Sharon K. Michelhaugh

The dopamine transporter gene (SLC6A3) variable number of tandem repeats domain enhances transcription in dopamine neurons.

The dopamine (DAT) and serotonin (SERT) transporter genes both contain variable number of tandem repeats (VNTR) in non‐coding gene regions which have been correlated with a predisposition to a variety of CNS disorders. There is considerable homology between individual DAT and SERT repeat DNA sequences, which is reflected in their ability to compete with each other for specific protein binding as demonstrated by electrophoretic mobility shift assay. The SERT VNTR has recently been shown to act as a transcriptional enhancer. Because of the similarities between SERT and DAT VNTRs, the DAT VNTR may also enhance transcription. This study demonstrates by lipid transfection into an immortalized dopaminergic cell line and biolistic transfection into dopamine neurons in neonatal rat midbrain slices that the human nine‐repeat DAT VNTR can enhance transcription. This enhancing activity suggests that the DAT VNTR may play a role in regulation of DAT gene expression.

The human dopamine transporter gene: gene organization, transcriptional regulation, and potential involvement in neuropsychiatric disorders

The dopamine transporter is a plasma membrane protein that controls the spatial and temporal domains of dopamine neurotransmission through the accumulation of extracellular dopamine. The dopamine transporter may play a role in numerous dopamine-linked neuropsychiatric disorders. We review the cloning and organization of the human dopamine transporter gene, polymorphisms in its coding and noncoding sequence, and emerging data on its transcriptional regulation.

Mining Affymetrix microarray data for long non-coding RNAs: altered expression in the nucleus accumbens of heroin abusers.

J. Neurochem. (2011) 116, 459–466.

SKCa channels mediate the medium but not the slow calcium-activated afterhyperpolarization in cortical neurons

Many neurons, including pyramidal cells of the cortex, express a slow afterhyperpolarization (sAHP) that regulates their firing. Although initial findings suggested that the current underlying the sAHP could be carried through SKCa channels, recent work has uncovered anomalies that are not congruent with this idea. Here, we used overexpression and dominant-negative strategies to assess the involvement of SKCa channels in mediating the current underlying the sAHP in pyramidal cells of the cerebral cortex. Pyramidal cells of layer V exhibit robust AHP currents composed of two kinetically and pharmacologically distinguishable currents known as the medium AHP current (ImAHP) and the slow AHP current (IsAHP). ImAHP is blocked by the SKCa channel blockers apamin and bicuculline, whereas IsAHP is resistant to these agents but is inhibited by activation of muscarinic receptors. To test for a role for SKCa channels, we overexpressed KCa2.1 (SK1) and KCa2.2 (SK2), the predominant SKCa subunits expressed in the cortex, in pyramidal cells of cultured brain slices. Overexpression of KCa2.1 and KCa2.2 resulted in a fourfold to fivefold increase in the amplitude of ImAHP but had no detectable effect on IsAHP. As an additional test, we examined IsAHP in a transgenic mouse expressing a truncated SKCa subunit (SK3-1B) capable of acting as a dominant negative for the entire family of SKCa–IKCa channels. Expression of SK3-1B profoundly inhibited ImAHP but again had no discernable effect on IsAHP. These results are inconsistent with the proposal that SKCa channels mediate IsAHP in pyramidal cells and indicate that a different potassium channel mediates this current.

Decreased expression of the transcription factor NURR1 in dopamine neurons of cocaine abusers

Chronic exposure to cocaine induces long-term adaptations that are likely to involve changes in transcription factor expression. This possibility has not been examined in the cocaine-exposed human brain. The transcription factor nurr1 is highly expressed in rodent midbrain dopamine neurons and is essential for their proper phenotypic development. Here we show that human NURR1 gene expression is robust within control subjects and reduced markedly within the dopamine neurons of human cocaine abusers. NURR1 is known to regulate transcription of the gene encoding the cocaine-sensitive dopamine transporter (DAT). We show here that DAT gene expression also is reduced markedly in the dopamine neurons of NURR1-deficient cocaine abusers, suggesting that NURR1 plays a critical role in vivo in controlling human DAT gene expression and adaptation to repeated exposure to cocaine.

