Gregory Kapatos

Gene expression profile of the nucleus accumbens of human cocaine abusers: evidence for dysregulation of myelin

Chronic cocaine abuse induces long‐term neural adaptations as a consequence of alterations in gene expression. This study was undertaken to identify those transcripts differentially regulated in the nucleus accumbens of human cocaine abusers. Affymetrix microarrays were used to measure transcript abundance in 10 cocaine abusers and 10 control subjects matched for age, race, sex, and brain pH. As expected, gene expression of cocaine‐ and amphetamine‐regulated transcript (CART) was increased in the nucleus accumbens of cocaine abusers. The most robust and consistent finding, however, was a decrease in the expression of a number of myelin‐related genes, including myelin basic protein (MBP), proteolipid protein (PLP), and myelin‐associated oligodendrocyte basic protein (MOBP). The differential expression seen by microarray for CART as well as MBP, MOBP, and PLP was verified by RT–PCR. In addition, immunohistochemical experiments revealed a decrease in the number of MBP‐immunoreactive oligodendrocytes present in the nucleus accumbens and surrounding white matter of cocaine abusers. These findings suggest a dysregulation of myelin in human cocaine abusers.

Gene Transfer of Human Guanosine 5′-Triphosphate Cyclohydrolase I Restores Vascular Tetrahydrobiopterin Level and Endothelial Function in Low Renin Hypertension

Background—We recently reported that arterial superoxide (O2−) is augmented by increased endothelin-1 (ET-1) in deoxycorticosterone acetate (DOCA)-salt hypertension, a model of low renin hypertension. Tetrahydrobiopterin (BH4), a potent reducing molecule with antioxidant properties and an essential cofactor for endothelial nitric oxide synthase, protects against O2−–induced vascular dysfunction. However, the interaction between O2− and BH4 on endothelial function and the underlying mechanisms are unknown. Methods and Results—The present study tested the hypothesis that BH4 deficiency due to ET-1–induced O2− leads to impaired endothelium-dependent relaxation and that gene transfer of human guanosine 5′-triphosphate (GTP) cyclohydrolase I (GTPCH I), the first and rate-limiting enzyme for BH4 biosynthesis, reverses such deficiency and endothelial dysfunction in carotid arteries of DOCA-salt rats. There were significantly increased arterial O2− levels and decreased GTPCH I activity and BH4 levels in DOCA-salt compared with sham rats. Treatment of arteries of DOCA-salt rats with the selective ETA receptor antagonist ABT-627, NADPH oxidase inhibitor apocynin, or superoxide dismutase (SOD) mimetic tempol abolished O2− and restored BH4 levels. Basal arterial NO release and endothelium-dependent relaxations were impaired in DOCA-salt rats, conditions that were improved by apocynin or tempol treatment. Gene transfer of GTPCH I restored arterial GTPCH I activity and BH4 levels, resulting in reduced O2− and improved endothelium-dependent relaxation and basal NO release in DOCA-salt rats. Conclusions—These results indicate that a BH4 deficiency resulting from ET-1–induced O2− via an ETA/NADPH oxidase pathway leads to endothelial dysfunction, and gene transfer of GTPCH I reverses the BH4 deficiency and endothelial dysfunction by reducing O2− in low renin mineralocorticoid hypertension.

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

J. Neurochem. (2011) 116, 459–466.

Effect of Aging on Tyrosine Hydroxylase Protein Content and the Relative Number of Dopamine Nerve Terminals in Human Caudate

Abstract: This study examined the effect of aging on the relative number of dopamine (DA) nerve terminals in human caudate nucleus, their content of tyrosine hydroxylase (TH) protein, and the relative abundance of TH monomers with different molecular weights. Preliminary studies on brain tissue cryopreservation, performed with rat striatum, indicated that intact synaptosomes can be prepared from fresh tissue slowly frozen in 0.32 M sucrose with 5% dimethyl sulfoxide and then thawed rapidly prior to synaptosome preparation. Synaptosomes were prepared in this manner from postmortem caudate nucleus tissue obtained from normal humans 1 month to 63 years of age. To determine the relative number of DA nerve terminals for each individual, dopaminergic synaptosomes were selectively labeled with a monoclonal antibody to TH and quantified by fluorescence‐activated cell sorting. To determine the relative amount of TH protein for each individual, the concentration of TH protein in the same synaptosomal preparations was determined using immunoblots. Our results suggest that caudate TH levels plateau soon after birth and tend to remain relatively stable during aging, since no changes in either the relative number of TH‐containing nerve terminals or the concentration of TH protein were found in subjects 15–63 years of age. In light of previous studies showing an age‐related loss of DA cell bodies, these findings suggest that remaining DA neurons compensate to maintain caudate levels of TH protein and TH‐containing nerve terminals. Immunoblot studies identified three forms of TH monomer (60.6, 61.7, and 65.1 kDa), indicating that mRNAs coding for high molecular mass forms of TH may be actively translated in human brain. No age‐related differences in the relative abundance of these forms were found.

