100 results on '"Denovan-Wright EM"'
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2. The dynamic nature of type 1 cannabinoid receptor ( CB1) gene transcription.
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Laprairie, RB, Kelly, MEM, and Denovan-Wright, EM
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CANNABINOID receptors ,GENETIC transcription ,HUNTINGTON disease ,INFLAMMATION ,PARKINSON'S disease ,MESSENGER RNA ,PATHOLOGY - Abstract
The type 1 cannabinoid receptor ( CB
1 ) is an integral component of the endocannabinoid system that modulates several functions in the CNS and periphery. The majority of our knowledge of the endocannabinoid system involves ligand-receptor binding, mechanisms of signal transduction, and protein-protein interactions. In contrast, comparatively little is known about regulation of CB1 gene expression. The levels and anatomical distribution of CB1 m RNA and protein are developmental stage-specific and are dysregulated in several pathological conditions. Moreover, exposure to a variety of drugs, including cannabinoids themselves, alters CB1 gene expression and m RNA levels. As such, alterations in CB1 gene expression are likely to affect the optimal response to cannabinoid-based therapies, which are being developed to treat a growing number of conditions. Here, we will examine the regulation of CB1 m RNA levels and the therapeutic potential inherent in manipulating expression of this gene. Linked Articles This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.167.issue-8 [ABSTRACT FROM AUTHOR]- Published
- 2012
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3. Enantiomeric Agonists of the Type 2 Cannabinoid Receptor Reduce Retinal Damage during Proliferative Vitreoretinopathy and Inhibit Hyperactive Microglia In Vitro .
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Young AP, Szczesniak AM, Hsu K, Kelly MEM, and Denovan-Wright EM
- Abstract
Microglia are resident immune cells of the central nervous system (CNS) and propagate inflammation following damage to the CNS, including the retina. Proliferative vitreoretinopathy (PVR) is a condition that can emerge following retinal detachment and is characterized by severe inflammation and microglial proliferation. The type 2 cannabinoid receptor (CB
2 ) is an emerging pharmacological target to suppress microglial-mediated inflammation when the eyes or brain are damaged. CB2 -knockout mice have exacerbated inflammation and retinal pathology during experimental PVR. We aimed to assess the anti-inflammatory effects of CB2 stimulation in the context of retinal damage and also explore the mechanistic roles of CB2 in microglia function. To target CB2 , we used a highly selective agonist, HU-308, as well as its enantiomer, HU-433, which is a putative selective agonist. First, β-arrestin2 and Gαi recruitment was measured to compare activation of human CB2 in an in vitro heterologous expression system. Both agonists were then utilized in a mouse model of PVR, and the effects on retinal damage, inflammation, and cell death were assessed. Finally, we used an in vitro model of microglia to determine the effects of HU-308 and HU-433 on phagocytosis, cytokine release, migration, and intracellular signaling. We observed that HU-308 more strongly recruited both β-arrestin2 and Gαi compared to HU-433. Stimulation of CB2 with either drug effectively blunted LPS- and IFNγ-mediated signaling as well as NO and TNF release from microglia. Furthermore, both drugs reduced IL-6 accumulation, total caspase-3 cleavage, and retinal pathology following the induction of PVR. Ultimately, this work supports that CB2 is a valuable target for drugs to suppress inflammation and cell death associated with infection or sterile retinopathy, although the magnitude of effector recruitment may not be predictive of anti-inflammatory capacity., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)- Published
- 2024
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4. JAK1/2 Regulates Synergy Between Interferon Gamma and Lipopolysaccharides in Microglia.
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Young AP and Denovan-Wright EM
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- Microglia, Signal Transduction, Cytokines metabolism, NF-kappa B metabolism, Interferon-gamma pharmacology, Lipopolysaccharides toxicity
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Microglia, the resident immune cells of the brain, regulate neuroinflammation which can lead to secondary neuronal damage and cognitive impairment under pathological conditions. Two of the many molecules that can elicit an inflammatory response from microglia are lipopolysaccharide (LPS), a component of gram-negative bacteria, and interferon gamma (IFNγ), an endogenous pro-inflammatory cytokine. We thoroughly examined the concentration-dependent relationship between LPS from multiple bacterial species and IFNγ in cultured microglia and macrophages. We measured the effects that these immunostimulatory molecules have on pro-inflammatory activity of microglia and used a battery of signaling inhibitors to identify the pathways that contribute to the microglial response. We found that LPS and IFNγ interacted synergistically to induce a pro-inflammatory phenotype in microglia, and that inhibition of JAK1/2 completely blunted the response. We determined that this synergistic action of LPS and IFNγ was likely dependent on JNK and Akt signaling rather than typical pro-inflammatory mediators such as NF-κB. Finally, we demonstrated that LPS derived from Escherichia coli, Klebsiella pneumoniae, and Akkermansia muciniphila can elicit different inflammatory responses from microglia and macrophages, but these responses could be consistently prevented using ruxolitinib, a JAK1/2 inhibitor. Collectively, this work reveals a mechanism by which microglia may become hyperactivated in response to the combination of LPS and IFNγ. Given that elevations in circulating LPS and IFNγ occur in a wide variety of pathological conditions, it is critical to understand the pharmacological interactions between these molecules to develop safe and effective treatments to suppress this process., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)
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- 2024
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5. The microglial endocannabinoid system is similarly regulated by lipopolysaccharide and interferon gamma.
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Young AP and Denovan-Wright EM
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- Endocannabinoids metabolism, Endocannabinoids pharmacology, Interferon-gamma metabolism, Interferon-gamma pharmacology, Lipopolysaccharides metabolism, Lipopolysaccharides pharmacology, Nitric Oxide metabolism, RNA, Messenger metabolism, Cannabinoids metabolism, Cannabinoids pharmacology, Microglia metabolism
- Abstract
Perturbation of the endocannabinoid system can have profound effects on immune function and synaptic plasticity. Microglia are one of few cell types with a self-contained endocannabinoid system and are positioned at the interface between the immune system and the central nervous system. Past work has produced conflicting results with respect to the effects of pro-inflammatory conditions on the microglial endocannabinoid system. Thus, we systematically investigated the relationship between the concentration of two distinct pro-inflammatory stimuli, lipopolysaccharide and interferon gamma, on the abundance of components of the endocannabinoid system within microglia. Here we show that lipopolysaccharide and interferon gamma influence messenger RNA abundances of the microglial endocannabinoid system in a concentration-dependent manner. Furthermore, we demonstrate that the efficacy of different synthetic cannabinoid treatments with respect to inhibition of microglia nitric oxide release is dependent on the concentration and type of pro-inflammatory stimuli presented to the microglia. This indicates that different pro-inflammatory stimuli influence the capacity of microglia to synthesize, degrade, and respond to cannabinoids which has implications for the development of cannabinoid-based treatments for neuroinflammation., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to report., (Copyright © 2022 Elsevier B.V. All rights reserved.)
- Published
- 2022
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6. Synthetic cannabinoids reduce the inflammatory activity of microglia and subsequently improve neuronal survival in vitro.
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Young AP and Denovan-Wright EM
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- Cells, Cultured, Endocannabinoids metabolism, Receptor, Cannabinoid, CB1 metabolism, Receptor, Cannabinoid, CB2 metabolism, Receptors, Cannabinoid metabolism, Cannabinoids pharmacology, Microglia metabolism
- Abstract
Microglia are resident immune cells of the brain that survey the microenvironment, provide trophic support to neurons, and clear debris to maintain homeostasis and healthy brain function. Microglia are also drivers of neuroinflammation in several neurodegenerative diseases. Microglia produce endocannabinoids and express both cannabinoid receptor subtypes suggesting that this system is a target to suppress neuroinflammation. We tested whether cannabinoid type 1 (CB
1 ) or type 2 (CB2 ) receptors could be targeted selectively or in combination to dampen the pro-inflammatory behavior of microglia, and whether this would have functional relevance to decrease secondary neuronal damage. We determined that components of the endocannabinoid system were altered when microglia are treated with lipopolysaccharide and interferon-gamma and shift to a pro-inflammatory phenotype. Furthermore, pro-inflammatory microglia released cytotoxic factors that induced cell death in cultured STHdhQ7/Q7 neurons. Treatment with synthetic cannabinoids that were selective for CB1 receptors (ACEA) or CB2 receptors (HU-308) dampened the release of nitric oxide (NO) and pro-inflammatory cytokines and decreased levels of mRNA for several pro-inflammatory markers. A nonselective agonist (CP 55,940) exhibited similar influence over NO release but to a lesser extent relative to ACEA or HU-308. All three classes of synthetic cannabinoids ultimately reduced the secondary damage to the cultured neurons. The mechanism for the observed neuroprotective effects appeared to be related to cannabinoid-mediated suppression of MAPK signaling in microglia. Taken together, the data indicate that activation of CB1 or CB2 receptors interfered with the pro-inflammatory activity of microglia in a manner that also reduced secondary damage to neurons., (Copyright © 2022 Elsevier Inc. All rights reserved.)- Published
- 2022
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7. The Dynamic Role of Microglia and the Endocannabinoid System in Neuroinflammation.
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Young AP and Denovan-Wright EM
- Abstract
Microglia, the resident immune cells of the brain, can take on a range of pro- or anti-inflammatory phenotypes to maintain homeostasis. However, the sustained activation of pro-inflammatory microglia can lead to a state of chronic neuroinflammation characterized by high concentrations of neurotoxic soluble factors throughout the brain. In healthy brains, the inflammatory processes cease and microglia transition to an anti-inflammatory phenotype, but failure to halt the pro-inflammatory processes is a characteristic of many neurological disorders. The endocannabinoid system has been identified as a promising therapeutic target for chronic neuroinflammation as there is evidence that synthetic and endogenously produced cannabinoids temper the pro-inflammatory response of microglia and may encourage a switch to an anti-inflammatory phenotype. Activation of cannabinoid type 2 (CB
2 ) receptors has been proposed as the mechanism of action responsible for these effects. The abundance of components of the endocannabinoid system in microglia also change dynamically in response to several brain pathologies. This can impact the ability of microglia to synthesize and degrade endocannabinoids or react to endogenous and exogenous cannabinoids. Cannabinoid receptors also participate in the formation of receptor heteromers which influences their function specifically in cells that express both receptors, such as microglia. This creates opportunities for drug-drug interactions between CB2 receptor-targeted therapies and other classes of drugs. In this article, we review the roles of pro- and anti-inflammatory microglia in the development and resolution of neuroinflammation. We also discuss the fluctuations observed in the components of the endocannabinoid in microglia and examine the potential of CB2 receptors as a therapeutic target in this context., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Young and Denovan-Wright.)- Published
- 2022
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8. Genetic counselling for the prevention of mental health consequences of cannabis use: A randomized controlled trial-within-cohort.
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Zwicker A, LeBlanc MA, Pavlova B, Alda M, Denovan-Wright EM, Uher R, and Austin JC
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- Adolescent, Child, Cohort Studies, Genetic Counseling, Humans, Mental Health, Cannabis, Psychotic Disorders genetics, Psychotic Disorders prevention & control
- Abstract
Background: Cannabis use is a risk factor for severe mental illness. However, cannabis does not affect everyone equally. Genetic information may help identify individuals who are more vulnerable to the harmful effects of cannabis on mental health. A common genetic variant within the AKT1 gene selectively increases risk of psychosis, only among those who use cannabis. Therapeutically oriented genetic counselling may enable us to reduce cannabis exposure among genetically sensitive individuals., Methods: Using a trial-within-cohort design, we aim to test if genetic counselling, including the option to receive AKT1 rs2494732 genotype, reduces cannabis use. To this end, we have designed a genetic counselling intervention: Interdisciplinary approach to Maximize Adolescent potential: Genetic counselling Intervention to reduce Negative Environmental effects (IMAGINE)., Results: IMAGINE will be implemented in a cohort of children and youth enriched for familial risk for major mood and psychotic disorders. Approximately 110 eligible individuals aged 12-21 years will be randomized in a 1:1 ratio to be offered a single genetic counselling session with a board-certified genetic counsellor, or not. Allocated youth will also be invited to attend a follow-up session approximately 1 month following the intervention. The primary outcome will be cannabis use (measured by self-report or urine screen) at subsequent annual assessments as part of the larger cohort study. Secondary outcomes include intervention acceptability and psychopathology., Conclusion: This study represents the first translational application of a gene-environment interaction to improve mental health and test an intervention with potential public health benefits. This study is registered with clinicaltrials.gov (NCT03601026)., (© 2020 John Wiley & Sons Australia, Ltd.)
- Published
- 2021
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9. Endothelin B receptor dysfunction mediates elevated myogenic tone in cerebral arteries from aged male Fischer 344 rats.
