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Re-expression of SynGAP protein in adulthood improves translatable measures of brain function and behavior

  1. Thomas K Creson
  2. Camilo Rojas
  3. Ernie Hwaun
  4. Thomas Vaissiere
  5. Murat Kilinc
  6. Andres Jimenez-Gomez
  7. Jimmy Lloyd Holder Jr
  8. Jianrong Tang
  9. Laura L Colgin
  10. Courtney A Miller
  11. Gavin Rumbaugh  Is a corresponding author
  1. The Scripps Research Institute, United States
  2. University of Texas at Austin, United States
  3. Baylor College of Medicine, United States
Research Article
Cite this article as: eLife 2019;8:e46752 doi: 10.7554/eLife.46752
5 figures and 5 additional files

Figures

Figure 1 with 1 supplement
Seizure threshold is improved after adult restoration of SynGAP expression.

(A) SyngapCre-;+/ls and SyngapCre+;+/ls mice exhibit hyperexcitability in two of the three events without Cre activation (No TMX) Main effects-1st clonus: Cre F(1,24)=2.13, p=0.157, Genotype F = 117.73, p=9.75E-11, Interaction F(1,24)=1.69, p=0.206); Cre- Cohen’s d = 3.855, Cre +Cohen’s d = 4.737. TC: Cre F(1,24)=722, p=0.404, Genotype F(1,24)=40.05, p=1.53E-6), Interaction F(1,24)=.257, p=0.617); Cre- Cohen’s d = 2.396, Cre+ Cohen’s d = 2.405. THE: Cre F(1,24)=9.99E-6, p=0.998, Genotype F(1,24)=.320, p=0.577), Interaction F(1,24)=.420, p=0.523. (B) SyngapCre+;+/ls mice exhibit thresholds comparable to those of SyngapCre-;+/ls mice after Cre activation (TMX-treated) in two of the three events Main effects-1st clonus: Cre F(1,71)=2.59, p=0.112; Genotype F(1,71)=58.328, p=7.86E-11, Interaction F = 1 (1,71)=18.84 p=4.62E-5; Cre- Cohen’s d = 3.329, Cre+ Cohen’s d = 0.674; TC: Cre F(1,71)=4.53, p=0.037, Genotype F(1,71)=26.15, p=2.57E-6, Interaction F(1,71)=6.50, p=0.013; Cre- Cohen’s d = 2.040; Cre+ Cohen's d = 0.540; THE: Cre F(1,71)=.037, p=0.847, Genotype F(1,71)=1.15E-5, p=0.997, Interaction F(1,71)=.049, p=0.826. Data points (and numbers) in bars represent biological replicates (animals). Data from panel B are pooled from two separate experiments.

https://doi.org/10.7554/eLife.46752.002
Figure 1—figure supplement 1
TMX-induced restoration of SynGAP protein levels in adult Syngap1Cre+;+/ls mice.

(A) Western blot demonstrating expression levels of total SynGAP in Cre(-) or Cre(+) heterozygous Lox-Stop mice and WT littermates. (B) Densitometric analysis of SynGAP. Band intensities were normalized to total protein levels and transformed to % of the Syngap1Cre-;+/+ group mean. Two-factor ANOVA. Main effects: Cre p=0.198, Genotype p=0.007, Interaction p=1.554E-4.=0.=0. Pairwise comparisons from posthoc tests can be found in Supplementary file 1. Data points (and numbers) in bars represent biological replicates (animals).

https://doi.org/10.7554/eLife.46752.003
Figure 2 with 1 supplement
Rectification of state-dependent paroxysmal spiking events in Syngap1 mutants after adult-initiated gene therapy.

(A) Representative EEG/LFP traces from a WT [Cre(+); +/+] and Syngap1 heterozygous mutant mouse [Cre(+); +/ls]. After initial recordings (pre-TMX), all animals were injected with TMX. Post-TMX recordings were acquired 30 days after the last TMX injection. TMX rescued low levels of SynGAP protein in +/ls animals (see Figure 1—figure supplement 1). Highlighted areas correspond to periods of sleep (see Materials and methods). Phase I and Phase II recordings are from the same animals. (B) Frequency of spiking events observed in the hippocampal LFP channel during the wake phase (i.e. non-highlighted areas in panel A) from both pre- and post-TMX recording sessions in each animal. Two-way repeated measures ANOVA.:Main genotype effects: F(1,11)=10.1, p=0.00879, Main TMX effects: F(1,11)=12.088, p=0.00518. Interaction between genotype and TMX: F(1,11)=9.777, p=0.00963. Cre(+);+/+n = 6, Cre(+);+/ls n = 7. (C) Comparison of the spiking frequency from the hLFP channel in Cre(+);+/ls mice during wake and sleep before TMX injections, paired-t test t(5)=-5.6007, p=0.002507 (n = 5). Data points in plots represent biological replicates (animals).

https://doi.org/10.7554/eLife.46752.007
Figure 2—figure supplement 1
Generalization of high-amplitude spikes across the forebrain.

Representative traces from all channels during a Phase I recording from a Cre(+) Lox-Stop mouse.

https://doi.org/10.7554/eLife.46752.008
Representative EEG recordings taken from SYNGAP1 patients during wake and sleep.

