Enhancing VTA Cav1.3 L-type Ca2+ channel activity promotes cocaine and mood-related behaviors via overlapping AMPA receptor mechanisms in the nucleus accumbens.

Genetic factors significantly influence susceptibility for substance abuse and mood disorders. Rodent studies have begun to elucidate a role of Cav1.3 L-type Ca2+ channels in neuropsychiatric-related behaviors, such as addictive and depressive-like behaviors. Human studies have also linked the CACNA1D gene, which codes for the Cav1.3 protein, with bipolar disorder. However, the neurocircuitry and the molecular mechanisms underlying the role of Cav1.3 in neuropsychiatric phenotypes are not well established. In the present study, we directly manipulated Cav1.3 channels in Cav1.2 dihydropyridine insensitive mutant mice and found that ventral tegmental area (VTA) Cav1.3 channels mediate cocaine-related and depressive-like behavior through a common nucleus accumbens (NAc) shell calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (CP-AMPAR) mechanism that requires GluA1 phosphorylation at S831. Selective activation of VTA Cav1.3 with (±)-BayK-8644 (BayK) enhanced cocaine conditioned place preference and cocaine psychomotor activity while inducing depressive-like behavior, an effect not observed in S831A phospho-mutant mice. Infusion of the CP-AMPAR-specific blocker Naspm into the NAc shell reversed the cocaine and depressive-like phenotypes. In addition, activation of VTA Cav1.3 channels resulted in social behavioral deficits. In contrast to the cocaine- and depression-related phenotypes, GluA1/A2 AMPARs in the NAc core mediated social deficits, independent of S831-GluA1 phosphorylation. Using a candidate gene analysis approach, we also identified single-nucleotide polymorphisms in the CACNA1D gene associated with cocaine dependence in human subjects. Together, our findings reveal novel, overlapping mechanisms through which VTA Cav1.3 mediates cocaine-related, depressive-like and social phenotypes, suggesting that Cav1.3 may serve as a target for the treatment of neuropsychiatric symptoms.

Induction of hepatitis B virus gene expression at low temperature.

There is a limited understanding of the cellular regulation of HBV gene expression in differentiated hepatocytes. We previously demonstrated that HBV replication inversely correlates with cell proliferation and DNA synthesis. In this report, temperature-induced modulation of cell growth was used as a novel approach to study HBV gene expression in the absence of indirect effects from drugs or serum deprivation. We observed markedly elevated levels of hepatic HBV mRNA expression from integrated and episomal HBV DNA at 32 degrees C. Additionally, hepatoblastoma cells cultured at 32 degrees C expressed increased levels of albumin mRNA and decreased levels of c-myc mRNA, which demonstrates that liver-derived cells cultured at low temperature exhibit characteristics of functional and differentiated hepatocytes. In transiently transfected HepG2 cells cultured at 32 degrees C, the HBV enhancer 1 activated the X promoter and core/pregenomic promoter by 7.3- and 28-fold, respectively. In the absence of enhancer 1, core/pregenomic promoter activity was 2.4-fold higher than the X promoter in HepG2 cells at 32 degrees C. In contrast, enhancer 1 exclusively activated the X promoter in transfected non-liver cells at 32 degrees C. Therefore, the core/pregenomic promoter exhibits strict liver-specificity at low temperature. This work supports the hypothesis that HBV replication and gene expression are optimal in non-activated hepatocytes, and provides a novel system for delineating molecular aspects of the HBV replication process.

Biochemical and functional properties of a palindromic sequence motif within the hepatitis B virus enhancer 1.

The hepatitis B virus (HBV) enhancer 1 is a transcriptional element that contributes to the liver-specific regulation of HBV gene expression. We previously identified a novel protein binding site within the enhancer that contains an 8-bp palindromic sequence motif. This motif partially overlaps the binding sites for nuclear factor 1 and hepatocyte nuclear factor 3beta (HNF3beta). Moreover, we demonstrated that this novel site is recognized by a protein or proteins, tentatively designated as palindrome-binding factor (PBF), that cooperatively interact with HNF3beta. In the present work, we have further examined the biochemical and functional attributes of PBF. Protein-DNA interaction studies indicate that three thymidine residues located at the 3'-end of the palindromic sequence motif are important for maximal PBF-binding activity. When protein-DNA complexes were photocrosslinked by exposure to ultraviolet (UV) light, a prominent polypeptide with an apparent molecular mass of 50 kDa was found to associate with the PBF-binding site. Furthermore, transient transfection studies support the hypothesis that PBF contributes to enhancer 1 activity by a combinatorial mechanism that involves at least one other cis-acting sequence motif, the HNF3beta-binding site.

Purification and properties of rat liver nuclear proteins that interact with the hepatitis B virus enhancer 1.

