A Single Dose of Lysergic Acid Diethylamide Influences Gene Expression Patterns Within the Mammalian Brain

Neuropsychopharmacology : Official Publication of the American College of Neuropsychopharmacology. May, 2002  |  Pubmed ID: 11927188

Hallucinogenic drugs such as lysergic acid diethylamide (LSD) have profound effects on humans including hallucinations and detachment from reality. These remarkable behavioral effects have many similarities to the debilitating symptoms of neuropsychiatric disorders such as schizophrenia. The effects of hallucinogens are thought to be mediated by serotonin receptor activation; however, how these drugs elicit the unusual behavioral effects remains largely a mystery, despite much research. We have undertaken the first comprehensive analysis of gene expression influenced by acute LSD administration in the mammalian brain. These studies represent a novel approach to elucidate the mechanism of action of this class of drugs. We have identified a number of genes that are predicted to be involved in the processes of synaptic plasticity, glutamatergic signaling and cytoskeletal architecture. Understanding these molecular events will lead to new insights into the etiology of disorders whose behavioral symptoms resemble the temporary effects of hallucinogenic drugs, and also may ultimately result in new therapies.

Yan Regulates Lozenge During Drosophila Eye Development

Development Genes and Evolution. Jul, 2002  |  Pubmed ID: 12111211

The Drosophila eye offers an excellent opportunity to understand how general developmental processes are subtly altered to result in specific cell fates. Numerous transcription factors have been characterized in the developing eye; most of these are active in overlapping subsets of cells. Mechanisms used to regulate transcription factors act at many levels, and include competition for cognate binding sites, post translational modification, transcriptional regulation and cofactor availability. In undifferentiated cells of the larval eye imaginal disc, the transcriptional repressor Yan outcompetes the transcriptional activator Pointed for ETS binding sites on the prosperoenhancer. During differentiation, the Ras signaling cascade alters the Yan/Pointed dynamic through protein phosphorylation, effecting a developmental switch. In this way, Yan and Pointed are essential for prospero regulation. Hyperstable Yan (ACT) cannot be phosphorylated and blocks prospero expression. Lozenge is expressed in undifferentiated cells, and is required for prospero regulation. We sequenced the eye-specific enhancer of lozenge in three Drosophila species spanning 17 million years of evolution and found complete conservation of three ETS consensus binding sites. We show that lozengeexpression increases as cells differentiate, and that Yan (ACT) blocks this upregulation at the level of transcription. We find that expression of Lozenge via an alternate enhancer alters the temporal expression of Prospero, and is sufficient to rescue Prospero expression in the presence of Yan (ACT). These results suggest that Lozenge is involved in the Yan/Pointed dynamic in a Ras-dependent manner. We propose that upregulated Lozenge acts as a cofactor to alter Pointed affinity, by a mechanism that is recapitulated in mammalian development.

Dynamic Changes in Prefrontal Cortex Gene Expression Following Lysergic Acid Diethylamide Administration

Brain Research. Molecular Brain Research. Mar, 2003  |  Pubmed ID: 12654518

Lysergic acid diethylamide (LSD) is a psychoactive drug that transiently alters human perception, behavior, and mood at extremely low doses. Certain aspects of the behavior elicited by acute doses of LSD closely resemble symptoms of mental disorders such as schizophrenia. Characterizing gene expression profiles after LSD will be important for understanding how it alters behavior, and will lead to novel insights into disorders, such as schizophrenia, whose behavioral symptoms resemble the temporary effects of hallucinogenic drugs. We previously identified a small collection of genes within the rat prefrontal cortex that respond to LSD. Many of the products of these genes are involved in the process of synaptic plasticity. In the current report, we present a detailed analysis of the expression of these genes within the brain using RNase protection analysis. We find that the gene response to LSD is quite dynamic. The expression of some genes increases rapidly and decreases rapidly, while other genes change more gradually. Dose-response studies show two classes of expression; gene expression maximally stimulated at lower doses, versus gene expression that continues to rise at the higher doses. The role of the 5-HT(1A) and 5-HT(2A) receptor in mediating the increases in gene expression was examined in a series of experiments using receptor specific antagonists. Most expression increases were due to activation of the 5-HT(2A) receptor, however expression of two genes had neither a 5-HT(1A) nor a 5-HT(2A) receptor component.