Ten second epochs of electroencephalograms from patients with SYNGAP1 pathogenic variants. (A) Patient S3-060 while awake (B) Patient S3-060 while asleep (C) Patient S3-080 while awake (D) Patient S3-080 while asleep. Shaded areas indicate bursts of generalized epileptiform activity.

https://doi.org/10.7554/eLife.46752.011
Long-term memory can be improved in adult mice with Syngap1 pathogenicity.

(A) Syngap1+/+ and Syngap1+/- ± were trained in the remote contextual fear conditioning paradigm and tested one month later for activity suppression levels. Activity of the Syngap1+/- was suppressed significantly less than that of the Syngap1+/+ group indicating compromised remote memory for the mutant group. Unpaired t test (t(19)=-2.567, p=0.019). Cohen’s d = 1.150. (B) Syngap1+/+ and Syngap1+/ls mice were trained in the contextual fear conditioning paradigm and tested one month later for activity suppression levels. Activity of the Syngap1+/ls group was suppressed significantly less than that of the Syngap1+/+ group indicating compromised remote memory for the mutant group. Wilcoxon rank sum test W = 19, p=2.82E-5, Cohen’s d = 1.676. (C) Syngap1+/+ and Syngap1+/- were tested, firstly, 1d after training, followed by another testing one month later. Activity suppression levels were not significantly different between the groups for either testing (unpaired t test,1-day t(13)=-0.033, p=0.974; 26 days t(13)=-1.068, p=0.305). (D) Experimental schematic depicting the breeding strategy for generation of Cre-inducible Syngap1Cre+;+/ls mice and Cre induction with TMX treatment for restoration of Syngap1 expression and subsequent remote fear conditioning testing. (E–F) Syngap1Cre-;+/+, Syngap1Cre-;+/ls, Syngap1Cre+;+/+, and Syngap1Cre+;+/ls mice were run in the remote contextual fear conditioning paradigm without (E) and with (F) TMX administration. Activity suppression values from mice without TMX administration (No TMX) were assessed (2-factor ANOVA: Main Effects-Cre F(1,90)=0.030, p=0.864, Genotype F(1,91)=46.78, p=9.28E-10, Interaction F(1,91)=6.81, p=0.011; Cre- Cohen’s d = 1.725, Cre+ Cohen’s d = 0.910. With TMX administration (2-factor ANOVA: Main Effects- Cre F=(1,73)=0.019, p=0.891, Genotype F(1,73)=27.49, p=1.48E-6, Interaction F(1,73)=14.75, p=2.59E-4; Cre- Cohen’s d = 2.167). Data points (and numbers) in bars represent biological replicates (animals). Data from panels E-F are pooled from at least two separate experiments.

https://doi.org/10.7554/eLife.46752.012
Figure 5 with 2 supplements
Increased amplitude of theta oscillations after SynGAP re-expression in adult Syngap1 mutant mice.

(A–B) CA1 LFP traces from a WT (A) and a Syngap1 mutant (B) mouse during Phase I and Phase II sessions. (C) Grand average of within-subjects changes in signal amplitude across the full spectrum of hippocampal rhythms. The amplitude change was normalized by the average amplitude during Phase I sessions. The shaded areas represent 95% bootstrapped confidence intervals. Significant increases in amplitude in Phase II were detected in the 6–12 Hz theta range (Permutation test: p=0.0128, 5000 shuffles). N’s are biological replicates (animals). Legends for Figure Supplements.

https://doi.org/10.7554/eLife.46752.018
Figure 5—figure supplement 1
Amplitude of theta oscillations in each mouse during Phase I and Phase II recording sessions.

(A–B) CA1 LFP recordings from WT (A) and Syngap1 mutant (B) mice during Phase I and Phase II sessions. (C–D) Average amplitude spectra for each mouse during Phase I (C) and Phase II (D) sessions. Individual mice are indicated with individual lines, and WT and Syngap1 mutant spectra are depicted in blue and red, respectively.

https://doi.org/10.7554/eLife.46752.019
Figure 5—figure supplement 2
Effect of genotype, but not phase, on horizontal activity during neurophysiological recordings.

Cre(+) WT and Cre(+) Lox-Stop mice were video tracked for distances traveled during the first ten minutes of recording during Phase I (TMX-) and Phase II (TMX+) sessions. RMANOVA- Group: F(1,12)=16.527, p=0.002; Phase: F(1,12)=0.164, p=0.692; Group x Phase: F(1,12)=3.521, p=0.085. Data points in bars represent biological replicates (animals). Legends for Supplementary Files.

https://doi.org/10.7554/eLife.46752.020

Data availability

Data used for generating figures are included in the manuscript and supporting files.

Additional files

Supplementary file 1

Pairwise comparison statistics for Figure 1.

Pairwise comparisons for each of the four groups in data presented in Figure 1A–B.

https://doi.org/10.7554/eLife.46752.022
Supplementary file 2

Pairwise comparison statistics for Figure 1—figure supplement 1.

Pairwise comparisons for each of the four groups in data presented in Figure 1—figure supplement 1B.

https://doi.org/10.7554/eLife.46752.023
Supplementary file 3

Summary of EEG data from MRD5 patients in the SYNGAP1 Registry.

Subset of entries from the SYNGAP1 patient registry noting EEG abnormalities.

https://doi.org/10.7554/eLife.46752.024
Supplementary file 4

Pairwise comparison statistics for Figure 4E–F.

Pairwise comparisons for each of the four groups in data presented in Figure 4E–F.

https://doi.org/10.7554/eLife.46752.025
Transparent reporting form
https://doi.org/10.7554/eLife.46752.026

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