Research ArticleNeuroepigenetics

DNA methylation regulates neuronal glutamatergic synaptic scaling

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Science Signaling  23 Jun 2015:
Vol. 8, Issue 382, pp. ra61
DOI: 10.1126/scisignal.aab0715
  • Fig. 1 Homeostatic upscaling of excitatory strength is associated with alterations in mRNAs for gene products associated with the regulation of DNA methylation.

    (A) Sample mEPSC records from cortical pyramidal neurons after 24 hours of exposure with control (CTL) or TTX (blue). (B and C) Cumulative probability distributions and mean mEPSC amplitudes (B) and mean mEPSC frequencies (C) from cortical pyramidal neurons treated with TTX. Bar graphs are means ± SEM from cells pooled from at least six experiments for each condition (CTL, n = 21 cells; TTX, n = 15 cells). (B) P < 0.001, Kolmogorov-Smirnov (K-S) test. Inset: *P < 0.001, Mann-Whitney (M-W) test. (D) Rank order plot of 1000 randomly selected mEPSC amplitudes from CTL and TTX (blue values). Fitting the data with y = ax (dashed black line) yielded a slope of 1.29, which was used to scale down amplitudes from TTX-treated cells in (B). The solid black line represents unity. (E) Cumulative probability distributions of scaled-down TTX-induced amplitudes compared to those from CTL cells; P = 0.1024, K-S test. (F) Polymerase chain reaction (PCR) detection of transcript expression relative to CTL after 24-hour TTX treatment. Dashed vertical line represents the average, normalized gene expression values of CTL cells. Data are means ± SEM from three experiments. *P < 0.05, Student’s unpaired t test.

  • Fig. 2 DNMT inhibition with RG108 multiplicatively upscales excitatory strength.

    (A) Sample mEPSC records from cortical pyramidal neurons after 24-hour treatment with control (CTL) or RG108 (red). (B and C) Cumulative probability distributions and mean mEPSC amplitudes (B) and mean mEPSC frequencies (C) from cortical pyramidal neurons treated with RG108. Bar graphs are means ± SEM from cells pooled from at least four experiments for each condition (CTL, n = 12 cells; RG108, n = 12 cells). (B) P < 0.001, K-S test. Inset: *P < 0.001, M-W test. (C) *P < 0.05, Student’s unpaired t test. (D) Rank order plot of 1000 randomly selected mEPSC amplitudes from CTL and RG108-treated cells (red values). Fitting the data with y = ax yielded a scaling factor of 1.23, which was used to scale down amplitudes from RG108-treated cells in (B). (E) Cumulative probability distributions of scaled-down RG108-induced amplitudes compared to those from CTL cells. P = 0.2645, K-S test. Inset: Testing a range of scaling factors (a) between 1.1 and 1.3 revealed that RG108-induced amplitudes from (B) scaled down with a = 1.23 resulted in the highest K-S P value (0.2645) (red dot) compared to CTL.

  • Fig. 3 Induction of excitatory synaptic scaling by inhibition of DNA methylation is activity-dependent.

    (A) Sample mEPSC traces from cortical pyramidal neurons after 24 hours of exposure to control (CTL), RG108 + APV (magenta), RG018 + APV + NBQX (purple), or RG108 + TTX (green). (B and C) Cumulative probability distributions and mean mEPSC amplitudes (B) and mean mEPSC frequencies (C) from cortical pyramidal neurons treated with RG108 and APV; RG108, APV, and NBQX; or RG108 and TTX. Bar graphs are means ± SEM. Data are cumulative of cells pooled from at least three experiments for each condition (CTL, n = 17 cells; RG108 + APV, n = 9 cells; RG108 + APV + NBQX, n = 6 cells; and RG108 + TTX, n = 12 cells). (B) *P < 0.001 compared to CTL, Kruskal-Wallis (K-W) test followed by Dunn’s test. (C) *P < 0.05 compared to CTL, analysis of variance (ANOVA) followed by Dunnett’s test.