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Young AP, Zhu J, Bagher AM, Denovan-Wright EM, Howlett SE, and Kelly MEM
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- Animals, Gene Knockout Techniques, Male, Rats, Rats, Inbred F344, Cerebral Arteries, Receptor, Endothelin B, Vasoconstriction
- Abstract
The human brain requires adequate cerebral blood flow to meet the high demand for nutrients and to clear waste products. With age, there is a chronic reduction in cerebral blood flow in small resistance arteries that can eventually limit proper brain function. The endothelin system is a key mediator in the regulation of cerebral blood flow, but the contributions of its constituent receptors in the endothelial and vascular smooth muscle layers of cerebral arteries have not been well defined in the context of aging. We isolated posterior cerebral arteries from young and aged Fischer 344 rats, as well as ET
B receptor knock-out rats and mounted the vessels in plexiglass pressure myograph chambers to measure myogenic tone in response to increasing pressure and targeted pharmacological treatments. We used an ETA receptor antagonist (BQ-123), an ETB receptor antagonist (BQ-788), endothelin-1, an endothelin-1 synthesis inhibitor (phosphoramidon), and vessel denudation to dissect the roles of each receptor in aging vasculature. Aged rats exhibited a higher myogenic tone than young rats, and the tone was sensitive to the ETA antagonist, BQ-123, but insensitive to the ETB antagonist, BQ-788. By contrast, the tone in the vessels from young rats was raised by BQ-788 but unaffected by BQ-123. When the endothelial layer that is normally enriched with ETB1 receptors was removed from young vessels, myogenic tone increased. However, denudation of the endothelial layer did not influence vessels from aged animals. This indicated that endothelial ETB1 receptors were not functional in the vessels from aged rats. There was also an increase in ETA receptor expression with age, whereas ETB receptor expression remained constant between young and aged animals. These results demonstrate that in young vessels, ETB1 receptors maintain a lower myogenic tone, but in aged vessels, a loss of ETB receptor activity allows ETA receptors in vascular smooth muscle cells to raise myogenic tone. Our findings have potentially important clinical implications for treatments to improve cerebral perfusion in older adults with diseases characterized by reduced cerebral blood flow.- Published
- 2021
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10. Antipsychotic potential of the type 1 cannabinoid receptor positive allosteric modulator GAT211: preclinical in vitro and in vivo studies.
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McElroy DL, Roebuck AJ, Scott GA, Greba Q, Garai S, Denovan-Wright EM, Thakur GA, Laprairie RB, and Howland JG
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- Animals, Behavior, Animal drug effects, Cell Line, Dizocilpine Maleate, Dose-Response Relationship, Drug, Dronabinol pharmacology, Excitatory Amino Acid Antagonists, Extracellular Signal-Regulated MAP Kinases metabolism, Male, Motor Activity drug effects, Phosphorylation, Prepulse Inhibition drug effects, Psychoses, Substance-Induced drug therapy, Psychoses, Substance-Induced psychology, Rats, Rats, Long-Evans, Antipsychotic Agents pharmacology, Indoles pharmacology, Receptor, Cannabinoid, CB1 drug effects
- Abstract
Rationale: Antipsychotics help alleviate the positive symptoms associated with schizophrenia; however, their debilitating side effects have spurred the search for better treatment options. Novel compounds can be screened for antipsychotic potential in neuronal cell cultures and following acute N-methyl-D-aspartate (NMDA) receptor blockade with non-competitive antagonists such as MK-801 in rodent behavioral models. Given the known interactions between NMDA receptors and type 1 cannabinoid receptors (CB1R), compounds that modulate CB1Rs may have therapeutic potential for schizophrenia., Objectives: This study assessed whether the CB1R positive allosteric modulator GAT211, when compared to ∆
9 -tetrahydrocannabinol (THC), has potential to reduce psychiatric behavioral phenotypes following acute MK-801 treatment in rats, and block hyperdopaminergic signalling associated with those behaviors., Methods: The effects of GAT211 and THC on cellular signaling were compared in Neuro2a cells, and behavioral effects of GAT211 and THC on altered locomotor activity and prepulse inhibition of the acoustic startle response caused by acute MK-801 treatment were assessed in male, Long Evans rats., Results: GAT211 limited dopamine D2 receptor-mediated extracellular regulated kinase (ERK) phosphorylation in Neuro2a cells, whereas THC did not. As expected, acute MK-801 (0.15 mg/kg) produced a significant increase in locomotor activity and impaired PPI. GAT211 treatment alone (0.3-3.0 mg/kg) dose-dependently reduced locomotor activity and the acoustic startle response. GAT211 (3.0 mg/kg) also prevented hyperlocomotion caused by MK-801 but did not significantly affect PPI impairments., Conclusion: Taken together, these findings support continued preclinical research regarding the usefulness of CB1R positive allosteric modulators as antipsychotics.- Published
- 2021
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11. Prevalence of methylenetetrahydrofolate reductase gene polymorphisms (C677T, and A1298C) among Saudi children receiving dental treatment.
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Bagher AM, Young AP, Neamatallah T, Al-Amoudi RM, Bagher SM, and Denovan-Wright EM
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- Child, Cross-Sectional Studies, Dental Care, Humans, Prevalence, Saudi Arabia epidemiology, Methylenetetrahydrofolate Reductase (NADPH2) genetics, Polymorphism, Genetic
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Background: Methylenetetrahydrofolate reductase, the encoded by the MTHFR gene, plays a crucial role in converting the amino acid homocysteine to methionine. Two polymorphisms of the MTHFR gene, C677T and A1298C, reportedly reduce enzyme activity, resulting in hyperhomocysteinemia. Patients with C677T and A1298C polymorphisms may be at higher risk for developing abnormal hyperhomocysteinemia, which has been linked to catastrophic neurological including fatal outcomes., Objective: Determine the prevalence of the MTHFR gene variants C677T and A1298C among pediatric dental patients treated at King Abdulaziz University Hospital., Design: Cross-sectional., Setting: Clinics of pediatric dentistry department., Subjects and Methods: Healthy Saudi children 6-12 years old with no known allergies were screened for eligibility between May and December 2019. A single investigator collected saliva samples. The MTHFR C677T and A1298C polymorphisms were analyzed using polymerase chain reaction and restriction fragment length polymorphism., Main Outcome Measure: The prevalence of MTHFR gene variants (C677T and A1298C) among the subjects., Sample Size: 138., Results: MTHFR C677T polymorphism was present in 36.2% of the sample and 90.0% of children carrying this allele were heterozygotes. MTHFR A1298C polymorphism was present in 91.3% of the sample and 77.0% of the children carrying this allele were heterozygotes. No linkage disequilibrium between MTHFR C677T and MTHFR A1298C was observed within this sample., Conclusions: Our study found a high frequency of the MTHFR A1298C genotype, which was substantially more abundant than expected based on a Hardy-Weinberg distribution. Therefore, caution is advised in using N
2 O in Saudi children as the increased prevalence of this MTHFR allele may increase the incidence of serious adverse effects among these children., Limitations: Further studies are recommended with a larger sample size from randomly selected hospitals from different regions of Saudi Arabia., Conflict of Interest: None.- Published
- 2021
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12. Heteromer formation between cannabinoid type 1 and dopamine type 2 receptors is altered by combination cannabinoid and antipsychotic treatments.
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Bagher AM, Young AP, Laprairie RB, Toguri JT, Kelly MEM, and Denovan-Wright EM
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- Animals, Cell Line, Transformed, Drug Therapy, Combination, HEK293 Cells, Humans, Male, Mice, Mice, Inbred C57BL, Receptor, Cannabinoid, CB1 agonists, Antipsychotic Agents administration & dosage, Cannabinoid Receptor Agonists administration & dosage, Cannabinoids administration & dosage, Receptor, Cannabinoid, CB1 metabolism, Receptors, Dopamine D2 metabolism
- Abstract
The cannabinoid type 1 (CB
1 ) receptor and the dopamine type 2 (D2 ) receptor are co-localized on medium spiny neuron terminals in the globus pallidus where they modulate neural circuits involved in voluntary movement. Physical interactions between the two receptors have been shown to alter receptor signaling in cell culture. The objectives of the current study were to identify the presence of CB1 /D2 heteromers in the globus pallidus of C57BL/6J male mice, define how CB1 /D2 heteromer levels are altered following treatment with cannabinoids and/or antipsychotics, and determine if fluctuating levels of CB1 /D2 heteromers have functional consequences. Using in situ proximity ligation assays, we observed CB1 /D2 heteromers in the globus pallidus of C57BL/6J mice. The abundance of the heteromers increased following treatment with the nonselective cannabinoid receptor agonist, CP55,940. In contrast, treatment with the typical antipsychotic haloperidol reduced the number of CB1 /D2 heteromers, whereas the atypical antipsychotic olanzapine treatment had no effect. Co-treatment with CP55,940 and haloperidol had similar effects to haloperidol alone, whereas co-treatment with CP55,940 and olanzapine had similar effects to CP55,940. The observed changes were found to have functional consequences as the differential effects of haloperidol and olanzapine also applied to γ-aminobutyric acid release in STHdhQ7/Q7 cells and motor function in C57BL/6J male mice. This work highlights the clinical relevance of co-exposure to cannabinoids and different antipsychotics over acute and prolonged time periods., (© 2020 Wiley Periodicals LLC.)- Published
- 2020
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13. Endothelin receptor heteromerization inhibits β-arrestin function in HEK293 cells.
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Zrein A, Bagher AM, Young AP, Denovan-Wright EM, and Kelly MEM
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- Extracellular Signal-Regulated MAP Kinases metabolism, HEK293 Cells, Humans, Phosphorylation, Protein Structure, Quaternary, Signal Transduction, Protein Multimerization, Receptor, Endothelin A chemistry, Receptor, Endothelin B chemistry, beta-Arrestins metabolism
- Abstract
The endothelin receptor A (ETA) and endothelin receptor B (ETB) are G protein-coupled receptors that are co-expressed in vascular smooth muscle cells. Endothelin-1 (ET-1) activates endothelin receptors to cause microvascular vasoconstriction. Previous studies have shown that heteromerization between ETA and ETB prolongs Ca
2+ transients, leading to prolongation of Gαq -dependent signaling and sustained vasoconstriction. We hypothesized that these effects are in part mediated by the resistance of ETA/ETB heteromers to β-arrestin recruitment and subsequent desensitization. Using bioluminescence resonance energy transfer 2 (BRET2 ), we found that ETB has a relatively equal affinity to form either homomers or heteromers with ETA when co-expressed in the human embryonic kidney 293 (HEK293) cells. When co-expressed, activation of ETA and ETB by ET-1 caused a heteromer-specific reduction and delay in β-arrestin-2 recruitment with a corresponding reduction and delay in ET-1-induced ETA/ETB co-internalization. Furthermore, the co-expression of ETA and ETB inhibited ET-1-induced β-arrestin-1-dependent extracellular signal-regulated kinase (ERK) phosphorylation while prolonging ET-1-induced Gαq -dependent ERK phosphorylation. ETA/ETB heteromerization mediates the long-lasting vasoconstrictor response to ET-1 by the prolongation of Gαq -dependent signaling and inhibition of β-arrestin function.- Published
- 2020
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14. Neurodevelopmental and genetic determinants of exposure to adversity among youth at risk for mental illness.
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Zwicker A, MacKenzie LE, Drobinin V, Bagher AM, Howes Vallis E, Propper L, Bagnell A, Abidi S, Pavlova B, Alda M, Denovan-Wright EM, and Uher R
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- Adolescent, Bullying, Child, Female, Humans, Intelligence genetics, Internal-External Control, Male, Mental Disorders physiopathology, Mental Disorders psychology, Multifactorial Inheritance genetics, Psychopathology, Socioeconomic Factors, Adverse Childhood Experiences psychology, Attention Deficit Disorder with Hyperactivity genetics, Genetic Predisposition to Disease, Mental Disorders etiology, Mental Disorders genetics
- Abstract
Background: Attention-deficit/hyperactivity disorder (ADHD) and lower cognitive ability have been linked with increased likelihood of exposure to adversity. We hypothesized that these associations may be partly due to genetic factors., Methods: We calculated polygenic scores for ADHD and intelligence and assessed psychopathology and general cognitive ability in a sample of 297 youth aged 5-27 years enriched for offspring of parents with mood and psychotic disorders. We calculated an adversity score as a mean of 10 indicators, including socio-economic disadvantage, childhood maltreatment and bullying. We tested the effects of polygenic scores, externalizing symptoms and IQ on adversity scores using mixed-effects linear regression., Results: Externalizing symptoms and general cognitive ability showed expected positive and negative relationships with adversity, respectively. Polygenic scores for intelligence were unrelated to adversity, but polygenic scores for ADHD were associated with adversity (β = 0.23, 95% CI 0.13 to 0.34, p < .0001). ADHD polygenic scores uniquely explained 4.0% of variance in adversity score. The relationship between polygenic scores for ADHD and adversity was independently significant among individuals with (β = 0.49, 95% CI 0.25 to 0.75, p < .0001) and without (β = 0.14, 95% CI 0.02 to 0.26, p = .022) ADHD., Conclusions: A genetic score indexing liability to ADHD was associated with exposure to adversity in early life. Previously observed associations between externalizing symptoms, lower cognitive ability and adversity may be partially attributed to genetic liability to ADHD., (© 2019 Association for Child and Adolescent Mental Health.)
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- 2020
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15. Application of Fluorine- and Nitrogen-Walk Approaches: Defining the Structural and Functional Diversity of 2-Phenylindole Class of Cannabinoid 1 Receptor Positive Allosteric Modulators.
