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1.
Neurobiol Dis ; 181: 106115, 2023 06 01.
Article in English | MEDLINE | ID: mdl-37037299

ABSTRACT

Parkinson's disease (PD) is a complex illness with a constellation of environmental insults and genetic vulnerabilities being implicated. Strikingly, many studies only focus on the cardinal motor symptoms of the disease and fail to appreciate the major non-motor features which typically occur early in the disease process and are debilitating. Common comorbid psychiatric features, notably clinical depression, as well as anxiety and sleep disorders are thought to emerge before the onset of prominent motor deficits. In this review, we will delve into the prodromal stage of PD and how early neuropsychiatric pathology might unfold, followed by later motor disturbances. It is also of interest to discuss how animal models of PD capture the complexity of the illness, including depressive-like characteristics along with motor impairment. It remains to be determined how the underlying PD disease processes contributes to such comorbidity. But some of the environmental toxicants and microbial pathogens implicated in PD might instigate pro-inflammatory effects favoring α-synuclein accumulation and damage to brainstem neurons fueling the evolution of mood disturbances. We posit that comprehensive animal-based research approaches are needed to capture the complexity and time-dependent nature of the primary and co-morbid symptoms. This will allow for the possibility of early intervention with more novel and targeted treatments that fit with not only individual patient variability, but also with changes that occur over time with the evolution of the disease.


Subject(s)
Parkinson Disease , Sleep Wake Disorders , Animals , Parkinson Disease/pathology , Models, Animal , Neurons/pathology , Anxiety Disorders , Prodromal Symptoms , Disease Models, Animal
2.
Transl Psychiatry ; 6(8): e863, 2016 08 02.
Article in English | MEDLINE | ID: mdl-27483381

ABSTRACT

Post-stroke depression (PSD) is a common outcome following stroke that is associated with poor recovery. To develop a preclinical model of PSD, we targeted a key node of the depression-anxiety circuitry by inducing a unilateral ischemic lesion to the medial prefrontal cortex (mPFC) stroke. Microinjection of male C57/BL6 mice with endothelin-1 (ET-1, 1600 pmol) induced a small (1 mm(3)) stroke consistently localized within the left mPFC. Compared with sham control mice, the stroke mice displayed a robust behavioral phenotype in four validated tests of anxiety including the elevated plus maze, light-dark, open-field and novelty-suppressed feeding tests. In addition, the stroke mice displayed depression-like behaviors in both the forced swim and tail suspension test. In contrast, there was no effect on locomotor activity or sensorimotor function in the horizontal ladder, or cylinder and home cage activity tests, indicating a silent stroke due to the absence of motor abnormalities. When re-tested at 6 weeks post stroke, the stroke mice retained both anxiety and depression phenotypes. Surprisingly, at 6 weeks post stroke the lesion site was infiltrated by neurons, suggesting that the ET-1-induced neuronal loss in the mPFC was reversible over time, but was insufficient to promote behavioral recovery. In summary, unilateral ischemic lesion of the mPFC results in a pronounced and persistent anxiety and depression phenotype with no evident sensorimotor deficits. This precise lesion of the depression circuitry provides a reproducible model to study adaptive cellular changes and preclinical efficacy of novel interventions to alleviate PSD symptoms.


Subject(s)
Anxiety/psychology , Behavior, Animal , Depression/psychology , Prefrontal Cortex/blood supply , Stroke/psychology , Animals , Anxiety/pathology , Body Dysmorphic Disorders , Depression/pathology , Endothelin-1/toxicity , Locomotion , Male , Mice , Neurons/pathology , Prefrontal Cortex/pathology , Stroke/chemically induced , Stroke/pathology
3.
Transl Psychiatry ; 6: e746, 2016 Mar 01.
Article in English | MEDLINE | ID: mdl-26926882

ABSTRACT

The G/C single-nucleotide polymorphism in the serotonin 1a receptor promoter, rs6295, has previously been linked with depression, suicide and antidepressant responsiveness. In vitro studies suggest that rs6295 may have functional effects on the expression of the serotonin 1a receptor gene (HTR1A) through altered binding of a number of transcription factors. To further explore the relationship between rs6295, mental illness and gene expression, we performed dual epidemiological and biological studies. First, we genotyped a cohort of 1412 individuals, randomly split into discovery and replication cohorts, to examine the relationship between rs6295 and five psychiatric outcomes: history of psychiatric hospitalization, history of suicide attempts, history of substance or alcohol abuse, current posttraumatic stress disorder (PTSD), current depression. We found that the rs6295G allele is associated with increased risk for substance abuse, psychiatric hospitalization and suicide attempts. Overall, exposure to either childhood or non-childhood trauma resulted in increased risk for all psychiatric outcomes, but we did not observe a significant interaction between rs6295 and trauma in modulating psychiatric outcomes. In conjunction, we also investigated the potential impact of rs6295 on HTR1A expression in postmortem human brain tissue using relative allelic expression assays. We found more mRNA produced from the C versus the G-allele of rs6295 in the prefrontal cortex (PFC), but not in the midbrain of nonpsychiatric control subjects. Further, in the fetal cortex, rs6295C allele exhibited increased relative expression as early as gestational week 18 in humans. Finally, we found that the C:G allelic expression ratio was significantly neutralized in the PFC of subjects with major depressive disorder (MDD) who committed suicide as compared with controls, indicating that normal patterns of transcription may be disrupted in MDD/suicide. These data provide a putative biological mechanism underlying the association between rs6295, trauma and mental illness. Moreover, our results suggest that rs6295 may affect transcription during both gestational development and adulthood in a region-specific manner, acting as a risk factor for psychiatric illness. These findings provide a critical framework for conceptualizing the effects of a common functional genetic variant, trauma exposure and their impact on mental health.


