Derek C. Norford

CB1 cannabinoid receptor-G protein association: a possible mechanism for differential signaling

Effects of cannabinoid compounds on neurons are predominantly mediated by the CB(1) cannabinoid receptor. Onset of signaling cascades in response to cannabimimetic drugs is triggered by the interaction of the cannabinoid receptor with G(i/o) proteins. Much work has been done to delineate the cannabinoid agonist-induced downstream signaling events; however, it remains to define the molecular basis of cannabinoid receptor-G protein interactions that stimulate these signaling pathways. In this review, we discuss several signal transduction pathways, focusing on studies that demonstrate the efficacy of CB(1) receptor agonists through G protein mediated pathways.

Cannabinoid receptor-mediated translocation of NO-sensitive guanylyl cyclase and production of cyclic GMP in neuronal cells

Cannabinoid agonists regulate NO and cyclic AMP production in N18TG2 neuroblastoma cells, leading to the hypothesis that neuronal cyclic GMP production could be regulated by CB(1) cannabinoid receptors. NO (nitric oxide)-sensitive guanylyl cyclase (GC) is a heterodimeric cytosolic protein that mediates the down-stream effects of NO. Genes of proteins in the cyclic GMP pathway (alpha(1), alpha(2), and beta(1) subunits of NO-sensitive GC and PKG1, but not PKG2) were expressed in N18TG2 cells, as was the CB(1) but not the CB(2) cannabinoid receptor. Stimulation of N18TG2 cells by cannabinoid agonists CP55940 and WIN55212-2 increased cyclic GMP levels in an ODQ-sensitive manner. GC-beta(1) in membrane fractions was increased after 5 or 20 min stimulation, and was significantly depleted in the cytosol by 1h. The cytosolic pool of GC-beta(1) was replenished after 48 h of continued cannabinoid drug treatment. Translocation of GC-beta(1) from the cytosol was blocked by the CB(1) antagonist rimonabant (SR141716) and by the Gi/o inactivator pertussis toxin, indicating that the CB(1) receptor and Gi/o proteins are required for translocation. Long-term treatment with rimonabant or pertussis toxin reduced the amount of GC-beta(1) in the cytosolic pool. We conclude that CB(1) receptors stimulate cyclic GMP production and that intracellular translocation of GC from cytosol to the membranes is intrinsic to the mechanism and may be a tonically active or endocannabinoid-regulated process.

Using BAC transgenesis in zebrafish to identify regulatory sequences of the amyloid precursor protein gene in humans

BackgroundNon-coding DNA in and around the human Amyloid Precursor Protein (APP) gene that is central to Alzheimer’s disease (AD) shares little sequence similarity with that of appb in zebrafish. Identifying DNA domains regulating expression of the gene in such situations becomes a challenge. Taking advantage of the zebrafish system that allows rapid functional analyses of gene regulatory sequences, we previously showed that two discontinuous DNA domains in zebrafish appb are important for expression of the gene in neurons: an enhancer in intron 1 and sequences 28–31 kb upstream of the gene. Here we identify the putative transcription factor binding sites responsible for this distal cis-acting regulation, and use that information to identify a regulatory region of the human APP gene.ResultsFunctional analyses of intron 1 enhancer mutations in enhancer-trap BACs expressed as transgenes in zebrafish identified putative binding sites of two known transcription factor proteins, E4BP4/ NFIL3 and Forkhead, to be required for expression of appb. A cluster of three E4BP4 sites at −31 kb is also shown to be essential for neuron-specific expression, suggesting that the dependence of expression on upstream sequences is mediated by these E4BP4 sites. E4BP4/ NFIL3 and XFD1 sites in the intron enhancer and E4BP4/ NFIL3 sites at −31 kb specifically and efficiently bind the corresponding zebrafish proteins in vitro. These sites are statistically over-represented in both the zebrafish appb and the human APP genes, although their locations are different. Remarkably, a cluster of four E4BP4 sites in intron 4 of human APP exists in actively transcribing chromatin in a human neuroblastoma cell-line, SHSY5Y, expressing APP as shown using chromatin immunoprecipitation (ChIP) experiments. Thus although the two genes share little sequence conservation, they appear to share the same regulatory logic and are regulated by a similar set of transcription factors.ConclusionThe results suggest that the clock-regulated and immune system modulator transcription factor E4BP4/ NFIL3 likely regulates the expression of both appb in zebrafish and APP in humans. It suggests potential human APP gene regulatory pathways, not on the basis of comparing DNA primary sequences with zebrafish appb but on the model of conservation of transcription factors.

Endocannabinoids and reactive nitrogen and oxygen species in neuropathologies.

Neuropathologies that affect our population include ischemic stroke and neurodegenerative diseases of immune origin, including multiple sclerosis. The endocannabinoid system in the brain, including agonists anandamide (arachidonyl ethanolamide) and 2-arachidonoylglycerol, and the CB1 and CB2 cannabinoid receptors, has been implicated in the pathophysiology of these disease states, and can be a target for therapeutic interventions. This review concentrates on cellular signal transduction pathways believed to be involved in the cellular damage.

