The nuclear receptor Nurr1 is preferentially expressed in human pro-inflammatory macrophages and limits their inflammatory profile.

Nurr1 is a member of the orphan nuclear receptor family NR4A (nuclear receptor subfamily 4 group A) that modulates inflammation in several cell lineages, both positively and negatively. Macrophages are key regulators of inflammatory responses, yet information about the role of Nurr1 in human macrophages is scarce. Here we examined Nurr1 expression and activity in steady state and activated human macrophages. Pro- and anti-inflammatory macrophages were generated in vitro by culture of blood monocytes with granulocyte/macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), respectively. Nurr1 expression was predominant in macrophages with the pro-inflammatory phenotype. Nurr1 activation with the agonists 1,1-bis(3'-indolyl)-1-(p-chlorophenyl) methane (C-DIM12) or isoxazolo-pyridinone 7e (IP7e) did not globally modify the polarization status of pro-inflammatory macrophages, but they decreased their production of TNF, IL-1ß, IL-6, IL-8, IL-12 p40, CCL2, IFN-ß, and reactive oxygen species, with variable potencies. Conversely, Nurr1 deficient macrophages increased the expression of transcripts encoding inflammatory mediators, particularly that of IL6, IFNB1, and CCL2. Mechanistically, endogenous Nurr1 interacted with NF-κB p65 in basal conditions and upon lipopolysaccharide (LPS)-mediated activation. C-DIM12 stabilized those complexes in cells exposed to LPS and concurrently decreased NF-κB transcriptional activity and p65 nuclear translocation. Expression of high levels of Nurr1 was associated with a subset of dermal macrophages that display enhanced levels of TNF and lower expression of the anti-inflammatory marker CD163L1 in skin lesions from patients with bullous pemphigoid (BP), a chronic inflammatory autoimmune blistering disorder. These results suggest that Nurr1 expression is linked with the pro-inflammatory phenotype of human macrophages, both in vivo and in vitro, where it may constitute a brake to attenuate the synthesis of inflammatory mediators.

Integrative analysis reveals structural basis for transcription activation of Nurr1 and Nurr1-RXRα heterodimer.

Orphan nuclear receptor Nurr1 plays important roles in the progression of various diseases, including Parkinson's disease, neuroinflammation, Alzheimer's disease, and multiple sclerosis. It can recognize DNA as a monomer or heterodimer with retinoid X receptor α (RXRα). But the molecular mechanism of its transcriptional activity regulation is still largely unknown. Here we obtained a crystal structure of monomer Nurr1 (DNA- and ligand-binding domains, DBD and LBD) bound to NGFI-B response element. The structure exhibited two different forms with distinct DBD orientations, unveiling the conformational flexibility of nuclear receptor monomer. We then generated an integrative model of Nurr1-RXRα heterodimer. In the context of heterodimer, the structural flexibility of Nurr1 would contribute to its transcriptional activity modulation. We demonstrated that the DNA sequence may specifically modulate the transcriptional activity of Nurr1 in the absence of RXRα agonist, but the modulation can be superseded when the agonist binds to RXRα. Together, we propose a set of signaling pathways for the constitutive transcriptional activation of Nurr1 and provide molecular mechanisms for therapeutic discovery targeting Nurr1 and Nurr1-RXRα heterodimer.

NURR1 expression regulates retinal pigment epithelial-mesenchymal transition and age-related macular degeneration phenotypes.

Phenotypic variations in the retinal pigment epithelial (RPE) layer are often a predecessor and driver of ocular degenerative diseases, such as age-related macular degeneration (AMD), the leading cause of vision loss in the elderly. We previously identified the orphan nuclear receptor-related 1 (NURR1), from a nuclear receptor atlas of human RPE cells, as a candidate transcription factor potentially involved in AMD development and progression. In the present study we characterized the expression of NURR1 as a function of age in RPE cells harvested from human donor eyes and in donor tissue from AMD patients. Mechanistically, we found an age-dependent shift in NURR1 dimerization from NURR1-RXRα heterodimers toward NURR1-NURR1 homodimers in primary human RPE cells. Additionally, overexpression and activation of NURR1 attenuated TNF-α-induced epithelial-to-mesenchymal transition (EMT) and migration, and modulated EMT-associated gene and protein expression in human RPE cells independent of age. In vivo, oral administration of IP7e, a potent NURR1 activator, ameliorated EMT in an experimental model of wet AMD and improved retinal function in a mouse model that presents with dry AMD features, impacting AMD phenotype, structure, and function of RPE cells, inhibiting accumulation of immune cells, and diminishing lipid accumulation. These results provide insight into the mechanisms of action of NURR1 in the aging eye, and demonstrate that the relative expression levels and activity of NURR1 is critical for both physiological and pathological functions of human RPE cells through RXRα-dependent regulation, and that targeting NURR1 may have therapeutic potential for AMD by modulating EMT, inflammation, and lipid homeostasis.