Molecular Genetic Responses to Lysergic Acid Diethylamide Include Transcriptional Activation of MAP Kinase Phosphatase-1, C/EBP-beta and ILAD-1, a Novel Gene with Homology to Arrestins

Journal of Neurochemistry. Aug, 2004  |  Pubmed ID: 15255935

We recently demonstrated that the potent hallucinogenic drug lysergic acid diethylamide (LSD) dynamically influences the expression of a small collection of genes within the mammalian prefrontal cortex. Towards generating a greater understanding of the molecular genetic effects of hallucinogens and how they may relate to alterations in behavior, we have identified and characterized expression patterns of a new collection of three genes increased in expression by acute LSD administration. These genes were identified through additional screens of Affymetrix DNA microarrays and examined in experiments to assess dose-response, time course and the receptor mediating the expression changes. The first induced gene, C/EBP-beta, is a transcription factor. The second gene, MKP-1, suggests that LSD activates the MAP (mitogen activated protein) kinase pathway. The third gene, ILAD-1, demonstrates sequence similarity to the arrestins. The increase in expression of each gene was partially mediated through LSD interactions at 5-HT2A (serotonin) receptors. There is evidence of alternative splicing at the ILAD-1 locus. Furthermore, data suggests that various splice isoforms of ILAD-1 respond differently at the transcriptional level to LSD. The genes thus far found to be responsive to LSD are beginning to give a more complete picture of the complex intracellular events initiated by hallucinogens.

Alternative Splicing Removes an Ets Interaction Domain from Lozenge During Drosophila Eye Development

Development Genes and Evolution. Aug, 2005  |  Pubmed ID: 15868204

Physical and functional characteristics of the RUNX family of transcription factors are conserved between vertebrates and the Drosophila protein Lozenge. The runt-homology domain responsible for DNA binding and also the C-terminus are both nearly identical between the two proteins. The mammalian and fly proteins heterodimerize with a non-DNA binding partner protein to form a core binding factor essential for gene regulation during cell differentiation. The mammalian protein RUNX1 (AML1/PEBP2alphaB) interacts with the transcription factor Ets-1 to increase DNA binding and transactivation potential. Alternative splicing of the mammalian RUNX1 removes a domain required for this cooperative transactivation. In this work we determine the structure of the lozenge transcription unit and map 21 mutations. We show that the lozenge transcript is alternatively spliced during eye development to remove an Ets interaction domain. Emphasis is placed on Pointed the Drosophila homolog of the vertebrate Ets-1 protein; both Lozenge and Pointed proteins are needed for the activation of prospero expression. We use site-directed mutagenesis and yeast two-hybrid analysis to show that conserved amino acids within the alternate Lozenge exon are important for interaction with Pointed. Furthermore, the ectopic expression of Lozenge is sufficient to rescue Prospero expression in the presence of the Pointed competitor, Yan(ACT). We show that both lozenge isoforms are expressed during eye development and that the relative ratio of the transcripts for the two isoforms is sensitive to changes in Ras activity. We suggest that during eye development, Lozenge isoforms function in divergent roles, either interacting with Pointed on downstream targets or by functioning independently to establish distinct cell fates.

Drosophila Melanogaster Neurobiology, Neuropharmacology, and How the Fly Can Inform Central Nervous System Drug Discovery

Pharmacology & Therapeutics. Dec, 2006  |  Pubmed ID: 16935347

Central nervous system (CNS) drug discovery in the post-genomic era is rapidly evolving. Older empirical methods are giving way to newer technologies that include bioinformatics, structural biology, genetics, and modern computational approaches. In the search for new medical therapies, and in particular treatments for disorders of the central nervous system, there has been increasing recognition that identification of a single biological target is unlikely to be a recipe for success; a broad perspective is required. Systems biology is one such approach, and has been increasingly recognized as a very important area of research, as it places specific molecular targets within a context of overall biochemical action. Understanding the complex interactions between the components within a given biological system that lead to modifications in output, such as changes in behavior or development, may be important avenues of discovery to identify new therapies. One avenue to drug discovery that holds tremendous potential is the use of model genetic organisms such as the fruit fly, Drosophila melanogaster. The similarity between mode of drug action, behavior, and gene response in D. melanogaster and mammalian systems, combined with the power of genetics, have recently made the fly a very attractive system to study fundamental neuropharmacological processes relevant to human diseases. The promise that the use of model organisms such as the fly offers is speed, high throughput, and dramatically reduced overall costs that together should result in an enhanced rate of discovery.

