Nuclear receptor Nurr1 agonists enhance its dual functions and improve behavioral deficits in an animal model of Parkinson's disease

Abstract

Parkinson's disease (PD), primarily caused by selective degeneration of midbrain dopamine (mDA) neurons, is the most prevalent movement disorder, affecting 1-2% of the global population over the age of 65. Currently available pharmacological treatments are largely symptomatic and lose their efficacy over time with accompanying severe side effects such as dyskinesia. Thus, there is an unmet clinical need to develop mechanism-based and/or disease-modifying treatments. Based on the unique dual role of the nuclear orphan receptor Nurr1 for development and maintenance of mDA neurons and their protection from inflammation-induced death, we hypothesize that Nurr1 can be a molecular target for neuroprotective therapeutic development for PD. Here we show successful identification of Nurr1 agonists sharing an identical chemical scaffold, 4-amino-7-chloroquinoline, suggesting a critical structure-activity relationship. In particular, we found that two antimalarial drugs, amodiaquine and chloroquine stimulate the transcriptional function of Nurr1 through physical interaction with its ligand binding domain (LBD). Remarkably, these compounds were able to enhance the contrasting dual functions of Nurr1 by further increasing transcriptional activation of mDA-specific genes and further enhancing transrepression of neurotoxic proinflammatory gene expression in microglia. Importantly, these compounds significantly improved behavioral deficits in 6-hydroxydopamine lesioned rat model of PD without any detectable signs of dyskinesia-like behavior. These findings offer proof of principle that small molecules targeting the Nurr1 LBD can be used as a mechanism-based and neuroprotective strategy for PD.

Keywords: NR4A2; Nurr1; Parkinson's disease; agonist; drug target.

Fig. 1.

Identification of AQ and CQ as Nurr1 activators. (A) Chemical structures of three hit compounds that activate Nurr1’s transcriptional function. Notably, all compounds contain an identical scaffold, 4-amino-7-chloroquinoline (highlighted by red color). (B) AQ, CQ, and glafenine increase the transcriptional activity of Nurr1-based reporter constructs: full-length Nurr1-dependent (fNurr; Left) and Nurr1 LBD-dependent (Right) transcriptional activities. (C) Effect of Nurr1-specific siRNA on Nurr1 LBD’s transactivation activity. A knockdown of Nurr1 expression by treatment with Nurr1-specific siRNA reduced the reporter gene activity of the Nurr1 LBD construct. (D) Effect of SRC proteins on AQ- and CQ-induced Nurr1 transactivation. AQ and CQ enhanced Nurr1’s transcriptional function in the presence of SRC-1/SRC-3 overexpression. The fold induction was derived by comparing each luciferase activity to basal level obtained by non-SRC transfection. (E) Target selectivity of AQ/CQ for LBDs of various NRs. A total of 30 μM AQ and 100 μM CQ robustly activates LBD function of Nurr1, but not other NRs tested here, indicating a high specificity. The positive reactivity of these NR constructs was confirmed by the known activators (2 nM dexamethasone, 20 nM retinoic acid, 2 μM GW3965, 50 nM GW7647, and 5 nM GW1929 for glucocorticoid receptor (GR), retinoid X receptor-α (RXRα), liver X receptor-α (LXRα), peroxisome proliferator-activated receptor-α (PPARα), and PPARγ, respectively). The basal level of transcriptional activity was normalized to 1. Bars represent means ± SEM from three independent experiments.

Fig. 2.

Interaction of AQ/CQ with the Nurr1-LBD protein. (A) Nurr1-LBD protein was incubated with increasing concentrations (3.9, 7.8, 15.5, 31, 62.5, 125, 250, 500, and 1,000 nM) of [³H]-CQ. The Inset indicates Scatchard analysis of the specific binding. (B) Competition of AQ, CQ, and primaquine (PQ) for binding of [³H]-CQ to Nurr1-LBD. Increasing concentrations of unlabeled AQ, CQ, or PQ were incubated with 500,nM [³H]-CQ and Nurr1-LBD. (C) Molecular interaction of the Nurr1-LBD and AQ by NMR titration experiments using uniformly ¹⁵N-labeled Nurr1-LBD. The 2D ¹H-¹⁵N TROSY-HSQC spectra of Nurr1-LBD were recorded on a Bruker Avance 700 spectrometer at 298K in the absence (red) and presence of AQ at molar ratios of 1–1 (magenta), 1–2 (black), and 1–5 (blue). Expanded sections of overlaid 2D ¹H-¹⁵N TROSY-HSQC spectra show concentration-dependent chemical shift perturbations upon AQ binding. Amino acids showing chemical shift perturbations with increasing concentration of AQ are indicated by arrows. Disappeared resonance of I403 by addition of AQ is marked as rectangular box. (D) Mapping of the interaction sites between Nurr1-LBD and AQ. (Left) Surface mapping of AQ binding site and interaction residues on the crystal structure of Nurr1-LBD based on 2D ¹H-¹⁵N HSQC titration data. Perturbed amino acid residues were displayed according to their chemical shift perturbation values: red (Δδ > 0.1), blue (0.08 < Δδ < 0.1), and green (disappeared), respectively. (Right) Expanded view of potential binding pocket for amodiaquine on the Nurr1-LBD. Perturbed amino acid residues were displayed by the same manner on the Right. (E) Functional effects of mutations in the potential AQ binding residues on Nurr1’s transcriptional activity. Wild-type and mutant constructs were tested by transient transfection assay with or without AQ. The mutations at I403, L409, Y575, or D580 significantly reduced both basal transcriptional activity and its activation by AQ.