Temporal origin of mouse claustrum and development of its cortical projections.

The claustrum is known for its extensive connectivity with many other forebrain regions, but its elongated shape and deep location have made further study difficult. We have sought to understand when mouse claustrum neurons are born, where they are located in developing brains, and when they develop their widespread connections to the cortex. We established that a well-characterized parvalbumin plexus, which identifies the claustrum in adults, is only present from postnatal day (P) 21. A myeloarchitectonic outline of the claustrum can be derived from a triangular fiber arrangement from P15. A dense patch of Nurr1+ cells is present at its core and is already evident at birth. Bromodeoxyuridine birth dating of forebrain progenitors reveals that the majority of claustrum neurons are born during a narrow time window centered on embryonic day 12.5, which is later than the adjacent subplate and endopiriform nucleus. Retrograde tracing revealed that claustrum projections to anterior cingulate (ACA) and retrosplenial cortex (RSP) follow distinct developmental trajectories. Claustrum-ACA connectivity matures rapidly and reaches adult-like innervation density by P10, whereas claustrum-RSP innervation emerges later over a protracted time window. This work establishes the timeline of claustrum development and provides a framework for understanding how the claustrum is built and develops its unique connectivity.

Albiflorin ameliorates thioacetamide-induced hepatic fibrosis: The involvement of NURR1-mediated inflammatory signaling cascades in hepatic stellate cells activation.

Thioacetamide (TAA) within the liver generates hepatotoxic metabolites that can be induce hepatic fibrosis, similar to the clinical pathological features of chronic human liver disease. The potential protective effect of Albiflorin (ALB), a monoterpenoid glycoside found in Paeonia lactiflora Pall, against hepatic fibrosis was investigated. The mouse hepatic fibrosis model was induced with an intraperitoneal injection of TAA. Hepatic stellate cells (HSCs) were subjected to treatment with transforming growth factor-beta (TGF-ß), while lipopolysaccharide/adenosine triphosphate (LPS/ATP) was added to stimulate mouse peritoneal macrophages (MPMs), leading to the acquisition of conditioned medium. For TAA-treated mice, ALB reduced ALT, AST, HYP levels in serum or liver. The administration of ALB reduced histopathological abnormalities, and significantly regulated the expressions of nuclear receptor-related 1 protein (NURR1) and the P2X purinoceptor 7 receptor (P2×7r) in liver. ALB could suppress HSCs epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) deposition, and pro-inflammatory factor level. ALB also remarkably up-regulated NURR1, inhibited P2×7r signaling pathway, and worked as working as C-DIM12, a NURR1 agonist. Moreover, deficiency of NURR1 in activated HSCs and Kupffer cells weakened the regulatory effect of ALB on P2×7r inhibition. NURR1-mediated inhibition of inflammatory contributed to the regulation of ALB ameliorates TAA-induced hepatic fibrosis, especially based on involving in the crosstalk of HSCs-macrophage. Therefore, ALB plays a significant part in the mitigation of TAA-induced hepatotoxicity this highlights the potential of ALB as a protective intervention for hepatic fibrosis.

A MED13-dependent skeletal muscle gene program controls systemic glucose homeostasis and hepatic metabolism.

The Mediator complex governs gene expression by linking upstream signaling pathways with the basal transcriptional machinery. However, how individual Mediator subunits may function in different tissues remains to be investigated. Through skeletal muscle-specific deletion of the Mediator subunit MED13 in mice, we discovered a gene regulatory mechanism by which skeletal muscle modulates the response of the liver to a high-fat diet. Skeletal muscle-specific deletion of MED13 in mice conferred resistance to hepatic steatosis by activating a metabolic gene program that enhances muscle glucose uptake and storage as glycogen. The consequent insulin-sensitizing effect within skeletal muscle lowered systemic glucose and insulin levels independently of weight gain and adiposity and prevented hepatic lipid accumulation. MED13 suppressed the expression of genes involved in glucose uptake and metabolism in skeletal muscle by inhibiting the nuclear receptor NURR1 and the MEF2 transcription factor. These findings reveal a fundamental molecular mechanism for the governance of glucose metabolism and the control of hepatic lipid accumulation by skeletal muscle. Intriguingly, MED13 exerts opposing metabolic actions in skeletal muscle and the heart, highlighting the customized, tissue-specific functions of the Mediator complex.