Distinctive profiles of gene expression in the human nucleus accumbens associated with cocaine and heroin abuse.

Drug abuse is thought to induce long-term cellular and behavioral adaptations as a result of alterations in gene expression. Understanding the molecular consequences of addiction may contribute to the development of better treatment strategies. This study utilized high-throughput Affymetrix microarrays to identify gene expression changes in the post-mortem nucleus accumbens of chronic heroin abusers. These data were analyzed independently and in relation to our previously reported data involving human cocaine abusers, in order to determine which expression changes were drug specific and which may be common to the phenomenon of addiction. A significant decrease in the expression of numerous genes encoding proteins involved in presynaptic release of neurotransmitter was seen in heroin abusers, a finding not seen in the cocaine-abusing cohort. Conversely, the striking decrease in myelin-related genes observed in cocaine abusers was not evident in our cohort of heroin subjects. Overall, little overlap in gene expression profiles was seen between the two drug-abusing cohorts: out of the approximately 39 000 transcripts investigated, the abundance of only 25 was significantly changed in both cocaine and heroin abusers, with nearly one-half of these being altered in opposite directions. These data suggest that the profiles of nucleus accumbens gene expression associated with chronic heroin or cocaine abuse are largely unique, despite what are thought to be common effects of these drugs on dopamine neurotransmission in this brain region. A re-examination of our current assumptions about the commonality of molecular mechanisms associated with substance abuse seems warranted.

Layer-Specific CREB Target Gene Induction in Human Neocortical Epilepsy

Epilepsy is a disorder of recurrent seizures that affects 1% of the population. To understand why some areas of cerebral cortex produce seizures and others do not, we identified differentially expressed genes in human epileptic neocortex compared with nearby regions that did not produce seizures. The transcriptome that emerged strongly implicates MAPK signaling and CREB-dependent transcription, with 74% of differentially expressed genes containing a cAMP response element (CRE) in their proximal promoter, more than half of which are conserved. Despite the absence of recent seizures in these patients, epileptic brain regions prone to seizures showed persistent activation of ERK and CREB. Persistent CREB activation was directly linked to CREB-dependent gene transcription by chromatin immunoprecipitation that showed phosphorylated CREB constitutively associated with the proximal promoters of many of the induced target genes involved in neuronal signaling, excitability, and synaptic plasticity. A distinct spatial pattern of ERK activation was seen in superficial axodendritic processes of epileptic neocortex that colocalized with both CREB phosphorylation and CREB target gene induction in well demarcated populations of layer 2/3 neurons. These same neuronal lamina showed a marked increase in synaptic density. The findings generated in this study generate a robust and spatially restricted pattern of epileptic biomarkers and associated synaptic changes that could lead to new mechanistic insights and potential therapeutic targets for human epilepsy.

Quantitation of rat dopamine transporter mrna effects of cocaine treatment and withdrawal

Abstract: Dopamine transporter mRNA levels in the rat substantia nigra were quantified using a sensitive nuclease protection assay with a highly homologous human dopamine transporter cDNA clone. The same probe was also used to visualize dopamine transporter mRNA in the substantia nigra by in situ hybridization. Repeated cocaine administration (15 mg/kg, twice a day for 6.5 days) resulted in a >40% decrease in nigral dopamine transporter mRNA levels. In contrast, dopamine transporter mRNA levels were unchanged after either acute treatment (4 h before death) or repeated cocaine treatment followed by a 72‐h withdrawal period. Thus, blockade of the dopamine transporter by repeated cocaine administration may result in the down‐regulation of dopamine transporter gene expression in dopamine neurons.

