MeCP2 and histone deacetylases 1 and 2 in dorsal striatum collectively suppress repetitive behaviors

Abstract

Class I histone deacetylases (HDACs) Hdac1 and Hdac2 can associate together in protein complexes with transcriptional factors such as methyl-CpG-binding protein 2 (MeCP2). Given their high degree of sequence identity, we examined whether Hdac1 and Hdac2 were functionally redundant in mature mouse brain. We demonstrate that postnatal forebrain-specific deletion of both Hdac1 and Hdac2 in mice impacts neuronal survival and results in an excessive grooming phenotype caused by dysregulation of Sap90/Psd95-associated protein 3 (Sapap3; also known as Dlgap3) in striatum. Moreover, Hdac1- and Hdac2-dependent regulation of Sapap3 expression requires MECP2, the gene involved in the pathophysiology of Rett syndrome. We show that postnatal forebrain-specific deletion of Mecp2 causes excessive grooming, which is rescued by restoring striatal Sapap3 expression. Our results provide new insight into the upstream regulation of Sapap3 and establish the essential role of striatal Hdac1, Hdac2 and MeCP2 for suppression of repetitive behaviors.

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Figure 1: Characterization of Hdac1;Hdac2 cDKO mice.
Figure 2: Conditional forebrain deletion of Hdac1 and Hdac2 leads to premature death.
Figure 3: Hdac1;Hdac2 cDKO mice have increased grooming behavior and dysregulation of Sapap3.
Figure 4: Striatal specific deletion of Hdac1 and Hdac2 recapitulates the excessive grooming phenotype observed in cDKO mice.
Figure 5: Loss of Mecp2 leads to excessive grooming and dysregulation of Sapap3.
Figure 6: Restoring Sapap3 in the striatum rescues grooming behavior in Mecp2 cKO mice.

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Acknowledgements

The authors are grateful to E. Olson, R. Bassel-Duby, W. Xu, and T.K. Kim for comments and suggestions. We also thank B. Trauterman for his excellent technical assistance. The authors thank E. Olson (University of Texas Southwestern Medical Center) for generously providing Hdac1loxP/loxP;Hdac2loxP/loxP, Hdac1loxP/loxP and Hdac2loxP/loxP mice, J. Richardson for performing the necropsy of the mice and G. Feng (Massachusetts Institute of Technology) for the initial vector construct of Sapap3. This work was supported by National Institute of Health grants MH081060 (L.M.M.) and MH66198 (E.T.K.). L.M. Monteggia holds the Ginny and John Eulich Professorship in Autism Spectrum Disorders at University of Texas Southwestern Medical Center.

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Authors

Contributions

M.M. and M.A. performed the behavioral experiments. M.M., M.A., K.S., M.H.C. and X.L. contributed to the molecular experiments. M.M. performed the statistical analyses. M.M. made the figures and wrote the corresponding sections of the paper. L.M.M. and E.T.K. designed the study, supervised the experiments and wrote the paper.

Corresponding author

Correspondence to Lisa M Monteggia.

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Competing interests

L.M. Monteggia is on the scientific advisory board for Rodin Therapeutics.

Integrated supplementary information

Supplementary Figure 1 Characterization of Hdac1 and Hdac2 cKO mice.

(a,b) Body weight was measured for a time period between 3 and 9 weeks of age. No differences in body weight were found between HDAC1 cKO mice and CTL littermates (a) or HDAC2 cKO mice and CTL littermates (b) (HDAC1 CTL n=5; cKO n=7; two-tailed t-test; t(10) = 2.08; P = 0.0642 for 3 weeks CTL versus cKO; t(10) = 0.0283; P = 0.9780 for 4 weeks CTL versus cKO; t(10) = 1.566; P = 0.1483 for 5 weeks CTL versus cKO; t(10) = 0.7404; P = 0.4761 for 6 weeks CTL versus cKO; t(10) = 1.028; P = 0.3281 for 7 weeks CTL versus cKO; t(10) = 1.015; P = 0.3343 for 8 weeks CTL versus cKO; t(10) = 0.1267; P = 0.9017 for 9 weeks CTL versus cKO; HDAC2 CTL n=5; cKO n=7; two-tailed t-test; t(10) = 1.166; P = 0.2707 for 3 weeks CTL versus cKO; t(10) = 1.125; P = 0.2869 for 4 weeks CTL versus cKO; t(10) = 2.011; P = 0.0720 for 5 weeks CTL versus cKO; t(10) = 0.4936; P = 0.6322 for 6 weeks CTL versus cKO; t(10) = 1.537; P = 0.1553 for 7 weeks CTL versus cKO; t(10) = 1.501; P = 0.1642 for 8 weeks CTL versus cKO; t(10) = 1.680; P = 0.1222 for 9 weeks CTL versus cKO). (c) HDAC1 cKO mice have a similar brain mass compared to CTL littermates at 8 weeks of age with no significant difference in weight (CTL n=6; cKO n=5; two-tailed t-test; t(9) =.0902; P = 0.9301 for CTL versus cKO). (d) HDAC2 cKO mice and CTL littermates have no measureable differences in brain mass at 8 weeks of age (n=5 mice per group; two-tailed t-test; t(8) = 1.822; P = 0.1059 for CTL versus cKO). (e) Brain weight of cDKO mice at 8 weeks of age was significantly reduced in comparison to littermate CTL mice (n=5 mice per group; two-tailed t-test; t(8) = 13.04; P < 0.0001 for CTL versus cDKO). Data are shown as median, 25th and 75th percentile, and min and max value (a-e). *P < 0.05.

