1Joint first authors.Abbreviations used: HI, H-Invitational; hNAcc, human nucleus accumbens; lncRNAs, long non-coding RNAs; MIAT, myocardial infarction associated transcript; MEG3, maternally expressed gene 3; NEAT1, nuclear enriched abundant transcript 1; NEAT2, nuclear enriched abundant transcript 2; qRT-PCR, quantitative real time-PCR. AbstractAlthough recent data suggest that some long non-coding RNAs (lncRNAs) exert widespread effects on gene expression and organelle formation, lncRNAs as a group constitute a sizable but poorly characterized fraction of the human transcriptome. We investigated whether some human lncRNA sequences were fortuitously represented on commonly used microarrays, then used this annotation to assess lncRNA expression in human brain. A computational and annotation pipeline was developed to identify lncRNA transcripts represented on Affymetrix U133 arrays. A previously published dataset derived from human nucleus accumbens was then examined for potential lncRNA expression. Twenty-three lncRNAs were determined to be represented on U133 arrays. Of these, dataset analysis revealed that five lncRNAs were consistently detected in samples of human nucleus accumbens. Strikingly, the abundance of these lncRNAs was upregulated in human heroin abusers compared to matched drug-free control subjects, a finding confirmed by quantitative PCR. This study presents a paradigm for examining existing Affymetrix datasets for the detection and potential regulation of lncRNA expression, including changes associated with human disease. The finding that all detected lncRNAs were up-regulated in heroin abusers is consonant with the proposed role of lncRNAs as mediators of widespread changes in gene expression as occur in drug abuse. In addition to sequence conservation, the precise temporal and cellular specificity of lncRNA expression is considered prima facie evidence of their functional importance. Nowhere is this more evident than in the CNS, where scores of lncRNAs exhibit robust and specific expression patterns and likely influence brain development, neurotransmission, and neuropsychiatric disorders (Mercer et al. 2008;Qureshi et al. 2010). Although our understanding in this regard is rudimentary, a recent study (Faghihi et al. 2008) found that one particular lncRNA is directly implicated in the increased CNS abundance of Ab 1-42 seen in Alzheimer's disease.In the course of our previous Affymetrix microarray studies of postmortem brain from human drug abusers (Albertson et al. 2006), we found that one of the transcripts most robustly increased by drug abuse was derived from a gene assumed to encode a hypothetical protein (LOC150271). Subsequent bioinformatic analysis has revealed that this transcript does not encode a protein but actually corresponds to a known lncRNA, myocardial infarction associated transcript (MIAT) (Ishii et al. 2006). Given the proposed role of some lncRNAs as top-down regulators of gene expression, and the widespread changes in gene expression seen in drug abuse (Albertson et al. 2...

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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 SK Ca 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 SK Ca 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 (I mAHP ) and the slow AHP current (I sAHP ). I mAHP is blocked by the SK Ca channel blockers apamin and bicuculline, whereas I sAHP is resistant to these agents but is inhibited by activation of muscarinic receptors. To test for a role for SK Ca channels, we overexpressed K Ca 2.1 (SK1) and K Ca 2.2 (SK2), the predominant SK Ca subunits expressed in the cortex, in pyramidal cells of cultured brain slices. Overexpression of K Ca 2.1 and K Ca 2.2 resulted in a fourfold to fivefold increase in the amplitude of I mAHP but had no detectable effect on I sAHP . As an additional test, we examined I sAHP in a transgenic mouse expressing a truncated SK Ca subunit (SK3-1B) capable of acting as a dominant negative for the entire family of SK Ca -IK Ca channels. Expression of SK3-1B profoundly inhibited I mAHP but again had no discernable effect on I sAHP . These results are inconsistent with the proposal that SK Ca channels mediate I sAHP in pyramidal cells and indicate that a different potassium channel mediates this current.

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 re¯ected in their ability to compete with each other for speci®c 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.

Chronic drug abuse, craving, and relapse are thought to be linked to long-lasting changes in neural gene expression arising through transcriptional and chromatin-related mechanisms. The key contributions of midbrain dopamine (DA)-synthesizing neurons throughout the addiction process provide a compelling rationale for determining the drug-induced molecular changes that occur in these cells. Yet our understanding of these processes remains rudimentary. The postmortem human brain constitutes a unique resource that can be exploited to gain insights into the pathophysiology of complex disorders such as drug addiction. In this study, we analyzed the profiles of midbrain gene expression in chronic cocaine abusers and well-matched drug-free control subjects using microarray and quantitative PCR. A small number of genes exhibited robust differential expression; many of these are involved in the regulation of transcription, chromatin, or DA cell phenotype. Transcript abundances for approximately half of these differentially expressed genes were diagnostic for assigning subjects to the cocaine-abusing vs control cohort. Identification of a molecular signature associated with pathophysiological changes occurring in cocaine abusers' midbrains should contribute to the development of biomarkers and novel therapeutic targets for drug addiction.

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.

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 cocainesensitive 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.

The knowledge of transporter protein expression and function at the human blood–brain barrier (BBB) is critical to prediction of drug BBB penetration and design of strategies for improving drug delivery to the brain or brain tumor. This study determined absolute transporter protein abundances in isolated microvessels of human normal brain (N = 30), glioblastoma (N = 47), rat (N = 10) and mouse brain (N = 10), and cell membranes of MDCKII cell lines, using targeted proteomics. In glioblastoma microvessels, efflux transporters (ABCB1 and ABCG2), monocarboxylate transporter 1 (MCT1), glucose transporter 1 (GLUT1), sodium–potassium pump (Na/K ATPase), and Claudin‐5 protein levels were significantly reduced, while large neutral amino acid transporter 1 (LAT1) was increased and GLU3 remained the same, as compared with human normal brain microvessels. ABCC4, OATP1A2, OATP2B1, and OAT3 were undetectable in microvessels of both human brain and glioblastoma. Species difference in BBB transporter abundances was noted. Cellular permeability experiments and modeling simulations suggested that not a single apical uptake transporter but a vectorial transport system consisting of an apical uptake transporter and basolateral efflux mechanism was required for efficient delivery of poor transmembrane permeability drugs from the blood to brain.