Comparison of Amino Acid Positron Emission Tomographic Radiotracers for Molecular Imaging of Primary and Metastatic Brain Tumors

Positron emission tomography (PET) is an imaging technology that can detect and characterize tumors based on their molecular and biochemical properties, such as altered glucose, nucleoside, or amino acid metabolism. PET plays a significant role in the diagnosis, prognostication, and treatment of various cancers, including brain tumors. In this article, we compare uptake mechanisms and the clinical performance of the amino acid PET radiotracers (l-[methyl-11C]methionine [MET], 18F-fluoroethyl-tyrosine [FET], 18F-fluoro-l-dihydroxy-phenylalanine [FDOPA], and 11C-alpha-methyl-l-tryptophan [AMT]) most commonly used for brain tumor imaging. First, we discuss and compare the mechanisms of tumoral transport and accumulation, the basis of differential performance of these radioligands in clinical studies. Then we summarize studies that provided direct comparisons among these amino acid tracers and to clinically used 2-deoxy-2[18F]fluoro-d-glucose [FDG] PET imaging. We also discuss how tracer kinetic analysis can enhance the clinical information obtained from amino acid PET images. We discuss both similarities and differences in potential clinical value for each radioligand. This comparative review can guide which radiotracer to favor in future clinical trials aimed at defining the role of these molecular imaging modalities in the clinical management of brain tumor patients.Positron emission tomography (PET) is an imaging technology that can detect and characterize tumors based on their molecular and biochemical properties, such as altered glucose, nucleoside, or amino acid metabolism. PET plays a significant role in the diagnosis, prognostication, and treatment of various cancers, including brain tumors. In this article, we compare uptake mechanisms and the clinical performance of the amino acid PET radiotracers (L-[methyl-11C]methionine [MET], 18F-fluoroethyl-tyrosine [FET], 18F-fluoro-L- dihydroxy-phenylalanine [FDOPA], and 11C-alpha-methyl-L-tryptophan [AMT]) most commonly used for brain tumor imaging. First, we discuss and compare the mechanisms of tumoral transport and accumulation, the basis of differential performance of these radioligands in clinical studies. Then we summarize studies that provided direct comparisons among these amino acid tracers and to clinically used 2-deoxy-2[18F]fluoro-D-glucose [FDG] PET imaging. We also discuss how tracer kinetic analysis can enhance the clinical information obtained from amino acid PET images. We discuss both similarities and differences in potential clinical value for each radioligand. This comparative review can guide which radiotracer to favor in future clinical trials aimed at defining the role of these molecular imaging modalities in the clinical management of brain tumor patients.

Valproate robustly increases Sp transcription factor-mediated expression of the dopamine transporter gene within dopamine cells

Several lines of evidence suggest that valproate, a drug used in the treatment of mania and bipolar disorders, epilepsies, and addictions, may modulate dopamine transporter (DAT) function, yet the effects of valproate on DAT gene expression have not been directly assessed. Utilizing a human dopaminergic cell line and rat midbrain dopamine (DA) neurons in organotypic culture, we found that valproate increased endogenous DAT gene expression in a concentration‐ and time‐dependent manner. Given previous data demonstrating that members of the specificity protein (Sp) family of transcription factors are strong trans‐activators of DAT gene transcription, we investigated the Sp‐dependence of valproate effects. Valproate‐induced transcription of a DAT reporter construct was significantly attenuated by coexpression of a dominant negative form of Sp, mutation of a Sp‐responsive cis‐element, or expression in a Sp‐null cellular background (SL‐2 cells). Valproate significantly altered Sp protein abundance in both dopaminergic model systems employed. In summary, valproate treatment significantly increased DAT gene expression in a Sp transcription factor‐dependent manner. Some of valproates therapeutic effects may involve activation of DAT gene expression.