Subject(s)
Mental Disorders/genetics , Receptor, Serotonin, 5-HT1A/genetics , Transcription Factors/genetics , Adolescent , Adult , Aged , Brain/metabolism , Female , Gene Expression/genetics , Genetic Predisposition to Disease/genetics , Humans , Male , Mental Disorders/metabolism , Middle Aged , Polymorphism, Single Nucleotide/genetics , Receptor, Serotonin, 5-HT1A/metabolism , Young Adult
4.
Genes Brain Behav ; 14(6): 466-76, 2015 Jul.
Article in English | MEDLINE | ID: mdl-26096691

ABSTRACT

Performance improvements in cognitive tasks requiring executive functions are evident with nicotinic acetylcholine receptor (nAChR) agonists, and activation of the underlying neural circuitry supporting these cognitive effects is thought to involve dopamine neurotransmission. As individual difference in response to nicotine may be related to a functional polymorphism in the gene encoding catechol-O-methyltransferase (COMT), an enzyme that strongly influences cortical dopamine metabolism, this study examined the modulatory effects of the COMT Val158Met polymorphism on the neural response to acute nicotine as measured with resting-state electroencephalographic (EEG) oscillations. In a sample of 62 healthy non-smoking adult males, a single dose (6 mg) of nicotine gum administered in a randomized, double-blind, placebo-controlled design was shown to affect α oscillatory activity, increasing power of upper α oscillations in frontocentral regions of Met/Met homozygotes and in parietal/occipital regions of Val/Met heterozygotes. Peak α frequency was also found to be faster with nicotine (vs. placebo) treatment in Val/Met heterozygotes, who exhibited a slower α frequency compared to Val/Val homozygotes. The data tentatively suggest that interindividual differences in brain α oscillations and their response to nicotinic agonist treatment are influenced by genetic mechanisms involving COMT.


Subject(s)
Catechol O-Methyltransferase/genetics , Electroencephalography/drug effects , Nicotine/pharmacology , Adult , Brain/drug effects , Brain/metabolism , Catechol O-Methyltransferase/metabolism , Cognition/drug effects , Cognition/physiology , Dopamine/metabolism , Double-Blind Method , Executive Function/drug effects , Genotype , Humans , Male , Nicotine/metabolism , Placebos , Polymorphism, Single Nucleotide , Receptors, Nicotinic/genetics , Receptors, Nicotinic/metabolism , Young Adult
5.
Brain Behav Immun ; 25(6): 1264-71, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21554945

ABSTRACT

OBJECTIVE: To assess serum brain derived neurotrophic factor (BDNF) concentrations as a correlate of cardiopulmonary fitness and as a predictor of cognitive performance in subjects with coronary artery disease (CAD). METHODS: Serum BDNF concentrations were assayed by ELISA and fitness was assessed using a standardized exercise stress test. The Mini Mental Status Examination (MMSE), California Verbal Learning Test 2nd Ed., Stroop, Trail Making Test B and the Digit Symbol-Coding task were administered. The val66met BDNF genotype and serum interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) concentrations were determined as potential confounders. RESULTS: In subjects with CAD (n=88; 85.2% male, mean age 62.8±10.5 yr), cardiopulmonary fitness was associated with higher serum BDNF concentrations (ß=.305, p=.013). Higher serum BDNF concentrations were associated with higher MMSE scores (F(1,87)=15.406, p<.0005) and better performance on the Digit Symbol-Coding task (F(1,87)=9.620, p=.003). IL-6, TNF-α and the val66met genotype did not influence these results. CONCLUSION: Serum BDNF concentrations were associated with cardiopulmonary fitness, psychomotor processing speed and overall cognition in subjects with CAD.


Subject(s)
Brain-Derived Neurotrophic Factor/blood , Cognition/physiology , Coronary Disease/blood , Physical Fitness/physiology , Aged , Brain-Derived Neurotrophic Factor/genetics , C-Reactive Protein/analysis , Confounding Factors, Epidemiologic , Coronary Disease/physiopathology , Coronary Disease/psychology , Coronary Disease/rehabilitation , Coronary Disease/therapy , Enzyme-Linked Immunosorbent Assay , Exercise Test , Female , Genotype , Humans , Interleukin-6/blood , Male , Middle Aged , Neuropsychological Tests , Polymorphism, Single Nucleotide , Psychomotor Performance , Risk Factors
6.
Mol Psychiatry ; 16(12): 1169-76, 2011 Dec.
Article in English | MEDLINE | ID: mdl-20856248

ABSTRACT

The brain serotonergic system has an essential role in the physiological functions of the central nervous system and dysregulation of serotonin (5-HT) homeostasis has been implicated in many neuropsychiatric disorders. The tryptophan hydroxylase-2 (TPH2) gene is the rate-limiting enzyme in brain 5-HT synthesis, and thus is an ideal candidate gene for understanding the role of dysregulation of brain serotonergic homeostasis. Here, we characterized a common, but functional single-nucleotide polymorphism (SNP rs1386493) in the TPH2 gene, which decreases efficiency of normal RNA splicing, resulting in a truncated TPH2 protein (TPH2-TR) by alternative splicing. TPH2-TR, which lacks TPH2 enzyme activity, dominant-negatively affects full-length TPH2 function, causing reduced 5-HT production. The predicted mRNA for TPH2-TR is present in postmortem brain of rs1386493 carriers. The rs13864923 variant does not appear to be overrepresented in either global or multiplex depression cohorts. However, in combination with other gene variants linked to 5-HT homeostasis, this variant may exhibit important epistatic influences.


