Shank3 modulates sleep and expression of circadian transcription factors

  1. Ashley M Ingiosi
  2. Hannah Schoch
  3. Taylor Wintler
  4. Kristan G Singletary
  5. Dario Righelli
  6. Leandro G Roser
  7. Elizabeth Medina
  8. Davide Risso
  9. Marcos G Frank  Is a corresponding author
  10. Lucia Peixoto  Is a corresponding author
  1. Washington State University, United States
  2. Consiglio Nazionale della Ricerche, Italy
  3. University of Fusciano, Italy
  4. University of Padova, Italy
  5. Weill Cornell Medicine, United States
5 figures, 4 tables and 4 additional files

Figures

Increased incidence of sleep problems reported in individuals with Phelan-McDermid syndrome (PMS) compared to typically developing (TD) individuals.

Dashed line indicates median incidence observed in TD (white marker) and ASD (black marker) populations (values from Figure 1—source data 1).

https://doi.org/10.7554/eLife.42819.003
Figure 1—source data 1

Incidence of sleep problems in Autism Spectrum Disorder (ASD) compared to typically developing (TD) individuals.

Survey of the literature, regardless of age. Median, first and third quartile values, age range, and included references reported for each phenotype.

https://doi.org/10.7554/eLife.42819.004
Figure 2 with 1 supplement
Shank3∆C mice sleep less during the dark period and show altered EEG spectral power under baseline conditions.

The rows represent the vigilance states of wakefulness (top), NREM sleep (middle), and REM sleep (bottom). (A) Time spent in wakefulness and sleep shown as percentage of recording time per hour. …

https://doi.org/10.7554/eLife.42819.005
Figure 2—source data 1

Source data and summary statistics used for generating plots for Figure 2.

https://doi.org/10.7554/eLife.42819.007
Figure 2—source data 2

Statistical results for Figure 2 and Figure 2—figure supplement 1. numDF, numerator degrees of freedom. denDF, denominator degrees of freedom.

https://doi.org/10.7554/eLife.42819.008
Figure 2—figure supplement 1
Baseline sleep bout analysis.

(A) Average number of state-specific bouts per hour shown in 6 hr blocks. (B) Average bout duration (min) per hour shown in 6 hr blocks. Repeated measures ANOVA with posthoc pairwise comparisons …

https://doi.org/10.7554/eLife.42819.006
Figure 3 with 2 supplements
Shank3∆C mice take longer to fall asleep and sleep less after sleep deprivation.

(A) Normalized NREM delta (δ; 0.5–4 Hz) power during recovery sleep post-sleep deprivation. The cross-hatched bar on the x-axis denotes the 5 hr sleep deprivation period and the open bar denotes the …

https://doi.org/10.7554/eLife.42819.010
Figure 3—source data 1

Source data and summary statistics used for generating plots for Figure 3.

https://doi.org/10.7554/eLife.42819.013
Figure 3—source data 2

Statistical results for Figure 3, Figure 3—figure supplement 1, and Figure 3—figure supplement 2.

numDF, numerator degrees of freedom. denDF, denominator degrees of freedom.

https://doi.org/10.7554/eLife.42819.014
Figure 3—figure supplement 1
Shank3∆C mice sleep less than wild type type after sleep deprivation.

The rows represent the vigilance states of wakefulness (top), NREM sleep (middle), and REM sleep (bottom). (A – B) Time in state shown as percentage of recording time per hour comparing baseline and …

https://doi.org/10.7554/eLife.42819.011
Figure 3—figure supplement 2
Bout analysis after sleep deprivation.

(A) Average number of state-specific bouts per hour (difference scores) shown in 7 hr (remaining light period) and 6 hr blocks. (B) Average bout duration (min) per hour (difference scores) shown in …

https://doi.org/10.7554/eLife.42819.012
Figure 4 with 1 supplement
Sleep deprivation induces a two-fold difference in gene expression between Shank3ΔC and wild type mice.

RNA-seq study of gene expression from prefrontal cortex obtained from adult male Shank3ΔC and wild type mice either under control homecage conditions (HC) or following 5 hr of sleep deprivation …

https://doi.org/10.7554/eLife.42819.015
Figure 4—source data 1

Genes differentially expressed between Shank3∆C vs. wild type mice.

RNA-seq study of gene expression from prefrontal cortex tissue obtained from adult male Shank3ΔC and wild type (WT) mice, either under control homecage conditions (HC) or following 5 hr of sleep deprivation (SD). N = 5 mice per group. False discovery rate (FDR) < 0.1.

https://doi.org/10.7554/eLife.42819.017
Figure 4—figure supplement 1
Recovery of positive control genes regulated by sleep deprivation.