5-HT2 Receptors in Drosophila Are Expressed in the Brain and Modulate Aspects of Circadian Behaviors

Developmental Neurobiology. May, 2007  |  Pubmed ID: 17443822

Dysregulation of 5-HT(2) receptor function has been strongly implicated in many neuropsychiatric disorders, including schizophrenia. At present, the molecular mechanisms linking 5-HT(2) receptor activation to behaviors is not well understood. In efforts to elucidate these processes, the fruit fly, Drosophila melanogaster, is proposed to serve as a powerful genetically tractable model organism to study 5-HT(2) receptor function. Data are presented here on the expression of the fly ortholog of the mammalian 5-HT(2) receptor, 5-HT(2)Dro, in the larval and adult brain of the fly, and on the involvement of these circuits in certain circadian behaviors. In the adult brain, 5-HT(2)Dro is expressed in the protocerebrum and ellipsoid body, areas believed to participate in higher order behaviors including learning, locomotion, and sensory perception. In the third instar larva, 5-HT(2)Dro receptor expression is detected in a specific pattern that markedly changes from early to late third instar. To probe the function of this receptor we have examined the effects of the 5-HT(2) receptor-specific agonist DOI in wild type and 5-HT(2)Dro hypomorphic flies on circadian behaviors. DOI was found to increase early day activity, eliminate anticipatory behavior, and reduce viability. The effects of DOI were significantly diminished in a 5-HT(2)Dro hypomorphic strain. Identifying the 5-HT(2)Dro receptor circuitry and behaviors they mediate are significant steps towards developing this model system to study conserved molecular mechanisms underlying behaviors mediated by 5-HT(2) receptors in mammalian systems.

Identification of Neuroprotective Compounds of Caenorhabditis Elegans Dopaminergic Neurons Against 6-OHDA

Journal of Molecular Neuroscience : MN. 2007  |  Pubmed ID: 17478886

Parkinson's disease (PD) is a severe debilitating disorder, characterized by progressive and selective dopaminergic (DAergic) neuron degeneration within the substantia nigra pars compacta. Although current pharmacological treatments are effective in early stages of the disease, with time, most patients fail to respond to medications and develop serious motor complications. Therefore, devising novel and efficacious therapeutics that address not only the symptoms of PD, but also the cause, are of great importance. Unfortunately, many obstacles are associated with current PD research in mammalian-based systems, which limit the rate of progress. One solution is to investigate mechanisms of PD in model genetic organisms like Caenorhabditis elegans. In general, striking and profound similarities underlie the basic cellular and molecular processes between the worm and humans. The use of C. elegans over traditional mammalian-based systems holds the promise of an enhanced rate of discovery with lower associated costs. Here, we have utilized C. elegans to screen a variety of compounds, including specific dopamine (DA), GABA, and NMDA receptor agonists, as well as antagonists to identify those that protect against 6-OHDA-induced DAergic toxicity. Two DA D2 receptor agonists, bromocriptine and quinpirole, were found to protect against 6-OHDA toxicity in a dose-dependent manner. Surprisingly, these protective effects appear to involve receptor-independent mechanisms. Given the high degree of conservation of cellular processes between the worm and mammalian systems, these results are likely relevant and important toward understanding potentially novel mechanisms leading to DAergic neuroprotection in mammalian systems and, ultimately, new therapeutics for PD.

