Kent E. Vrana

Twenty-five years of quantitative PCR for gene expression analysis

Following its invention 25 years ago, PCR has been adapted for numerous molecular biology applications. Gene expression analysis by reverse-transcription quantitative PCR (RT-qPCR) has been a key enabling technology of the post-genome era. Since the founding of BioTechniques, this journal has been a resource for the improvements in qPCR technology, experimental design, and data analysis. qPCR and, more specifically, real-time qPCR has become a routine and robust approach for measuring the expression of genes of interest, validating microarray experiments, and monitoring biomarkers. The use of real-time qPCR has nearly supplanted other approaches (e.g., Northern blotting, RNase protection assays). This review examines the current state of qPCR for gene expression analysis now that the method has reached a mature stage of development and implementation. Specifically, the different fluorescent reporter technologies of real-time qPCR are discussed as well as the selection of endogenous controls. The conceptual framework for data analysis methods is also presented to demystify these analysis techniques. The future of qPCR remains bright as the technology becomes more rapid, cost-effective, easier to use, and capable of higher throughput.

Intricate regulation of tyrosine hydroxylase activity and gene expression.

Abstract: Tyrosine hydroxylase catalyzes the rate‐limiting step in the biosynthesis of the catecholamines dopamine, norepinephrine, and epinephrine. Therefore, the regulation of tyrosine hydroxylase enzyme number and intrinsic enzyme activity represents the central means for controlling the synthesis of these important biogenic amines. An intricate scheme has evolved whereby tyrosine hydroxylase activity is modulated by nearly every documented form of regulation. Beginning with the genomic DNA, evidence exists for the transcriptional regulation of tyrosine hydroxylase mRNA levels, alternative RNA processing, and the regulation of RNA stability. There is also experimental support for the role of both translational control and enzyme stability in establishing steady‐state levels of active tyrosine hydroxylase protein. Finally, mechanisms have been proposed for feedback inhibition of the enzyme by catecholamine products, allosteric modulation of enzyme activity, and phosphorylation‐dependent activation of the enzyme by various different kinase systems. Given the growing literature suggesting that different tissues regulate tyrosine hydroxylase mRNA levels and activity in different ways, regulatory mechanisms provide not only redundancy but also diversity in the control of catecholamine biosynthesis.

Cytotoxic and genotoxic potential of dopamine

A variety of in vitro and in vivo studies demonstrate that dopamine is a toxic molecule that may contribute to neurodegenerative disorders such as Parkinsons disease and ischemia‐induced striatal damage. While much attention has focused on the fact that the metabolism of dopamine produces reactive oxygen species (peroxide, superoxide, and hydroxyl radical), growing evidence suggests that the neurotransmitter itself may play a direct role in the neurodegenerative process. Oxidation of the dopamine molecule produces a reactive quinone moiety that is capable of covalently modifying and damaging cellular macromolecules. This quinone formation occurs spontaneously, can be accelerated by metal ions (manganese or iron), and also arises from selected enzyme‐catalyzed reactions. Macromolecular damage, combined with increased oxidant stress, may trigger cellular responses that eventually lead to cell death. Reactive quinones have long been known to represent environmental toxicants and, within the context of dopamine metabolism, may also play a role in pathological processes associated with neurodegeneration. The present discussion will review the oxidative metabolism of dopamine and describe experimental evidence suggesting that dopamine quinone may contribute to the cytotoxic and genotoxic potential of this essential neurotransmitter. J. Neurosci. Res. 55:659–665, 1999. 

Dopamine, in the presence of tyrosinase, covalently modifies and inactivates tyrosine hydroxylase

Dopamine has been implicated as a potential mediating factor in a variety of neurodegenerative disorders. Dopamine can be oxidized to form a reactive dopamine quinone that can covalently modify cellular macromolecules including protein and DNA. This oxidation can be enhanced through various enzymes including tyrosinase and/or prostaglandin H synthase. One of the potential targets in brain for dopamine quinone damage is tyrosine hydroxylase, the rate‐limiting enzyme in catecholamine biosynthesis. The present studies demonstrated that dopamine quinone, the formation of which was enhanced through the activity of the melanin biosynthetic enzyme, tyrosinase, covalently modified and inactivated tyrosine hydroxylase.