Fig. 3.

Functional effects of AQ and CQ. (A and B) AQ stimulated the generation and gene expression of DA neurons from neural progenitors isolated from E14.5 rat cortex in a dose-dependent manner. Differentiation was induced by withdrawal of bFGF and AQ was added for 2 h during the differentiation. Immunocytochemical analyses for TH (A) and yields of TH⁺/DAPI cells (B) for each treatment group were obtained following in vitro differentiation for 3 d and 9 d. (C) Real-time PCR analysis shows that AQ treatment enhances expression of the mDA-specific genes TH, dopamine transporter (DAT), vesicular monoamine transporter (VMAT), and aromatic amino acid decarboxylase (AADC) during in vitro differentiation of neural stem cells at 9 d. (D) Rat PC12 cells were treated with 20 μM AQ or 70 μM CQ. ChIP assay shows AQ- or CQ-dependent Nurr1 recruitment to the TH promoter. *P < 0.05 and **P < 0.005 versus untreated. Results are expressed as the average of three independent experiments. Error bars represent SDs.

Fig. 4.

Neuroprotective effects of AQ and CQ. (A–C) Primary cultures of rat mesencephalic DA neurons were treated with 20 μM 6-OHDA for 24 h in the presence or absence of 5 μM AQ and 20 μM CQ. (A) The number of TH⁺ neurons and (B) the rate of [³H]DA uptake were measured. (C) Cell survival was measured by the MTT reduction assay in PC12 cells treated with 6-OHDA alone or in combination with AQ. Values from each treatment expressed as a percentage of untreated control for the MTT assay. (D) AQ suppresses LPS-induced expression of proinflammatory cytokines. Primary microglia from P1 rat brains were treated with 10 ng/mL LPS for 4 h in the presence or absence of AQ (10, 15, and 20 μM). Levels of mRNA expression were analyzed by quantitative real-time PCR and normalized with GAPDH. This experiment has been repeated three times in triplicate using independently prepared RNAs. Each bar represents means ± SEM of n = 4–5. *P < 0.05, **P < 0.01,***P < 0.001, compared with the LPS-only–treated group.

Fig. 5.

Effects of AQ treatments on a 6-OHDA–lesioned rat model of PD. (A) Schematic representation of the administration of AQ to 6-OHDA–lesioned rats. Unilateral striatal 6-OHDA–lesioned rats were treated with AQ or saline for 2 wk, starting from 1 d before lesioning. The gray shade indicates l-DOPA treatment for 2 wk as control of AIMs test. (B) Amphetamine-induced rotational test was performed at 4 and 6 wk post–6-OHDA lesion. (C) 6-OHDA–lesioned rats treated with l-DOPA, but not with AQ, exhibited severe side effects, as measured by AIMs scores. Four types of AIMs were monitored (axial, forelimb, orolingual, and locomotor) at 2 and 6 wk postlesioning. Each AIM behavior was monitored and scored on a 0–4 scale and summed. Bars represent the mean + SEM (^#P < 0.06, *P < 0.01, ***P < 0.0001).

Fig. 6.

Immunocytochemical analysis of 6-OHDA–lesioned rat administrated by AQ. (A) TH⁺ fibers and neurons were plentiful in the STR and SN of normal site, whereas their marked depletion was observed in the right STR and SN by 6-OHDA lesioning. In contrast, abundant TH-immunopositive cells were spared not only in the striatum but also in the SN in the AQ-treated group. [Scale bar, 200 μm (black), 100 μm (white), 20 μm (red).] (B–E) AQ reduces microglial activation in 6-OHDA–injected rat brains. Brain sections including the SN (B and D) and STR (C and E) regions were immunostained with anti–Iba-1 antibody, and Iba-1⁺ cells were counted in both lesion and intact sides. Data represent the mean ± SEM (**P < 0.01, ***P < 0.001). (Scale bar, 200 μm.)