Serotonin Receptors

Chemical Reviews. May, 2008  |  Pubmed ID: 18476671

Serotonin 5-hydroxytryptamine(2A) Receptor Activation Suppresses Tumor Necrosis Factor-alpha-induced Inflammation with Extraordinary Potency

The Journal of Pharmacology and Experimental Therapeutics. Nov, 2008  |  Pubmed ID: 18708586

The G protein-coupled serotonin 5-hydroxytryptamine (5-HT)(2A) receptor is primarily recognized for its role in brain neurotransmission, where it mediates a wide variety of functions, including certain aspects of cognition. However, there is significant expression of this receptor in peripheral tissues, where its importance is largely unknown. We have now discovered that activation of 5-HT(2A) receptors in primary aortic smooth muscle cells provides a previously unknown and extremely potent inhibition of tumor necrosis factor (TNF)-alpha-mediated inflammation. 5-HT(2A) receptor stimulation with the agonist (R)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane [(R)-DOI] rapidly inhibits a variety of TNF-alpha-mediated proinflammatory markers, including intracellular adhesion molecule 1 (ICAM-1), vascular adhesion molecule 1 (VCAM-1), and interleukin (IL)-6 gene expression, nitric-oxide synthase activity, and nuclear translocation of nuclear factor kappaB, with IC(50) values of only 10 to 20 pM. It is significant that proinflammatory markers can also be inhibited by (R)-DOI hours after treatment with TNF-alpha. With the exception of a few natural toxins, no current drugs or small molecule therapeutics demonstrate a comparable potency for any physiological effect. TNF-alpha-mediated inflammatory pathways have been strongly implicated in a number of diseases, including atherosclerosis, rheumatoid arthritis, psoriasis, type II diabetes, depression, schizophrenia, and Alzheimer's disease. Our results indicate that activation of 5-HT(2A) receptors represents a novel, and extraordinarily potent, potential therapeutic avenue for the treatment of disorders involving TNF-alpha-mediated inflammation. Note that because (R)-DOI can significantly inhibit the effects of TNF-alpha many hours after the administration of TNF-alpha, potential therapies could be aimed not only at preventing inflammation but also treating inflammatory injury that has already occurred or is ongoing.

Anterograde Trafficking of G Protein-coupled Receptors: Function of the C-terminal F(X)6LL Motif in Export from the Endoplasmic Reticulum

Molecular Pharmacology. Apr, 2009  |  Pubmed ID: 19118123

We have reported previously that the F(X)(6)LL motif in the C termini is essential for export of alpha(2B)-adrenergic (alpha(2B)-AR) and angiotensin II type 1 receptors (AT1Rs) from the endoplasmic reticulum (ER). Here, we further demonstrate that mutation of the F(X)(6)LL motif similarly abolished the cell-surface expression of alpha(2B)-AR, AT1R, alpha(1B)-AR, and beta(2)-AR, suggesting that the F(X)(6)LL motif plays a general role in ER export of G protein-coupled receptors (GPCRs). Mutation of Phe to Val, Leu, Trp, and Tyr, and mutation of LL to FF and VV, markedly inhibited alpha(2B)-AR transport, indicating that the F(X)(6)LL function cannot be fully substituted by other hydrophobic residues. The structural analysis revealed that the Phe residue in the F(X)(6)LL motif is buried in the transmembrane domains and possibly interacts with Ile58 in beta(2)-AR and Val42 in alpha(2B)-AR, whereas the LL motif is exposed to the cytosolic space. Indeed, mutation of Ile58 in beta(2)-AR and Val42 in alpha(2B)-AR markedly disrupted cell surface transport of the receptors. It is noteworthy that the Val and Ile residues are highly conserved among the GPCRs carrying the F(X)(6)LL motif. Furthermore, the Phe mutant exhibited a stronger interaction with ER chaperones and was more potently rescued by physical and chemical treatments than the LL mutant. These data suggest that the Phe residue is probably involved in folding of alpha(2B)-AR and beta(2)-AR, possibly through interaction with other hydrophobic residues in neighboring domains. These data also provide the first evidence implying crucial roles of the C termini possibly through modulating multiple events in anterograde trafficking of GPCRs.