The hepatitis B virus enhancer 1 element plays a fundamental role in the liver-specific regulation of hepatitis B virus gene expression. A central region of enhancer 1, the enhancer core domain, contains at least four cis-acting sequence motifs that are essential for enhancer 1 activity. In this study, we have investigated an essential motif within the core domain previously defined as footprint V (FPV). Transient transfection analyses demonstrate that FPV is capable of independently functioning in a liver-specific manner to activate transcription. Therefore, to further examine the liver-specific properties of FPV-mediated enhancer 1 activity, we have carried out the biochemical purification and characterization of FPV binding activity from rat liver nuclei. This study has conclusively identified hepatocyte nuclear factor 3beta (HNF-3beta), a liver-enriched member of the HNF-3/forkhead gene family, as the predominant purified protein that interacts with the FPV motif. Moreover, a cellular protein(s) that copurified with HNF-3beta specifically interacts with a novel sequence motif that partially overlaps FPV. Since this novel motif contains a palindromic sequence, we have tentatively referred to the protein(s) that binds to this site as palindrome-binding factor (PBF). Additional evidence indicates that HNF-3beta and PBF cooperatively interact with enhancer 1. Therefore, this study supports the hypothesis that FPV-mediated enhancer activity involves a cooperative interplay between HNF-3beta and at least one other enhancer 1-binding protein, PBF.

Retinoid X receptor alpha transactivates the hepatitis B virus enhancer 1 element by forming a heterodimeric complex with the peroxisome proliferator-activated receptor.

The hepatitis B virus enhancer 1 contains a retinoic acid responsive element (RARE). We have previously demonstrated that retinoid X receptor alpha (RXR alpha) transactivates enhancer 1 by binding to the RARE. The present study has revealed that a heterodimeric complex composed of RXR alpha and peroxisome proliferator-activated receptor (PPAR) interacts with the hepatitis B virus RARE. Transient transfection studies, in conjunction with in vitro DNA binding data, support the hypothesis that the RXR alpha-PPAR heterodimer transactivates enhancer 1.

Immunological identification of human platelet mitochondrial DNA topoisomerase I.

A nuclear human blood platelets have been used to study mitochondrial topoisomerase activity in the absence of nuclear contamination. Previous work utilizing this novel system demonstrated that platelet mitochondria contain type-I topoisomerase (Kosovsky, M.J. and Soslau, G. (1991) Biochim. Biophys. Acta 1078, 56-62). The present work has demonstrated that mitochondrial topoisomerase activity was inhibited by the specific topoisomerase-I inhibitor, topotecan, yet was not affected by a specific topoisomerase-II inhibitor, VM-26. These results confirm that platelet mitochondria contain topoisomerase I, yet do not contain a detectable level of topoisomerase-II activity. It has been demonstrated for the first time that antibodies directed against nuclear topo I cross-react with mitochondria topo I. Furthermore, immunoblot analysis of platelet mitochondrial proteins, in conjunction with renaturation studies, has led to the identification of a catalytically active 60-kDa form of mitochondrial topoisomerase I.

Mitochondrial DNA topoisomerase I from human platelets.

An anucleated cell system has been used for the first time to study mitochondrial topoisomerase activity. Mitochondrial extracts from human blood platelets contained type I topoisomerase. The type I classification was based on ATP-independent activity, inhibition by ATP or camptothecin, and the lack of inhibition by novobiocin. Platelet mitochondrial topoisomerase I relaxation activity was inhibited linearly by increasing concentrations of EGTA. Topoisomerase activity greater than 90% inhibited by 175 microM EGTA was partially restored to 16 and 50% of the initial level of activity by the subsequent addition of 50 and 100 microM Ca2+, respectively. Additionally, results from studies of partially purified platelet mitochondrial topoisomerase I were consistent with the crude extract data. This work supports the hypothesis that platelet mitochondria contain a type I topoisomerase that is biochemically distinct from that previously isolated and characterized from cell nuclei.

Isolation and characterization of herpes simplex virus type 1 host range mutants defective in viral DNA synthesis.

Cell lines were generated by cotransfection of Vero cells with pSV2neo and a plasmid containing the herpes simplex virus type 1 (HSV-1) EcoRI D fragment (coordinates 0.086 to 0.194). One such cell line (S22) contained the genes for alkaline exonuclease and several uncharacterized functions. Three mutant isolates of HSV-1 strain KOS which grew on S22 cells but not on normal Vero cells were isolated and characterized. All three mutants (hr27, hr48, and hr156) were defective in the synthesis of viral DNA and late proteins when grown in nonpermissive Vero cells. Early gene expression in cells infected with these host range mutants appeared to be normal at the nonpermissive condition. The mutations were mapped by marker rescue to a 1.5-kilobase fragment (coordinates 0.145 to 0.155). The mutation of one of these mutants, hr27, was more finely mapped to an 800-base-pair region (coordinates 0.145 to 0.151). This position of these mutations is consistent with the map location of a putative 94-kilodalton polypeptide as determined by sequence analysis (D. McGeoch, personal communication). Complementation studies demonstrated that these mutants formed a new complementation group, designated 1-36. The results presented in this report indicate that the 94-kilodalton gene product affected by these mutations may have a direct role in viral DNA synthesis.