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Preventing proteostasis diseases by selective inhibition of a phosphatase regulatory subunit

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Science  10 Apr 2015:
Vol. 348, Issue 6231, pp. 239-242
DOI: 10.1126/science.aaa4484
  • Fig. 1 Sephin1 is a selective inhibitor of PPP1R15A.

    (A) Structures of GBZ and Sephin1. (B) Coomassie-stained gel showing recombinant PPP1R15A325-636 and PPP1R15B340-698 (input). Biotinylated Sephin1 selectively captured PPP1R15A on neutravidin beads (bound). (C) Immunoprecipitations of PPP1R15 complexes from cells treated with vehicle, 50 μM GBZ, or Sephin1 for 6 hours analyzed with immunoblotting. (D) Immunoblots of the indicated proteins in lysates of HeLa cells treated with 2.5 μg/ml tunicamycin (Tm) in the presence or absence of 50 μM Sephin1 for the indicated time. (E) Newly synthesized proteins labeled with 35S-methionine in HeLa cells treated with Tm or with or without 50 μM GBZ or Sephin1 and revealed with autoradiography. (Bottom) Coomassie-stained gel. (F) Dose-dependent protection by Sephin1 of wild-type but not Ppp1r15a mutant (mut/mut) cells from Tm (2.5 μg/ml). Data are means ± SEM (n = 4 replicates). **P ≤ 0.001. (G) Adrenergic activity of GBZ and Sephin1 in cells expressing recombinant human adrenergic α2A receptor. Representative results of at least three independent experiments are shown in each panel.

  • Fig. 2 Sephin1 is devoid of adverse effects on rotarod performances, total body weight gain, or memory.

    (A) Motor performance of mice on an accelerating rotarod before or after the indicated treatments. Data are means ± SEM. (n = 5 mice). (B) Total body weight gain of mice treated orally with Sephin1 (1 mg/kg) or vehicle twice a day, from postnatal days 28 to 61. Data are means ± SEM (n = 6 mice). (C and D) Distance and latency to locate a hidden platform in the Morris water maze, in five trials a day for 5 consecutive days. (E) Quadrant occupancy after training and removal of the platform. (F) Freezing response during the conditioning session, where a light/tone [conditioned stimulus (CS)] and foot shock [aversive unconditioned stimulus (US)] were applied. (G and H) Freezing responses expressed as percentage of total time (2 min) mice spent immobile during context and auditory cue testing. In (C) to (H), data are means ± SEM (n = 12 mice). Mice were treated with 1 mg/kg Sephin1 or vehicle twice a day for 4 weeks. No statistical differences were found between Sephin1- or vehicle-treated mice [(B) to (H)].

  • Fig. 3 Sephin1 prevents the defects caused by misfolding-prone myelin MPZmutant protein ex vivo and in vivo.

    (A and B) Representative images (A) and quantification (B) of myelin internodes revealed with immunostaining for myelin basic protein (MBP, red) from cultured DRG of the indicated genotype treated for 2 weeks with vehicle or Sephin1 (100 nM). In green, neurofilament (NF). Data are means ± SEM (n = 4 to 6 mice). (C) mRNA levels [by means of quantitative polymerase chain reaction (PCR)] of the indicated ER stress markers in cultures as (A). Data are means ± SEM (n = 5 to 7 mice). (D) Motor performance of 4-month-old wild-type or MPZmutant mice on a rotarod after a 3-month oral treatment with Sephin1 (1 mg/kg) or vehicle twice a day. Data are means ± SEM (n = 14 to 16 mice). (E) Myelin thickness revealed on toluidine blue–stained semithin sciatic nerve sections of 6-month-old mice of the indicated genotype after 5 months of oral Sephin1 treatment (1 mg/kg) or vehicle twice a day. Data are means ± SEM (n = 6 to 8 mice). (F) Demyelination expressed as increased g-ratio (ratio of axon diameter to fiber diameter) over wild-type mice. Data are means ± SEM (n = 5 or 6 mice). (G) Immunoblots on sciatic nerve lysates from mice. (H) mRNA levels (quantitative PCR) in sciatic nerves of 6-month-old mice as in (E). Data are means ± SEM (n = 5 to 7 mice). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Scale bars, 100 μm.

  • Fig. 4 Sephin1 prevents motor deficits, motor neuron loss, and the molecular defects in SOD1mutant mice.

    (A) Total body weight gain of wild-type or SOD1mutant mice treated orally with Sephin1 (5 mg/kg) or vehicle once a day from 4 to 11 weeks of age. Data are means ± SEM (n = 4 to 6 mice). (B) Rotarod analysis of 110-day-old wild-type or SOD1mutant mice treated as in (A). Data are means ± SEM (n = 4 to 6 mice). (C and D) Representative motor neuron staining (NeuN, green) and quantification (D) of sections of anterior horn of the lumbar region of spinal cord of 110-day-old mice treated as in (A). Nuclei, H33258 (blue). (D) Data are means ± SEM (n = 6 to 8 mice). (E) SOD1 immunoblots on soluble and insoluble fractions of spinal cord extracts from SOD1mutant mice treated as in (A). (F) mRNA levels (quantitative PCR) in lumbar spinal cord from 4-month-old mice of indicated genotype, after treatment with Sephin1 or vehicle as in (A). Data are means ± SEM (n = 4 to 6 mice). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Scale bar, 50 μm.

Supplementary Materials

  • Preventing proteostasis diseases by selective inhibition of a phosphatase regulatory subunit

    Indrajit Das, Agnieszka Krzyzosiak, Kim Schneider, Lawrence Wrabetz, Maurizio D’Antonio, Nicholas Barry, Anna Sigurdardottir, Anne Bertolotti

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S6
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