Engineered G-protein Coupled Receptors Are Powerful Tools to Investigate Biological Processes and Behaviors

Frontiers in Molecular Neuroscience. 2009  |  Pubmed ID: 19893765

Understanding how discreet tissues and neuronal circuits function in relation to the whole organism to regulate physiological processes and behaviors is a fundamental goal of modern biological science. Powerful and important new tools in this discovery process are modified G-protein coupled receptors (GPCRs) known as 'Receptors Activated Solely by Synthetic Ligands (RASSLs),' and 'Designer Receptors Exclusively Activated by a Designer Drug (DREADDs).' Collectively, these are GPCRs modified either through rational design (RASSLs) or directed molecular evolution (DREADDs), that do not respond to native ligand, but functionally respond only to synthetic ligands. Importantly, the utility of these receptors is not limited to examination of the role of GPCR-coupled effector signal transduction pathways. Due to the near ubiquitous expression of GPCRs throughout an organism, this technology, combined with whole animal transgenics to selectively target expression, has the ability to regulate activity of discreet tissues and neuronal circuits through effector pathway modulation to study function and behavior throughout the organism. Advantages over other systems currently used to modify in vivo function include the ability to rapidly, selectively and reversibly manipulate defined signal transduction pathways both in short term and long term studies, and no need for specialized equipment due to convenient systemic treatment with activating ligand.

Serotonin 5-HT(2A) Receptor Function As a Contributing Factor to Both Neuropsychiatric and Cardiovascular Diseases

Cardiovascular Psychiatry and Neurology. 2009  |  Pubmed ID: 20029624

There are high levels of comorbidity between neuropsychiatric and cardiovascular disorders. A key molecule central to both cognitive and cardiovascular function is the molecule serotonin. In the brain, serotonin modulates neuronal activity and is actively involved in mediating many cognitive functions and behaviors. In the periphery, serotonin is involved in vasoconstriction, inflammation, and cell growth, among other processes. It is hypothesized that one component of the serotonin system, the 5-HT(2A) receptor, is a common and contributing factor underlying aspects of the comorbidity between neuropsychiatric and cardiovascular disorders. Within the brain this receptor participates in processes such as cognition and working memory, been implicated in effective disorders such as schizophrenia, and mediate the primary effects of hallucinogenic drugs. In the periphery, 5-HT(2A) receptors have been linked to vasoconstriction and hypertension, and to inflammatory processes that can lead to atherosclerosis.

An Animal Model of Schizophrenia Based on Chronic LSD Administration: Old Idea, New Results

Neuropharmacology. Sep, 2011  |  Pubmed ID: 21352832

Many people who take LSD experience a second temporal phase of LSD intoxication that is qualitatively different, and was described by Daniel Freedman as "clearly a paranoid state." We have previously shown that the discriminative stimulus effects of LSD in rats also occur in two temporal phases, with initial effects mediated by activation of 5-HT(2A) receptors (LSD30), and the later temporal phase mediated by dopamine D2-like receptors (LSD90). Surprisingly, we have now found that non-competitive NMDA antagonists produced full substitution in LSD90 rats, but only in older animals, whereas in LSD30, or in younger animals, these drugs did not mimic LSD. Chronic administration of low doses of LSD (>3 months, 0.16 mg/kg every other day) induces a behavioral state characterized by hyperactivity and hyperirritability, increased locomotor activity, anhedonia, and impairment in social interaction that persists at the same magnitude for at least three months after cessation of LSD treatment. These behaviors, which closely resemble those associated with psychosis in humans, are not induced by withdrawal from LSD; rather, they are the result of neuroadaptive changes occurring in the brain during the chronic administration of LSD. These persistent behaviors are transiently reversed by haloperidol and olanzapine, but are insensitive to MDL-100907. Gene expression analysis data show that chronic LSD treatment produced significant changes in multiple neurotransmitter system-related genes, including those for serotonin and dopamine. Thus, we propose that chronic treatment of rats with low doses of LSD can serve as a new animal model of psychosis that may mimic the development and progression of schizophrenia, as well as model the established disease better than current acute drug administration models utilizing amphetamine or NMDA antagonists such as PCP.

Human Disease Models in Drosophila Melanogaster and the Role of the Fly in Therapeutic Drug Discovery