NR4A2 as a Novel Target Gene for Developmental and Epileptic Encephalopathy: A Systematic Review of Related Disorders and Therapeutic Strategies.

The NR4A2 gene encodes an orphan transcription factor of the steroid-thyroid hormone-retinoid receptor superfamily. This review focuses on the clinical findings associated with the pathogenic variants so far reported, including three unreported cases. Also, its role in neurodegenerative diseases, such as Parkinson's or Alzheimer's disease, is examined, as well as a brief exploration on recent proposals to develop novel therapies for these neurological diseases based on small molecules that could modulate NR4A2 transcriptional activity. The main characteristic shared by all patients is mild to severe developmental delay/intellectual disability. Moderate to severe disorder of the expressive and receptive language is present in at least 42%, while neuro-psychiatric issues were reported in 53% of patients. Movement disorders, including dystonia, chorea or ataxia, are described in 37% patients, although probably underestimated because of its frequent onset in late adolescence-young adulthood. Finally, epilepsy was surprisingly present in 42% of patients, being drug-resistant in three of them. The age at onset varied widely, from five months to twenty-six years, as did the classification of epilepsy, which ranged from focal epilepsy to infantile spasms or Lennox-Gastaut syndrome. Accordingly, we propose that NR4A2 should be considered as a first-tier target gene for the genetic diagnosis of developmental and epileptic encephalopathy.

Mechanistic insights into the role of vitamin D and computational identification of potential lead compounds for Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder that affects dopaminergic neurons in the midbrain. A recent study suggests that Orphan Nuclear Receptor 1 (NURR1) impairment may contribute to PD pathogenesis. Our study found three potent agonists for NURR1 protein based on structural and ligand-based screening methods. The pharmacophore is comprised of a hydrogen bond donor, a hydrophobic group, and two aromatic rings (DHRR). The Pharmacophore screening method screened 3142 compounds, of which 3 were screened using structure-based screening. An analysis of the molecules using Molecular Mechanics-Generalized Born Surface Area (binding free energy) revealed a range of -46.77 to -59.06 Kcal/mol. After that, chemical reactivity was investigated by density functional theory, and molecular dynamics simulation was performed (protein-ligand stability). Based on the computational studies, Lifechemical_16901310, Maybridge_2815310, and NPACT_392450 are promising agonists with respect to NURR1. To confirm the potency of the identified compounds, further validation and experiments must be conducted.

Timing of MeCP2 Expression Determines Midbrain Dopamine Neuron Phenotype Specification.

Midbrain dopamine (DA) neurons are associated with locomotor and psychiatric disorders. DA phenotype is specified in ancestral neural precursor cells (NPCs) and maintained throughout neuronal differentiation. Here we show that endogenous expression of MeCP2 coincides with DA phenotype specification in mouse mesencephalon, and premature expression of MeCP2 prevents in vitro cultured NPCs from acquiring DA phenotype through interfering NURR1 transactivation of DA phenotype genes. By contrast, ectopic MeCP2 expression does not disturb DA phenotype in the DA neurons. By analyzing the dynamic change of DNA methylation along DA neuronal differentiation at the promoter of DA phenotype gene tyrosine hydroxylase (Th), we show that Th expression is determined by TET1-mediated de-methylation of NURR1 binding sites within Th promoter. Chromatin immunoprecipitation assays demonstrate that premature MeCP2 dominates the DNA binding of the corresponding sites thereby blocking TET1 function in DA NPCs, whereas TET1-mediated de-methylation prevents excessive MeCP2 binding in DA neurons. The significance of temporal DNA methylation status is further confirmed by targeted methylation/demethylation experiments showing that targeted de-methylation in DA NPCs protects DA phenotype specification from ectopic MeCP2 expression, whereas targeted methylation disturbs phenotype maintenance in MeCP2-overexpressed DA neurons. These findings suggest the appropriate timing of MeCP2 expression as a novel determining factor for guiding NPCs into DA lineage.