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Garai S, Kulkarni PM, Schaffer PC, Leo LM, Brandt AL, Zagzoog A, Black T, Lin X, Hurst DP, Janero DR, Abood ME, Zimmowitch A, Straiker A, Pertwee RG, Kelly M, Szczesniak AM, Denovan-Wright EM, Mackie K, Hohmann AG, Reggio PH, Laprairie RB, and Thakur GA
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- Allosteric Regulation drug effects, Animals, Anti-Inflammatory Agents, Non-Steroidal pharmacokinetics, Anti-Inflammatory Agents, Non-Steroidal pharmacology, Biotransformation, Freund's Adjuvant, HEK293 Cells, Humans, Indoles pharmacokinetics, Indoles pharmacology, Inflammation chemically induced, Inflammation prevention & control, Male, Mice, Mice, Inbred C57BL, Neurons drug effects, Receptor, Cannabinoid, CB1 agonists, Stereoisomerism, Structure-Activity Relationship, Anti-Inflammatory Agents, Non-Steroidal chemical synthesis, Fluorine chemistry, Indoles chemistry, Nitrogen chemistry, Receptor, Cannabinoid, CB1 drug effects
- Abstract
Cannabinoid 1 receptor (CB1R) allosteric ligands hold a far-reaching therapeutic promise. We report the application of fluoro- and nitrogen-walk approaches to enhance the drug-like properties of GAT211, a prototype CB1R allosteric agonist-positive allosteric modulator (ago-PAM). Several analogs exhibited improved functional potency (cAMP, β-arrestin 2), metabolic stability, and aqueous solubility. Two key analogs, GAT591 ( 6r ) and GAT593 ( 6s ), exhibited augmented allosteric-agonist and PAM activities in neuronal cultures, improved metabolic stability, and enhanced orthosteric agonist binding (CP55,940). Both analogs also exhibited good analgesic potency in the CFA inflammatory-pain model with longer duration of action over GAT211 while being devoid of adverse cannabimimetic effects. Another analog, GAT592 ( 9j ), exhibited moderate ago-PAM potency and improved aqueous solubility with therapeutic reduction of intraocular pressure in murine glaucoma models. The SAR findings and the enhanced allosteric activity in this class of allosteric modulators were accounted for in our recently developed computational model for CB1R allosteric activation and positive allosteric modulation.
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- 2020
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16. Indomethacin Enhances Type 1 Cannabinoid Receptor Signaling.
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Laprairie RB, Mohamed KA, Zagzoog A, Kelly MEM, Stevenson LA, Pertwee R, Denovan-Wright EM, and Thakur GA
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In addition to its known actions as a non-selective cyclooxygenase (COX) 1 and 2 inhibitor, we hypothesized that indomethacin can act as an allosteric modulator of the type 1 cannabinoid receptor (CB1R) because of its shared structural features with the known allosteric modulators of CB1R. Indomethacin enhanced the binding of [
3 H]CP55940 to hCB1R and enhanced AEA-dependent [35 S]GTPγS binding to hCB1R in Chinese hamster ovary (CHO) cell membranes. Indomethacin (1 μM) also enhanced CP55940-dependent βarrestin1 recruitment, cAMP inhibition, ERK1/2 and PLCβ3 phosphorylation in HEK293A cells expressing hCB1R, but not in cells expressing hCB2R. Finally, indomethacin enhanced the magnitude and duration of CP55940-induced hypolocomotion, immobility, hypothermia, and anti-nociception in C57BL/6J mice. Together, these data support the hypothesis that indomethacin acted as a positive allosteric modulator of hCB1R. The identification of structural and functional features shared amongst allosteric modulators of CB1R may lead to the development of novel compounds designed for greater CB1R or COX selectivity and compounds designed to modulate both the prostaglandin and endocannabinoid systems., (Copyright © 2019 Laprairie, Mohamed, Zagzoog, Kelly, Stevenson, Pertwee, Denovan-Wright and Thakur.)- Published
- 2019
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17. Positive allosteric modulation of the type 1 cannabinoid receptor reduces the signs and symptoms of Huntington's disease in the R6/2 mouse model.
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Laprairie RB, Bagher AM, Rourke JL, Zrein A, Cairns EA, Kelly MEM, Sinal CJ, Kulkarni PM, Thakur GA, and Denovan-Wright EM
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- Animals, Cannabinoid Receptor Agonists pharmacology, Cell Survival drug effects, Corpus Striatum metabolism, Disease Models, Animal, Disease Progression, Gene Expression drug effects, Huntington Disease metabolism, Huntington Disease physiopathology, Indoles pharmacology, Male, Mice, Mice, Transgenic, Motor Activity drug effects, Motor Activity physiology, Allosteric Regulation drug effects, Cannabinoid Receptor Agonists therapeutic use, Corpus Striatum drug effects, Huntington Disease drug therapy, Indoles therapeutic use, Receptor, Cannabinoid, CB1 metabolism
- Abstract
Huntington's disease (HD) is an inherited progressive neurodegenerative disease characterized by motor, cognitive, and behavioural changes. One of the earliest changes to occur in HD is a reduction in cannabinoid 1 receptor (CB
1 ) levels in the striatum, which is strongly correlated with HD pathogenesis. CB1 positive allosteric modulators (PAM) enhance receptor affinity for, and efficacy of activation by, orthosteric ligands, including the endocannabinoids anandamide and 2-arachidonoylglycerol. The goal of this study was to determine whether the recently characterized CB1 allosteric modulators GAT211 (racemic), GAT228 (R-enantiomer), and GAT229 (S-enantiomer), affected the signs and symptoms of HD. GAT211, GAT228, and GAT229 were evaluated in normal and HD cell models, and in a transgenic mouse model of HD (7-week-old male R6/2 mice, 10 mg/kg/d, 21 d, i.p.). GAT229 was a CB1 PAM that improved cell viability in HD cells and improved motor coordination, delayed symptom onset, and normalized gene expression in R6/2 HD mice. GAT228 was an allosteric agonist that did not enhance endocannabinoid signaling or change symptom progression in R6/2 mice. GAT211 displayed intermediate effects between its enantiomers. The compounds used here are not drugs, but probe compounds used to determine the potential utility of CB1 PAMs in HD. Changes in gene expression, and not protein, were quantified in R6/2 HD mice because HD pathogenesis is associated with dysregulation of mRNA levels. The data presented here provide the first proof of principle for the use of CB1 PAMs to treat the signs and symptoms of HD., (Copyright © 2019. Published by Elsevier Ltd.)- Published
- 2019
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18. Allosteric and orthosteric pharmacology of cannabidiol and cannabidiol-dimethylheptyl at the type 1 and type 2 cannabinoid receptors.
- Author
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Tham M, Yilmaz O, Alaverdashvili M, Kelly MEM, Denovan-Wright EM, and Laprairie RB
- Subjects
- Camphanes pharmacology, Cyclohexanols pharmacology, HEK293 Cells, Humans, Indoles pharmacology, Ligands, Molecular Docking Simulation, Piperidines pharmacology, Protein Binding, Pyrazoles pharmacology, Receptor, Cannabinoid, CB1 genetics, Receptor, Cannabinoid, CB2 genetics, Transfection, Allosteric Regulation drug effects, Cannabidiol pharmacology, Receptor, Cannabinoid, CB1 metabolism, Receptor, Cannabinoid, CB2 metabolism
- Abstract
Background and Purpose: We sought to understand why (-)-cannabidiol (CBD) and (-)-cannabidiol-dimethylheptyl (CBD-DMH) exhibit distinct pharmacology, despite near identical structures., Experimental Approach: HEK293A cells expressing either human type 1 cannabinoid (CB
1 ) receptors or CB2 receptors were treated with CBD or CBD-DMH with or without the CB1 and CB2 receptor agonist CP55,940, CB1 receptor allosteric modulator Org27569 or CB2 receptor inverse agonist SR144528. Ligand binding, cAMP levels and βarrestin1 recruitment were measured. CBD and CBD-DMH binding was simulated with models of human CB1 or CB2 receptors, based on the recently published crystal structures of agonist-bound (5XRA) or antagonist-bound (5TGZ) human CB1 receptors., Key Results: At CB1 receptors, CBD was a negative allosteric modulator (NAM), and CBD-DMH was a mixed agonist/positive allosteric modulator. CBD and Org27569 shared multiple interacting residues in the antagonist-bound model of CB1 receptors (5TGZ) but shared a binding site with CP55,940 in the agonist-bound model of CB1 receptors (5XRA). The binding site for CBD-DMH in the CB1 receptor models overlapped with CP55,940 and Org27569. At CB2 receptors, CBD was a partial agonist, and CBD-DMH was a positive allosteric modulator of cAMP modulation but a NAM of βarrestin1 recruitment. CBD, CP55,940 and SR144528 shared a binding site in the CB2 receptor models that was separate from CBD-DMH., Conclusion and Implications: The pharmacological activity of CBD and CBD-DMH in HEK293A cells and their modelled binding sites at CB1 and CB2 receptors may explain their in vivo effects and illuminates the difficulties associated with the development of allosteric modulators for CB1 and CB2 receptors., Linked Articles: This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc., (© 2018 The British Pharmacological Society.)- Published
- 2019
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19. Huntington's disease pattern of transcriptional dysregulation in the absence of mutant huntingtin is produced by knockout of neuronal GLT-1.
- Author
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Laprairie RB, Petr GT, Sun Y, Fischer KD, Denovan-Wright EM, and Rosenberg PA
- Subjects
- Animals, Astrocytes metabolism, Cerebral Cortex metabolism, Corpus Striatum metabolism, Hippocampus metabolism, Homeostasis physiology, Mice, Inbred C57BL, Mice, Knockout, Excitatory Amino Acid Transporter 2 genetics, Huntington Disease genetics, Huntington Disease metabolism, Neurons metabolism
- Abstract
GLT-1 is the major glutamate transporter in the brain, and is expressed in astrocytes and in axon terminals in the hippocampus, cortex, and striatum. Neuronal GLT-1 accounts for only 5-10% of total brain GLT-1 protein, and its function is uncertain. In HD, synaptic dysfunction of the corticostriate synapse is well-established. Transcriptional dysregulation is a key feature of HD. We hypothesized that deletion of neuronal GLT-1, because it is expressed in axon terminals in the striatum, might produce a synaptopathy similar to that present in HD. If true, then some of the gene expression changes observed in HD might also be observed in the neuronal GLT-1 knockout. In situ hybridization using
33 P labeled oligonucleotide probes was carried out to assess localization and expression of a panel of genes known to be altered in expression in HD. We found changes in the expression of cannabinoid receptors 1 and 2, preproenkaphalin, and PDE10A in the striatum of mice in which the GLT-1 gene was inactivated in neurons by expression of synapsin-Cre, compared to wild-type littermates. These changes in expression were observed at 12 weeks of age but not at 6 weeks of age. No changes in DARPP-32, PDE1B, NGFIA, or β-actin expression were observed. In addition, we found widespread alteration in expression of the dynamin 1 gene. The changes in expression in the neuronal GLT-1 knockout of genes thought to exemplify HD transcriptional dysregulation suggest an overlap in the synaptopathy caused by neuronal GLT-1 deletion and HD. These data further suggest that specific changes in expression of cannabinoid receptors, preproenkephalin, and PDE10A, considered to be the hallmark of HD transcriptional dysregulation, may be produced by an abnormality of glutamate homeostasis under the regulation of neuronal GLT-1, or a synaptic disturbance caused by that abnormality, independently of mutation in huntingtin., (Copyright © 2018 Elsevier Ltd. All rights reserved.)- Published
- 2019
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20. Combining SRET 2 and BiFC to Study GPCR Heteromerization and Protein-Protein Interactions.
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Bagher AM, Kelly MEM, and Denovan-Wright EM
- Subjects
- Fluorescence, Humans, Microscopy, Fluorescence, Bioluminescence Resonance Energy Transfer Techniques methods, Fluorescence Resonance Energy Transfer methods, Fluorescent Antibody Technique methods, Luciferases, Renilla metabolism, Protein Multimerization, Receptors, G-Protein-Coupled chemistry, Receptors, G-Protein-Coupled metabolism
- Abstract
G protein-coupled receptors (GPCRs) are the target for many drugs. Evidence continues to accumulate demonstrating that multiple receptors form homo- and heteromeric complexes, which in turn dynamically couple with G proteins, and other interacting proteins. Here, we describe a method to simultaneously determine the identity of up to four distinct constituents of GPCR complexes using a combination of sequential bioluminescence resonance energy transfer 2-fluorescence resonance energy transfer (SRET
2 ) with bimolecular fluorescence complementation (BiFC). The method is amenable to moderate throughput screening of changes in response to ligands and time-course analysis of protein-protein oligomerization.- Published
- 2019
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21. Gene-environment interplay in the etiology of psychosis.
- Author
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Zwicker A, Denovan-Wright EM, and Uher R
- Subjects
- Humans, Gene-Environment Interaction, Psychotic Disorders epidemiology, Psychotic Disorders etiology, Psychotic Disorders genetics, Schizophrenia epidemiology, Schizophrenia etiology, Schizophrenia genetics
- Abstract
Schizophrenia and other types of psychosis incur suffering, high health care costs and loss of human potential, due to the combination of early onset and poor response to treatment. Our ability to prevent or cure psychosis depends on knowledge of causal mechanisms. Molecular genetic studies show that thousands of common and rare variants contribute to the genetic risk for psychosis. Epidemiological studies have identified many environmental factors associated with increased risk of psychosis. However, no single genetic or environmental factor is sufficient to cause psychosis on its own. The risk of developing psychosis increases with the accumulation of many genetic risk variants and exposures to multiple adverse environmental factors. Additionally, the impact of environmental exposures likely depends on genetic factors, through gene-environment interactions. Only a few specific gene-environment combinations that lead to increased risk of psychosis have been identified to date. An example of replicable gene-environment interaction is a common polymorphism in the AKT1 gene that makes its carriers sensitive to developing psychosis with regular cannabis use. A synthesis of results from twin studies, molecular genetics, and epidemiological research outlines the many genetic and environmental factors contributing to psychosis. The interplay between these factors needs to be considered to draw a complete picture of etiology. To reach a more complete explanation of psychosis that can inform preventive strategies, future research should focus on longitudinal assessments of multiple environmental exposures within large, genotyped cohorts beginning early in life.
- Published
- 2018
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22. Methods to Quantify Cell Signaling and GPCR Receptor Ligand Bias: Characterization of Drugs that Target the Endocannabinoid Receptors in Huntington's Disease.
