Research

NR4A2 Association with Parkinson's Disease

Parkinson's disease (PD) is a common neurodegenerative movement disorder, with prevalence that increases with age and afflicts 1% of the population at the age of 65. PD patients exhibit classical clinical symptoms including bradykinesia, resting tremor, rigidity, and postural instability. The major motor disabilities of PD are associated with the extensive loss of dopaminergic neurons in the substantia nigra. The physiological changes and biochemical pathways involved in the selective demise of these neurons are still unclear. Epidemiologic and genetic data indicate that multiple pathways may be involved in predisposing or causing PD (Fahn 2003).

A growing body of evidence indicates that in many cases PD is caused by genetic defects or by environmental insults. Genetic factors that have been linked to familial PD include a-synuclein, Parkin, DJ-1, MAPT, UCHL1, PINK LRRK2 and several other undefined genes (Dawson, 2003). Accumulation and aggregation of a-synuclein may contribute to the death of dopamine neurons through impairments in protein handling and detoxification. Dysfunction of parkin and UCHL1 contribute to these deficits. Strategies aimed at reducing oxidative stress and α-synuclein aggregation, and enhancing protein degradation may hold particular promise as powerful neuroprotective agents in the treatment of PD (Giasson, 2003).

Taking a different point of view we suggested that genes that regulate the development and maintenance of midbrain dopaminergic neurons could be associated with PD. Several studies have indicated that NR4A2 (NURR1), an orphan nuclear receptor, may be a potential susceptibility gene for PD: NR4A2 is highly expressed in the midbrain dopaminergic neurons and NR4A2 elimination from mice results in selective agenesis of mesencephalic dopaminergic neurons. NR4A2 +/- mice show greater susceptibility to nigral injury induced by the dopaminergic neurotoxin MPTP (Zetterstrom, 1997; Saucedo-Cardenas, 1998; Le, 1999). Endogenous ligands that regulate RXR function as well as synthetic ligands that activate NR4A2-RXR heterodimers have been shown to mediate RXR ligand-induced signaling in neuronal cells. These RXR ligands require the presence of NR4A2 and increase the number of surviving dopaminergic cells and other neurons (Wallen-Mackenzie 2003). NR4A2 can activate the expression of tyrosine hydroxylase (TH), an enzyme required for dopamine biosynthesis, and enhance transcription of the dopamine transporter (DAT).

To determine whether NR4A2 is a susceptibility gene for PD, we carried out genetic analyses in 201 individuals affected with PD and 221 age-matched unaffected controls. We identified two mutations (-291Tdel and -245T->G) in NR4A2 associated with PD. The mutations map to the first untranslated exon of NR4A2 and affect one allele in 10 of 107 individuals with familial PD but not in any individuals with sporadic PD (n = 94) or in unaffected controls (n = 221) (Figure1) (Le 2003). The affected individuals were of European descent and had a clear family history of Parkinson disease involving at least two generations. The onset of disease in the affected individuals ranged from 45 to 67 years with a mean of 54 7 years. The clinical features of these individuals were not different from those of individuals with typical Parkinson's disease.

Figure 1
A. Heteroduplex analysis of a pedigree available members with familial Parkinson disease. Filled symbols represent affected individuals, who also showed abnormal heteroduplex bands.
B. Sequence analysis of the first exon of the NR4A2 mutations -291Tdel and -245T->G w, wild-type; m, mutated.

Because the mutations map to the untranslated first exon of the gene and do not affect the protein sequence we wanted to determine whether mutations in NR4A2 could cause PD. We established an assay to elucidate whether the -291Tdel and -245T->G mutations affected expression of NR4A2 and dopaminergic function. We incorporated the -291Tdel or -245T->G mutations into full-length NR4A2 sequence using PCR site-directed mutagenesis. We transfected expression vectors harboring the mutated or wild-type NR4A2 alleles into HEK293 or SHSY-5Y cell lines and measured the expression levels of the transcripts arising from the transfected genes by real-time PCR. The cells transfected with the mutated NR4A2 gene produced 87-95% less NR4A2 mRNA (Fig. 2A). Taking advantage of the fact that NR4A2 is expressed in human lymphocytes20, we measured the levels of NR4A2 mRNA in the lymphocytes of two individuals with Parkinson disease who had the -291Tdel mutation to determine whether the mutation could affect gene expression in vivo. We detected a significant reduction of NR4A2mRNA in these two individuals (Fig. 2B).To verify that the two mutations could affect downstream gene transcription, we measured the expression levels of the dopamine biosynthesis enzyme tyrosine hydroxylase in NR4A2-transfected SHSY-5Y cells, and found that the levels of TH mRNA were significantly lower in the cells transfected with -291Tdel or -245T->G mutated alleles than in those transfected with wild-type NR4A2 (Fig. 2C).

Figure 2
A. Real-time RT-PCR determination of NR4A2 mRNA levels in HEK293 or SHSY-5Y cells transfected with NR4A2: wild-type (lane 1), -291Tdel (lane 2), -245T->G (lane 3), wild-type + -291Tdel (lane 4), vector alone (lane 5) or without transfection (lane 6). Values represent the mean +/-s.d. of three experiments. *P < 0.01, **P < 0.005 and ***P < 0.001 versus wild-type (Student's t-test).
B. Relative NR4A2 mRNA levels determined by cycle threshold after normalization with GAPD. Orange line and lane 5, 54-yr-old proband; purple line and lane 1, proband's 52-yr-old unaffected sister (used as control); red line and lane 2, proband's 57-yr-old unaffected sister; yellow line and lane 3, proband's 82-yr-old unaffected father; green line and lane 4, proband's 84-yr-old mother affected with Parkinson disease. Error bars represent s.d.
C. Measurement of luciferase activity in the extracts of SHSY-5Y cells co-transfected with the luciferase reporter plasmid and NR4A2: wildtype (lane 1), -291Tdel (lane 2), -245T->G (lane 3) or vector alone (lane 4). Values represent the mean +/- s.d. of three experiments. *P < 0.01 versus wild-type (Student's t-test).

Current Projects:
    1.    Mechanism by which NR4A2 mutations affect mRNA levels.
    2.    Creation of Hellenic PD Genetic Database
    3.    Identification of neuroprotective gene network in dopaminergic cells.
    4.    Creation of conditional and reversable mouse models for neurotoxicity and neuroprotection.
    5.    Interaction of protein toxicity and neuroprotection.