Abnormal pontine activation in pathological laughing as shown by functional magnetic resonance imaging

To explore the aetiology of pathological laughing, a 65-year-old woman with pathological laughing was examined by 3-T functional magnetic resonance imaging (fMRI) before and after treatment with drugs. Here, we report that the patient consistently showed exaggerated pontine activation during the performance of three tasks before treatment, whereas abnormal pontine activation was no longer found after successful treatment with the selective serotonin reuptake inhibitor, paroxetine. Our findings in this first fMRI study of pathological laughing suggest that serotonergic replacement decreases the aberrant activity in a circuit that involves the pons

日本における『アフリカン・ダンス』の広がり : 東海地区のダンス・クラスを事例に

2007-03-31departmental bulletin pape

Yeast strains used in this study.

a<p>NA, not applicable.</p

Accumulation of a Threonine Biosynthetic Intermediate Attenuates General Amino Acid Control by Accelerating Degradation of Gcn4 via Pho85 and Cdk8

<div><p>Gcn4 is a master transcriptional regulator of amino acid and vitamin biosynthetic enzymes subject to the general amino acid control (GAAC), whose expression is upregulated in response to amino acid starvation in <i>Saccharomyces cerevisiae</i>. We found that accumulation of the threonine pathway intermediate β-aspartate semialdehyde (ASA), substrate of homoserine dehydrogenase (Hom6), attenuates the GAAC transcriptional response by accelerating degradation of Gcn4, already an exceedingly unstable protein, in cells starved for isoleucine and valine. The reduction in Gcn4 abundance on ASA accumulation requires Cdk8/Srb10 and Pho85, cyclin-dependent kinases (CDKs) known to mediate rapid turnover of Gcn4 by the proteasome via phosphorylation of the Gcn4 activation domain under nonstarvation conditions. Interestingly, rescue of Gcn4 abundance in <i>hom6</i> cells by elimination of <i>SRB10</i> is not accompanied by recovery of transcriptional activation, while equivalent rescue of UAS-bound Gcn4 in <i>hom6 pho85</i> cells restores greater than wild-type activation of Gcn4 target genes. These and other findings suggest that the two CDKs target different populations of Gcn4 on ASA accumulation, with Srb10 clearing mostly inactive Gcn4 molecules at the promoter that are enriched for sumoylation of the activation domain, and Pho85 clearing molecules unbound to the UAS that include both fully functional and inactive Gcn4 species.</p></div

The DNA binding domain is dispensable, but CDK phosphorylation site Thr-165 is required, for depletion of Gcn4 in <i>hom6Δ</i> cells treated with SM.

