Systemic Regulation of RAS/MAPK Signaling by the Serotonin Metabolite 5-HIAA.
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2015 May 15;11(5):e1005236. doi: 10.1371/journal.pgen.1005236. eCollection 2015.Systemic Regulation of RAS/MAPK Signaling by the Serotonin Metabolite 5-HIAA.1, 2, 3, 2, 2, 3.1University of Zurich, Institute of Molecular Life Sciences, Zurich, S PhD Program in Molecular Life Sciences, University and ETH Zurich, Zurich, Switzerland.2Laboratory of Nematology, Wageningen University, Wageningen, The Netherlands.3University of Zurich, Institute of Molecular Life Sciences, Zurich, Switzerland.AbstractHuman cancer is caused by the interplay of mutations in oncogenes and tumor suppressor genes and inherited variations in cancer susceptibility genes. While many of the tumor initiating mutations are well characterized, the effect of genetic background variation on disease onset and progression is less understood. We have used C. elegans genetics to identify genetic modifiers of the oncogenic RAS/MAPK signaling pathway. Quantitative trait locus analysis of two highly diverged C. elegans isolates combined with allele swapping experiments identified the polymorphic monoamine oxidase A (MAOA) gene amx-2 as a negative regulator of RAS/MAPK signaling. We further show that the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA), which is a product of MAOA catalysis, systemically inhibits RAS/MAPK signaling in different organs of C. elegans. Thus, MAOA activity sets a global threshold for MAPK activation by controlling 5-HIAA levels. To our knowledge, 5-HIAA is the first endogenous small molecule that acts as a systemic inhibitor of RAS/MAPK signaling. PMID:
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(A) RAS/MAPK signaling induces three VPCs. P6.p receives most of the inductive EGF signal from the anchor cell and activates the EGFR/RAS/MAPK pathway inducing the 1° cell fate (green arrows). Lateral signaling via the Notch pathway induces the 2° cell fate in the neighboring VPCs P5.p and P7.p (red arrows). The remaining VPCs (blue) adopt the non-vulval 3° cell fate. (B) Crossing scheme to generate the let-60(n1046gf) miRILs. Hawaii males (red) were crossed with Bristol let-60(n1046gf) mutants (blue). For each example animal, the two chromosomes IV carrying the n1046 mutation and another arbitrary chromosome pair are shown. Random segregation of the two parental genomes was allowed except for the let-60(gf) mutation that was kept homozygous from F2 generation onwards. After ten generations of self-fertilization to drive all regions to homozygosity, 228 independent miRILs were obtained. (C) Genotypes and phenotypes of the let-60(gf) miRILs sorted by increasing VI. Genotypes determined by FLP mapping [] are plotted on the y-axis versus the miRIL numbers on the x-axis. Hawaii genotypes are indicated with red, Bristol genotypes with blue and missing genotypes with gray colors. The VIs for each miRIL are shown below the genotypes. Error bars indicate the standard error of the mean. (D) QTL mapping identified three regions (QTL1 through QTL3) above the threshold LOD score of 3 (dotted red line). In each of the panels showing chromosomes I through X, the locations of the FLP markers used for genotyping are indicated on the x-axis with vertical lines. For the exact locations of the FLPs used, see
and [].PLoS Genet. ):e1005236. (A) Fine-mapping of QTL1 with ILs. For each IL, the regions containing the Hawaii (black) genome in the Bristol (grey) background are indicated, and the corresponding VIs are plotted below. Black columns indicate the average VI of three independent lines carrying an introgression and gray columns the average VI of three sibling lines without introgression. Dashed boxes indicate the QTL1a and QTL1b sub-regions. (B) Allele-specific effects of amx-2 RNAi compared to empty vector controls. (C) Two copies of Bristol but not Hawaii amx-2 rescue the increased VI of amx-2(ok1235); let-60(n1046gf) double mutants. (D) Epistasis analysis of amx-2(ok1235). The dashed line indicates the wild-type VI of 3. (E-H) Expression pattern of a transcriptional P amx-2::gfp reporter in the pharynx and head neurons (E), the adult vulva (F), the intestine (G) and some rectal cells (H) of L4 larvae. The scale bar is 10μm. (I) Tissue-specific amx-2 RNAi. Knock-down in the intestine but not the vulval cells increases the VI of let-60(n1046gf) mutants (J) Quantitative PCR of amx-2 and amx-1. Expression levels were normalized to the N2 wild-type Bristol strain. Error bars in (A) to (I) indicate the standard error of the mean and in (J) the