- Author
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Bagher AM, Laprairie RB, Kelly MEM, and Denovan-Wright EM
- Subjects
- Animals, Cannabinoid Receptor Agonists therapeutic use, Cell Culture Techniques instrumentation, Cell Culture Techniques methods, Cell Survival drug effects, Corpus Striatum pathology, Endocannabinoids metabolism, Fluorescent Antibody Technique instrumentation, HEK293 Cells, Humans, Huntingtin Protein genetics, Huntingtin Protein metabolism, Huntington Disease genetics, Huntington Disease pathology, Immunoassay instrumentation, Immunoassay methods, Ligands, Mice, Receptor, Cannabinoid, CB1 agonists, Cannabinoid Receptor Agonists pharmacology, Fluorescent Antibody Technique methods, Huntington Disease drug therapy, Receptor, Cannabinoid, CB1 metabolism, Signal Transduction drug effects
- Abstract
G protein-coupled receptors (GPCRs) interact with multiple intracellular effector proteins such that different ligands may preferentially activate one signal pathway over others, a phenomenon known as signaling bias. Signaling bias can be quantified to optimize drug selection for preclinical research. Here, we describe moderate-throughput methods to quantify signaling bias of known and novel compounds. In the example provided, we describe a method to define cannabinoid-signaling bias in a cell culture model of Huntington's disease (HD). Decreasing type 1 cannabinoid receptor (CB
1 ) levels is correlated with chorea and cognitive deficits in HD. There is evidence that elevating CB1 levels and/or signaling may be beneficial for HD patients while decreasing CB1 levels and/or signaling may be detrimental. Recent studies have found that Gαi/o -biased CB1 agonists activate extracellular signal-regulated kinase (ERK), increase CB1 protein levels, and improve viability of cells expressing mutant huntingtin. In contrast, CB1 agonists that are β-arrestin1-biased were found to reduce CB1 protein levels and cell viability. Measuring agonist bias of known and novel CB1 agonists will provide important data that predict CB1 -specific agonists that might be beneficial in animal models of HD and, following animal testing, in HD patients. This method can also be applied to study signaling bias for other GPCRs.- Published
- 2018
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23. Differential regulation of the duplicated fabp7, fabp10 and fabp11 genes of zebrafish by peroxisome proliferator activated receptors.
- Author
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Laprairie RB, Denovan-Wright EM, and Wright JM
- Subjects
- Animals, Fatty Acid-Binding Protein 7 genetics, Fatty Acid-Binding Proteins genetics, HEK293 Cells, Humans, NF-kappa B p50 Subunit genetics, NF-kappa B p50 Subunit metabolism, Peroxisome Proliferator-Activated Receptors genetics, Promoter Regions, Genetic physiology, Zebrafish genetics, Zebrafish Proteins genetics, Fatty Acid-Binding Protein 7 biosynthesis, Fatty Acid-Binding Proteins biosynthesis, Gene Duplication, Gene Expression Regulation physiology, Peroxisome Proliferator-Activated Receptors metabolism, Zebrafish metabolism, Zebrafish Proteins biosynthesis
- Abstract
In the duplication-degeneration-complementation model, duplicated gene-pairs undergo nonfunctionalization (loss from the genome), subfunctionalization (the functions of the ancestral gene are sub-divided between duplicate genes), or neofunctionalization (one of the duplicate genes acquires a new function). These processes occur by loss or gain of regulatory elements in gene promoters. Fatty acid-binding proteins (Fabp) belong to a multigene family composed of orthologous proteins that are highly conserved in sequence and function, but differ in their gene regulation. We previously reported that the zebrafish fabp1a, fabp1b.1, and fabp1b.2 promoters underwent subfunctionalization of PPAR responsiveness. Here, we describe the regulation at the duplicated zebrafish fabp7a/fabp7b, fabp10a/fabp10b and fabp11a/fabp11b gene promoters. Differential control at the duplicated fabp promoters was assessed by DNA sequence analysis, responsiveness to PPAR-isoform specific agonists and NF-κB p50 antagonists in zebrafish liver and intestine explant tissue, and in HEK293A cells transfected with fabp promoter-reporter constructs. Each zebrafish fabp gene displayed unique transcriptional regulation compared to its paralogous duplicate. This work provides a framework to account for the evolutionary trajectories that led to the high retention (57%) of duplicated fabp genes in the zebrafish genome compared to only ~3% of all duplicated genes in the zebrafish genome., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2017
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24. Bidirectional allosteric interactions between cannabinoid receptor 1 (CB 1 ) and dopamine receptor 2 long (D 2L ) heterotetramers.
- Author
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Bagher AM, Laprairie RB, Toguri JT, Kelly MEM, and Denovan-Wright EM
- Subjects
- Allosteric Site, Arachidonic Acids pharmacology, Extracellular Signal-Regulated MAP Kinases metabolism, GTP-Binding Protein alpha Subunits metabolism, HEK293 Cells, Humans, Phosphorylation drug effects, Protein Structure, Quaternary, Quinpirole pharmacology, Receptor, Cannabinoid, CB1 agonists, Receptors, Dopamine D2 agonists, Signal Transduction drug effects, beta-Arrestin 1 metabolism, Protein Multimerization drug effects, Receptor, Cannabinoid, CB1 chemistry, Receptor, Cannabinoid, CB1 metabolism, Receptors, Dopamine D2 chemistry, Receptors, Dopamine D2 metabolism
- Abstract
Type 1 cannabinoid (CB
1 ) and dopamine 2 long form (D2L ) receptors can physically interact to form heteromers that display unique pharmacology in vitro compared to homomeric complexes. Co-expression of CB1 and D2L and co-application of CB1 and D2 agonists increases cAMP levels while administration of either agonist alone decreases cAMP levels. To understand the observed co-agonist response, our first goal of the current study was to define the stoichiometry of CB1 /D2L /Gα protein complexes. Using bioluminescence resonance energy transfer 2 (BRET2 ), we confirmed that, CB1 homodimers, D2L homodimers, and CB1 /D2L heteromers are formed. By using sequential resonance energy transfer 2 (SRET2 ) combined with bimolecular fluorescence complementation (BiFC), we were able to demonstrate that CB1 /D2L form heterotetramers consisting of CB1 and D2L homodimers. We demonstrated that CB1 /D2L heterotetramers are coupled to at least two Gα proteins. The second aim of the study was to investigate allosteric effects of a D2L agonist (quinpirole) on CB1 receptor function and to investigate the effects of a CB1 agonist [arachidonyl-2-chloroethylamide (ACEA)] on D2L receptor function within CB1 /D2L heterotetramers. Treating cells co-expressing CB1 and D2L with both ACEA and quinpirole switched CB1 and D2L receptor coupling and signaling from Gαi to Gαs proteins, enhanced β-arrestin1 recruitment and receptor co-internalization. The concept of bidirectional allosteric interaction within CB1 /D2 heterotetramers has important implications for understanding the activity of receptor complexes in native tissues and under pathological conditions., (Copyright © 2017 Elsevier B.V. All rights reserved.)- Published
- 2017
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25. Enantiospecific Allosteric Modulation of Cannabinoid 1 Receptor.
- Author
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Laprairie RB, Kulkarni PM, Deschamps JR, Kelly MEM, Janero DR, Cascio MG, Stevenson LA, Pertwee RG, Kenakin TP, Denovan-Wright EM, and Thakur GA
- Subjects
- Allosteric Regulation drug effects, Allosteric Site drug effects, Animals, HEK293 Cells, Humans, Isomerism, Mice, Cannabinoid Receptor Agonists chemical synthesis, Cannabinoid Receptor Agonists chemistry, Cannabinoid Receptor Agonists pharmacology, Indoles chemical synthesis, Indoles chemistry, Indoles pharmacology, Receptor, Cannabinoid, CB1 agonists, Receptor, Cannabinoid, CB1 drug effects
- Abstract
The cannabinoid 1 receptor (CB1R) is one of the most widely expressed metabotropic G protein-coupled receptors in brain, and its participation in various (patho)physiological processes has made CB1R activation a viable therapeutic modality. Adverse psychotropic effects limit the clinical utility of CB1R orthosteric agonists and have promoted the search for CB1R positive allosteric modulators (PAMs) with the promise of improved drug-like pharmacology and enhanced safety over typical CB1R agonists. In this study, we describe the synthesis and in vitro and ex vivo pharmacology of the novel allosteric CB1R modulator GAT211 (racemic) and its resolved enantiomers, GAT228 (R) and GAT229 (S). GAT211 engages CB1R allosteric site(s), enhances the binding of the orthosteric full agonist [
3 H]CP55,490, and reduces the binding of the orthosteric antagonist/inverse agonist [3 H]SR141716A. GAT211 displayed both PAM and agonist activity in HEK293A and Neuro2a cells expressing human recombinant CB1R (hCB1R) and in mouse-brain membranes rich in native CB1R. GAT211 also exhibited a strong PAM effect in isolated vas deferens endogenously expressing CB1R. Each resolved and crystallized GAT211 enantiomer showed a markedly distinctive pharmacology as a CB1R allosteric modulator. In all biological systems examined, GAT211's allosteric agonist activity resided with the R-(+)-enantiomer (GAT228), whereas its PAM activity resided with the S-(-)-enantiomer (GAT229), which lacked intrinsic activity. These results constitute the first demonstration of enantiomer-selective CB1R positive allosteric modulation and set a precedent whereby enantiomeric resolution can decisively define the molecular pharmacology of a CB1R allosteric ligand.- Published
- 2017
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26. Cannabinoid receptor ligand bias: implications in the central nervous system.
- Author
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Laprairie RB, Bagher AM, and Denovan-Wright EM
- Subjects
- Animals, Central Nervous System metabolism, Central Nervous System physiopathology, Humans, Huntington Disease drug therapy, Ligands, Receptor, Cannabinoid, CB1 metabolism, Receptor, Cannabinoid, CB2 metabolism, Receptors, Cannabinoid, Signal Transduction, Huntington Disease physiopathology, Molecular Targeted Therapy, Receptors, G-Protein-Coupled metabolism
- Abstract
The G protein-coupled cannabinoid receptors CB
1 , CB2 , GPR18, and GPR55 regulate neurotransmission, pain, and inflammation and have been intensively investigated as potential drug targets. Each of these GPCRs is coupled to multiple effector proteins mediating divergent cellular signals. The ligand bias of cannabinoid-targeted compounds is only beginning to be quantified. Research into cannabinoid bias is now revealing correlations between bias in cell culture and functional outcomes in vivo. We present an example study of cannabinoid bias in the context of Huntington disease. In future, an understanding of cannabinoid receptor structure and quantification of ligand bias will optimize drug selection matched to patient population and disease., (Copyright © 2016 Elsevier Ltd. All rights reserved.)- Published
- 2017
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27. Subfunctionalization of peroxisome proliferator response elements accounts for retention of duplicated fabp1 genes in zebrafish.
- Author
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Laprairie RB, Denovan-Wright EM, and Wright JM
- Subjects
- Animals, Gene Expression Regulation, Humans, Mutation, PPAR alpha genetics, Promoter Regions, Genetic, Response Elements, Zebrafish, Fatty Acid-Binding Proteins genetics, Gene Duplication, Genes, Duplicate, Peroxisome Proliferators pharmacology, Zebrafish Proteins genetics
- Abstract
Background: In the duplication-degeneration-complementation (DDC) model, a duplicated gene has three possible fates: it may lose functionality through the accumulation of mutations (nonfunctionalization), acquire a new function (neofunctionalization), or each duplicate gene may retain a subset of functions of the ancestral gene (subfunctionalization). The role that promoter evolution plays in retention of duplicated genes in eukaryotic genomes is not well understood. Fatty acid-binding proteins (Fabp) belong to a multigene family that are highly conserved in sequence and function, but differ in their gene regulation, suggesting selective pressure is exerted via regulatory elements in the promoter., Results: In this study, we describe the PPAR regulation of zebrafish fabp1a, fabp1b.1, and fabp1b.2 promoters and compare them to the PPAR regulation of the spotted gar fabp1 promoter, representative of the ancestral fabp1 gene. Evolution of the fabp1 promoter was inferred by sequence analysis, and differential PPAR-agonist activation of fabp1 promoter activity in zebrafish liver and intestine explant cells, and in HEK293A cells transiently transfected with wild-type and mutated fabp1promoter-reporter gene constructs. The promoter activity of spotted gar fabp1, representative of the ancestral fabp1, was induced by both PPARα- and PPARγ-specific agonists, but displayed a biphasic response to PPARα activation. Zebrafish fabp1a was PPARα-selective, fabp1b.1 was PPARγ-selective, and fabp1b.2 was not regulated by PPAR., Conclusions: The zebrafish fabp1 promoters underwent two successive rounds of subfunctionalization with respect to PPAR regulation leading to retention of three zebrafish fabp1 genes with stimuli-specific regulation. Using a pharmacological approach, we demonstrated here the divergent regulation of the zebrafish fabp1a, fabp1b.1, and fabp1b.2 with regard to subfunctionalization of PPAR regulation following two rounds of gene duplication.
- Published
- 2016
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28. Mapping Cannabinoid 1 Receptor Allosteric Site(s): Critical Molecular Determinant and Signaling Profile of GAT100, a Novel, Potent, and Irreversibly Binding Probe.