Subject(s)
Alternative Splicing , Depression/genetics , Genetic Predisposition to Disease/genetics , Serotonin/biosynthesis , Tryptophan Hydroxylase/genetics , Animals , Brain Stem/metabolism , Cell Line, Transformed , Female , Genetic Predisposition to Disease/psychology , Genotype , Humans , Male , PC12 Cells , Pedigree , Polymorphism, Single Nucleotide/genetics , Rats
7.
Neuroscience ; 163(4): 1119-27, 2009 Nov 10.
Article in English | MEDLINE | ID: mdl-19647046

ABSTRACT

Chronic stress is known to affect brain areas involved in learning and emotional responses. These changes, thought to be related to the development of cognitive deficits are evident in major depressive disorder and other stress-related pathophysiologies. The serotonin-related transcription factors (Freud-1/CC2D1A; five prime repressor element under dual repression/coiled-coil C2 domain 1a, and NUDR/Deaf-1; nuclear-deformed epidermal autoregulatory factor) are two important regulators of the 5-HT1A receptor. Using Western blotting and quantitative real-time polymerase chain reaction (qPCR) we examined the expression of mRNA and proteins for Freud-1, NUDR, and the 5-HT1A receptor in the prefrontal cortex (PFC) of male rats exposed to chronic restraint stress (CRS; 6 h/day for 21 days). After 21 days of CRS, significant reductions in both Freud-1 mRNA and protein were observed in the PFC (36.8% and 32%, respectively; P<0.001), while the levels of both NUDR protein and mRNA did not change significantly. Consistent with reduced Freud-1 protein, 5-HT1A receptor mRNA levels were equally upregulated in the PFC, while protein levels actually declined, suggesting post-transcriptional receptor downregulation. The data suggest that CRS produces distinct alterations in the serotonin system specifically altering Freud-1 and the 5-HT1A receptor in the PFC of the male rat while having no effect on NUDR. These results point to the importance of understanding the mechanism for the differential regulation of Freud-1 and NUDR in the PFC as a basis for understanding the related effects of chronic stress on the serotonin system (serotonin-related transcription factors) and stress-related disorders like depression.


Subject(s)
Nuclear Proteins/metabolism , Prefrontal Cortex/metabolism , Receptor, Serotonin, 5-HT1A/metabolism , Repressor Proteins/metabolism , Stress, Psychological/metabolism , Animals , Blotting, Western , Chronic Disease , Corticosterone/blood , Gene Expression , Male , Nuclear Proteins/genetics , Polymerase Chain Reaction , RNA, Messenger/metabolism , Radioimmunoassay , Rats , Rats, Sprague-Dawley , Receptor, Serotonin, 5-HT1A/genetics , Repressor Proteins/genetics , Restraint, Physical , Stress, Psychological/blood , Stress, Psychological/genetics , Transcription Factors
8.
J Physiol ; 538(Pt 1): 41-51, 2002 Jan 01.
Article in English | MEDLINE | ID: mdl-11773315

ABSTRACT

The importance of specific protein kinase C (PKC) sites for modulation of the inhibitory coupling of 5-HT(1A) receptors to N-type Ca(2+) channels was examined using patch-clamp techniques in F11 rat dorsal root ganglion x mouse neuroblastoma hybrid cells. The PKC activator phorbol 12-myristate 13-acetate (PMA, 10 nM) reduced by 28.6 +/- 6.8 % 5-HT-mediated, but not GTP-gamma-S-induced, inhibition of Ca(2+) current, whereas a higher concentration of PMA (500 nM) inhibited both the actions of 5-HT and GTP-gamma-S. 5-HT(1A) receptor expression plasmids with or without mutation of a single PKC site in the second intracellular loop (i2, T149A) or of three PKC sites located in the third intracellular loop (i3, T229A-S253G-T343A) were stably transfected into F11 cells. The T149A 5 HT(1A) receptor inhibited forskolin-stimulated cyclic AMP levels but was largely uncoupled from Ca(2+) channel modulation. In one (i2) clone a response rate to 5-HT of 31.6 % was obtained. The T149A mutant displayed markedly reduced sensitivity to PMA (10 nM) compared to wild-type 5-HT(1A) receptors, with only a 13.4 +/- 3 % reduction in 5-HT-induced channel inhibition; when exposed to 500 nM PMA, reductions in the action of 5-HT were comparable to those of the wild-type receptor. By contrast, the i3 mutant displayed comparable sensitivity to the wild-type 5-HT(1A) receptor to either concentration of PMA. PMA at 10 nM exhibited a similar uncoupling effect on the response of the endogenous opiate receptor to the agonist D-alanine-5-leucine-enkephalin (DADLE) in wild-type and T149A mutant-expressing clones. The T149 site of the 5-HT(1A) receptor is crucial for receptor uncoupling by sub-maximal PKC activation while at maximal PKC activation, downstream sites uncouple G proteins from the N-type Ca(2+) channel.


