ABSTRACT
OBJECTIVE: We performed a mutation screen of NR4A2 (also known as NURR1) in 409 Parkinson's disease (PD) patients. We identified a novel single base substitution in the 5'UTR of the NR4A2 (also known as NURR1) gene (c.-309C>T). RESULTS: We have performed expression studies in neuronal cell lines showing that the c.-309C>T mutation reduces NR4A2 mRNA expression in vitro. We have confirmed this finding in vivo by performing allele specific real-time PCR from brain tissue harbouring the 309C>T mutation and show a 3.48+/-1.62 fold reduction in mRNA expression of the mutant allele compared to wild-type. In addition we have undertaken genome wide expression analysis of the mutant NR4A2 brain and shown underexpressed genes were significantly enriched for gene ontology categories in nervous system development and synaptic transmission and overexpressed genes were enriched for unfolded protein response and morphogenesis. Lastly we have shown that the c.-309C>T mutation abrogates the protective effect of wild-type NR4A2 against apoptopic stress. CONCLUSIONS: Our findings indicate the c.-309C>T mutation reduces NR4A2 expression resulting in the downregulation of genes involved in the development and maintenance of the nervous system and synaptic transmission. These downregulated pathways contained genes known to be transactivated by NR4A2 and were not disrupted in idiopathic PD brain suggesting causality of the mutation.
Subject(s)
Brain/physiology , DNA-Binding Proteins/genetics , Genetic Predisposition to Disease/genetics , Parkinson Disease/genetics , Transcription Factors/genetics , Cell Line , DNA Mutational Analysis , Gene Expression , Gene Expression Profiling , Humans , Mutation , Neurons/physiology , Nuclear Receptor Subfamily 4, Group A, Member 2 , Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The mesocorticolimbic system is the reward centre of the brain and the major target for drugs of abuse including alcohol. Neuroadaptive changes in this region are thought to underlie the process of tolerance and dependence. Recently, several research groups have searched for alcohol-responsive genes using high-throughput microarrays and well-characterized human post-mortem material. Comparison of data from these studies of cortical regions highlights the differences in experimental approach and selection of cases. However, alcohol-responsive gene sets associated with transcription, oxidative stress and energy production were common to these studies. In marked contrast, alcohol-responsive genes in the nucleus accumbens and the ventral tegmental area are primarily associated with changes in neurotransmission and signal transduction. These data support the concept that, within cortical regions, changes in gene expression are associated with alcoholism-related pathology. In the dopaminergic tract of the mesocorticolimbic system, alcohol-responsive gene sets suggest long-term neuroplastic changes in synaptic transmission.
Subject(s)
Alcoholism/genetics , Alcoholism/metabolism , Cerebral Cortex/metabolism , Gene Expression Profiling , Nerve Tissue Proteins/metabolism , Functional Laterality/genetics , Functional Laterality/physiology , Gene Expression , Humans , Limbic System/metabolism , Motor Cortex/metabolism , Nerve Tissue Proteins/genetics , Oligonucleotide Array Sequence Analysis , Prefrontal Cortex/metabolism , Temporal Lobe/metabolismABSTRACT
Polymerase chain reaction (PCR)-based differential display was used to screen for alterations in gene expression in the mesolimbic system of the human alcoholic brain. Total RNA was extracted from the nucleus accumbens of five alcoholic and five control brains. A selected subpopulation of mRNA was reverse-transcribed to cDNA and amplified by PCR. A differentially expressed cDNA fragment was recovered, cloned, and sequenced. Full sequence analysis of this 467 bp fragment revealed 98.2% homology with the human mitochondrial 12S rRNA gene. Dot-blot analysis showed increased expression of this gene in nucleus accumbens and hippocampus, but not in the superior frontal cortex, primary motor cortex, caudate, and pallidus/putamen in a total of eight human alcoholic brains, compared with seven control brains. A similar increased expression was observed by dot-blot analysis, using RNA from the cerebral cortex of rats chronically treated with alcohol vapor. Hybridization of a 16S rRNA oligonucleotide probe indicated that the expression of both rRNAs genes was significantly increased in nucleus accumbens. These results indicate that chronic alcohol consumption induces alteration in expression of mitochondrial genes in selected brain regions. The altered gene expression may reflect mitochondrial dysfunction in the alcohol-affected brain.