<p>(A) <i>gcn4Δ</i> (F731) and <i>hom6Δ gcn4Δ</i> (YR009) strains transformed with sc plasmids with WT <i>GCN4</i> (p164), mutant alleles <i>gcn4-Δ235-250</i> (pCD114-1) or <i>gcn4-Δ251-281</i> (pCD115-1), or empty vector (YCplac33), were subjected to Western analysis as in Fig. 4A, after SM treatment for 2 h. (B) WT (BY4741) and <i>hom6Δ</i> (YR001) strains transformed with vector YCplac33 or sc mutant allele <i>gcn4-Δ251-281</i> (pCD115-1) were analyzed as in (A). * indicates mutant gcn4-<i>Δ</i>251-281 protein, displaying slightly greater electrophoretic mobility than WT Gcn4. (C) WT (BY4741), <i>hom6Δ</i> (YR001), <i>hom6Δ hom3Δ</i> (YR003), <i>srb10Δ</i> (F736), <i>hom6Δ srb10Δ</i> (YR004), <i>pho85Δ</i> (F947) and <i>hom6Δ pho85Δ</i> (YR006) strains transformed with vector (pRS313) or <i>HOM3^fbr</i> plasmid pYPR030; were analyzed as in (A). * indicates putative phosphorylated isoforms of Gcn4. In <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004534#pgen.1004534.s005" target="_blank">Fig. S5</a>, we verified that the level of Gcn4 measured by Western analysis is indistinguishable between the <i>hom6Δ hom3Δ HOM3^fbr</i> strain examined here in lane 4 and a <i>hom6Δ HOM3^fbr</i> strain that represents the most appropriate WT control for the mutants analyzed here in lanes 8 and 12, as would be expected from the dominance of <i>HOM3^fbr</i>. (D) Western signals from (C) were quantified and the mean and S.E.M. Gcn4/Gcd6 ratios were calculated from 3 independent transformants. (E) <i>gcn4Δ</i> (F731) and <i>hom6Δ gcn4Δ</i> (YR009) strains transformed with sc plasmids harboring WT <i>GCN4</i> (pYPR013), <i>gcn4-T165A</i> (pYPR047), or vector (YCplac111) were analyzed as in (A). (F) Western signals from (E) were analyzed as in (D). (G) Transformants of the strains in (E) harboring pHYC2 were analyzed for <i>UAS_GCRE-CYC1-lacZ</i> expression as in Fig. 1C. Means and S.E.Ms were calculated from three independent transformants of each strain. (H) Strains in (E) were analyzed for <i>HIS4</i> and <i>ARG1</i> mRNA levels as in Fig. 1D, after SM treatment for 2 h. Mean and S.D. values determined from two independent cultures were plotted relative to the value determined for WT cells.</p

Model for the roles of Pho85 and Srb10 in accelerated turnover of Gcn4 in response to ASA accumulation in <i>hom6Δ</i> cells.

<p>(A) Starvation of WT (<i>HOM6</i>) cells for Ile/Val evokes increased synthesis of Gcn4 at the translational level and subsequent increased binding of Gcn4 to UAS elements of genes subject to GAAC. Gcn4 recruits coactivators, including Mediator (green and red shapes connecting Gcn4 to Pol II), and Pol II to the promoter (TATA) for increased transcription of target gene coding sequences (CDS). As Srb10 is associated with Mediator and recruited by UAS-bound Gcn4, we propose that Srb10 phosphorylates the bulk of UAS-bound Gcn4 molecules (orange balls labeled with “P”), stimulating their ubiquitylation and attendant degradation by the proteasome. UAS-bound Gcn4 is also sumoylated by Ubc9 (white balls labeled with “S”) and the sumoylated molecules are targeted for degradation by Srb10. Pho85 is responsible for the majority of Gcn4 turnover, and we propose it phosphorylates (green balls labeled with “P”) and triggers degradation of both active and defective non-UAS bound Gcn4 species. (B) Starvation of <i>hom6Δ</i> cells for Ile/Val also evokes increased synthesis of Gcn4 and attendant increased binding of Gcn4 to UAS elements. However, we hypothesize that ASA accumulation evokes damage or inactivating modifications of Gcn4. Damage/modification restricted to the activation domain, disengages UAS-bound Gcn4 from coactivators and increases its rate of phosphorylation by Srb10 at the promoter, with attendant increased degradation by the proteasome. (Gcn4 opacity is reduced to depict its decreased occupancy of the UAS.) Damage/modification that extends to the DNA binding or dimerization domain disengages Gcn4 from the UAS and makes it susceptible to phosphorylation by Pho85 and subsequent proteasomal degradation. Pho85 also targets functional Gcn4 molecules when they disengage from the UAS, just as in <i>HOM6</i> cells. Hence, the absence of Srb10 in <i>srb10Δ</i> cells spares from degradation defective Gcn4 molecules capable of UAS-binding and thereby reduces the specific activity of UAS-bound Gcn4. The absence of Pho85 in <i>pho85Δ</i> cells spares both fully functional Gcn4 molecules and inactive species incapable of stable UAS-binding and thereby increases the specific activity of UAS-bound Gcn4 while simultaneously decreasing the fraction of Gcn4 capable of UAS binding.</p

Plasmids used in this study.

a<p>sc, single copy.</p>b<p>lc, low copy.</p>c<p>hc, high copy.</p

<i>hom6Δ</i> impairs GAAC in cells starved for Ile/Val.