Subject(s)
Calcium Channels, N-Type/metabolism , Protein Kinase C/metabolism , Receptors, Serotonin/metabolism , Animals , Binding Sites/physiology , Calcium Channels/metabolism , Carbazoles/pharmacology , Cells, Cultured , Dose-Response Relationship, Drug , Enkephalin, Leucine-2-Alanine/pharmacology , Enzyme Inhibitors/pharmacology , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , Hybrid Cells , Indoles/pharmacology , Mice , Mutation/physiology , Osmolar Concentration , Phosphorylation , Protein Kinase C/antagonists & inhibitors , Rats , Receptors, Serotonin/genetics , Receptors, Serotonin, 5-HT1 , Tetradecanoylphorbol Acetate/administration & dosage , Tetradecanoylphorbol Acetate/pharmacology
9.
Trends Endocrinol Metab ; 12(10): 453-60, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11701344

ABSTRACT

Studies of the serotonin (5-HT) receptors have illustrated several important concepts in G-protein-mediated signaling. These concepts include G-protein specificity and cellular specificity of signaling; mechanisms of transactivation; receptor states and constitutive receptor activity; and the structural basis of coupling. The 5-HT1 receptors couple via specific G(i)/G(o) proteins to inhibitory pathways [inhibition of adenylyl cyclase (AC) activity and regulation of ion channels], but also to stimulate phospholipase C, ACII, and the mitogen-activated protein kinase (MAPK) growth-signaling pathway. 5-HT1 receptors initiate novel endocytotic and Ca(2+)-dependent pathways to activate MAPK acutely, but can downregulate MAPK on chronic activation. These pathways are often mediated via distinct G(i)/G(o)-protein subtypes. Desensitization by multiple protein kinases via receptor phosphorylation is pathway selective. Structural determination of 5-HT1 receptor and G-protein domains that mediate G-protein-specific coupling and desensitization could lead to the development of highly selective ligands that directly regulate receptor-G-protein coupling.


Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Heterotrimeric GTP-Binding Proteins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Receptors, Serotonin/metabolism , Signal Transduction/physiology , Animals , GTP-Binding Proteins/metabolism , Humans , Receptors, Serotonin, 5-HT1 , Sensitivity and Specificity
10.
J Cell Biol ; 155(2): 207-16, 2001 Oct 15.
Article in English | MEDLINE | ID: mdl-11591730

ABSTRACT

p53 is a transcriptional activator which has been implicated as a key regulator of neuronal cell death after acute injury. We have shown previously that p53-mediated neuronal cell death involves a Bax-dependent activation of caspase 3; however, the transcriptional targets involved in the regulation of this process have not been identified. In the present study, we demonstrate that p53 directly upregulates Apaf1 transcription as a critical step in the induction of neuronal cell death. Using DNA microarray analysis of total RNA isolated from neurons undergoing p53-induced apoptosis a 5-6-fold upregulation of Apaf1 mRNA was detected. Induction of neuronal cell death by camptothecin, a DNA-damaging agent that functions through a p53-dependent mechanism, resulted in increased Apaf1 mRNA in p53-positive, but not p53-deficient neurons. In both in vitro and in vivo neuronal cell death processes of p53-induced cell death, Apaf1 protein levels were increased. We addressed whether p53 directly regulates Apaf1 transcription via the two p53 consensus binding sites in the Apaf1 promoter. Electrophoretic mobility shift assays demonstrated p53-DNA binding activity at both p53 consensus binding sequences in extracts obtained from neurons undergoing p53-induced cell death, but not in healthy control cultures or when p53 or the p53 binding sites were inactivated by mutation. In transient transfections in a neuronal cell line with p53 and Apaf1 promoter-luciferase constructs, p53 directly activated the Apaf1 promoter via both p53 sites. The importance of Apaf1 as a p53 target gene in neuronal cell death was evaluated by examining p53-induced apoptotic pathways in primary cultures of Apaf1-deficient neurons. Neurons treated with camptothecin were significantly protected in the absence of Apaf1 relative to those derived from wild-type littermates. Together, these results demonstrate that Apaf1 is a key transcriptional target for p53 that plays a pivotal role in the regulation of apoptosis after neuronal injury.


Subject(s)
Apoptosis , Neurons/metabolism , Proteins/genetics , Transcriptional Activation , Tumor Suppressor Protein p53/physiology , Animals , Apoptotic Protease-Activating Factor 1 , Base Sequence , Brain Ischemia/metabolism , Brain Ischemia/pathology , Camptothecin/pharmacology , Cell Line , Cells, Cultured , Mice , Mice, Transgenic , Neurons/pathology , Promoter Regions, Genetic , Protein Biosynthesis , Proteins/physiology , RNA, Messenger/biosynthesis
11.
J Biol Chem ; 276(17): 14299-307, 2001 Apr 27.
Article in English | MEDLINE | ID: mdl-11278286