- Author
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Laprairie RB, Kulkarni AR, Kulkarni PM, Hurst DP, Lynch D, Reggio PH, Janero DR, Pertwee RG, Stevenson LA, Kelly ME, Denovan-Wright EM, and Thakur GA
- Subjects
- Allosteric Regulation, Cannabinoids pharmacology, HEK293 Cells, Humans, Indoles chemistry, Indoles pharmacology, Isothiocyanates chemistry, Phenylurea Compounds chemistry, Phenylurea Compounds pharmacology, Piperidines chemistry, Piperidines pharmacology, Protein Binding, Pyridines chemistry, Pyridines pharmacology, Receptor, Cannabinoid, CB1 metabolism, Allosteric Site physiology, Isothiocyanates pharmacology, Receptor, Cannabinoid, CB1 chemistry, Signal Transduction drug effects
- Abstract
One of the most abundant G-protein coupled receptors (GPCRs) in brain, the cannabinoid 1 receptor (CB1R), is a tractable therapeutic target for treating diverse psychobehavioral and somatic disorders. Adverse on-target effects associated with small-molecule CB1R orthosteric agonists and inverse agonists/antagonists have plagued their translational potential. Allosteric CB1R modulators offer a potentially safer modality through which CB1R signaling may be directed for therapeutic benefit. Rational design of candidate, druglike CB1R allosteric modulators requires greater understanding of the architecture of the CB1R allosteric endodomain(s) and the capacity of CB1R allosteric ligands to tune the receptor's information output. We have recently reported the synthesis of a focused library of rationally designed, covalent analogues of Org27569 and PSNCBAM-1, two prototypic CB1R negative allosteric modulators (NAMs). Among the novel, pharmacologically active CB1R NAMs reported, the isothiocyanate GAT100 emerged as the lead by virtue of its exceptional potency in the [(35)S]GTPγS and β-arrestin signaling assays and its ability to label CB1R as a covalent allosteric probe with significantly reduced inverse agonism in the [(35)S]GTPγS assay as compared to Org27569. We report here a comprehensive functional profiling of GAT100 across an array of important downstream cell-signaling pathways and analysis of its potential orthosteric probe-dependence and signaling bias. The results demonstrate that GAT100 is a NAM of the orthosteric CB1R agonist CP55,940 and the endocannabinoids 2-arachidonoylglycerol and anandamide for β-arrestin1 recruitment, PLCβ3 and ERK1/2 phosphorylation, cAMP accumulation, and CB1R internalization in HEK293A cells overexpressing CB1R and in Neuro2a and STHdh(Q7/Q7) cells endogenously expressing CB1R. Distinctively, GAT100 was a more potent and efficacious CB1R NAM than Org27569 and PSNCBAM-1 in all signaling assays and did not exhibit the inverse agonism associated with Org27569 and PSNCBAM-1. Computational docking studies implicate C7.38(382) as a key feature of GAT100 ligand-binding motif. These data help inform the engineering of newer-generation, druggable CB1R allosteric modulators and demonstrate the utility of GAT100 as a covalent probe for mapping structure-function correlates characteristic of the druggable CB1R allosteric space.
- Published
- 2016
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29. Antagonism of Dopamine Receptor 2 Long Affects Cannabinoid Receptor 1 Signaling in a Cell Culture Model of Striatal Medium Spiny Projection Neurons.
- Author
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Bagher AM, Laprairie RB, Kelly ME, and Denovan-Wright EM
- Subjects
- Animals, Arachidonic Acids pharmacology, Cells, Cultured, Cyclic AMP Response Element-Binding Protein metabolism, Endocytosis drug effects, Extracellular Signal-Regulated MAP Kinases metabolism, GTP-Binding Protein alpha Subunits, Gi-Go metabolism, Haloperidol pharmacology, Humans, Kinetics, Male, Mice, Inbred C57BL, Models, Biological, Neurons drug effects, Phosphorylation drug effects, Protein Binding drug effects, Protein Isoforms metabolism, Protein Multimerization drug effects, beta-Arrestins metabolism, Corpus Striatum cytology, Dopamine D2 Receptor Antagonists pharmacology, Neurons metabolism, Receptor, Cannabinoid, CB1 metabolism, Receptors, Dopamine D2 metabolism
- Abstract
Activation of dopamine receptor 2 long (D2L) switches the signaling of type 1 cannabinoid receptor (CB1) from Gαi to Gαs, a process thought to be mediated through CB1-D2L heteromerization. Given the clinical importance of D2 antagonists, the goal of this study was to determine if D2 antagonists could modulate CB1 signaling. Interactions between CB1 and D2L, Gαi, Gαs, and β-arrestin1 were studied using bioluminescence resonance energy transfer 2 (BRET(2)) in STHdh(Q7/Q7) cells. CB1-dependent extracellular regulated kinase (ERK)1/2, CREB phosphorylation, and CB1 internalization following cotreatment of CB1 agonist and D2 antagonist were quantified. Preassembled CB1-Gαi complexes were detected by BRET(2) Arachidonyl-2'-chloroethylamide (ACEA), a selective CB1 agonist, caused a rapid and transient increase in BRET efficiency (BRETEff) between Gαi-Rluc and CB1-green fluorescent protein 2 (GFP(2)), and a Gαi-dependent increase in ERK phosphorylation. Physical interactions between CB1 and D2L were observed using BRET(2) Cotreatment of STHdh(Q7/Q7) cells with ACEA and haloperidol, a D2 antagonist, inhibited BRETEff signals between Gαi-Rluc and CB1-GFP(2) and reduced the EMax and pEC50 of ACEA-mediated Gαi-dependent ERK phosphorylation. ACEA and haloperidol cotreatments produced a delayed and sustained increase in BRETEff between Gαs-Rluc and CB1-GFP(2) and increased the EMax and pEC50 of ACEA-induced Gαs-dependent cAMP response element-binding protein phosphorylation. In cells expressing CB1 and D2L treated with ACEA, binding of haloperidol to D2 receptors switched CB1 coupling from Gαi to Gαs In addition, haloperidol treatment reduced ACEA-induced β-arrestin1 recruitment to CB1 and CB1 internalization. D2 antagonists allosterically modulate cannabinoid-induced CB1 coupling, signaling, and β-arrestin1 recruitment through binding to CB1-D2L heteromers. These findings indicate that D2 antagonism, like D2 agonists, change agonist-mediated CB1 coupling and signaling., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)
- Published
- 2016
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30. Divergent evolution of cis-acting peroxisome proliferator-activated receptor elements that differentially control the tandemly duplicated fatty acid-binding protein genes, fabp1b.1 and fabp1b.2, in zebrafish.
- Author
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Laprairie RB, Denovan-Wright EM, and Wright JM
- Subjects
- Animals, Base Sequence, Evolution, Molecular, Gene Duplication, Genes, Duplicate, Genetic Variation, HEK293 Cells, Humans, Mutation, Promoter Regions, Genetic, RNA, Messenger genetics, Transfection, Fatty Acid-Binding Proteins genetics, Peroxisome Proliferator-Activated Receptors genetics, Zebrafish genetics, Zebrafish Proteins genetics
- Abstract
Gene duplication is thought to facilitate increasing complexity in the evolution of life. The fate of most duplicated genes is nonfunctionalization: functional decay resulting from the accumulation of mutations. According to the duplication-degeneration-complementation (DDC) model, duplicated genes are retained by subfunctionalization, where the functions of the ancestral gene are sub-divided between duplicate genes, or by neofunctionalization, where one of the duplicates acquires a new function. Here, we report the differential regulation of the zebrafish tandemly duplicated fatty acid-binding protein genes, fabp1b.1 and fabp1b.2, by peroxisome proliferator-activated receptors (PPAR). fabp1b.1 mRNA levels were induced in tissue explants of liver, but not intestine, by PPAR agonists. fabp1b.1 promoter activity was induced to a greater extent by rosiglitazone (PPARγ-selective agonist) compared to WY 14,643 (PPARα-selective agonist) in HEK293A cells. Mutation of a peroxisome proliferator response element (PPRE) at -1232 bp in the fabp1b.1 promoter reduced PPAR-dependent activation. fabp1b.2 promoter activity was not affected by PPAR agonists. Differential regulation of the duplicated fabp1b promoters may be the result of PPRE loss in fabp1b.2 during a meiotic crossing-over event. Retention of PPAR inducibility in fabp1b.1 and not fabp1b.2 suggests unique regulation and function of the fabp1b duplicates.
- Published
- 2016
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31. Biased Type 1 Cannabinoid Receptor Signaling Influences Neuronal Viability in a Cell Culture Model of Huntington Disease.
- Author
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Laprairie RB, Bagher AM, Kelly ME, and Denovan-Wright EM
- Subjects
- Animals, Cannabidiol metabolism, Cannabidiol pharmacology, Cannabinoids metabolism, Cannabinoids pharmacology, Cell Line, Transformed, Cell Survival drug effects, Dose-Response Relationship, Drug, Humans, Mice, Mice, Inbred C57BL, Neurons drug effects, Receptor, Cannabinoid, CB1 agonists, Signal Transduction drug effects, Signal Transduction physiology, Cell Survival physiology, Huntington Disease metabolism, Neurons metabolism, Receptor, Cannabinoid, CB1 metabolism
- Abstract
Huntington disease (HD) is an inherited, autosomal dominant, neurodegenerative disorder with limited treatment options. Prior to motor symptom onset or neuronal cell loss in HD, levels of the type 1 cannabinoid receptor (CB1) decrease in the basal ganglia. Decreasing CB1 levels are strongly correlated with chorea and cognitive deficit. CB1 agonists are functionally selective (biased) for divergent signaling pathways. In this study, six cannabinoids were tested for signaling bias in in vitro models of medium spiny projection neurons expressing wild-type (STHdh(Q7/Q7)) or mutant huntingtin protein (STHdh(Q111/Q111)). Signaling bias was assessed using the Black and Leff operational model. Relative activity [ΔlogR (τ/KA)] and system bias (ΔΔlogR) were calculated relative to the reference compound WIN55,212-2 for Gαi/o, Gαs, Gαq, Gβγ, and β-arrestin1 signaling following treatment with 2-arachidonoylglycerol (2-AG), anandamide (AEA), CP55,940, Δ(9)-tetrahydrocannabinol (THC), cannabidiol (CBD), and THC+CBD (1:1), and compared between wild-type and HD cells. The Emax of Gαi/o-dependent extracellular signal-regulated kinase (ERK) signaling was 50% lower in HD cells compared with wild-type cells. 2-AG and AEA displayed Gαi/o/Gβγ bias and normalized CB1 protein levels and improved cell viability, whereas CP55,940 and THC displayed β-arrestin1 bias and reduced CB1 protein levels and cell viability in HD cells. CBD was not a CB1 agonist but inhibited THC-dependent signaling (THC+CBD). Therefore, enhancing Gαi/o-biased endocannabinoid signaling may be therapeutically beneficial in HD. In contrast, cannabinoids that are β-arrestin-biased--such as THC found at high levels in modern varieties of marijuana--may be detrimental to CB1 signaling, particularly in HD where CB1 levels are already reduced., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)
- Published
- 2016
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32. Novel Electrophilic and Photoaffinity Covalent Probes for Mapping the Cannabinoid 1 Receptor Allosteric Site(s).
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Kulkarni PM, Kulkarni AR, Korde A, Tichkule RB, Laprairie RB, Denovan-Wright EM, Zhou H, Janero DR, Zvonok N, Makriyannis A, Cascio MG, Pertwee RG, and Thakur GA
- Subjects
- Affinity Labels, Allosteric Site, Animals, Arrestins drug effects, Arrestins metabolism, Binding Sites drug effects, CHO Cells, Cricetinae, Cricetulus, Cyclic AMP antagonists & inhibitors, Cyclohexanols pharmacology, Drug Discovery methods, Guanosine 5'-O-(3-Thiotriphosphate) metabolism, Humans, Indoles pharmacology, Ligands, Models, Molecular, Phenylurea Compounds pharmacology, Piperidines pharmacology, Pyridines pharmacology, Radioligand Assay, Rats, Structure-Activity Relationship, Receptor, Cannabinoid, CB1 chemistry, Receptor, Cannabinoid, CB1 drug effects
- Abstract
Undesirable side effects associated with orthosteric agonists/antagonists of cannabinoid 1 receptor (CB1R), a tractable target for treating several pathologies affecting humans, have greatly limited their translational potential. Recent discovery of CB1R negative allosteric modulators (NAMs) has renewed interest in CB1R by offering a potentially safer therapeutic avenue. To elucidate the CB1R allosteric binding motif and thereby facilitate rational drug discovery, we report the synthesis and biochemical characterization of first covalent ligands designed to bind irreversibly to the CB1R allosteric site. Either an electrophilic or a photoactivatable group was introduced at key positions of two classical CB1R NAMs: Org27569 (1) and PSNCBAM-1 (2). Among these, 20 (GAT100) emerged as the most potent NAM in functional assays, did not exhibit inverse agonism, and behaved as a robust positive allosteric modulator of binding of orthosteric agonist CP55,940. This novel covalent probe can serve as a useful tool for characterizing CB1R allosteric ligand-binding motifs.
- Published
- 2016
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33. Cannabidiol is a negative allosteric modulator of the cannabinoid CB1 receptor.
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Laprairie RB, Bagher AM, Kelly ME, and Denovan-Wright EM
- Subjects
- Animals, Arrestins metabolism, Cell Line, HEK293 Cells, Humans, MAP Kinase Signaling System physiology, Mice, Phospholipase C beta metabolism, Cannabidiol pharmacology, Cannabinoid Receptor Modulators pharmacology, Receptor, Cannabinoid, CB1 metabolism
- Abstract
Background and Purpose: Cannabidiol has been reported to act as an antagonist at cannabinoid CB1 receptors. We hypothesized that cannabidiol would inhibit cannabinoid agonist activity through negative allosteric modulation of CB1 receptors., Experimental Approach: Internalization of CB1 receptors, arrestin2 recruitment, and PLCβ3 and ERK1/2 phosphorylation, were quantified in HEK 293A cells heterologously expressing CB1 receptors and in the STHdh(Q7/Q7) cell model of striatal neurons endogenously expressing CB1 receptors. Cells were treated with 2-arachidonylglycerol or Δ(9)-tetrahydrocannabinol alone and in combination with different concentrations of cannabidiol., Key Results: Cannabidiol reduced the efficacy and potency of 2-arachidonylglycerol and Δ(9)-tetrahydrocannabinol on PLCβ3- and ERK1/2-dependent signalling in cells heterologously (HEK 293A) or endogenously (STHdh(Q7/Q7)) expressing CB1 receptors. By reducing arrestin2 recruitment to CB1 receptors, cannabidiol treatment prevented internalization of these receptors. The allosteric activity of cannabidiol depended upon polar residues being present at positions 98 and 107 in the extracellular amino terminus of the CB1 receptor., Conclusions and Implications: Cannabidiol behaved as a non-competitive negative allosteric modulator of CB1 receptors. Allosteric modulation, in conjunction with effects not mediated by CB1 receptors, may explain the in vivo effects of cannabidiol. Allosteric modulators of CB1 receptors have the potential to treat CNS and peripheral disorders while avoiding the adverse effects associated with orthosteric agonism or antagonism of these receptors., (© 2015 The British Pharmacological Society.)