Mechanisms of NURR1 Regulation: Consequences for Its Biological Activity and Involvement in Pathology.

NURR1 (Nuclear receptor-related 1 protein or NR4A2) is a nuclear protein receptor transcription factor with an essential role in the development, regulation, and maintenance of dopaminergic neurons and mediates the response to stressful stimuli during the perinatal period in mammalian brain development. The dysregulation of NURR1 activity may play a role in various diseases, including the onset and progression of neurodegenerative diseases, and several other pathologies. NURR1 is regulated by multiple mechanisms, among which phosphorylation by kinases or SUMOylation are the best characterized. Both post-translational modifications can regulate the activity of NURR1, affecting its stability and transcriptional activity. Other non-post-translational regulatory mechanisms include changes in its subcellular distribution or interaction with other protein partners by heterodimerization, also affecting its transcription activity. Here, we summarize the currently known regulatory mechanisms of NURR1 and provide a brief overview of its participation in pathological alterations.

The Selective Agonist for Sphingosine-1-Phosphate Receptors Siponimod Increases the Expression Level of NR4A Genes in Microglia Cell Line.

Fingolimod (FTY720) and siponimod (BAF312) are selective agonists for sphingosine-1-phosphate (S1P) receptors approved for the treatment of relapsing-remitting (RR) and secondary progressive (SP) multiple sclerosis (MS), respectively. BAF312 exerts pro-myelination and neuro-protective functions on CNS resident cells, although the underlying molecular mechanism is not yet fully understood. NR4A2 is an anti-inflammatory gene, belonging to the NR4A family, whose expression is reduced in blood from treatment-naïve patients with RRMS, but is restored in patients treated with FTY720 for more than two years. We performed an in vitro study to investigate the potential involvement of the NR4A genes in the protective and restorative effects of BAF312. We showed that BAF312 enhances the expression of NR4A1 and NR4A2 in the N9 microglial cell line, but has no effect in the peripheral blood mononuclear cells and oligodendrocytes. This study suggests a novel molecular mechanism of action for the selective agonists for S1P receptors within the CNS.

NURR1-deficient mice have age- and sex-specific behavioral phenotypes.

The transcription factor NURR1 is essential to the generation and maintenance of midbrain dopaminergic (mDA) neurons and its deregulation is involved in the development of dopamine (DA)-associated brain disorders, such as Parkinson's disease (PD). The old male NURR1 heterozygous knockout (NURR1-KO) mouse has been proposed as a model of PD due to its altered motor performance that was, however, not confirmed in a subsequent study. Based on these controversial results, we explored the effects of the NURR1 deficiency on locomotor activity, motor coordination, brain and plasma DA levels, blood pressure and heart rate of old mice, also focusing on the potential effect of sex. As a probable consequence of the role of NURR1 in DA pathway, we observed that the old NURR1-KO mouse is characterized by motor impairment, and increased brain DA level and heart rate, independently from sex. However, we also observed an alteration in spontaneous locomotor activity that only affects males. In conclusion, NURR1 deficiency triggers sex- and age-specific alterations of behavioral responses, of DA levels and cardiovascular abnormalities. Further studies in simplified systems will be necessary to dissect the mechanism underlying these observations.

CB2 receptor activation inhibits the phagocytic function of microglia through activating ERK/AKT-Nurr1 signal pathways.