standard deviation measured in three independent experiments. The numbers of animals scored are shown inside the columns. *** indicates p&0.001, ** p&0.01, *&0.05 and n.s. p&0.1 in a Student’s t-test.PLoS Genet. ):e1005236. (A) Function of MAOA in DA and 5-HT degradation. (B) 5-HT levels in total extracts of wild-type and amx-2(ok1235) animals. (C) Effect of DA, 5-HT and its metabolites on the VI of let-60(n1046gf) single and amx-2(ok1235); let-60(n1046gf) double mutants. (D) Examples of (top) a 5-HIAA treated and (bottom) an untreated let-60(n1046gf) L4 larva. The normal vulva and the ectopically induced cells are underlined. (E) Dose-dependent reduction of the VI by 5-HT and (F) 5-HIAA treatments. Note in (E) the different sensitivities of the two strains to 1μM 5-HT. (G) Effect of 5-HIAA on mutations activating the EGFR/RAS/MAPK pathway at different levels. (H) Resistance of some 5-HT pathway mutants to 5-HIAA treatment. Error bars indicate the standard error of the mean. The numbers of animals scored are indicated in brackets or inside the columns. *** indicates p&0.001, ** p&0.01, and n.s. p&0.1 in a Student’s t-test.PLoS Genet. ):e1005236. (A) Partial suppression of the germline defect in let-60(ga89ts) mutants treated with 5-HIAA and grown at 25°C. The images show the gonads of 5-HIAA treated (top) and untreated (bottom) young adults. Note the regularly stacked oocytes in 5-HIAA treated and the irregularly stacked and smaller oocytes in untreated animals. (B) Partial suppression of the duct cell duplication phenotype in let-60(n1046gf) mutants by 5-HIAA. The images show the single duct cell in a 5-HIAA treated let-60(n1046gf) L4 larva (top) and the two duct cells in an untreated larva (bottom). The arrows point at the nuclei of the duct cells expressing the lin-48::gfp marker. (C) MPK-1 phosphorylation in total extracts of let-60(n1046gf) single and amx-2(ok1235); let-60(n1046gf) double mutant larvae treated with 4mM 5-HT or 5-HIAA. The ratios of phosphoMPK-1 to total MPK-1 levels were determined in three independent experiments as described in [] and Materials and Methods. Values were normalized to the ratios in untreated animals. The numbers of animals scored are indicated in brackets or inside the columns. *** indicates p&0.001 and ** p&0.01 in a Student’s t-testPLoS Genet. ):e1005236.Publication TypesMeSH TermsSubstancesFull Text Sources
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External link. Please review our .Effects and side effects associated with the non-nutritional use of tryptophan by humans.
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):S. doi: 10.3945/jn.111.157065. Epub
2012 Oct 17.Effects and side effects associated with the non-nutritional use of tryptophan by humans.1.1Department of Psychiatry, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. fernstromjd@upmc.eduAbstractThe daily nutritional requirement for L-tryptophan (Trp) is modest (5 mg/kg). However, many adults choose to consume much more, up to 4-5 g/d (60-70 mg/kg), typically to improve mood or sleep. Ingesting L-Trp raises brain tryptophan levels and stimulates its conversion to serotonin in neurons, which is thought to mediate its actions. Are there side effects from Trp supplementation? Some consider drowsiness a side effect, but not those who use it to improve sleep. Though the literature is thin, occasional side effects, seen mainly at higher doses (70-200 mg/kg), include tremor, nausea, and dizziness, and may occur when Trp is taken alone or with a drug that enhances serotonin function (e.g., antidepressants). In rare cases, the "serotonin syndrome" occurs, the result of too much serotonin stimulation when Trp is combined with serotonin drugs. Symptoms include delirium, myoclonus, hyperthermia, and coma. In 1989 a new syndrome appeared, dubbed eosinophilia myalgia syndrome (EMS), and was quickly linked to supplemental Trp use. Key symptoms included debilitating myalgia (muscle pain) and a high peripheral eosinophil count. The cause was shown not to be Trp but a contaminant in certain production batches. This is not surprising, because side effects long associated with Trp use were not those associated with the EMS. Over 5 decades, Trp has been taken as a supplement and as an adjunct to medications with occasional modest, short-lived side effects. Still, the database is small and largely anecdotal. A thorough, dose-related assessment of side effects remains to be conducted.PMID:
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External link. Please review our .Electric foot shock stress: a useful tool in neuropsychiatric studies.