- Published
- 2015
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34. Components of the endocannabinoid and dopamine systems are dysregulated in Huntington's disease: analysis of publicly available microarray datasets.
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Laprairie RB, Bagher AM, Precious SV, and Denovan-Wright EM
- Abstract
The endocannabinoid system (ECS) and the dopaminergic system (DAS) are two major regulators of basal ganglia function. During Huntington's disease (HD) pathogenesis, the expression of genes in both the ECS and DAS is dysregulated. The purpose of this study was to determine the changes that were consistently observed in the ECS and DAS during HD progression in the central nervous system (CNS) and in the periphery in different models of HD and human HD tissue. To do this, we conducted a meta-analysis of differential gene expression in the ECS and DAS using publicly available microarray data. The consolidated data were summarized as observed changes in gene expression (OCGE) using a weighted sum for each gene. In addition, consolidated data were compared to previously published studies that were not available in the gene expression omnibus (GEO) database. The resulting data confirm gene expression changes observed using different approaches and provide novel insights into the consistency between changes observed in human tissue and various models, as well as disease stage- and tissue-specific transcriptional dysregulation in HD. The major implication of the systems-wide data presented here is that therapeutic strategies targeting the ECS or DAS must consider the dynamic changes in gene expression over time and in different body areas, which occur during HD progression and the interconnectedness of the two systems.
- Published
- 2015
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35. Type 1 cannabinoid receptor ligands display functional selectivity in a cell culture model of striatal medium spiny projection neurons.
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Laprairie RB, Bagher AM, Kelly ME, Dupré DJ, and Denovan-Wright EM
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- Animals, Arachidonic Acids pharmacology, Arrestin genetics, Arrestin metabolism, Benzoxazines pharmacology, Blotting, Western, Cells, Cultured, Corpus Striatum cytology, Corpus Striatum metabolism, Cyclohexanols pharmacology, Dendritic Spines metabolism, Dronabinol pharmacology, Endocannabinoids pharmacology, Fluorescence Resonance Energy Transfer, GTP-Binding Proteins metabolism, Glycerides pharmacology, Ligands, Luminescent Proteins genetics, Luminescent Proteins metabolism, Mice, Models, Biological, Morpholines pharmacology, Naphthalenes pharmacology, Neurons cytology, Polyunsaturated Alkamides pharmacology, Receptor, Cannabinoid, CB1 genetics, Signal Transduction drug effects, Cannabinoid Receptor Agonists pharmacology, Cannabinoids pharmacology, Neurons metabolism, Receptor, Cannabinoid, CB1 agonists, Receptor, Cannabinoid, CB1 metabolism
- Abstract
Modulation of type 1 cannabinoid receptor (CB1) activity has been touted as a potential means of treating addiction, anxiety, depression, and neurodegeneration. Different agonists of CB1 are known to evoke varied responses in vivo. Functional selectivity is the ligand-specific activation of certain signal transduction pathways at a receptor that can signal through multiple pathways. To understand cannabinoid-specific functional selectivity, different groups have examined the effect of individual cannabinoids on various signaling pathways in heterologous expression systems. In the current study, we compared the functional selectivity of six cannabinoids, including two endocannabinoids (2-arachidonyl glycerol (2-AG) and anandamide (AEA)), two synthetic cannabinoids (WIN55,212-2 and CP55,940), and two phytocannabinoids (cannabidiol (CBD) and Δ(9)-tetrahydrocannabinol (THC)) on arrestin2-, Gα(i/o)-, Gβγ-, Gα(s)-, and Gα(q)-mediated intracellular signaling in the mouse STHdh(Q7/Q7) cell culture model of striatal medium spiny projection neurons that endogenously express CB1. In this system, 2-AG, THC, and CP55,940 were more potent mediators of arrestin2 recruitment than other cannabinoids tested. 2-AG, AEA, and WIN55,212-2, enhanced Gα(i/o) and Gβγ signaling, with 2-AG and AEA treatment leading to increased total CB1 levels. 2-AG, AEA, THC, and WIN55,212-2 also activated Gα(q)-dependent pathways. CP55,940 and CBD both signaled through Gα(s). CP55,940, but not CBD, activated downstream Gα(s) pathways via CB1 targets. THC and CP55,940 promoted CB1 internalization and decreased CB1 protein levels over an 18-h period. These data demonstrate that individual cannabinoids display functional selectivity at CB1 leading to activation of distinct signaling pathways. To effectively match cannabinoids with therapeutic goals, these compounds must be screened for their signaling bias., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2014
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36. Anti-inflammatory effects of cannabinoid CB(2) receptor activation in endotoxin-induced uveitis.
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Toguri JT, Lehmann C, Laprairie RB, Szczesniak AM, Zhou J, Denovan-Wright EM, and Kelly ME
- Subjects
- Animals, Cannabinoids pharmacology, Male, NF-kappa B genetics, RNA, Messenger genetics, Rats, Rats, Inbred Lew, Receptor, Cannabinoid, CB2 metabolism, Reverse Transcriptase Polymerase Chain Reaction, Transcription Factor AP-1 genetics, Uveitis chemically induced, Uveitis drug therapy, Cannabinoids therapeutic use, Lipopolysaccharides toxicity, Receptor, Cannabinoid, CB2 agonists, Uveitis metabolism
- Abstract
Background and Purpose: Cannabinoid CB2 receptors mediate immunomodulation. Here, we investigated the effects of CB2 receptor ligands on leukocyte-endothelial adhesion and inflammatory mediator release in experimental endotoxin-induced uveitis (EIU)., Experimental Approach: EIU was induced by intraocular injection of lipopolysaccharide (LPS, 20 ng·μL(-1) ). Effects of the CB2 receptor agonist, HU308 (1.5% topical), the CB2 receptor antagonist, AM630 (2.5 mg·kg(-1) i.v.), or a combination of both compounds on leukocyte-endothelial interactions were measured hourly for 6 h in rat iridial vasculature using intravital microscopy. Anti-inflammatory actions of HU308 were compared with those of clinical treatments for uveitis - dexamethasone, prednisolone and nepafenac. Transcription factors (NF-κB, AP-1) and inflammatory mediators (cytokines, chemokines and adhesion molecules) were measured in iris and ciliary body tissue., Key Results: Leukocyte-endothelium adherence was increased in iridial microvasculature between 4-6 h after LPS. HU308 reduced this effect after LPS injection and decreased pro-inflammatory mediators: TNF-α, IL-1β, IL-6, CCL5 and CXCL2. AM630 blocked the actions of HU-308, and increased leukocyte-endothelium adhesion. HU-308 decreased levels of the transcription factors NF-κB and AP-1, while AM630 increased levels of NF-κB. Topical treatments with dexamethasone, prednisolone or nepafenac, failed to alter leukocyte adhesion or mitigate LPS-induced increases in inflammatory mediators during the 6 h of EIU., Conclusion and Implications: Activation of CB2 receptors was anti-inflammatory in a model of acute EIU and involved a reduction in NF-κB, AP-1 and inflammatory mediators. CB2 receptors may be promising drug targets for the development of novel ocular anti-inflammatory agents., Linked Articles: This article is part of a themed section on Cannabinoids 2013. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-6., (© 2013 The British Pharmacological Society.)
- Published
- 2014
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37. The cytokine and endocannabinoid systems are co-regulated by NF-κB p65/RelA in cell culture and transgenic mouse models of Huntington's disease and in striatal tissue from Huntington's disease patients.
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Laprairie RB, Warford JR, Hutchings S, Robertson GS, Kelly ME, and Denovan-Wright EM
- Subjects
- Adult, Age Factors, Aged, Amidohydrolases genetics, Amidohydrolases metabolism, Animals, Arachidonic Acids pharmacology, Cannabinoids pharmacology, Cells, Cultured, Corpus Striatum cytology, Cytokines genetics, Disease Models, Animal, Endocannabinoids agonists, Endocannabinoids antagonists & inhibitors, Enzyme Inhibitors pharmacology, Female, Gene Expression Regulation drug effects, Gene Expression Regulation physiology, Humans, Huntingtin Protein, Indoles pharmacology, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Middle Aged, NF-kappa B genetics, Nerve Tissue Proteins genetics, Neurons drug effects, Neurons metabolism, Nuclear Proteins genetics, Receptor, Cannabinoid, CB1 genetics, Receptor, Cannabinoid, CB1 metabolism, Transcription Factor RelA genetics, Trinucleotide Repeats genetics, Young Adult, Corpus Striatum metabolism, Cytokines metabolism, Endocannabinoids metabolism, Huntington Disease pathology, NF-kappa B metabolism, Transcription Factor RelA metabolism
- Abstract
Transcriptional dysregulation is a major pathological feature of Huntington's disease (HD). The goal of this study was to understand how p65/RelA co-regulated genes, specifically those of the cytokine and endocannabinoid systems, were affected in HD. p65/RelA levels were lower in human HD tissue and R6/2 HD mice, as were the levels of the type 1 cannabinoid receptor (CB1), IL-1β, IL-8, CCL5, GM-CSF, MIP-1β, and TNFα, all of which may be regulated by p65/RelA. Activation of p65/RelA restored CB1 and CCL5 expression in STHdh cell models of HD. Therefore, p65/RelA activation may normalize the expression of some genes in HD., (Copyright © 2013 Elsevier B.V. All rights reserved.)
- Published
- 2014
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38. Co-expression of the human cannabinoid receptor coding region splice variants (hCB₁) affects the function of hCB₁ receptor complexes.
- Author
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Bagher AM, Laprairie RB, Kelly ME, and Denovan-Wright EM
- Subjects
- Aged, Animals, Brain cytology, Cell Membrane drug effects, Cell Membrane metabolism, Female, Gene Expression, HEK293 Cells, Humans, Macaca fascicularis, Neurons cytology, Neurons metabolism, Pertussis Toxin toxicity, Protein Isoforms chemistry, Protein Isoforms genetics, Protein Isoforms metabolism, Protein Multimerization, Protein Structure, Quaternary, Protein Transport drug effects, RNA, Messenger genetics, RNA, Messenger metabolism, Receptor, Cannabinoid, CB1 chemistry, Receptor, Cannabinoid, CB1 genetics, Receptor, Cannabinoid, CB1 metabolism
- Abstract
The human type 1 cannabinoid (hCB1) receptor is expressed at high levels in the central nervous system. mRNA variants of the coding region of this receptor, human cannabinoid hCB1a and hCB1b receptors, have been identified, their biological function remains unclear. The present study demonstrated that the three human cannabinoid hCB1 coding region variants are expressed in the human and monkey (Macaca fascicularis) brain. Western blot analyses of homogenates from different regions of the monkey brain demonstrated that proteins with the expected molecular weights of the cannabinoid CB1, CB1a and CB1b receptors were co-expressed throughout the brain. Given the co-localization of these receptors, we hypothesized that physical interactions between the three splice variants may affect cannabinoid pharmacology. The human cannabinoid hCB1, hCB1a, and hCB1b receptors formed homodimers and heterodimers, as determined by BRET in transiently transfected HEK 293A cells. We found that the co-expression of the human cannabinoid hCB1 and each of the splice variants increased cell surface expression of the human cannabinoid hCB1 receptor and increased Gi/o-dependent ERK phosphorylation in response to cannabinoid agonists. Therefore, the human cannabinoid hCB1 coding region splice variants play an important physiological role in the activity of the endocannabinoid system., (© 2013 Elsevier B.V. All rights reserved.)
- Published
- 2013
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39. The impact of ovariectomy on calcium homeostasis and myofilament calcium sensitivity in the aging mouse heart.
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Fares E, Pyle WG, Ray G, Rose RA, Denovan-Wright EM, Chen RP, and Howlett SE
- Subjects
- Action Potentials, Animals, Calcium Signaling, Excitation Contraction Coupling, Female, Heart Rate physiology, Mice, Mice, Inbred C57BL, Myocardial Contraction, Myocytes, Cardiac metabolism, Sarcoplasmic Reticulum metabolism, Aging metabolism, Calcium metabolism, Homeostasis, Myocardium metabolism, Myofibrils metabolism, Ovariectomy
- Abstract
This study determined whether deficiency of ovarian estrogen starting very early in life promoted age-associated Ca(2+) dysregulation and contractile dysfunction in isolated ventricular myocytes. Myocytes were isolated from anesthetized C57BL/6 female mice. Animals received an ovariectomy or sham-operation at one month and were aged to ~24 months. Excitation-contraction coupling parameters were compared in fura-2 loaded myocytes (37°C). While Ca(2+) transients were larger and faster in field-stimulated myocytes from ovariectomized mice, ovariectomy had no effect on peak fractional shortening. Similarly, ovariectomy had no effect on fractional shortening measured in vivo by echocardiography (values were 60.5 ± 2.9 vs. 60.3 ± 2.5% in sham and ovariectomized, respectively; n=5 mice/group). Ovariectomy did decrease myofilament Ca(2+) sensitivity, as evidenced by a 26% increase in the Ca(2+) required to activate actomyosin MgATPase in ovariectomized hearts. Larger Ca(2+) transients were attributable to a 48% increase in peak Ca(2+) current, along with an increase in the amplitude, width and frequency of Ca(2+) sparks measured in fluo-4 loaded myocytes. These changes in Ca(2+) handling were not due to increased expression of Ca(2+) channels (Cav1.2), sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) or Na(+)-Ca(2+) exchanger in ovariectomized hearts. However, ovariectomy increased sarcoplasmic reticulum Ca(2+) stores by ~90% and promoted spontaneous Ca(2+) release from the sarcoplasmic reticulum when compared to sham controls. These observations demonstrate that long-term ovariectomy promotes intracellular Ca(2+) dysregulation, reduces myofilament Ca(2+) sensitivity and increases spontaneous Ca(2+) release in the aging female heart.
