Against this backdrop we set
Against this backdrop, we set out to identify synthetic and endogenous ligands that bind directly to and regulate the activity of Nurr1. Owing to the pivotal role Nurr1 plays in producing and processing dopamine, and the need for neurons to tightly regulate dopamine levels, we postulated that the receptor might be regulated by dopamine, its biosynthetic precursor L-DOPA, or metabolites of these molecules (Figure 1B). Outside of an acidic environment (e.g., synaptic vesicle), dopamine is unstable (Segura-Aguilar et al., 2014). Following release into the synaptic cleft, excess dopamine is rapidly taken back up into the nerve terminal, traveling through the dopamine transporter, and then repacked into synaptic vesicles, via the vesicular monoamine transporter. Dopamine that is not processed in this way is typically either enzymatically (COMT, MAO) converted to inactive (but oxidatively unstable) metabolites or auto-oxidized into reactive species, including 5,6-dihydroxyindole (DHI) and 5,6-indolequinone (IQ) (Figure 1B), which are the focus of this work (Meiser et al., 2013). The reactive compounds polymerize to form neuromelanin, a chromogenic pigment of uncertain function that accumulates in and stains midbrain dopaminergic neurons black in healthy individuals (Fedorow et al., 2006, Zucca et al., 2017), or are otherwise quenched by direct conjugation to scavenging small molecules (e.g., glutathione) or protein thiols (Sulzer and Zecca, 2000). Using biophysical, structural, and biological assays, we evaluated the interaction of Nurr1 with oxidative metabolites of L-DOPA and dopamine. We found that DHI binds directly to Nurr1 in a non-canonical ligand-binding pocket, forming a covalent adduct by reacting as the IQ with an endogenous cysteine residue (Cys566). In both cultured ML-099 synthesis and zebrafish, DHI stimulates Nurr1 transcription, including upregulating the target gene underlying the management of excess cytoplasmic dopamine (i.e., VMAT2).
Discussion Considerable evidence suggests that dysregulation of dopamine is both a contributor to and consequence of PD (Burbulla et al., 2017, Hastings, 2009, Jenner, 2003, Lotharius and Brundin, 2002, Sulzer et al., 2000). Metabolism of dopamine produces reactive oxygen species (ROS) and quinones, and the formation of these toxins is exacerbated by excessive levels of cytoplasmic dopamine (VMAT2 dysfunction), increased levels of ROS (mitochondrial dysfunction), and other forms of oxidative stress—all conditions associated with PD. The transcriptional regulator Nurr1 plays a pivotal role in maintaining dopamine homeostasis, regulating the synthesis, packaging, and reuptake of the neurotransmitter. The regulation of Nurr1 itself is incompletely understood, however, partly owing to the absence of a well-defined ligand-binding pocket within the receptor. Delineating a binding site for small molecules within Nurr1 is a critical step toward understanding this receptor\'s role in and potential effect on PD. In this study, we used biophysical and structural assays to identify a binding site for a specific dopamine metabolite within the Nurr1 LBD. We found that DHI binds to Nurr1 within a non-canonical binding site, forming a covalent adduct as the IQ with Cys566. The interaction with DHI/IQ is detectable by SPR in the high-nanomolar range (>0.25 μM), a concentration consistent with sensing cytoplasmic dopamine under conditions of oxidative stress (Eisenhofer et al., 2004, Mosharov et al., 2003, Mosharov et al., 2006, Olefirowicz and Ewing, 1990, Omiatek et al., 2013, Pifl et al., 2014), and starts to show evidence of saturable binding at low-micromolar concentrations (2.5 μM, Figure 3). In functional assays, DHI stimulates Nurr1 activity, driving the transcription of genes controlling dopamine homeostasis. These data suggest that Nurr1 functions as a sensor for oxidative stress in dopaminergic neurons, responding directly to a specific oxidative metabolite of dopamine.