<p>(<b>A</b>) Schematic diagram of the threonine biosynthesis pathway in <i>Saccharomyces cerevisiae</i>. Feedback inhibition by threonine of aspartate kinase (<i>HOM3</i>) and homoserine kinase (<i>THR1</i>) and transcriptional induction of certain pathway genes by Gcn4 under the GAAC are shown. (<b>B</b>) SM-sensitivity of <i>hom6Δ</i>, <i>thr1Δ</i>, and <i>thr4Δ</i> mutants. Parental WT strain BY4741/F729, <i>gcn4Δ</i> mutant F731, and deletion mutants lacking the indicated threonine biosynthetic gene (F2057, F1929, F941, F2056 and F926) were cultured overnight in SC-Ile/Val containing 2.5 mM threonine, washed and resuspended in sterile water at A₆₀₀ = 1.0, and 10-fold serial dilations were spotted on agar plates of the same growth medium (SC) or medium also containing 0.5 µg/ml sulfomeutron methyl (SC+SM) and incubated at 30°C for 2d. (<b>C</b>) Yeast strains from (B) were transformed with pHYC2 (<i>UAS_GCRE-CYC1-lacZ</i>) or p367 (<i>HIS4-lacZ</i>) and cultured overnight in SC-Ura/Ile/Val containing 2.5 mM threonine. Duplicate cultures were diluted at A₆₀₀ = 0.5 in SC-Ura/Ile/Val medium containing 1 mM threonine, and one set was harvested after 6 h at 30°C (Unstarved). For the duplicate set, SM was added to 0.5 µg/mL after 2.5 h and incubation continued another 6 h before harvesting. β-galactosidase activity (nmole of ONPG cleaved per min per mg) was measured in WCEs for three independent transformants of each strain, and mean and S.E.M. values are plotted. (<b>D</b>) WT (BY4741), <i>gcn4Δ</i> (F731), <i>hom3Δ</i> (F1929), <i>hom6Δ</i> (F941) and <i>thr1Δ</i> (F2056) strains were cultured as in (C) except that cultures were harvested at A₆₀₀ = 0.4–0.6 after doubling at least twice (Unstarved) or treated with SM and cultured an additional 30 min or 120 min. Total RNA was purified and used for cDNA synthesis and using the appropriate fluorescently-labeled Taqman probes <i>HIS4, ARG1</i>, and <i>ACT1</i> mRNAs were quantified by real-time qPCR. The levels of <i>HIS4</i> or <i>ARG1</i> mRNAs were normalized to those of <i>ACT1</i> mRNA and expressed relative to the value determined for WT unstarved cells. Mean and S.D. values determined from two independent cultures are plotted.</p

Hom6 catalytic activity is required for robust GAAC in Ile/Val-starved cells.

<p>(<b>A</b>) The growth of WT (BY4741) and <i>hom6Δ</i> (F941) strains transformed with vector (YCplac111) and F941 (<i>hom6Δ</i>) transformed with single-copy plasmids carrying WT <i>HOM6</i> (pYPR010) or active-site <i>hom6</i> alleles <i>K117L</i> (pYPR018), <i>E208D</i> (pYPR020), <i>E208L</i> (pYPR022), or <i>D219L</i> (pYPR024) was analyzed essentially as in Fig. 1B except that growth on SC-Leu/Thr medium (SC-Thr) was also examined. (<b>B</b>) Transformants of the strains in (A) harboring pHYC2 were analyzed for β-galactosidase activity as in Fig. 1C. Means and S.E.Ms were calculated from three independent transformants of each strain.</p