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):655-77. doi: 10.1515/revneuro-.Electric foot shock stress: a useful tool in neuropsychiatric studies., .AbstractElectric foot shock is a complex stressor with both physical and emotional components. It has been employed as an important tool to develop diverse animal models in the field of psychopharmacology. The electric foot shock paradigm includes acute or chronic exposures of shocks of varying intensity and duration on an electrified grid floor in an electric foot shock apparatus. Research evidence reveals that foot shocks of varying intensity produce behavioral and neurochemical changes reflecting depression, anxiety, and post-traumatic stress disorder (PTSD) in humans. Animals generally do not habituate to foot shocks in comparison to other stressors, including loud noise, bright light, and hot and cold temperatures. Additionally, it offers an experimental advantage of control over in therefore, by varying its application parameters, different disorder models have been created. Electric foot shock fear conditioning-induced ultrasonic vocalization and fear-potentiated startle have been explored to develop models of anxiety and panic. Similarly, fear conditioning in the form of foot shock exposure followed by situational reminders has been used to develop a model of PTSD. Electric foot shock-induced conflict has been explored to develop operant conflict models (Geller-Seifter and Vogel tests), which in turn are pharmacologically validated to screen potential anti-anxiety agents. Inescapable electric shock-induced 'learned helplessness' mimics the symptomology of depression, and this phenomenon has been employed to develop the model of depression. The present review describes the pharmacologically validated models of anxiety, depression, and PTSD involving electric foot shock as an aversive stimulus. PMID:
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External link. Please review our .Autism gene variant causes hyperserotonemia, serotonin receptor hypersensitivity, social impairment and repetitive behavior.
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2012 Apr 3;109(14):5469-74. doi: 10.1073/pnas.. Epub
2012 Mar 19.Autism gene variant causes hyperserotonemia, serotonin receptor hypersensitivity, social impairment and repetitive behavior.1, , , , , , , , , , , , , , , , , , , .1Department of Psychiatry, Vanderbilt University, Nashville, TN 37232, USA. j.vvw@vanderbilt.eduAbstractFifty years ago, increased whole-blood serotonin levels, or hyperserotonemia, first linked disrupted 5-HT homeostasis to Autism Spectrum Disorders (ASDs). The 5-HT transporter (SERT) gene (SLC6A4) has been associated with whole blood 5-HT levels and ASD susceptibility. Previously, we identified multiple gain-of-function SERT coding variants in children with ASD. Here we establish that transgenic mice expressing the most common of these variants, SERT Ala56, exhibit elevated, p38 MAPK-dependent transporter phosphorylation, enhanced 5-HT clearance rates and hyperserotonemia. These effects are accompanied by altered basal firing of raphe 5-HT neurons, as well as 5HT(1A) and 5HT(2A) receptor hypersensitivity. Strikingly, SERT Ala56 mice display alterations in social function, communication, and repetitive behavior. Our efforts provide strong support for the hypothesis that altered 5-HT homeostasis can impact risk for ASD traits and provide a model with construct and face validity that can support further analysis of ASD mechanisms and potentially novel treatments.PMID:
[PubMed - indexed for MEDLINE] Dysregulated SERT phosphorylation, increased 5-HT uptake, and hyperserotonemia in the SERT Ala56 knock-in mice. (A) Representative gel and cumulative graph of basal phosphorylation of SERT [Bartlett test statistic, 26.49, showing unequal variances (P & 0.0001); therefore, nonparametric Mann–Whitney test was used, U = 6.00, P = 0.0002, n = 12 per genotype]. (B) Representative gel and cumulative graph of 8-Br-cGMP–induced phosphorylation (two-way repeated-measures ANOVA interaction of cGMP treatment by genotype, F = 17.51, P = 0.0013; Bonferroni post-test in WT for cGMP treatment, t = 3.66, P & 0.01; Bonferroni post-test in Ala56 for cGMP, t = 2.26, P & 0.05; WT, n = 7; Ala56, n = 7). (C) Representative gel and cumulative graph of PD169316 inhibition of phosphorylation (two-way repeated-measures ANOVA interaction of PD169316 treatment by genotype, F = 8.815, P = 0.018; Bonferroni post-test of genotype difference in basal condition, t = 4.83, P & 0.01; Bonferroni post-test of genotype difference after PD169316 treatment, t = 0.63, P & 0.05; WT, n = 5; Ala56, n = 5). (D) 5-HT clearance rates in the CA3 region of the hippocampus as a function of increasing extracellular 5-HT concentrations. Mean clearance values from multiple 5-HT pulses ± SEM with three to six mice per point. For purposes of clarity, the SEMs for signal amplitudes are not shown, but they were always within 10% of the mean. Clearance of 5-HT was significantly