophys. Acta 1125: 56-61.[Medline]
Baker, K.D., Shewchuk, L.M., Kozlova, T., Makishima, M., Hassell, A., Wisely, B., Caravella, J.A., Lambert, M.H., Reinking, J.L., Krause, H., et al. 2003. The Drosophila orphan nuclear receptor DHR38 mediates an atypical ecdysteroid signaling pathway. Cell 113: 731-742.[CrossRef][Medline]
Boehm, M.F., Zhang, L., Zhi, L., McClurg, M.R., Berger, E., Wagoner, M., Mais, D.E., Suto, C.M., Davies, P.J.A., Heyman, R.A., et al. 1995. Design and synthesis of potent retinoid X receptor selective ligands that induce apoptosis in leukemia cells. J. Med. Chem. 38: 3146-3155.[CrossRef][Medline]
Botling, J., Castro, D.S., Oberg, F., Nilsson, K., and Perlmann, T. 1997. Retinoic acid receptor/retinoid X receptor heterodimers can be activated through both subunits providing a basis for synergistic transactivation and cellular differentiation. J. Biol. Chem. 272: 9443-9449.
Branton, R.L. and Clarke, D.J. 1999. Apoptosis in primary cultures of E14 rat ventral mesencephala: Time course of dopaminergic cell death and implications for neural transplantation. Exp. Neurol. 160: 88-98.[CrossRef][Medline]
Canan Koch, S.S., Dardashti, L.J., Hebert, J.J., White, S.K., Croston, G.E., Flatten, K.S., Heyman, R.A., and Nadzan, A.M. 1996. Identification of the first retinoid X receptor homodimer antagonist. J. Med. Chem. 39: 3229-3234.[CrossRef][Medline]
Canan Koch, S.S., Dardashti, L.J., Cesario, R.M., Croston, G.E., Boehm, M.F., Heyman, R.A., and Nadzan, A.M. 1999. Synthesis of retinoid X receptor-specific ligands that are potent inducers of adipogenesis in 3T3-L1 cells. J. Med. Chem. 42: 742-750.[CrossRef][Medline]
Castillo, S.O., Baffi, J.S., Palkovits, M., Goldstein, D.S., Kopin, I.J., Witta, J., Magnuson, M.A., and Nikodem, V.M. 1998. Dopamine biosynthesis is selectively abolished in substantia nigra/ventral tegmental area but not in hypothalamic neurons in mice with targeted disruption of the Nurr1 gene. Mol. Cell. Neurosci. 11: 36-46.[CrossRef][Medline]
Castro, D.S., Hermanson, E., Joseph, B., Wallen, A., Aarnisalo, P., Heller, A., and Perlmann, T. 2001. Induction of cell cycle arrest and morphological differentiation by Nurr1 and retinoids in dopamine MN9D cells. J. Biol. Chem. 276: 43277-43284.
Crispino, M., Tocco, G., Feldman, J.D., Herschman, H.R., and Baudry, M. 1998. Nurr1 mRNA expression in neonatal and adult rat brain following kainic acid-induced seizure activity. Brain Res. Mol. Brain Res. 59: 178-188.[Medline]
Dolle, P., Fraulob, V., Kastner, P., and Chambon, P. 1994. Developmental expression of murine retinoid X receptor (RXR) genes. Mech. Dev. 45: 91-104.[CrossRef][Medline]
Eells, J.B., Yeung, S.K., and Nikodem, V.M. 2002. Dopamine neurons heterozygous for the Nurr1-null allele have reduced survival in vitro. Neurosci. Res. Comm. 30: 173-183.
Forman, B.M., Umesono, K., Chen, J., and Evans, R.M. 1995. Unique response pathways are established by allosteric interactions among nuclear hormone receptors. Cell 81: 541-550.[CrossRef][Medline]
Germain, P., Iyer, J., Zechel, C., and Gronemeyer, H. 2002. Co-regulator recruitment and the mechanism of retinoic acid receptor synergy. Nature 415: 187-192.[CrossRef][Medline]
Giguère, V. 1999. Orphan nuclear receptors: From gene to function. Endocr. Rev. 20: 689-725.