ABSTRACT

Negative regulation of neuronal serotonin (5-HT1A) receptor levels by glucocorticoids in vivo may contribute to depression. Both types I (mineralocorticoid) and II (glucocorticoid) receptors (MR and GR, respectively) participate in corticosteroid-induced transcriptional repression of the 5-HT1A gene; however, the precise mechanism is unclear. A direct repeat 6-base pair glucocorticoid response element (GRE) half-site 5'-TGTCCT separated by 6 nucleotides was conserved in human, mouse, and rat 5-HT1A receptor promoters. In SN-48 neuronal cells that express MR, GR, and 5-HT1A receptors, deletion or inactivation of the nGRE (negative GRE) eliminated negative regulation of the rat 5-HT1A or heterologous promoters by corticosteroids, whereas its inclusion conferred corticosteroid-induced inhibition to a heterologous promoter. Bacterially expressed recombinant MR and GR preferentially bound to the nGRE as a heterodimer, as identified in nuclear extracts of MR/GR-transfected COS-7 cells, and with higher affinity than MR or GR homodimers. In SN48 and COS-7 cells, concentration-dependent coactivation of MR and GR was required for maximal inhibitory action by corticosteroids and was abrogated in the L501P-GR mutant lacking DNA binding activity. Corticosteroid-mediated transcriptional inhibition was greater for MR/GR in combination than for MR or GR alone. These data represent the first identification of an nMRE/GRE and indicate that heterodimerization of MR and GR mediates direct corticosteroid-induced transrepression of the 5-HT1A receptor promoter.


Subject(s)
Receptors, Glucocorticoid/chemistry , Receptors, Mineralocorticoid/chemistry , Receptors, Serotonin/genetics , Response Elements , Transcription, Genetic , Animals , Blotting, Western , COS Cells , Cell Line , Cell Nucleus/metabolism , Conserved Sequence , DNA-Binding Proteins/metabolism , Deoxyribonuclease I/metabolism , Dexamethasone/pharmacology , Dimerization , Dose-Response Relationship, Drug , Gene Deletion , Glucocorticoids/pharmacology , Glutathione Transferase/metabolism , Humans , Kinetics , Mice , Plasmids/metabolism , Promoter Regions, Genetic , Rats , Receptors, Serotonin, 5-HT1 , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/metabolism , Transfection
12.
J Biol Chem ; 276(6): 4382-8, 2001 Feb 09.
Article in English | MEDLINE | ID: mdl-11080494

ABSTRACT

Serotonin (5-HT) up-regulates B and T lymphocyte proliferation by activating mitogen-induced cell surface 5-HT(1A) receptors. The mechanism of 5-HT(1A) receptor induction by B and T cell mitogens at the mRNA and protein levels in mouse splenocytes was addressed. Quantitation by RNase protection assay showed maximal increases of 3.4-, 3.0-, 3.8-, and 4.9-fold in relative 5-HT(1A) mRNA levels after 48 h of stimulation of splenocytes with lipopolysaccharide, phytohemagglutinin, concanavalin A, or phorbol 12-myristate 13-acetate plus ionomycin, respectively, as compared with unstimulated cells. Mitogens did not alter 5-HT(1A) mRNA stability (t(12) = 26 h), but induction of 5-HT(1A) mRNA was blocked by the transcriptional inhibitor actinomycin D (10 microgram/ml) and by inhibition of nuclear factor-kappaB signaling. Additionally, mitogenic stimulation of transcription was paralleled by increased cell surface 5-HT(1A) receptor immunoreactivity in splenocytes. Thus, mitogen-induced 5-HT(1A) receptor expression appears to involve transcriptional regulation by the nuclear factor-kappaB signaling cascade. Increased expression of the 5-HT(1A) receptor in activated B and T lymphocytes may enhance the immune response and provide therapeutic target for tissue inflammation and immune stimulation.


Subject(s)
B-Lymphocytes/metabolism , RNA, Messenger/genetics , Receptors, Serotonin/biosynthesis , Receptors, Serotonin/genetics , T-Lymphocytes/metabolism , Transcription, Genetic , Animals , B-Lymphocytes/cytology , Base Sequence , Cell Division , DNA Primers , Female , Immunohistochemistry , Lymphocyte Activation , Mice , Mice, Inbred BALB C , NF-kappa B/metabolism , Protein Transport , Receptors, Serotonin, 5-HT1 , T-Lymphocytes/cytology , Up-Regulation
13.
Curr Protoc Neurosci ; Chapter 4: Unit 4.1, 2001 May.
Article in English | MEDLINE | ID: mdl-18428473

ABSTRACT

The principle of cloning utilizes hybridization of single-stranded DNA probes to denatured DNA fixed on membranes to detect the identical DNA molecules present in a large population of diverse DNAs. In homology or low-stringency cloning, hybridization of oligonucleotide, cDNA, or genomic DNA probes to denatured DNA from plasmid or bacteriophage libraries is carried out under low-stringency conditions that promote hybridization in the presence of sequence mismatch (i.e., mispaired nucleotides). The procedures used are identical to those for screening libraries at high stringency, with the exception that hybridization and washing conditions are altered to permit hybridization with mismatched sequence; salt concentration is increased and the hybridization and wash temperatures are decreased. This unit describes conditions that have been used to clone and identify novel genes and cDNA clones using low-stringency hybridization of known probes to membranes that contain libraries of bacterial or bacteriophage DNA.