Neuroinflammation is closely related to the pathogenesis of neurodegenerative diseases. Activation of microglia, the resident immune cells in CNS, induces inflammatory responses, resulting in the release of neurotoxic molecules, which favors neuronal death and neurodegeneration. Nuclear receptor-related 1 (Nurr1) protein, one of the orphan nuclear receptor superfamilies, is an emerging target for neuroprotective therapy. In addition, the anti-inflammatory function of cannabinoid (CB) receptors has attracted increasing interest. As both CB receptors (especially CB2 receptor) and Nurr1 exist in microglia, and regulate a number of same molecular points such as NF-κB, we herein explored the interplay between the CB2 receptor and Nurr1 as well as the regulatory mechanisms in microglial cells. We showed that the application of CB2 receptor agonists JWH015 (1, 10 µM) significantly increased the nuclear Nurr1 protein in BV-2 cells and primary midbrain microglia. Overexpression of Nurr1 or application of Nurr1 agonist C-DIM12 (10 µM) significantly increased the mRNA level of CB2 receptor in BV-2 cells, suggesting that positive expression feedback existing between the CB2 receptor and Nurr1. After 2-AG and JWH015 activated the CB2 receptors, the levels of p-ERK, p-AKT, p-GSK-3ß in BV-2 cells were significantly increased. Using ERK1/2 inhibitor U0126 and PI3K/AKT inhibitor LY294002, we revealed that the amount of Nurr1 in the nucleus was upregulated through ß-arrestin2/ERK1/2 and PI3K/AKT/GSK-3ß signaling pathways. With these inhibitors, we found a cross-talk interaction between the two pathways, and the ERK1/2 signaling pathway played a more dominant regulatory role. Furthermore, we demonstrated that when the CB2 receptor was activated, the phagocytic function of BV-2 cells was significantly weakened; the activation of Nurr1 also inhibited the phagocytic function of BV-2 cells. Pretreatment with the signaling pathway inhibitors, especially U0126, reversed the inhibitory effect of 2-AG on phagocytosis, suggesting that CB2 receptor may regulate the phagocytic function of microglia by activating Nurr1. In conclusion, CB2 receptor or/and Nurr1-mediated signal pathways play instrumental roles in the progress of phagocytosis, which are expected to open up new treatment strategies for neurodegenerative diseases.

Intranasal infusion of GD3 and GM1 gangliosides downregulates alpha-synuclein and controls tyrosine hydroxylase gene in a PD model mouse.

Parkinson's disease (PD) is characterized by Lewy bodies (composed predominantly of alpha-synuclein [aSyn]) and loss of pigmented midbrain dopaminergic neurons comprising the nigrostriatal pathway. Most PD patients show significant deficiency of gangliosides, including GM1, in the brain, and GM1 ganglioside appears to keep dopaminergic neurons functioning properly. Thus, supplementation of GM1 could potentially provide some rescuing effects. In this study, we demonstrate that intranasal infusion of GD3 and GM1 gangliosides reduces intracellular aSyn levels. GM1 also significantly enhances expression of tyrosine hydroxylase (TH) in the substantia nigra pars compacta of the A53T aSyn overexpressing mouse, following restored nuclear expression of nuclear receptor related 1 (Nurr1, also known as NR4A2), an essential transcription factor for differentiation, maturation, and maintenance of midbrain dopaminergic neurons. GM1 induces epigenetic activation of the TH gene, including augmentation of acetylated histones and recruitment of Nurr1 to the TH promoter region. Our data indicate that intranasal administration of gangliosides could reduce neurotoxic proteins and restore functional neurons via modulating chromatin status by nuclear gangliosides.

A Proposed TUSC7/miR-211/Nurr1 ceRNET Might Potentially be Disturbed by a cer-SNP rs2615499 in Breast Cancer.

Evidence and in silico analyses showed that TUSC7, miR-211, and Nurr1 may be involved in BC pathogenesis by ceRNET signaling axis. This study aimed to investigate the potential role of TUSC7/miR-211/Nurr1 ceRNET and rs2615499 variant as a novel cer-SNP in BC subjects. The expression assays were conducted by qPCR on tumor tissues (n = 50), tumor-adjacent normal tissues (TANTs) (n = 50), and clinically healthy control tissues (n = 50). The expression of TUSC7 and Nurr1 significantly decreased, but the level of miR-211 significantly increased in tumor tissues compared to TANTs and healthy normal tissues. Altered expression of TUSC7 and miR-211 was associated with poor prognosis of patients. The Nurr1 exhibited a double-edged sword-like activity in BC. In addition, TUSC7, Nurr1, and miR-211 expressions were significantly related to a novel BC-associated rs2615499 (A > C) located in the miR-211 binding site on Nurr1 3'-UTR. In the second part of the study, a case-control study was performed on BC patients (n = 100) and matched healthy controls (n = 100). The genomic DNA was isolated and genotyping was performed using Tetra-Primer ARMS PCR. The CC and AC genotypes were associated with higher expression levels of Nurr1 and worse outcomes of the disease. Our findings revealed that TUSC7 functions as a tumor suppressor in BC potentially via miR-211/Nurr1, which might be disturbed by the cer-SNP rs2615499. However, functional studies are needed to validate these results.

Lmx1a-Dependent Activation of miR-204/211 Controls the Timing of Nurr1-Mediated Dopaminergic Differentiation.