Dopamine neurons express multiple isoforms of the nuclear receptor nurr1 with diminished transcriptional activity

Nurr1 (NR4A2) is an orphan nuclear receptor required for the development and maintenance of the dopaminergic phenotype in neurons of the ventral midbrain. This study demonstrates that multiple splice variants of nurr1 are produced in rat and human dopamine neurons. Formed by alternative RNA splicing in exon 7, nurr1a has a truncated carboxy‐terminus, nurr1b has an internal deletion in the ligand‐binding domain and nurr1c, newly identified in this study, has a novel carboxy‐terminus produced by a frame shift downstream of the splice junction. Alternative RNA splicing in exon 3 produces the isoform known as the transcriptionally‐inducible nuclear receptor (TINUR), lacking the amino‐terminus. Nurr2 and the newly identified nurr2c are produced by utilization of both exon 3 and exon 7 alternative splice sites. In rat midbrain, variants other than full‐length nurr1 constitute 20–35% of NR4A2 transcripts. Transfection studies in dopaminergic SK‐N‐AS cells demonstrate that nurr1a, nurr1b, nurr1c and TINUR have significantly reduced transcriptional activities compared with full‐length nurr1, while nurr2 and nurr2c are inactive. Furthermore, in these experiments, nurr2 and nurr2c both act as dominant negatives. Production of these nurr1 variants in vivo as demonstrated here could represent a novel regulatory mechanism of nurr1 transcriptional activity and therefore, dopaminergic phenotype.

The transcription factor NURR1 exerts concentration-dependent effects on target genes mediating distinct biological processes

The transcription factor NURR1 plays a pivotal role in the development and maintenance of neurotransmitter phenotype in midbrain dopamine neurons. Conversely, decreased NURR1 expression is associated with a number of dopamine-related CNS disorders, including Parkinson’s disease and drug addiction. In order to better understand the nature of NURR1-responsive genes and their potential roles in dopamine neuron differentiation and survival, we used a human neural cellular background (SK-N-AS cells) in which to generate a number of stable clonal lines with graded NURR1 gene expression that approximated that seen in DA cell-rich human substantia nigra. Gene expression profiling data from these NURR1-expressing clonal lines were validated by quantitative RT-PCR and subjected to bioinformatic analyses. The present study identified a large number of NURR1-responsive genes and demonstrated the potential importance of concentration-dependent NURR1 effects in the differential regulation of distinct NURR1 target genes and biological pathways. These data support the promise of NURR1-based CNS therapeutics for the neuroprotection and/or functional restoration of DA neurons.

Ventral midbrain correlation between genetic variation and expression of the dopamine transporter gene in cocaine-abusing versus non-abusing subjects

Altered activity of the human dopamine transporter gene (hDAT) is associated with several common and severe brain disorders, including cocaine abuse. However, there is little a priori information on whether such alterations are due to nature (genetic variation) or nurture (human behaviors such as cocaine abuse). This study investigated the correlation between seven markers throughout hDAT and its mRNA levels in postmortem ventral midbrain tissues from 18 cocaine abusers and 18 strictly matched drug‐free controls in the African‐American population. Here, we show that one major haplotype with the same frequency in cocaine abusers versus drug‐free controls displays a 37.1% reduction of expression levels in cocaine abusers compared with matched controls (P = 0.0057). The most studied genetic marker, variable number tandem repeats (VNTR) located in Exon 15 (3′VNTR), is not correlated with hDAT mRNA levels. A 5′ upstream VNTR (rs70957367) has repeat numbers that are positively correlated with expression levels in controls (r2 = 0.9536, P = 0.0235), but this positive correlation disappears in cocaine abusers. The findings suggest that varying hDAT activity is attributable to both genetics and cocaine abuse.