Subject(s)
Cloning, Molecular/methods , DNA, Complementary/genetics , Nucleic Acid Hybridization/methods , Sequence Alignment/methods , Sequence Homology, Nucleic Acid , DNA/analysis , DNA/genetics , DNA, Complementary/analysis , Nucleic Acid Hybridization/genetics
14.
J Biol Chem ; 275(11): 8161-8, 2000 Mar 17.
Article in English | MEDLINE | ID: mdl-10713139

ABSTRACT

The level of expression of the 5-HT1A receptor in the raphe and limbic systems is implicated in the etiology and treatment of major depression and anxiety disorders. The rat 5-HT1A receptor gene is regulated by a proximal TATA-driven promoter and by upstream repressors that inhibit gene expression. Deletion of a 71-base pair (bp) segment between -1590/-1519 bp of the 5-HT1A receptor gene induced over 10-fold enhancement of transcriptional activity in both 5-HT1A receptor-expressing (RN46A raphe and SN48 septal) cells and receptor-negative (L6 myoblast and C6 glioma) cells. A 31-bp segment of the repressor was protected from DNase I digestion by RN46A or L6 nuclear extracts. Within the 31-bp segment, a single protein complex was present in receptor-expressing cells that bound a novel 14-bp DNA element; in receptor-negative cells, an additional complex bound an adjacent 12-bp sequence. In receptor-positive but not receptor-negative cells, mutation of the 14-bp element to eliminate protein binding abrogated repression to nearly the same extent as deletion of the -1590/-1519 bp segment. Additional mutation of both 14-bp and 12-bp elements abolished protein binding and repressor activity in receptor-negative cells. Thus a single protein-DNA complex at the 14-bp element represses the 5-HT1A receptor gene in 5-HT1A receptor-positive neuronal cells, whereas adjacent DNA elements provide a dual repression mechanism in 5-HT1A receptor-negative cells.


Subject(s)
Neurons/metabolism , Raphe Nuclei/metabolism , Receptors, Serotonin/genetics , Regulatory Sequences, Nucleic Acid , Animals , Anxiety Disorders/etiology , DNA-Binding Proteins/metabolism , Depressive Disorder/etiology , Enhancer Elements, Genetic , Gene Expression Regulation , Molecular Sequence Data , Promoter Regions, Genetic , Protein Binding , Raphe Nuclei/cytology , Rats , Receptors, Serotonin/biosynthesis , Receptors, Serotonin, 5-HT1 , Septum of Brain/cytology , Septum of Brain/metabolism , Transcription, Genetic
15.
Mol Cell Biol ; 20(5): 1497-506, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10669727

ABSTRACT

Control of cell proliferation depends on intracellular mediators that determine the cellular response to external cues. In neuroendocrine cells, the dopamine D2 receptor short form (D2S receptor) inhibits cell proliferation, whereas in mesenchymal cells the same receptor enhances cell proliferation. Nontransformed BALB/c 3T3 fibroblast cells were stably transfected with the D2S receptor cDNA to study the G proteins that direct D2S signaling to stimulate cell proliferation. Pertussis toxin inactivates G(i) and G(o) proteins and blocks signaling of the D2S receptor in these cells. D2S receptor signaling was reconstituted by individually transfecting pertussis toxin-resistant Galpha(i/o) subunit mutants and measuring D2-induced responses in pertussis toxin-treated cells. This approach identified Galpha(i)2 and Galpha(i)3 as mediators of the D2S receptor-mediated inhibition of forskolin-stimulated adenylyl cyclase activity; Galpha(i)2-mediated D2S-induced stimulation of p42 and p44 mitogen-activated kinase (MAPK) and DNA synthesis, whereas Galpha(i)3 was required for formation of transformed foci. Transfection of toxin-resistant Galpha(i)1 cDNA induced abnormal cell growth independent of D2S receptor activation, while Galpha(o) inhibited dopamine-induced transformation. The role of Gbetagamma subunits was assessed by ectopic expression of the carboxyl-terminal domain of G protein receptor kinase to selectively antagonize Gbetagamma activity. Mobilization of Gbetagamma subunits was required for D2S-induced calcium mobilization, MAPK activation, and DNA synthesis. These findings reveal a remarkable and distinct G protein specificity for D2S receptor-mediated signaling to initiate DNA synthesis (Galpha(i)2 and Gbetagamma) and oncogenic transformation (Galpha(i)3), and they indicate that acute activation of MAPK correlates with enhanced DNA synthesis but not with transformation.


Subject(s)
Cell Transformation, Neoplastic , DNA Replication , GTP-Binding Protein alpha Subunits, Gi-Go , GTP-Binding Protein beta Subunits , GTP-Binding Protein gamma Subunits , GTP-Binding Proteins/physiology , Heterotrimeric GTP-Binding Proteins/physiology , Proto-Oncogene Proteins/physiology , Receptors, Dopamine D2/physiology , Signal Transduction , 3T3 Cells , Animals , Cell Division/physiology , GTP-Binding Protein alpha Subunit, Gi2 , Mice , Mice, Inbred BALB C
16.
J Biol Chem ; 274(50): 35469-74, 1999 Dec 10.
Article in English | MEDLINE | ID: mdl-10585418

ABSTRACT

The 5-HT1A receptor is implicated in depression and anxiety. This receptor couples to G(i) proteins to inhibit adenylyl cyclase (AC) activity but can stimulate AC in tissues (e.g. hippocampus) that express ACII. The role of ACII in receptor-mediated stimulation of cAMP formation was examined in HEK-293 cells transfected with the 5-HT1A receptor, which mediated inhibition of basal and G(s)-induced cAMP formation in the absence of ACII. In cells cotransfected with 5-HT1A receptor and ACII plasmids, 5-HT1A agonists induced a 1. 5-fold increase in cAMP level. Cotransfection of 5-HT1A receptor, ACII, and Galpha(i2), but not Galpha(i1), Galpha(i3), or Galpha(o), resulted in an agonist-independent 6-fold increase in the basal cAMP level, suggesting that G(i2) preferentially coupled the receptor to ACII. The 5-HT1B receptor also constitutively activated ACII. Constitutive activity of the 5-HT1A receptor was blocked by pertussis toxin and the Gbetagamma antagonist, betaCT, suggesting an important role for Gbetagamma-mediated activation of ACII. The Thr-149 --> Ala mutation in the second intracellular domain of the 5-HT1A receptor disrupted Gbetagamma-selective activation of ACII. Spontaneous 5-HT1A receptor activity was partially attenuated by 5-HT1A receptor partial agonists with anxiolytic activity (e.g. buspirone and flesinoxan) but was not altered by full agonists or antagonists. Thus, anxiolytic activity may involve inhibition of spontaneous 5-HT1A receptor activity.