The development of midbrain dopaminergic (DA) neurons requires a fine temporal and spatial regulation of a very specific gene expression program. Here, we report that during mouse brain development, the microRNA (miR-) 204/211 is present at a high level in a subset of DA precursors expressing the transcription factor Lmx1a, an early determinant for DA-commitment, but not in more mature neurons expressing Th or Pitx3. By combining different in vitro model systems of DA differentiation, we show that the levels of Lmx1a influence the expression of miR-204/211. Using published transcriptomic data, we found a significant enrichment of miR-204/211 target genes in midbrain dopaminergic neurons where Lmx1a was selectively deleted at embryonic stages. We further demonstrated that miR-204/211 controls the timing of the DA differentiation by directly downregulating the expression of Nurr1, a late DA differentiation master gene. Thus, our data indicate the Lmx1a-miR-204/211-Nurr1 axis as a key component in the cascade of events that ultimately lead to mature midbrain dopaminergic neurons differentiation and point to miR-204/211 as the molecular switch regulating the timing of Nurr1 expression.

Nurr1 Is Not an Essential Regulator of BDNF in Mouse Cortical Neurons.

Nurr1 and brain-derived neurotrophic factor (BDNF) play major roles in cognition. Nurr1 regulates BDNF in midbrain dopaminergic neurons and cerebellar granule cells. Nurr1 and BDNF are also highly expressed in the cerebral cortex, a brain area important in cognition. Due to Nurr1 and BDNF tissue specificity, the regulatory effect of Nurr1 on BDNF in different brain areas cannot be generalized. The relationship between Nurr1 and BDNF in the cortex has not been investigated previously. Therefore, we examined Nurr1-mediated BDNF regulation in cortical neurons in activity-dependent and activity-independent states. Mouse primary cortical neurons were treated with the Nurr1 agonist, amodiaquine (AQ). Membrane depolarization was induced by KCl or veratridine and reversed by nimodipine. AQ and membrane depolarization significantly increased Nurr1 (p < 0.001) and BDNF (pAQ < 0.001, pKCl < 0.01) as assessed by real-time qRT-PCR. However, Nurr1 knockdown did not affect BDNF gene expression in resting or depolarized neurons. Accordingly, the positive correlation between Nurr1 and BDNF expression in AQ and membrane depolarization experiments does not imply co-regulation because Nurr1 knockdown did not affect BDNF gene expression in resting or depolarized cortical neurons. Therefore, in contrast to midbrain dopaminergic neurons and cerebellar granule cells, Nurr1 does not regulate BDNF in cortical neurons.

YM750, an ACAT Inhibitor, Acts on Adrenocortical Cells to Inhibit Aldosterone Secretion Due to Depolarization.

Primary aldosteronism (PA) is considered the most common form of secondary hypertension, which is associated with excessive aldosterone secretion in the adrenal cortex. The cause of excessive aldosterone secretion is the induction of aldosterone synthase gene (CYP11B2) expression by depolarization of adrenocortical cells. In this study, we found that YM750, an Acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitor, acts on adrenocortical cells to suppress CYP11B2 gene expression and aldosterone secretion. YM750 inhibited the induction of CYP11B2 gene expression by KCl stimulation, but not by angiotensin II and forskolin stimulation. Interestingly, YM750 did not inhibit KCl-stimulated depolarization via an increase in intracellular calcium ion concentration. Moreover, ACAT1 expression was relatively abundant in the zona glomerulosa (ZG) including these CYP11B2-positive cells. Thus, YM750 suppresses CYP11B2 gene expression by suppressing intracellular signaling activated by depolarization. In addition, ACAT1 was suggested to play an important role in steroidogenesis in the ZG. YM750 suppresses CYP11B2 gene expression and aldosterone secretion in the adrenal cortex, suggesting that it may be a potential therapeutic agent for PA.

Transcriptional Regulation of the Synaptic Vesicle Protein Synaptogyrin-3 (SYNGR3) Gene: The Effects of NURR1 on Its Expression.