Glozman, S., Green, P., and Yavin, E. 1998. Intraamniotic ethyl docosahexaenoate administration protects fetal rat brain from ischemic stress. J. Neurochem. 70: 2484-2491.[Medline]
Heyman, R.A., Mangelsdorf, D.J., Dyck, J.A., Stein, R.B., Eichele, G., Evans, R.M., and Thaller, C. 1992. 9-cis retinoic acid is a high affinity ligand for the retinoid X receptor. Cell 68: 397-406.[CrossRef][Medline]
Honkaniemi, J. and Sharp, F.R. 1996. Global ischemia induces immediate-early genes encoding zinc finger transcription factors. J. Cereb. Blood Flow Metab. 16: 557-565.[CrossRef][Medline]
____. 1999. Prolonged expression of zinc finger immediate-early gene mRNAs and decreased protein synthesis following kainic acid induced seizures. Eur. J. Neurosci. 11: 10-17.[CrossRef][Medline]
Horton, C. and Maden, M. 1995. Endogenous distribution of retinoids during normal development and teratogenesis in the mouse embryo. Dev. Dyn. 202: 312-323.[Medline]
Hyman, C., Hofer, M., Barde, Y.A., Yuhasz, M., Yancopoulos, G.D., Squinto, S.P., and Lindsay, R.M. 1991. BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra. Nature 350: 230-232.[CrossRef][Medline]
Hynes, M. and Rosenthal, A. 1999. Specification of dopaminergic and serotonergic neurons in the vertebrate CNS. Curr. Op. Neurobiol. 9: 26-36.[CrossRef][Medline]
Johansson, I.M., Wester, P., Hakova, M., Gu, W., Seckl, J.R., and Olsson, T. 2000. Early and delayed induction of immediate early gene expression in a novel focal cerebral ischemia model in the rat. Eur. J. Neurosci. 12: 3615-3625.[CrossRef][Medline]
Kitareewan, S., Burka, L.T., Tomer, K.B., Parker, C.E., Deterding, L.J., Stevens, R.D., Forman, B.M., Mais, D.E., Heyman, R.A., McMorris, T., et al. 1996. Phytol metabolites are circulating dietary factors that activate the nuclear receptor RXR. Mol. Biol. Cell 7: 1153-1166.[Abstract]
Lauritzen, I., Blondeau, N., Heurteaux, C., Widmann, C., Romey, G., and Lazdunski, M. 2000. Polyunsaturated fatty acids are potent neuroprotectors. EMBO J. 19: 1784-1793.[CrossRef][Medline]
Law, S.W., Conneely, O.M., DeMayo, F.J., and O'Malley, B.W. 1992. Identification of a new brain-specific transcription factor, NURR1. Mol. Endocrinol. 6: 2129-2135.[Abstract]
Le, W., Conneely, O.M., Zou, L., He, Y., Saucedo-Cardenas, O., Jankovic, J., Mosier, D.R., and Appel, S.H. 1999a. Selective agenesis of mesencephalic dopaminergic neurons in Nurr1-deficient mice. Exp. Neurol. 159: 451-458.[CrossRef][Medline]
Le, W.-D., Conneely, O.M., He, Y., Jankovic, J., and Appel, S.H. 1999b. Reduced Nurr1 expression increases the vulnerability of mesencephalic dopamine neurons to MPTP-induced injury. J. Neurochem. 73: 2218-2221.[Medline]
Le, W.D., Xu, P., Jankovic, J., Jiang, H., Appel, S.H., Smith, R.G., and Vassilatis, D.K. 2003. Mutations in NR4A2 associated with familial Parkinson disease. Nat. Genet. 3: 385-389.