Subject(s)
Adenylyl Cyclases/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Receptors, Serotonin/physiology , Adenylate Cyclase Toxin , Adenylyl Cyclases/genetics , Buspirone/pharmacology , Cell Line , Cyclic AMP/metabolism , Dopamine/pharmacology , Humans , Isoenzymes/metabolism , Kidney , Kinetics , Pertussis Toxin , Receptor, Serotonin, 5-HT1B , Receptors, Serotonin/drug effects , Receptors, Serotonin/genetics , Receptors, Serotonin, 5-HT1 , Recombinant Proteins/drug effects , Recombinant Proteins/metabolism , Serotonin Antagonists/pharmacology , Serotonin Receptor Agonists/pharmacology , Spiperone/pharmacology , Transfection , Virulence Factors, Bordetella/pharmacology
17.
J Neurochem ; 72(6): 2238-47, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10349831

ABSTRACT

The transcriptional initiation and regulation of the rat serotonin 5-HT1A receptor gene were characterized. By three types of analyses, a single brain-specific site of transcriptional initiation was localized to -967 bp upstream of the translation initiation codon that is utilized both in hippocampus and in the rat raphe RN46A cell line. This major site of transcriptional initiation was located 58 bp downstream from a consensus TATA element, suggesting TATA-driven transcription of the rat 5-HT1A receptor. To identify the promoter activity of the receptor gene, progressive 5' deletions of the -2,719/-117-bp fragment of the 5-HT1A promoter linked to luciferase gene were transfected into 5-HT1A-negative (pituitary GH4C1, L6 myoblast, and C6 glioma) and 5-HT1A-positive (septal SN-48 and raphe RN46A) cell lines. Enhancer regions were identified within a fragment between nucleotides -426 and -117 that selectively enhanced transcription in 5-HT1A-positive cells. A nonselective enhancer/promoter that mediated expression in all cell lines was located upstream between -1,519 and -426 bp in a DNA segment containing consensus TATA, CCAAT, SP-1, and AP-1 elements as well as a poly-GT26 dinucleotide repeat. Strong repression of transcription in all cell lines was conferred by the region upstream of -1,519 bp that contains a 152-bp DNA segment with >80% identity to RANTES, tumor necrosis factor-beta, and other immune system genes. Our results indicate that TATA-driven expression of the 5-HT1A receptor is regulated by a novel proximal tissue-specific enhancer region, a nonselective promoter, and an upstream repressor region that is distinct from previously identified neuron-specific repressors.


Subject(s)
Gene Expression Regulation , Receptors, Serotonin/genetics , TATA Box , Transcription, Genetic , Animals , Base Sequence , Brain/metabolism , Cell Line , Cloning, Molecular , Consensus Sequence , Genes, Reporter , Glioma , Luciferases/biosynthesis , Luciferases/genetics , Mice , Molecular Sequence Data , Raphe Nuclei/metabolism , Rats , Receptors, Serotonin/biosynthesis , Receptors, Serotonin, 5-HT1 , Recombinant Fusion Proteins/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Deletion , Sequence Homology, Nucleic Acid , Transfection , Tumor Cells, Cultured , beta-Galactosidase/biosynthesis , beta-Galactosidase/genetics
18.
J Biol Chem ; 274(23): 16444-50, 1999 Jun 04.
Article in English | MEDLINE | ID: mdl-10347206

ABSTRACT

The three Galphai subunits were independently depleted from rat pituitary GH4C1 cells by stable transfection of each Galphai antisense rat cDNA construct. Depletion of any Galphai subunit eliminated receptor-induced inhibition of basal cAMP production, indicating that all Galphai subunits are required for this response. By contrast, receptor-mediated inhibition of vasoactive intestinal peptide (VIP)-stimulated cAMP production was blocked by selective depletions for responses induced by the transfected serotonin 1A (5-HT1A) (Galphai2 or Galphai3) or endogenous muscarinic-M4 (Galphai1 or Galphai2) receptors. Strikingly, receptor activation in Galphai1-depleted clones (for the 5-HT1A receptor) or Galphai3-depleted clones (for the muscarinic receptor) induced a pertussis toxin-sensitive increase in basal cAMP production, whereas the inhibitory action on VIP-stimulated cAMP synthesis remained. Finally, in Galphai2-depleted clones, activation of 5-HT1A receptors increased VIP-stimulated cAMP synthesis. Thus, 5-HT1A and muscarinic M4 receptor may couple dominantly to Galphai1 and Galphai3, respectively, to inhibit cAMP production. Upon removal of these Galphai subunits to reduce inhibitory coupling, stimulatory receptor coupling is revealed that may involve Gbetagamma-induced activation of adenylyl cyclase II, a Gi-stimulated cyclase that is predominantly expressed in GH4C1 cells. Thus Gi-coupled receptor activation involves integration of both inhibitory and stimulatory outputs that can be modulated by specific changes in alphai subunit expression level.