Tyrosinase mRNA is expressed in human substantia nigra

Dopamine acts, under appropriate conditions, as a selective neurotoxin. This toxicity is attributed to the autoxidation of the neurotransmitter into a reactive quinone that covalently modifies cellular macromolecules (i.e. proteins and nucleic acids). The oxidation of the catecholamine to a quinone is greatly accelerated by the enzyme tyrosinase. There is controversy, however, as to whether or not tyrosinase is expressed in human brain. In the present study, RT-PCR was utilized to demonstrate the presence of tyrosinase mRNA in post-mortem human brain tissues. Using gene-specific amplification primers, specific tyrosinase amplicons were detected following analysis of RNA from substantia nigra of four individuals. Analysis of cerebellar RNA from the same individuals produced no amplification products. Control reactions performed in the absence of reverse transcriptase failed to generate PCR products for any tissue tested. Three amplicons were subjected to direct DNA sequencing and all proved to be identical with tyrosinase sequences, thus obviating the possibility of amplification of a related gene. It is clear, therefore, that the tyrosinase gene is expressed in the human substantia nigra, lending support to previous studies describing tyrosinase-like activity and immunoreactive protein in the brain. This enzyme could be central to dopamine neurotoxicity as well as contribute to the neurodegeneration associated with Parkinsons disease.

Chronic cocaine-mediated changes in non-human primate nucleus accumbens gene expression.

Chronic cocaine use elicits changes in the pattern of gene expression within reinforcement‐related, dopaminergic regions. cDNA hybridization arrays were used to illuminate cocaine‐regulated genes in the nucleus accumbens (NAcc) of non‐human primates (Macaca fascicularis; cynomolgus macaque), treated daily with escalating doses of cocaine over one year. Changes seen in mRNA levels by hybridization array analysis were confirmed at the level of protein (via specific immunoblots). Significantly up‐regulated genes included: protein kinase A α catalytic subunit (PKAcα); cell adhesion tyrosine kinase beta (PYK2); mitogen activated protein kinase kinase 1 (MEK1); and β‐catenin. While some of these changes exist in previously described cocaine‐responsive models, others are novel to any model of cocaine use. All of these adaptive responses coexist within a signaling scheme that could account for known inductions of genes(e.g. fos and jun proteins, and cyclic AMP response element binding protein) previously shown to be relevant to cocaines behavioral actions. The complete data set from this experiment has been posted to the newly created Drug and Alcohol Abuse Array Data Consortium (http://www.arraydata.org) for mining by the general research community.

Chronic Cocaine Administration Increases CNS Tyrosine Hydroxylase Enzyme Activity and mRNA Levels and Tryptophan Hydroxylase Enzyme Activity Levels

Cocaine is an inhibitor of dopamine and serotonin reuptake by synaptic terminals and has potent reinforcing effects that lead to its abuse. Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) catalyze the rate‐limiting steps in dopamine and serotonin biosynthesis, respectively, and are the subject of dynamic regulatory mechanisms that could be sensitive to the actions of cocaine. This study assessed the effects of chronic cocaine on brain TH and TPH activities. Cocaine was administered (0.33 mg/infusion, i.v.) to rats for 7 days every 8 min for 6 h per day. This administration schedule is similar to patterns of self‐administration by rats when given ad libitum access to this dose. This chronic, response‐independent administration increased TH enzyme activity in the substantia nigra (30%) and ventral tegmental area (43%). Moreover, TH mRNA levels were also increased (45 and 50%, respectively). In contrast to the enzymatic and molecular biological changes in the cell bodies, TH activity was unchanged in the terminal fields (corpus striaturn and nucleus accumbens). Similarly, TPH activity was increased by 50% in the raphe nucleus (serotonergic cell bodies). In summary, the chronic response‐independent administration of cocaine produces increases in the expression of TH mRNA and activity in both the cell bodies of motor (nigrostriatal) and reinforcement (mesolimbic) dopamine pathways. These increases are not manifested in the terminal fields of these pathways.