  • Fig. 4 Transcription inhibition blocks the effect of chronic inhibition of DNA methylation on excitatory transmission.

    (A) Sample mEPSC traces from cortical pyramidal neurons after 24-hour exposure to control (CTL), actinomycin D (Act-D; green), or Act-D + RG108 (blue). (B and C) Cumulative probability distributions and mean mEPSC amplitudes (B) and mean mEPSC frequencies (C) in cortical pyramidal neurons treated with Act-D alone or co-incubation with RG108. Bar graphs are means ± SEM. Data are cumulative of cells pooled from at least three experiments for each condition (CTL, n = 9 cells; Act-D, n = 4 cells; Act-D + RG108, n = 7 cells). (B) K-W test, P = 0.0059. (C) ANOVA, P = 0.3439.

  • Fig. 5 Combined Dnmt1 and Dnmt3a knockdown multiplicatively upscales excitatory strength.

    (A) Depiction of ASO base pairing with target Dnmt mRNA. ASO-binding positions are listed under the target transcript. Underlined ASO sequences represent 2′-OMe–modified nucleotide wings. (B) Bar graphs show relative expression of Dnmt1 and Dnmt3a mRNA after ASO treatment. Data are means ± SEM from three experiments; *P < 0.05 compared to scrambled CTL, ANOVA followed by Dunnett’s test. (C and D) Cumulative probability distributions and mean mEPSC amplitudes (C) and mean mEPSC frequencies (D) in cortical pyramidal neurons treated with Dnmt-targeted ASOs. Bar graphs are means ± SEM. Data are cumulative of cells pooled from at least two experiments for each condition (scrambled CTL, n = 16 cells; combined ASOs, n = 8 cells; Dnmt1 ASO, n = 5 cells; and Dnmt3a ASO, n = 5 cells). (C) *P < 0.001 compared to scrambled CTL, K-W test followed by Dunn’s test. (D) *P < 0.05 compared to scrambled CTL, ANOVA followed by Dunnett’s test. (E) Rank order plot of 1000 randomly selected mEPSC amplitudes from scrambled CTL and combined ASO treatment (orange values). Linear regression yielded a scaling factor of 1.34, which was used to scale down amplitudes from combined ASO-treated cells from (C). (F) Scaled-down amplitudes from combined ASO-treated cells compared to those in scrambled CTL cells; P = 0.1323 compared to scrambled CTL, K-S test.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/382/ra61/DC1

    Fig. S1. Indirect DNMT inhibition with the nucleoside analog zebularine decreases mEPSC frequency with no effect on amplitude.

    Fig. S2. Tet1 knockdown blocks RG108-induced upscaling of excitatory synaptic strength.

    Table S1. Primers used in this study.

  • Supplementary Materials for:

    DNA methylation regulates neuronal glutamatergic synaptic scaling

    Jarrod P. Meadows, Mikael C. Guzman-Karlsson, Scott Phillips, Cassie Holleman, Jessica L. Posey, Jeremy J. Day, John J. Hablitz,* J. David Sweatt*

    *Corresponding author. E-mail: jhablitz{at}uab.edu (J.J.H.); dsweatt{at}uab.edu (J.D.S.)

    This PDF file includes:

    • Fig. S1. Indirect DNMT inhibition with the nucleoside analog zebularine decreases mEPSC frequency with no effect on amplitude.
    • Fig. S2. Tet1 knockdown blocks RG108-induced upscaling of excitatory synaptic strength.
    • Table S1. Primers used in this study.

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    Citation: J. P. Meadows, M. C. Guzman-Karlsson, S. Phillips, C. Holleman, J. L. Posey, J. J. Day, J. J. Hablitz, J. D. Sweatt, DNA methylation regulates neuronal glutamatergic synaptic scaling. Sci. Signal. 8, ra61 (2015).

    © 2015 American Association for the Advancement of Science

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