Synaptogyrin-3 (SYNGR3) is a synaptic vesicular membrane protein. Amongst four homologues (SYNGR1 to 4), SYNGR1 and 3 are especially abundant in the brain. SYNGR3 interacts with the dopamine transporter (DAT) to facilitate dopamine (DA) uptake and synaptic DA turnover in dopaminergic transmission. Perturbed SYNGR3 expression is observed in Parkinson's disease (PD). The regulatory elements which affect SYNGR3 expression are unknown. Nuclear-receptor-related-1 protein (NURR1) can regulate dopaminergic neuronal differentiation and maintenance via binding to NGFI-B response elements (NBRE). We explored whether NURR1 can regulate SYNGR3 expression using an in silico analysis of the 5'-flanking region of the human SYNGR3 gene, reporter gene activity and an electrophoretic mobility shift assay (EMSA) of potential cis-acting sites. In silico analysis of two genomic DNA segments (1870 bp 5'-flanking region and 1870 + 159 bp of first exon) revealed one X Core Promoter Element 1 (XCPE1), two SP1, and three potential non-canonical NBRE response elements (ncNBRE) but no CAAT or TATA box. The longer segment exhibited gene promoter activity in luciferase reporter assays. Site-directed mutagenesis of XCPE1 decreased promoter activity in human neuroblastoma SH-SY5Y (↓43.2%) and human embryonic kidney HEK293 cells (↓39.7%). EMSA demonstrated NURR1 binding to these three ncNBRE. Site-directed mutagenesis of these ncNBRE reduced promoter activity by 11-17% in SH-SY5Y (neuronal) but not in HEK293 (non-neuronal) cells. C-DIM12 (Nurr1 activator) increased SYNGR3 protein expression in SH-SY5Y cells and its promoter activity using a real-time luciferase assay. As perturbed vesicular function is a feature of major neurodegenerative diseases, inducing SYNGR3 expression by NURR1 activators may be a potential therapeutic target to attenuate synaptic dysfunction in PD.

Striatal Nurr1 Facilitates the Dyskinetic State and Exacerbates Levodopa-Induced Dyskinesia in a Rat Model of Parkinson's Disease.

The transcription factor Nurr1 has been identified to be ectopically induced in the striatum of rodents expressing l-DOPA-induced dyskinesia (LID). In the present study, we sought to characterize Nurr1 as a causative factor in LID expression. We used rAAV2/5 to overexpress Nurr1 or GFP in the parkinsonian striatum of LID-resistant Lewis or LID-prone Fischer-344 (F344) male rats. In a second cohort, rats received the Nurr1 agonist amodiaquine (AQ) together with l-DOPA or ropinirole. All rats received a chronic DA agonist and were evaluated for LID severity. Finally, we performed single-unit recordings and dendritic spine analyses on striatal medium spiny neurons (MSNs) in drug-naïve rAAV-injected male parkinsonian rats. rAAV-GFP injected LID-resistant hemi-parkinsonian Lewis rats displayed mild LID and no induction of striatal Nurr1 despite receiving a high dose of l-DOPA. However, Lewis rats overexpressing Nurr1 developed severe LID. Nurr1 agonism with AQ exacerbated LID in F344 rats. We additionally determined that in l-DOPA-naïve rats striatal rAAV-Nurr1 overexpression (1) increased cortically-evoked firing in a subpopulation of identified striatonigral MSNs, and (2) altered spine density and thin-spine morphology on striatal MSNs; both phenomena mimicking changes seen in dyskinetic rats. Finally, we provide postmortem evidence of Nurr1 expression in striatal neurons of l-DOPA-treated PD patients. Our data demonstrate that ectopic induction of striatal Nurr1 is capable of inducing LID behavior and associated neuropathology, even in resistant subjects. These data support a direct role of Nurr1 in aberrant neuronal plasticity and LID induction, providing a potential novel target for therapeutic development.SIGNIFICANCE STATEMENT The transcription factor Nurr1 is ectopically induced in striatal neurons of rats exhibiting levodopa-induced dyskinesia [LID; a side-effect to dopamine replacement strategies in Parkinson's disease (PD)]. Here we asked whether Nurr1 is causing LID. Indeed, rAAV-mediated expression of Nurr1 in striatal neurons was sufficient to overcome LID-resistance, and Nurr1 agonism exacerbated LID severity in dyskinetic rats. Moreover, we found that expression of Nurr1 in l-DOPA naïve hemi-parkinsonian rats resulted in the formation of morphologic and electrophysiological signatures of maladaptive neuronal plasticity; a phenomenon associated with LID. Finally, we determined that ectopic Nurr1 expression can be found in the putamen of l-DOPA-treated PD patients. These data suggest that striatal Nurr1 is an important mediator of the formation of LID.