Circulating Cytokines as Biomarkers of Alcohol Abuse and Alcoholism

There are currently no consistent objective biochemical markers of alcohol abuse and alcoholism. Development of reliable diagnostic biomarkers that permit accurate assessment of alcohol intake and patterns of drinking is of prime importance to treatment and research fields. Diagnostic biomarker development in other diseases has demonstrated the utility of both open, systems biology, screening for biomarkers and more rational focused efforts on specific biomolecules or families of biomolecules. Long-term alcohol consumption leads to altered inflammatory cell and adaptive immune responses with associated pathologies and increased incidence of infections. This has led researchers to focus attention on identifying cytokine biomarkers in models of alcohol abuse. Alcohol is known to alter cytokine levels in plasma and a variety of tissues including lung, liver, and very importantly brain. A number of cytokine biomarker candidates have been identified, including: tumor necrosis factor-alpha, interleukin (IL)-1-alpha, IL-1-beta, IL-6, IL-8, IL-12, and monocyte chemoattractant protein-1. This is an emerging and potentially exciting avenue of research in that circulating cytokines may contribute to diagnostic biomarker panels, and a combination of multiple biomarkers may significantly increase the sensitivity and specificity of the biochemical tests aiding reliable and accurate detection of excessive alcohol intake.

Persistent alterations in mesolimbic gene expression with abstinence from cocaine self-administration

Cocaine-responsive gene expression changes have been described after either no drug abstinence or short periods of abstinence. Little data exist on the persistence of these changes after long-term abstinence. Previously, we reported that after discrete-trial cocaine self-administration and 10 days of forced abstinence, incubation of cocaine reinforcement was observable by a progressive ratio schedule. The present study used rat discrete-trial cocaine self-administration and long-term forced abstinence to examine extinction responding, mRNA abundance of known cocaine-responsive genes, and chromatin remodeling. At 30 and 100 days of abstinence, extinction responding increased compared to 3-day abstinent rats. Decreases in both medial prefrontal cortex (mPFC) and nucleus accumbens c-fos, Nr4a1, Arc, and EGR1 mRNA were observed, and in most cases persisted, for 100 days of abstinence. The signaling peptides CART and neuropeptide Y (NPY) transiently increased in the mPFC, but returned to baseline levels following 10 days of abstinence. To investigate a potential regulatory mechanism for these persistent mRNA changes, levels of histone H3 acetylation at promoters for genes with altered mRNA expression were examined. In the mPFC, histone H3 acetylation decreased after 1 and 10 days of abstinence at the promoter for EGR1. H3 acetylation increased for NPY after 1 day of abstinence and returned to control levels by 10 days of abstinence. Behaviorally, these results demonstrate incubation after discrete-trial cocaine self-administration and prolonged forced abstinence. This incubation is accompanied by changes in gene expression that persist long after cessation of drug administration and may be regulated by chromatin remodeling.

Dopamine toxicity in neuroblastoma cells: role of glutathione depletion by L-BSO and apoptosis

Dopamine (DA), while an essential neurotransmitter, is also a known neurotoxin that potentially plays an etiologic role in several neurodegenerative diseases. DA metabolism and oxidation readily produce reactive oxygen species (ROS) and DA can also be oxidized to a reactive quinone via spontaneous, enzyme-catalyzed or metal-enhanced reactions. A number of these reactions are cytotoxic, yet the precise mechanisms by which DA leads to cell death remain unknown. In this study, the neuroblastoma cell line, SK-N-SH, was utilized to examine DA toxicity under varying oxidant states. Cells pretreated with the glutathione (GSH)-depleting compound, L-buthionine sulfoximine (L-BSO), exhibited enhanced sensitivity to DA compared to controls (non-GSH-depleted cells). Furthermore, in cells pretreated with L-BSO, the addition of ascorbate (250 microM) afforded significant protection against DA-induced toxicity, while pyruvate (500 microM) had no protective effect. To further characterize the possibility that DA is associated with oxidative stress, additional studies were carried out with manganese (30 microM) as a pro-oxidant. Manganese and DA (200 microM), although not cytotoxic when individually administered to SK-N-SH cells, had a synergistic action on cytotoxicity. Finally, morphological and molecular markers of programmed cell death (apoptosis) were observed in cells treated with DA and L-BSO. These markers included membrane blebbing and internucleosomal DNA fragmentation. These results suggest that DA toxicity is tightly linked to intracellular oxidant/antioxidant levels, and that environmental factors, such as excessive Mn exposure, may modulate cellular sensitivity to DA.