Volcano plot of differential expression (−log10 P-value versus log2 fold change) caused by sleep deprivation in wild type animals (HC5 vs SD5). Genes with an FDR < 0.05 are highlighted in black. …

https://doi.org/10.7554/eLife.42819.016
Figure 5 with 1 supplement
Shank3∆C mice show disruption of running wheel activity in constant darkness.

Representative actograms and periodograms for two wild type and two Shank3∆C mice. Mice were entrained to a 12:12 hr light:dark cycle (LD, 559 ± 4 : 0 ± 0 lux) for two weeks prior to 3 weeks …

https://doi.org/10.7554/eLife.42819.022
Figure 5—figure supplement 1
Actograms for all wild type and Shank3∆C mice.

Mice were entrained to a 12:12 hr light:dark cycle (LD, 559 ± 4 : 0 ± 0 lux) for two weeks prior to 3 weeks constant darkness (DD, 0 ± 0 lux). Gray shading is representative of the dark period. (A) …

https://doi.org/10.7554/eLife.42819.023

Tables

Table 1
Shank3ΔC mice sleep less than wild type mice during the dark period.

Repeated measures ANOVA with posthoc pairwise comparisons using Sidak correction. Values are means ± SEM for wild type (n = 10) and Shank3ΔC (n = 10). *p<0.05, difference from wild type.

https://doi.org/10.7554/eLife.42819.009
Total Sleep Time (%)Wild TypeShank3ΔCp-value
Light Period62.90 ± 1.5762.96 ± 1.650.980
Dark Period28.72 ± 2.4121.16 ± 2.540.045*
Table 2
Results of functional bioinformatics analysis of the genes differentially expressed between Shank3ΔCmice relative to wild type mice.

Functional annotation and clustering analysis was performed using DAVID (https://david.ncifcrf.gov) and functional information was obtained from the following databases: GO (Biological process and …

https://doi.org/10.7554/eLife.42819.018
Shank3ΔC vs. Wild Type
HomecageSleep Deprivation
Up-regulatedUp-regulated
Cholesterol Metabolism: Hmgcr, Insig1Potassium Ion Transport: Kcnv1, Kcnk1, Kcnk2
Transcription: Jun, Fosl2, Nfil3, Stat4Dephosphorylation: Dusp10, Dusp3, Ptprj
Neuron Projection Development: Cntn1, Ntrk2, Reln, Sema3a
Down-regulatedDown-regulated
MAPK Signaling: Mapk3 (ERK1), Elk1GnRH Signaling: Mapk1 (ERK2), Elk1, Mapk11 (p38)
Circadian Rhythms: Per3, Tef, HlfCircadian Rhythms: Per3, Nr1d1 (REV-ERBα), Tef, Hlf, Prkab2 (AMPK), Bhlhe41 (DEC2)
Transcription: Tef, HlfSodium Ion Transport: Slc6a15, Slc22a4, Slc24a4
Table 2—source data 1

Functional annotation clustering analysis of genes in Figure 4—source data 1.

https://doi.org/10.7554/eLife.42819.019
Table 3
Shank3∆C mice show reduced wheel-running activity during constant darkness.

Summary of wheel-running behavior measured for each week of 12:12 hr light dark cycle (LD, 559 ± 4 : 0 ± 0 lux) or constant darkness (DD, 0 ± 0 lux) for both wild type and Shank3∆C mice. A linear …

https://doi.org/10.7554/eLife.42819.020
Wild TypeShank3∆C
Period (h)
LD Week 224.0 ± 0.024.0 ± 0.0
LD Week 324.0 ± 0.023.9 ± 0.1
DD Week 123.9 ± 0.123.7 ± 0.1
DD Week 223.7 ± 0.120.3 ± 3.4
DD Week 323.5 ± 0.225.8 ± 1.5
Alpha (h)
LD Week 210.0 ± 0.410.7 ± 0.4
LD Week 310.3 ± 0.410.7 ± 0.5
DD Week 110.5 ± 0.29.0 ± 0.4
DD Week 210.4 ± 0.48.2 ± 0.7
DD Week 39.0 ± 1.09.9 ± 1.0
Activity (rev/day)*
LD Week 233985.2 ± 565.923258.2 ± 861.2
LD Week 340306.7 ± 1257.125365.1 ± 964.0
DD Week 137942.6 ± 795.423027.7 ± 669.6
DD Week 238250.7 ± 431.116820.7 ± 1302.5
DD Week 338586.3 ± 231.16961.5 ± 383.9
Table 3—source data 1

Results of genotype x time interaction analysis for LD week three through DD week 3.