Lehmann, J.M., Jong, L., Fanjul, A., Cameron, J.F., Lu, X.P., Haefner, P., Dawson, I., and Pfahl, M. 1992. Retinoids selective for retinoid X receptor response pathways. Science 258: 1944-1946.
Lemotte, P.K., Keidel, S., and Apfel, C.M. 1996. Phytanic acid is a retinoid X receptor ligand. Eur. J. Biochem. 236: 328-333.[Medline]
Levin, A.A., Sturzenbecker, L.J., Kazmer, S., Bosakowski, T., Huselton, C., Allenby, G., Speck, J., Kratzeisen, C., Rosenberger, M., Lovey, A., et al. 1992. 9-cis retinoic acid steroisomer binds and activates the nuclear receptor RXR . Nature 355: 359-361.[CrossRef][Medline]
Lin, L.F., Doherty, D.H., Lile, J.D., Bektesh, S., and Collins, F. 1993. GDNF: A glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science 260: 1130-1132.
Lin, T.N., Chen, J.J., Wang, S.J., Cheng, J.T., Chi, S.I., Shyu, A.B., Sun, G.Y., and Hsu, C.Y. 1996. Expression of NGFI-B mRNA in a rat focal cerebral ischemia-reperfusion model. Brain Res. Mol. Brain Res. 43: 149-156.[Medline]
Lu, H.C., Eichele, G., and Thaller, C. 1997. Ligand-bound RXR can mediate retinoid signal transduction during embryogenesis. Development 124: 195-203.[Abstract]
Mangelsdorf, D.J. and Evans, R.M. 1995. The RXR heterodimers and orphan receptors. Cell 83: 841-850.[CrossRef][Medline]
Mangelsdorf, D.J., Ong, E.S., Dyck, J.A., and Evans, R.M. 1990. Nuclear receptor that identifies a novel retinoic acid response pathway. Nature 345: 224-229.[CrossRef][Medline]
Mangelsdorf, D.J., Borgmeyer, U., Heyman, R.A., Zhou, J.Y., Ong, E.S., Oro, A.E., Kakizuka, A., and Evans, R.M. 1992. Characterization of three RXR genes that mediate the action of 9-cis retinoic acid. Genes & Dev. 6: 329-344.
Mascrez, B., Mark, M., Dierich, A., Ghyselinck, N.B., Kastner, P., and Chambon, P. 1998. The RXR ligand-dependent activation function 2 (AF-2) is important for mouse development. Development 125: 4691-4707.[Abstract]
Mata de Urquiza, A., Solomin, L., and Perlmann, T. 1999. Feedback-inducible nuclear-receptor-driven reporter gene expression in transgenic mice. Proc. Natl. Acad. Sci. 96: 13270-13275.
Mata de Urquiza, A., Liu, S., Sjöberg, M., Zetterström, R.H., Griffiths, W., Sjövall, J., and Perlmann, T. 2000. Docosahexaenoic acid: A ligand for the retinoid X receptor in the mouse brain. Science 290: 2140-2144.
Neumann-Haefelin, T., Wiessner, C., Vogel, P., Back, T., and Hossmann, K.A. 1994. Differential expression of the immediate early genes c-fos, c-jun, junB, and NGFI-B in the rat brain following transient forebrain ischemia. J. Cereb. Blood Flow Metab. 14: 206-216.[Medline]
Neuringer, M., Anderson, G.J., and Connor, W.E. 1988. The essentiality of n - 3 fatty acids for the development and function of the retina and brain. Annu. Rev. Nutr. 8: 517-541.[CrossRef][Medline]
Perlmann, T. and Jansson, L. 1995. A novel pathway for vitamin A signaling mediated by RXR heterodimerization with NGFI-B and NURR1. Genes & Dev. 9: 769-782.
Perlmann, T., Rangarajan, P.N., Umesono, K., and Evans, R.M. 1993. Determinants for selective RAR and TR recognition of direct repeat HREs. Genes & Dev. 7: 1411-142