Study of a Swiss dopa-responsive dystonia family with a deletion in GCH1: Redefining DYT14 as DYT5

Objective: To report the study of a multigenerational Swiss family with dopa-responsive dystonia (DRD). Methods: Clinical investigation was made of available family members, including historical and chart reviews. Subject examinations were video recorded. Genetic analysis included a genome-wide linkage study with microsatellite markers (STR), GTP cyclohydrolase I (GCH1) gene sequencing, and dosage analysis. Results: We evaluated 32 individuals, of whom 6 were clinically diagnosed with DRD, with childhood-onset progressive foot dystonia, later generalizing, followed by parkinsonism in the two older patients. The response to levodopa was very good. Two additional patients had late onset dopa-responsive parkinsonism. Three other subjects had DRD symptoms on historical grounds. We found suggestive linkage to the previously reported DYT14 locus, which excluded GCH1. However, further study with more stringent criteria for disease status attribution showed linkage to a larger region, which included GCH1. No mutation was found in GCH1 by gene sequencing but dosage methods identified a novel heterozygous deletion of exons 3 to 6 of GCH1. The mutation was found in seven subjects. One of the patients with dystonia represented a phenocopy. Conclusions: This study rules out the previously reported DYT14 locus as a cause of disease, as a novel multiexonic deletion was identified in GCH1. This work highlights the necessity of an accurate clinical diagnosis in linkage studies as well as the need for appropriate allele frequencies, penetrance, and phenocopy estimates. Comprehensive sequencing and dosage analysis of known genes is recommended prior to genome-wide linkage analysis. GLOSSARY: DRD = dopa-responsive dystonia; GCH1 = GTP cyclohydrolase I; SNP = single nucleotide polymorphisms; STR = short tandem repeats.

GTP cyclohydrolase I gene expression in the brains of male and female hph-1 mice.

Abstract : The hph‐1 mouse is characterized by low levels of GTP cyclohydrolase I (GTPCH) and tetrahydrobiopterin. A quantitative double‐lable in situ hybridization technique was used to examine CNS GTPCH mRNA expression within serotonin, dopamine, and norepinephrine neurons of male and female wild‐type and hph‐1 mice. In wild‐type male and female animals the highest levels of GTPCH mRNA expression were observed within serotonin neurons, followed by norepinephrine and then dopamine neurons. Wild‐type female animals were found to express lower levels of GTPCH mRNA in each cell type when compared with levels seen in wild‐type males. GTPCH mRNA abundance in all three cell types was lower in hph‐1 male than in wild‐type male mice, with the greatest reduction in serotonin neurons. GTPCH mRNA levels were also lower in hph‐1 female than in wild‐type female mice, again with the greatest reduction occurring in serotonin neurons. Comparison of hph‐1 male and hph‐1 female mice revealed that the sex‐linked difference in GTPCH mRNA expression observed in wild‐type neurons was only present within female dopamine neurons. Overall, these results indicate that not only are basal levels of GTPCH mRNA expression heterogeneous across wild‐type murine monoamine cell types but that gene expression is also modified in a sex‐linked and cell‐specific fashion by the hph‐1 gene locus. The hph‐1 mutation does not lie within the GT‐PCH mRNA coding region. The 5′ flanking region of the GTPCH gene was cloned and sequenced and shown to be identical for both wild‐type and hph‐1 genomic DNA. Transient transfection assays performed in PC12 cells demonstrated that this 5′ flanking region was sufficient to initiate transcription of a luciferase reporter gene. Although the hph‐1 mutation does not lie within the 5′ flanking region of the GTPCH gene, this region of the gene can function as a core promoter and is thus crucial to the control of GTPCH gene expression.

Effect of Cyclic AMP‐Dependent Protein Phosphorylating Conditions on the pH‐Dependent Activity of Tyrosine Hydroxylase from Beef and Rat Striata

Abstract Tyrosine hydroxylase (TH, EC 1.14.16.2) from beef brain striata was purified 23‐fold from an extract of an acetone powder. If this enzyme preparation is treated with a cyclic AMP‐dependent protein phosphorylation system, there is a change in the pH dependence of the enzyme activity. The pH optimum at saturating tetrahydrobiopterin (BH4) concentration is shifted from below pH 6 to about pH 6.7. At pH 7, activation is expressed mainly as an increase in Vmax, whereas at pH 6, activation is expressed mainly as a decrease in Km for the pterin cofactor. Further, even with the control enzyme the Km for pterin cofactor declines precipitously as the pH is increased from 6 toward neutrality. Similar data were obtained with G‐25 Sephadex‐treated rat striatal TH. Experiments in which rat striatal synaptosomes were used demonstrated that the in situ activation of TH by phosphorylating conditions is expressed primarily as an increase in the maximum rate of dopamine synthesis. These results indicate that changes in TH activity caused by cyclic AMP‐dependent protein phosphorylation will depend to a large extent on the pH of the TH environment.