Linear mixed-effects ANOVA for LD week three through DD week 3. The model was fitted using genotype, time, and their interaction as fixed effects and the individual mice labeled as random effect. There is a significant effect of genotype x time for activity (p<0.0001) and no significant effect for genotype x time or genotype for period and alpha. Significance at p<0.05. numDF, numerator degrees of freedom. denDF, denominator degrees of freedom.

https://doi.org/10.7554/eLife.42819.021
Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
Identifiers
Gene (Mus musculus)Shank3; SH3 and multiple ankyrin repeat domains 3NAENTREZ_ID: 58234
Gene (Mus musculus)Per3; period circadian clock 3NAENTREZ_ID: 18628
Gene (Mus musculus)Hlf; hepatic leukemia factorNAENTREZ_ID: 217082
Gene (Mus musculus)Tef; thyrotroph embryonic factorNAENTREZ_ID: 21685
Gene (Mus musculus)Nr1d1; nuclear receptor subfamily 1, group D, member 1;NAENTREZ_ID: 217166
Gene (Mus musculus)Bhlhe41; basic helix-loop-helix family, member e41;NAENTREZ_ID: 79362
Strain, strain background (Mus musculus)Shank3ΔC; Shank3tm1.1Pfw/JKouser et al., 2013; The Jackson LaboratoryRRID:IMSR_JAX:018398
Commercial assay or kitRNAeasy kitQiagen74104
Commercial assay or kitHigh Sensitivity RNA Analysis KitAdvanced Analytical TechnologiesDNF-472
Commercial assay or kitHigh Sensitivity NGS Fragment Analysis KitAdvanced Analytical TechnologiesDNF-474
Commercial assay or kitKAPA Library Quantification KitKapabiosystemsKK4824
Commercial assay or kitTruSeq Stranded mRNA Library Prep KitIlluminaRS-122–2101
Software, algorithmHiSeq Control Software version (v. 2.2.68)Illumina15073358 Rev A
Software, algorithmbcl2fastq (v. 2.17.1.14)R/BioconductorSCR_015058
Software, algorithmRsubread (v. 1.26.1)R/BioconductorSCR_016945
Software, algorithmNOISeq (v.2.22.1)R/BioconductorSCR_003002
Software, algorithmRUVSeq (v. 1.12.0)R/BioconductorSCR_006263
Software, algorithmedgeR (v. 3.20.9)R/BioconductorSCR_012802
Software, algorithmR package nlme (v. 3.5.0)CRANSCR_015655
Software, algorithmR package mgcf (v. 3.5.0)CRAN
Software, algorithmR package pheatmap (v. 1.0.10)CRANSCR_016418
Software, algorithmDatabase for annotation, visualization, and integrated discovery (v 6.7)DAVID; https://david.ncifcrf.gov
Software, algorithmVitalRecorderKissei Comtec3, 0, 0, 0
Software, algorithmSleepSign for AnimalKissei Comtec3, 0, 0, 812
Software, algorithmWheel managerMed Associates IncSOF-860
Software, algorithmClockLab (v. 3.5.0)Actimetrics6.0.50

Additional files

Source data 1

Results of genotype x time interaction analysis for 5 weeks of LD.

Linear mixed-effects ANOVA for LD week 1–5. The model was fitted using genotype, time, and their interaction as fixed effects and the individual mice labeled as random effect. There is a significant effect for genotype x time in activity (p<0.0001), a significant effect of genotype for period (p=0.047), and no significant effect for genotype x time or genotype for alpha. Significance at p<0.05. numDF, numerator degrees of freedom. denDF, denominator degrees of freedom.

https://doi.org/10.7554/eLife.42819.024
Source code 1

Detailed report of statistical analysis including R code to reproduce the RNA-seq and circadian wheel running analysis.

https://doi.org/10.7554/eLife.42819.025
Supplementary file 1

Wheel-running behavior in Shank3∆C mice across 5 weeks of continuous light:dark (LD) cycles.

Summary of wheel-running behavior measured for each week of 12:12 hr light:dark cycles for both wild type and Shank3∆C mice. A linear mixed-effects ANOVA was used to estimate the contributions of genotype and the interaction between genotype and time for period, alpha, and wheel-running activity for LD week 1 through 5. There is no significant interaction effect for genotype x time for period (p=0.41), but there is a significant effect of genotype (p=0.047, indicated by *). There is no significant interaction effect for genotype x time for alpha (p=0.43), but there is a significant effect of time (p=0.004, indicated by ). There is a significant interaction effect (indicated by vertical bar) for genotype x time for activity (p<0.0001). See Source data 1 for details. Values are means ± SEM for wild type (n = 8) and Shank3∆C (n = 8) mice.

https://doi.org/10.7554/eLife.42819.026
Transparent reporting form
https://doi.org/10.7554/eLife.42819.027

Download links