faster in Ala56 mice than that in WT controls (main effect genotype, F1,73 = 64.69, P & 0.0001; main effect 5-HT concentration, F7,73 = 15.86, P & 0.0001, two-way ANOVA with Bonferroni post hoc comparisons). Kinetic analysis reveals an approximate twofold increase in the apparent Vmax for 5-HT clearance (t10 = 7.248, P & 0.0001; Ala56, 82 ± 20 nM/s vs. Gly56, 41 ± 15 nM/s) with no change in apparent transporter affinity (KT) (corrected for volume fraction, α = 0.02; Ala56, 64 ± 42 nM vs. Gly56, 64 ± 28 nM). (E) Time to clear 20% (T20) and 80% (T80) of the peak 5-HT signal amplitude 10 min after application of 0.5 pmol of 8-Br-cGMP, normalized to baseline 5-HT clearance. T20 reflects 5-HT at a concentration at which SERT-mediated 5-HT clearance is near Vmax, whereas T80 provides an index of 5-HT clearance at a concentration approximating the Km for SERT-mediated 5-HT uptake. 8-Br-cGMP significantly shortened both T20 and T80 for 5-HT clearance in WT mice but was without effect in Ala56 mice (T20, t = 2.65, P = 0.022; T80: t = 3.195, P = 0.009; WT, n = 7; Ala56 n = 6). (F) HPLC measurement of 5-HT in whole blood. Unpaired t test revealed a significant increase in whole-blood 5-HT in the Ala56 animals compared with WT controls (t = 2.55, P = 0.02; WT, n = 11; Ala56, n = 9).Proc Natl Acad Sci U S A. 2012 April 3;109(14):.Increased receptor sensitivity and altered social, communication, and repetitive behavior in SERT Ala56 knock-in mice. (A) Head twitches recorded by two observers blind to genotype over 15 min following injection of saline solution, the 5-HT2 agonist DOI, or the specific 5-HT2A antagonist M-100907 followed by DOI. Two-way repeated-measures ANOVA revealed a significant genotype–drug interaction (F = 6.88, P = 0.0029, n = 10 per genotype), with a significant Bonferroni posttest result only for the difference between WT and SERT Ala56 animals in the DOI condition (P & 0.01). (B) Change in rectal temperature from baseline after administration of the 5-HT1A/5-HT7 receptor agonist 8-OH-DPAT. Piecewise mixed linear model analysis revealed a significant genotype–drug–time interaction over the 30 min from baseline to maximal hypothermia response (F1,177, P & 0.0001, n = 12 per genotype), reflecting a steeper slope in the SERT Ala56 animals compared with the WT controls. (C) Example traces with basal firing rates are shown for cell-attached extracellular recordings of dorsal raphe neurons in midbrain slices (n = 16 per genotype). Unpaired t test with Welch correction for unequal variances (F test to compare variances, F15,15 = 4.345, P = 0.0036) revealed a significant decrease in firing rate in the Ala56 animals compared with the WT controls (t = 2.92, P = 0.032). (D) Percent inhibition of firing of dorsal raphe neurons as a function of varying, bath-applied 5-HT concentration. Curve fit analysis against log(5-HT concentration) with variable slope reveals a significant increase in sensitivity to inhibition of firing by 5-HT in the Ala56 animals compared with the WT controls (F2,6 = 292.3, P & 0.0001). (E) Pup vocalizations upon separation from the dam for 5 min at postnatal day 7. Mann–Whitney test revealed a significant decrease in ultrasonic vocalizations in the SERT Ala56 animals in contrast with WT littermate controls (U = 85.5, P = 0.015; WT, n = 15; Ala56, n = 22). (F) Time in each chamber of the three-chamber Crawley sociability test is shown. Animals with four or fewer total entries were excluded from the analysis as a result of inactivity (Fig. S5C). Two-way repeated measures ANOVA revealed a main effect for chamber (F = 23.25, P = 0.0006) and a trend for an interaction between genotype and stimulus (F = 3.92, P = 0.058; WT, n = 11; Ala56, n = 17). Bonferroni posttest revealed a significant preference for the social chamber in the WT (P & 0.01) but not the SERT Ala56 animals (P & 0.05). (G) Wins (frontward exit) for male animals on the tube test. McNemar exact test revealed a significant decrease in wins in the SERT Ala56 animals in contrast with WT littermate controls (P & 0.0001, n = 140 pairings). (H) Time spent performing individual behaviors over 24 h in the home cage. To allow better visualization, time spent sleeping is not shown, but did not differ by genotype. Two-way repeated measures ANOVA of log10(time) revealed a significant genotype effect (F = 5.84, P = 0.027, n = 10 per genotype), with Bonferroni posttest showing a significant genotype difference only for time spent hanging (P & 0.05). (I) Number of bouts of hanging behavior in 24 h in the home cage. t test of log10(bouts) revealed a significant increase in bouts of hanging in Ala56 SERT animals in comparison with WT littermate controls (t = 2.567, P = 0.019), with a significant correlation between log10(time) and log10(bouts) (Pearson R = 0.749, P & 0.001).Proc Natl Acad Sci U S A. 2012 April 3;109(14):.Publication TypesMeSH TermsSubstancesGrant SupportFull Text SourcesOther Literature SourcesMolecular Biology DatabasesMiscellaneous
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