Identifying Distal cis-acting Gene-Regulatory Sequences by Expressing BACs Functionalized with loxP-Tn10 Transposons in Zebrafish

Bacterial Artificial Chromosomes (BACs) are large pieces of DNA from the chromosomes of organisms propagated faithfully in bacteria as large extra-chromosomal plasmids. Expression of genes contained in BACs can be monitored after functionalizing the BAC DNA with reporter genes and other sequences that allow stable maintenance and propagation of the DNA in the new host organism. The DNA in BACs can be altered within its bacterial host in several ways. Here we discuss one such approach, using Tn10 mini-transposons, to introduce exogenous sequences into BACs for a variety of purposes. The largely random insertions of Tn10 transposons carrying lox sites have been used to position mammalian cell-selectable antibiotic resistance genes, enhancer-traps and inverted repeat ends of the vertebrate transposon Tol2 precisely at the ends of the genomic DNA insert in BACs. These modified BACs are suitable for expression in zebrafish or mouse, and have been used to functionally identify important long-range gene regulatory sequences in both species. Enhancer-trapping using BACs should prove uniquely useful in analyzing multiple discontinuous DNA domains that act in concert to regulate expression of a gene, and is not limited by genome accessibility issues of traditional enhancer-trapping methods.

Long range gene-regulatory sequences identified by transgenic expression of bacterially-engineered enhancer-trap BACs in zebrafish

Abstract Large pieces of DNA from the chromosomes of numerous organisms, including the human, are faithfully propagated in bacteria as large extra-chromosomal plasmids known as Bacterial Artificial Chromosomes (BACs). Because they

Harnessing mobile genetic elements to explore gene regulation

Sequences that regulate expression of a gene in cis but are located at large distances along the DNA from the gene, as found with most developmentally regulated genes in higher vertebrates, are difficult to identify if those sequences are not conserved across species. Mutating suspected gene-regulatory sequences to alter expression then becomes a hit-or-miss affair. The relaxed specificity of transposon insertions offers an opportunity to develop alternate strategies, to scan in an unbiased manner, pieces of chromosomal DNA cloned in BACs for transcription enhancing elements. This article illustrates how insertions of Tn10 with enhancer-traps into BAC DNA containing the gene, and its germ-line expression in zebrafish, have identified distal regulatory elements functionally. Transposition of Tn10 first introduces the enhancer-trap with a loxP site randomly into BAC DNA. Cre-recombination between the inserted loxP and the loxP endogenous to a BAC-end positions the enhancer-trap to the newly created truncated end of BAC DNA. The procedure generates a library of integration-ready enhancer-trap BACs with progressive truncations from an end in a single experiment. Individual enhancer-trap BACs from the library can be evaluated functionally in zebrafish or mice. Furthermore, the ability to readily alter sequences in a small transposon plasmid containing a regulatory domain of the gene allows re-introduction of altered parts of a BAC back into itself. It serves as a useful strategy to functionally dissect multiple discontinuous regulatory domains of a gene quickly. These methodologies have been successfully used in identifying novel regulatory domains of the Amyloid Precursor Protein (appb) gene in zebrafish, and provided important clues for regulation of the gene in humans.

Trapping Enhancers by Transgenic Expression of BACs Engineered in Bacteria with loxP Transposons

Bacterial Artificial Chromosomes (BACs) are large extra-chromosomal plasmids in bacteria that faithfully propagate large pieces of DNA from the chromosomes of organisms. Because they represent tiny contiguous pieces of the chromosome, BACs are ideally suited for expression of genes in their chromosomal contexts. Genes in BACs can be monitored for expression after the DNA is modified with reporter genes and other sequences that allow it to be stably propagated in the new host. Several methods have been developed to alter BAC DNA within its bacterial host. One approach uses Tn10 mini-transposons to introduce exogenous DNA into BACs. The random insertions of Tn10 carrying lox sites have directed mammalian cell-selectable antibiotic resistance genes, enhancer-traps and inverted repeat ends of the vertebrate transposon Tol2 precisely to the ends of genomic DNA inserts in BACs. Reporter gene expression from BAC DNA integrated into zebrafish or mouse chromosomes have resulted from such retrofitting. The methodology has been used extensively to analyze regulation of the Amyloid Precursor Protein (appb) gene in zebrafish. Functional identification of long-range regulatory sequences of appb has provided important clues for regulation of the APP gene in humans.

Functionalizing Bacterial Artificial Chromosomes with Transposons to Explore Gene Regulation

Strategies for altering sequences in large DNA inserts in BACs are fundamentally different from those traditionally used for small plasmids. Two factors are primarily responsible for this: the existence of a high multiplicity of sites in BACs recognized by DNA modifying enzymes, such as restriction endo-nucleases, as well as the brittle nature of large duplex DNA that is not packaged with DNA-binding proteins in the test-tube. The large number of DNA fragments generated by the common restriction enzymes, and the unavailability of robust separation techniques to isolate and keep track of the relative order of the pieces; precludes using the “cut-and-paste” mechanism to alter DNA sequences in BACs. Instead DNA recombination in vivo has become the method of choice for modifying BACs. Although the net result again is cutting and re-joining of DNA, the entire process is concerted in the bacterial host with no free ends of DNA to go astray; and occurs primarily in a nucleoprotein complex that protects it from shear forces which otherwise would break the large DNA during manipulations in vitro.