Subject(s)
Adenylyl Cyclases/metabolism , Cyclic AMP/biosynthesis , GTP-Binding Protein alpha Subunits, Gi-Go , GTP-Binding Proteins/biosynthesis , Proto-Oncogene Proteins/biosynthesis , Receptors, Serotonin/metabolism , Adenylate Cyclase Toxin , Animals , Blotting, Western , Calcium/metabolism , Cells, Cultured , GTP-Binding Protein alpha Subunit, Gi2 , GTP-Binding Proteins/genetics , Gene Expression Regulation , Pertussis Toxin , Proto-Oncogene Proteins/genetics , Rats , Receptor, Muscarinic M4 , Receptors, Muscarinic/metabolism , Receptors, Serotonin, 5-HT1 , Virulence Factors, Bordetella/pharmacology
19.
J Biol Chem ; 274(14): 9238-45, 1999 Apr 02.
Article in English | MEDLINE | ID: mdl-10092597

ABSTRACT

Previous studies have shown that a single G protein-coupled receptor can regulate different effector systems by signaling through multiple subtypes of heterotrimeric G proteins. In LD2S fibroblast cells, the dopamine D2S receptor couples to pertussis toxin (PTX)-sensitive Gi/Go proteins to inhibit forskolin- or prostaglandin E1-stimulated cAMP production and to stimulate calcium mobilization. To analyze the role of distinct Galphai/o protein subtypes, LD2S cells were stably transfected with a series of PTX-insensitive Galphai/o protein Cys --> Ser point mutants and assayed for D2S receptor signaling after PTX treatment. The level of expression of the transfected Galpha mutant subunits was similar to the endogenous level of the most abundant Galphai/o proteins (Galphao, Galphai3). D2S receptor-mediated inhibition of forskolin-stimulated cAMP production was retained only in clones expressing mutant Galphai2. In contrast, the D2S receptor utilized Galphai3 to inhibit PGE1-induced (Gs-coupled) enhancement of cAMP production. Following stable or transient transfection, no single or pair set of mutant Galphai/o subtypes rescued the D2S-mediated calcium response following PTX pretreatment. On the other hand, in LD2S cells stably transfected with GRK-CT, a receptor kinase fragment that specifically antagonizes Gbeta gamma subunit activity, D2S receptor-mediated calcium mobilization was blocked. The observed specificity of Galphai2 and Galphai3 for different states of adenylyl cyclase activation suggests a higher level of specificity for interaction of Galphai subunits with forskolin- versus Gs-activated states of adenylyl cyclase than has been previously appreciated.


Subject(s)
Calcium/metabolism , Colforsin/metabolism , Cyclic AMP/metabolism , GTP-Binding Proteins/physiology , Receptors, Dopamine D2/metabolism , Adenylate Cyclase Toxin , Adenylyl Cyclase Inhibitors , Animals , Cells, Cultured , GTP-Binding Protein alpha Subunits, Gs/physiology , Mice , Mutagenesis, Site-Directed , Pertussis Toxin , Transfection , Virulence Factors, Bordetella/pharmacology
20.
Mol Endocrinol ; 13(1): 138-47, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9892019

ABSTRACT

To characterize the specificity of endogenously expressed G protein-coupled receptor kinases (GRKs) for endogenous Gi-coupled alpha2C-adrenergic and serotonin 1B (5-HT1B) receptors in the opossum kidney (OK) cell line, we have isolated a 3.073-kb OK-GRK2 clone encoding a 689-amino acid protein that shares 94.2% amino acid identity with rat GRK2. Northern blot analysis revealed the presence of GRK2 mRNA transcripts of 5.0 and 3.0 kb in OK cells. In intact OK cells, preincubation (45 min) with agonist (5-HT or UK 14304, 1 microM) reduced the maximal inhibition of forskolin-induced cAMP accumulation mediated by endogenous 5-HT1B and alpha2C-adrenergic receptors by 12 +/- 2% or 17 +/- 4%, respectively. In transfected OK cells overexpressing OK-GRK2, agonist-induced desensitization of the alpha2C-adrenergic receptor, but not the 5-HT1B receptor, was enhanced by 2- to 4-fold. Conversely, in cells overexpressing the kinase-inactive mutant OK-GRK2-K220R, alpha2C-adrenergic receptor desensitization was selectively abolished, whereas desensitization of the 5-HT1B receptor was slightly enhanced. Similarly, depletion of GRK-2 protein by stable transfection of full-length antisense OK-GRK2 cDNA blocked the desensitization of alpha2C-adrenergic receptors but not of 5-HT1B receptors. These results represent the first evidence of the coexistence of GRK2-dependent (for alpha2C receptors) and GRK2-independent (for 5-HT1B receptors) mechanisms of desensitization in intact cells and demonstrate the selectivity of GRK2 for distinct Gi-coupled receptors.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Kidney/metabolism , Receptors, Adrenergic, alpha-2/metabolism , Receptors, Serotonin/metabolism , Adrenergic alpha-Agonists/pharmacology , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Brimonidine Tartrate , Cell Line , Cloning, Molecular , Colforsin/pharmacology , G-Protein-Coupled Receptor Kinase 2 , Kidney/cytology , Kidney/drug effects , Molecular Sequence Data , Mutation , Opossums , Quinoxalines/pharmacology , Rats , Receptor, Serotonin, 5-HT1B , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , beta-Adrenergic Receptor Kinases
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