- Published
- 2013
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40. Cannabinoids increase type 1 cannabinoid receptor expression in a cell culture model of striatal neurons: implications for Huntington's disease.
- Author
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Laprairie RB, Kelly ME, and Denovan-Wright EM
- Subjects
- Adenosine Triphosphate metabolism, Animals, Cells, Cultured, Dose-Response Relationship, Drug, Dronabinol analogs & derivatives, Dronabinol pharmacology, Enzyme Inhibitors pharmacology, Gene Expression Regulation genetics, Huntingtin Protein, MAP Kinase Signaling System drug effects, MAP Kinase Signaling System physiology, Mice, Mice, Transgenic, Morpholines pharmacology, Mutation genetics, NF-kappa B genetics, NF-kappa B metabolism, Nerve Tissue Proteins genetics, Nuclear Proteins genetics, Pyrans pharmacology, Pyrazoles pharmacology, Receptor, Cannabinoid, CB1 genetics, gamma-Aminobutyric Acid metabolism, Cannabinoids pharmacology, Corpus Striatum cytology, Gene Expression Regulation drug effects, Neurons drug effects, Neurons metabolism, Receptor, Cannabinoid, CB1 metabolism
- Abstract
The type 1 cannabinoid receptor (CB1) is a G protein-coupled receptor that is expressed at high levels in the striatum. Activation of CB1 increases expression of neuronal trophic factors and inhibits neurotransmitter release from GABA-ergic striatal neurons. CB1 mRNA levels can be elevated by treatment with cannabinoids in non-neuronal cells. We wanted to determine whether cannabinoid treatment could induce CB1 expression in a cell culture model of striatal neurons and, if possible, determine the molecular mechanism by which this occurred. We found that treatment of STHdh(7/7) cells with the cannabinoids ACEA, mAEA, and AEA produced a CB1receptor-dependent increase in CB1 promoter activity, mRNA, and protein expression. This response was Akt- and NF-κB-dependent. Because decreased CB1 expression is thought to contribute to the pathogenesis of Huntington's disease (HD), we wanted to determine whether cannabinoids could increase CB1 expression in STHdh(7/111) and (111/111) cells expressing the mutant huntingtin protein. We observed that cannabinoid treatment increased CB1 mRNA levels approximately 10-fold in STHdh(7/111) and (111/111) cells, compared to vehicle treatment. Importantly, cannabinoid treatment improved ATP production, increased the expression of the trophic factor BDNF-2, and the mitochondrial regulator PGC1α, and reduced spontaneous GABA release, in HD cells. Therefore, cannabinoid-mediated increases in CB1 levels could reduce the severity of some molecular pathologies observed in HD., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2013
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41. Tissue-specific differential induction of duplicated fatty acid-binding protein genes by the peroxisome proliferator, clofibrate, in zebrafish (Danio rerio).
- Author
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Venkatachalam AB, Lall SP, Denovan-Wright EM, and Wright JM
- Subjects
- Animals, Genes, Duplicate, Mitochondria, Heart drug effects, Mitochondria, Liver drug effects, Organ Specificity, RNA, Heterogeneous Nuclear genetics, RNA, Messenger genetics, Response Elements, Transcription Initiation, Genetic, Up-Regulation, Zebrafish metabolism, Clofibrate pharmacology, Fatty Acid-Binding Proteins genetics, Gene Expression Regulation, Developmental drug effects, Peroxisome Proliferators pharmacology, Zebrafish genetics
- Abstract
Background: Force, Lynch and Conery proposed the duplication-degeneration-complementation (DDC) model in which partitioning of ancestral functions (subfunctionalization) and acquisition of novel functions (neofunctionalization) were the two primary mechanisms for the retention of duplicated genes. The DDC model was tested by analyzing the transcriptional induction of the duplicated fatty acid-binding protein (fabp) genes by clofibrate in zebrafish. Clofibrate is a specific ligand of the peroxisome proliferator-activated receptor (PPAR); it activates PPAR which then binds to a peroxisome proliferator response element (PPRE) to induce the transcriptional initiation of genes primarily involved in lipid homeostasis. Zebrafish was chosen as our model organism as it has many duplicated genes owing to a whole genome duplication (WGD) event that occurred ~230-400 million years ago in the teleost fish lineage. We assayed the steady-state levels of fabp mRNA and heterogeneous nuclear RNA (hnRNA) transcripts in liver, intestine, muscle, brain and heart for four sets of duplicated fabp genes, fabp1a/fabp1b.1/fabp1b.2, fabp7a/fabp7b, fabp10a/fabp10b and fabp11a/fabp11b in zebrafish fed different concentrations of clofibrate., Result: Electron microscopy showed an increase in the number of peroxisomes and mitochondria in liver and heart, respectively, in zebrafish fed clofibrate. Clofibrate also increased the steady-state level of acox1 mRNA and hnRNA transcripts in different tissues, a gene with a functional PPRE. These results demonstrate that zebrafish is responsive to clofibrate, unlike some other fishes. The levels of fabp mRNA and hnRNA transcripts for the four sets of duplicated fabp genes was determined by reverse transcription, quantitative polymerase chain reaction (RT-qPCR). The level of hnRNA coded by a gene is an indirect estimate of the rate of transcriptional initiation of that gene. Clofibrate increased the steady-state level of fabp mRNAs and hnRNAs for both the duplicated copies of fabp1a/fabp1b.1, and fabp7a/fabp7b, but in different tissues. Clofibrate also increased the steady-state level of fabp10a and fabp11a mRNAs and hnRNAs in liver, but not for fabp10b and fabp11b., Conclusion: Some duplicated fabp genes have, most likely, retained PPREs, but induction by clofibrate is over-ridden by an, as yet, unknown tissue-specific mechanism(s). Regardless of the tissue-specific mechanism(s), transcriptional control of duplicated zebrafish fabp genes by clofibrate has markedly diverged since the WGD event.
- Published
- 2012
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42. Promoters are differentially sensitive to N-terminal mutant huntingtin-mediated transcriptional repression.
- Author
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Hogel M, Laprairie RB, and Denovan-Wright EM
- Subjects
- Animals, Cell Nucleus metabolism, DNA Primers genetics, Disease Progression, Humans, Huntingtin Protein, Mutagenesis, Nerve Tissue Proteins physiology, Nuclear Proteins physiology, Protein Binding, Protein Structure, Tertiary, Rats, Sequence Analysis, DNA, Gene Expression Regulation, Mutation, Nerve Tissue Proteins genetics, Nuclear Proteins genetics, Promoter Regions, Genetic, Transcription, Genetic
- Abstract
Huntington's disease (HD) is a neurodegenerative disorder caused by the inheritance of one mutant copy of the huntingtin gene. Mutant huntingtin protein (mHtt) contains an expanded polyglutamine repeat region near the N-terminus. Cleavage of mHtt releases an N-terminal fragment (N-mHtt) which accumulates in the nucleus. Nuclear accumulation of N-mHtt has been directly associated with cellular toxicity. Decreased transcription is among the earliest detected changes that occur in the brains of HD patients, animal and cellular models of HD. Transcriptional dysregulation may trigger many of the perturbations that occur later in disease progression. An understanding of the effects of mHtt may lead to strategies to slow the progression of HD. Current models of N-mHtt-mediated transcriptional dysregulation suggest that abnormal interactions between N-mHtt and transcription factors impair the ability of these transcription factors to associate at N-mHtt-affected promoters and properly regulate gene expression. We tested various aspects of the current models using two N-mHtt-affected promoters in two cell models of HD using overexpression of known N-mHtt-interacting transcription factors, promoter deletion and mutation analyses and in vitro promoter binding assays. Consequently, we proposed a new model of N-mHtt-mediated transcriptional dysregulation centered on the presence of N-mHtt at promoters. In this model, N-mHtt interacts with multiple partners whose presence and affinity for N-mHtt influence the severity of gene dysregulation. We concluded that simultaneous interaction of N-mHtt with multiple binding partners within the transcriptional machinery would explain the gene-specificity of N-mHtt-mediated transcriptional dysregulation, as well as why some genes are affected early in disease progression while others are affected later. Our model explains why alleviating N-mHtt-mediated transcriptional dysregulation through overexpression of N-mHtt-interacting proteins has proven to be difficult and suggests that the most realistic strategy for restoring gene expression across the spectrum of N-mHtt affected genes is by reducing the amount of soluble nuclear N-mHtt.
- Published
- 2012
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43. The silence of the limbs.
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Denovan-Wright EM and Currie RW
- Subjects
- Animals, Male, Eukaryotic Initiation Factor-3 genetics, Gene Silencing, Genetic Therapy methods, Ischemia therapy, Muscle, Skeletal blood supply, Neovascularization, Physiologic, Peripheral Arterial Disease therapy, RNA, Small Interfering administration & dosage
- Published
- 2011
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44. Transgenic mice expressing caspase-6-derived N-terminal fragments of mutant huntingtin develop neurologic abnormalities with predominant cytoplasmic inclusion pathology composed largely of a smaller proteolytic derivative.
- Author
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Tebbenkamp AT, Green C, Xu G, Denovan-Wright EM, Rising AC, Fromholt SE, Brown HH, Swing D, Mandel RJ, Tessarollo L, and Borchelt DR
- Subjects
- Amino Acid Substitution, Animals, Ataxia genetics, Ataxia metabolism, Ataxia pathology, Caspase 6, Corpus Striatum pathology, Humans, Huntingtin Protein, Huntington Disease genetics, Huntington Disease pathology, Inclusion Bodies pathology, Mice, Mice, Transgenic, Nerve Tissue Proteins genetics, Nuclear Proteins genetics, RNA, Messenger biosynthesis, RNA, Messenger genetics, Rotarod Performance Test, Corpus Striatum metabolism, Gene Expression, Huntington Disease metabolism, Inclusion Bodies metabolism, Mutation, Missense, Nerve Tissue Proteins biosynthesis, Nuclear Proteins biosynthesis
- Abstract
Recent studies have implicated an N-terminal caspase-6 cleavage product of mutant huntingtin (htt) as an important mediator of toxicity in Huntington's disease (HD). To directly assess the consequences of such fragments on neurologic function, we produced transgenic mice that express a caspase-6 length N-terminal fragment of mutant htt (N586) with both normal (23Q) and disease (82Q) length glutamine repeats. In contrast to mice expressing N586-23Q, mice expressing N586-82Q accumulate large cytoplasmic inclusion bodies that can be visualized with antibodies to epitopes throughout the N586 protein. However, biochemical analyses of aggregated mutant huntingtin in these mice demonstrated that the inclusion bodies are composed largely of a much smaller htt fragment (terminating before residue 115), with lesser amounts of full-length N586-82Q fragments. Mice expressing the N586-82Q fragment show symptoms typical of previously generated mice expressing mutant huntingtin fragments, including failure to maintain weight, small brain weight and reductions in specific mRNAs in the striatum. Uniquely, these N586-82Q mice develop a progressive movement disorder that includes dramatic deficits in motor performance on the rotarod and ataxia. Our findings suggest that caspase-6-derived fragments of mutant htt are capable of inducing novel HD-related phenotypes, but these fragments are not terminal cleavage products as they are subject to further proteolysis. In this scenario, mutant htt fragments derived from caspase 6, or possibly other proteases, could mediate HD pathogenesis via a 'hit and run' type of mechanism in which caspase-6, or other larger N-terminal fragments, mediate a neurotoxic process before being cleaved to a smaller fragment that accumulates pathologically.
- Published
- 2011
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45. Longitudinal behavioral, cross-sectional transcriptional and histopathological characterization of a knock-in mouse model of Huntington's disease with 140 CAG repeats.
- Author
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Rising AC, Xu J, Carlson A, Napoli VV, Denovan-Wright EM, and Mandel RJ
- Subjects
- Animals, Cross-Sectional Studies, Disease Models, Animal, Female, Gait genetics, Genetic Carrier Screening methods, Homozygote, Humans, Huntingtin Protein, Huntington Disease psychology, Longitudinal Studies, Male, Mice, Mice, Inbred C57BL, Motor Activity genetics, Rotarod Performance Test, Behavior, Animal physiology, Gene Knock-In Techniques methods, Huntington Disease genetics, Huntington Disease pathology, Nerve Tissue Proteins genetics, Nuclear Proteins genetics, Trinucleotide Repeat Expansion genetics
- Abstract
The discovery of the gene mutation responsible for Huntington's disease (HD), huntingtin, in 1993 allowed for a better understanding of the pathology of and enabled the development of animal models. HD is caused by the expansion of a polyglutamine repeat region in the N-terminal of the huntingtin protein. Here we examine the behavioral, transcriptional, histopathological and anatomical characteristics of a knock-in HD mouse model with a 140 polyglutamine expansion in the huntingtin protein. This CAG 140 model contains a portion of the human exon 1 with 140 CAG repeats knocked into the mouse huntingtin gene. We have longitudinally examined the rearing behavior, accelerating rotarod, constant speed rotarod and gait for age-matched heterozygote, homozygote and non-transgenic mice and have found a significant difference in the afflicted mice. However, while there were significant differences between the non-transgenic and the knock-in mice, these behaviors were not progressive. As in HD, we show that the CAG 140 mice also have a significant decrease in striatally enriched mRNA transcripts. In addition, striatal neuronal intranuclear inclusion density increases with age. Lastly these CAG 140 mice show slight cortical thinning compared to non-transgenic mice, similarly to the cortical thinning recently reported in HD., (Copyright © 2010 Elsevier Inc. All rights reserved.)