Pharmacological Reviews. Jun, 2011  |  Pubmed ID: 21415126

The common fruit fly, Drosophila melanogaster, is a well studied and highly tractable genetic model organism for understanding molecular mechanisms of human diseases. Many basic biological, physiological, and neurological properties are conserved between mammals and D. melanogaster, and nearly 75% of human disease-causing genes are believed to have a functional homolog in the fly. In the discovery process for therapeutics, traditional approaches employ high-throughput screening for small molecules that is based primarily on in vitro cell culture, enzymatic assays, or receptor binding assays. The majority of positive hits identified through these types of in vitro screens, unfortunately, are found to be ineffective and/or toxic in subsequent validation experiments in whole-animal models. New tools and platforms are needed in the discovery arena to overcome these limitations. The incorporation of D. melanogaster into the therapeutic discovery process holds tremendous promise for an enhanced rate of discovery of higher quality leads. D. melanogaster models of human diseases provide several unique features such as powerful genetics, highly conserved disease pathways, and very low comparative costs. The fly can effectively be used for low- to high-throughput drug screens as well as in target discovery. Here, we review the basic biology of the fly and discuss models of human diseases and opportunities for therapeutic discovery for central nervous system disorders, inflammatory disorders, cardiovascular disease, cancer, and diabetes. We also provide information and resources for those interested in pursuing fly models of human disease, as well as those interested in using D. melanogaster in the drug discovery process.

The Serotonin 5-HT7Dro Receptor is Expressed in the Brain of Drosophila, and is Essential for Normal Courtship and Mating

PloS One. 2011  |  Pubmed ID: 21674056

The 5-HT(7) receptor remains one of the less well characterized serotonin receptors. Although it has been demonstrated to be involved in the regulation of mood, sleep, and circadian rhythms, as well as relaxation of vascular smooth muscles in mammals, the precise mechanisms underlying these functions remain largely unknown. The fruit fly, Drosophila melanogaster, is an attractive model organism to study neuropharmacological, molecular, and behavioral processes that are largely conserved with mammals. Drosophila express a homolog of the mammalian 5-HT(7) receptor, as well as homologs for the mammalian 5-HT(1A), and 5-HT(2), receptors. Each fly receptor couples to the same effector pathway as their mammalian counterpart and have been demonstrated to mediate similar behavioral responses. Here, we report on the expression and function of the 5-HT(7)Dro receptor in Drosophila. In the larval central nervous system, expression is detected postsynaptically in discreet cells and neuronal circuits. In the adult brain there is strong expression in all large-field R neurons that innervate the ellipsoid body, as well as in a small group of cells that cluster with the PDF-positive LNvs neurons that mediate circadian activity. Following both pharmacological and genetic approaches, we have found that 5-HT(7)Dro activity is essential for normal courtship and mating behaviors in the fly, where it appears to mediate levels of interest in both males and females. This is the first reported evidence of direct involvement of a particular serotonin receptor subtype in courtship and mating in the fly.

Insulin-producing Cells in the Brain of Adult Drosophila Are Regulated by the Serotonin 5-HT1A Receptor

Cellular and Molecular Life Sciences : CMLS. Feb, 2012  |  Pubmed ID: 21818550

Insulin signaling regulates lifespan, reproduction, metabolic homeostasis, and resistance to stress in the adult organism. In Drosophila, there are seven insulin-like peptides (DILP1-7). Three of these (DILP2, 3 and 5) are produced in median neurosecretory cells of the brain, designated IPCs. Previous work has suggested that production or release of DILPs in IPCs can be regulated by a factor secreted from the fat body as well as by neuronal GABA or short neuropeptide F. There is also evidence that serotonergic neurons may regulate IPCs. Here, we investigated mechanisms by which serotonin may regulate the IPCs. We show that the IPCs in adult flies express the 5-HT(1A), but not the 5-HT(1B) or 5-HT(7) receptors, and that processes of serotonergic neurons impinge on the IPC branches. Knockdown of 5-HT(1A) in IPCs by targeted RNA interference (RNAi) leads to increased sensitivity to heat, prolonged recovery after cold knockdown and decreased resistance to starvation. Lipid metabolism is also affected, but no effect on growth was seen. Furthermore, we show that DILP2-immunolevels in IPCs increase after 5-HT(1A) knockdown; this is accentuated by starvation. Heterozygous 5-HT(1A) mutant flies display the same phenotype in all assays, as seen after targeted 5-HT(1A) RNAi, and flies fed the 5-HT(1A) antagonist WAY100635 display reduced lifespan at starvation. Our findings suggest that serotonin acts on brain IPCs via the 5-HT(1A) receptor, thereby affecting their activity and probably insulin signaling. Thus, we have identified a second inhibitory pathway regulating IPC activity in the Drosophila brain.