- Published
- 2011
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46. Tandem duplication of the fabp1b gene and subsequent divergence of the tissue-specific distribution of fabp1b.1 and fabp1b.2 transcripts in zebrafish (Danio rerio).
- Author
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Karanth S, Denovan-Wright EM, Thisse C, Thisse B, and Wright JM
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Embryo, Nonmammalian embryology, Embryo, Nonmammalian metabolism, Fatty Acid-Binding Proteins classification, Female, Gene Expression Regulation, Developmental, In Situ Hybridization, Male, Molecular Sequence Data, Phylogeny, Protein Isoforms genetics, Radiation Hybrid Mapping, Reverse Transcriptase Polymerase Chain Reaction, Tandem Repeat Sequences, Transcription, Genetic, Zebrafish embryology, Zebrafish growth & development, Zebrafish Proteins classification, Fatty Acid-Binding Proteins genetics, Gene Duplication, Gene Expression Profiling methods, Genetic Variation, Zebrafish genetics, Zebrafish Proteins genetics
- Abstract
We describe a fatty acid-binding protein 1 (fabp1b.2) gene and its tissue-specific expression in zebrafish embryos and adults. The 3.5 kb zebrafish fabp1b.2 gene is the paralog of the previously described zebrafish fabp1a and fabp1b genes. Using the LN54 radiation hybrid mapping panel, we assigned the zebrafish fabp1b.2 gene to linkage group 8, the same linkage group to which fabp1b.1 was mapped. fabp1b.1 and fabp1b.2 appear to have arisen by a tandem duplication event. Whole-mount in situ hybridization of a riboprobe to embryos and larvae detected fabp1b.2 transcripts in the diencephalon and as spots in the periphery of the yolk sac. In adult zebrafish, in situ hybridization revealed fabp1b.2 transcripts in the anterior intestine and skin, and reverse transcription PCR (RT-PCR) detected fabp1b.2 transcripts in the intestine, brain, heart, ovary, skin, and eye. By contrast, fabp1b.1 transcripts were detected by RT-PCR in the liver, intestine, heart, testis, ovary, and gills. The tissue-specific distribution of transcripts for the tandemly duplicated fabp1b.1 and fabp1b.2 genes in adult tissues and during development suggests that the duplicated fabp1b genes of zebrafish have acquired additional functions compared with the ancestral fabp1 gene, i.e., by neofunctionalization. Furthermore, these functions were subsequently divided between fabp1b.1 and fabp1b.2 owing to subfunctionalization.
- Published
- 2009
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- View/download PDF
47. Differential transcriptional modulation of duplicated fatty acid-binding protein genes by dietary fatty acids in zebrafish (Danio rerio): evidence for subfunctionalization or neofunctionalization of duplicated genes.
- Author
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Karanth S, Lall SP, Denovan-Wright EM, and Wright JM
- Subjects
- Animals, Diet, Fatty Acid-Binding Proteins metabolism, Female, Gene Expression Regulation, Developmental, Male, Promoter Regions, Genetic, RNA, Heterogeneous Nuclear metabolism, RNA, Messenger metabolism, Transcription, Genetic, Zebrafish Proteins metabolism, Fatty Acid-Binding Proteins genetics, Fatty Acids metabolism, Zebrafish genetics, Zebrafish Proteins genetics
- Abstract
Background: In the Duplication-Degeneration-Complementation (DDC) model, subfunctionalization and neofunctionalization have been proposed as important processes driving the retention of duplicated genes in the genome. These processes are thought to occur by gain or loss of regulatory elements in the promoters of duplicated genes. We tested the DDC model by determining the transcriptional induction of fatty acid-binding proteins (Fabps) genes by dietary fatty acids (FAs) in zebrafish. We chose zebrafish for this study for two reasons: extensive bioinformatics resources are available for zebrafish at zfin.org and zebrafish contains many duplicated genes owing to a whole genome duplication event that occurred early in the ray-finned fish lineage approximately 230-400 million years ago. Adult zebrafish were fed diets containing either fish oil (12% lipid, rich in highly unsaturated fatty acid), sunflower oil (12% lipid, rich in linoleic acid), linseed oil (12% lipid, rich in linolenic acid), or low fat (4% lipid, low fat diet) for 10 weeks. FA profiles and the steady-state levels of fabp mRNA and heterogeneous nuclear RNA in intestine, liver, muscle and brain of zebrafish were determined., Result: FA profiles assayed by gas chromatography differed in the intestine, brain, muscle and liver depending on diet. The steady-state level of mRNA for three sets of duplicated genes, fabp1a/fabp1b.1/fabp1b.2, fabp7a/fabp7b, and fabp11a/fabp11b, was determined by reverse transcription, quantitative polymerase chain reaction (RT-qPCR). In brain, the steady-state level of fabp7b mRNAs was induced in fish fed the linoleic acid-rich diet; in intestine, the transcript level of fabp1b.1 and fabp7b were elevated in fish fed the linolenic acid-rich diet; in liver, the level of fabp7a mRNAs was elevated in fish fed the low fat diet; and in muscle, the level of fabp7a and fabp11a mRNAs were elevated in fish fed the linolenic acid-rich or the low fat diets. In all cases, induction of the steady-state level of fabp mRNAs by dietary FAs correlated with induced levels of hnRNA for a given fabp gene. As such, up-regulation of the steady-state level of fabp mRNAs by FAs occurred at the level of initiation of transcription. None of the sister duplicates of these fabp genes exhibited an increase in their steady-state transcript levels in a specific tissue following feeding zebrafish any of the four experimental diets., Conclusion: Differential induction of only one of the sister pair of duplicated fabp genes by FAs provides evidence to support the DDC model for retention of duplicated genes in the zebrafish genome by either subfunctionalization or neofunctionalization.
- Published
- 2009
- Full Text
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48. Spatio-temporal distribution of fatty acid-binding protein 6 (fabp6) gene transcripts in the developing and adult zebrafish (Danio rerio).
- Author
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Alves-Costa FA, Denovan-Wright EM, Thisse C, Thisse B, and Wright JM
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, DNA, Complementary metabolism, Gene Expression Regulation, Developmental, In Situ Hybridization, Molecular Sequence Data, Multigene Family, Phylogeny, Sequence Homology, Amino Acid, Time Factors, Tissue Distribution, Zebrafish, Fatty Acid-Binding Proteins metabolism, Gastrointestinal Hormones metabolism, Transcription, Genetic
- Abstract
We have determined the structure of the fatty acid-binding protein 6 (fabp6) gene and the tissue-specific distribution of its transcripts in embryos, larvae and adult zebrafish (Danio rerio). Like most members of the vertebrate FABP multigene family, the zebrafish fabp6 gene contains four exons separated by three introns. The coding region of the gene and expressed sequence tags code for a polypeptide of 131 amino acids (14 kDa, pI 6.59). The putative zebrafish Fabp6 protein shared greatest sequence identity with human FABP6 (55.3%) compared to other orthologous mammalian FABPs and paralogous zebrafish Fabps. Phylogenetic analysis showed that the zebrafish Fabp6 formed a distinct clade with the mammalian FABP6s. The zebrafish fabp6 gene was assigned to linkage group (chromosome) 21 by radiation hybrid mapping. Conserved gene synteny was evident between the zebrafish fabp6 gene on chromosome 21 and the FABP6/Fabp6 genes on human chromosome 5, rat chromosome 10 and mouse chromosome 11. Zebrafish fabp6 transcripts were first detected in the distal region of the intestine of embryos at 72 h postfertilization. This spatial distribution remained constant to 7-day-old larvae, the last stage assayed during larval development. In adult zebrafish, fabp6 transcripts were detected by RT-PCR in RNA extracted from liver, heart, intestine, ovary and kidney (most likely adrenal tissue), but not in RNA from skin, brain, gill, eye or muscle. In situ hybridization of a fabp6 riboprobe to adult zebrafish sections revealed intense hybridization signals in the adrenal homolog of the kidney and the distal region of the intestine, and to a lesser extent in ovary and liver, a transcript distribution that is similar, but not identical, to that seen for the mammalian FABP6/Fabp6 gene.
- Published
- 2008
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49. The evolutionary relationship between the duplicated copies of the zebrafish fabp11 gene and the tetrapod FABP4, FABP5, FABP8 and FABP9 genes.
- Author
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Karanth S, Denovan-Wright EM, Thisse C, Thisse B, and Wright JM
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Embryo, Nonmammalian metabolism, Fatty Acid-Binding Proteins classification, Fatty Acid-Binding Proteins metabolism, Genome, Larva metabolism, Molecular Sequence Data, Phylogeny, RNA, Messenger metabolism, Retina embryology, Retina growth & development, Retina metabolism, Tissue Distribution, Zebrafish embryology, Zebrafish genetics, Zebrafish growth & development, Zebrafish Proteins classification, Zebrafish Proteins metabolism, Evolution, Molecular, Fatty Acid-Binding Proteins genetics, Gene Duplication, Multigene Family, Zebrafish Proteins genetics
- Abstract
We describe the structure of a fatty acid-binding protein 11 (fabp11b) gene and its tissue-specific expression in zebrafish. The 3.4 kb zebrafish fabp11b is the paralog of the previously described zebrafish fabp11a, with a deduced amino acid sequence for Fabp11B exhibiting 65% identity with that of Fabp11A. Whole mount in situ hybridization of a riboprobe to embryos and larvae showed that zebrafish fabp11b transcripts were restricted solely to the retina and were first detected at 24 h postfertilization. In situ hybridization revealed fabp11b transcripts along the spinal cord in adult zebrafish. However, the highly sensitive RT-PCR assay detected fabp11b transcripts in the brain, heart, ovary and eye in adult tissues. By contrast, fabp11a transcripts had been previously detected in the liver, brain, heart, testis, muscle, ovary and skin of adult zebrafish. Using the LN54 radiation hybrid panel, we assigned zebrafish fabp11b to linkage group 16. Phylogenetic analysis and conserved gene synteny with tetrapod genes indicated that the emergence of two copies of fabp11 in the zebrafish genome may have resulted from a fish-specific whole genome duplication event. Furthermore, we propose that the FABP4-FABP5-FABP8-FABP9 (PERF15) gene cluster on a single chromosome in the tetrapod genome and the fabp11 genes in the zebrafish genome originated from a common ancestral gene, which, following their divergence, gave rise to the fabp11 genes of zebrafish, and the progenitor of the FABP4, FABP5, FABP8 and FABP9 genes in tetrapods after the separation of the fish and tetrapod lineages.
- Published
- 2008
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50. Sustained striatal ciliary neurotrophic factor expression negatively affects behavior and gene expression in normal and R6/1 mice.
- Author
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Denovan-Wright EM, Attis M, Rodriguez-Lebron E, and Mandel RJ
- Subjects
- Animals, Behavior, Animal physiology, Ciliary Neurotrophic Factor genetics, Ciliary Neurotrophic Factor physiology, Down-Regulation genetics, Humans, Huntington Disease complications, Huntington Disease genetics, Huntington Disease metabolism, Male, Mice, Mice, Inbred C57BL, Mice, Inbred CBA, Mice, Transgenic, Motor Skills Disorders etiology, Motor Skills Disorders genetics, Time Factors, Ciliary Neurotrophic Factor biosynthesis, Corpus Striatum metabolism, Down-Regulation physiology, Motor Skills Disorders metabolism
- Abstract
Huntington's disease (HD) is a neurodegenerative disorder caused by an elongation of CAG repeats in the HD gene, which encodes a mutant copy of huntingtin with an expanded polyglutatmine repeat. Individuals who are affected by the disease suffer from motor, cognitive, and emotional impairments. Levels of certain striatal-enriched mRNAs decrease in both HD patients and transgenic HD mice prior to the development of motor symptoms and neuronal cell death. Ciliary neurotrophic factor (CNTF) has been shown to protect neurons against chemically induced toxic insults in vitro and in vivo. To test the hypothesis that CNTF might protect neurons from the negative effects of the mutant huntingtin protein in vivo, CNTF was continuously expressed following transduction of the striatum by recombinant adeno-associated viral vectors (rAAV2). Wild-type and R6/1 HD transgenic (R6/1) mice that received bilateral or unilateral intrastriatal injections of rAAV2-CNTF experienced weight loss. The CNTF-treated R6/1 HD transgenic mice experienced motor impairments at an earlier age than expected compared with age-matched control R6/1 HD transgenic animals. CNTF also caused abnormal behavior in WT mice. In addition to behavioral impairments, in situ hybridization showed that, in both WT and R6/1 mice, CNTF expression caused a significant decrease in the levels of striatal-enriched transcripts. Overall, continuous expression of striatal CNTF at the dose mediated by the expression cassette used in this study was detrimental to HD and wild-type mice., ((c) 2008 Wiley-Liss, Inc.)
- Published
- 2008
- Full Text
- View/download PDF
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