Supplementary Figure 2 cDKO mice display neuronal apoptosis.

TUNEL positive neurons are labeled in green, and red staining represents a propidium iodide (PI) nuclear counterstain. No TUNEL positive neurons appear in CTL mice indicating an absence of neuronal apoptosis. cDKO mice have several TUNEL positive cells in the cortex (CTX) and dentate gyrus region of the hippocampus (DG) suggestive of cell death occurring in the forebrain region as a result of apoptosis. No TUNEL positive neurons are detected in the striatum (STR) or cerebellum (CBL) of cDKO mice. Images were captured using an Olympus BX51 epifluorescence microscope and Olympus DP70 software. Scale bar represents 100 µm.

Supplementary Figure 3 cDKO mice are hypoactive and display heightened activity.

(a,d) Locomotor activity of 3 week (a) and 6 week (d) old animals was assessed by horizontal beams breaks for a 2 hour testing period and the data are shown in 5 minute increments. (Inset) The total number of beam breaks shows that cDKO mice have significantly less total locomotor activity in comparison to CTL mice at both 3 weeks (a) and 6 weeks (d) of age (3 week n=5 mice per group; two-tailed t-test; t(8) = 2.939; P = 0.0187 for CTL versus cDKO; 6 week n=6 mice per group; two-tailed t-test; t(10) = 4.571; P = 0.0010 for CTL versus cDKO). (b,e) The loss of HDAC1 and HDAC2 in the forebrain resulted in increased anxiety-like behavior in the open field paradigm. 3 week old (b) and 6 week old (e) cDKO mice spent significantly less time in the center field and more time in periphery of the arena, compared to CTL mice (3 week CTL n=11; cDKO n=9; two-tailed t-test; t(18) = 4.775; P = 0.0002 for complete center and periphery CTL versus cDKO; 6 week CTL n=7; cDKO n=6; two-tailed t-test; t(11) = 2.575; P = 0.0258 for complete center and periphery CTL versus cDKO). (c) cDKO mice spend significantly more time grooming at 3 weeks of age compared to CTL mice (CTL n=7; cDKO n=5; two-tailed t-test; t(10) = 2.286; P = 0.0453 for 0-10 min CTL versus cDKO; two-tailed t-test; t(10) = 2.112; P = 0.0608 for 0-20 min CTL versus cDKO; two-tailed t-test; t(10) = 2.333; P = 0.0419 for 0-30 min CTL versus cDKO). Data are shown as median, 25th and 75th percentile, and min and max value (a,d inset, b-e) and mean value ± s.e.m. (a,d). *P < 0.05.

Supplementary Figure 4 Grooming behavior was assessed in Hdac1 and Hdac2 cKO mice and the total time spent grooming over a 30 minute testing period was recorded.

No significant differences were found in the total amount of time spent grooming between the HDAC1 cKO (a) or HDAC2 cKO mice (b) and their respective CTLs (n=6 mice per group for HDAC1 and HDAC2; HDAC1 two-tailed t-test; t(10) = 1.061; P = 0.3137 for 0-10 min CTL versus cKO; two-tailed t-test; t(10) = 0.2714; P = 0.7916 for 0-20 min CTL versus cKO; two-tailed t-test; t(10) = 0.4703; P = 0.6483 for 0-30 min CTL versus cKO; HDAC2 two-tailed t-test; t(10) = 2.05; P = 0.675 for 0-10 min CTL versus cKO; two-tailed t-test; t(10) = 0.6498; P = 0.5305 for 0-20 min CTL versus cKO; two-tailed t-test; t(10) = 1.452; P = 0.1773 for 0-30 min CTL versus cKO). (c) Seven day treatment of fluoxetine (Flx) did not reduce the excessive grooming seen in cDKO mice (CTL Saline n=5; CTL Flx n=6; cDKO Saline n=4; cDKO Flx n=6; two-way ANOVA F(1,17) = 18.97, P = 0.0004 for genotype; Tukey’s post hoc analysis for CTL Saline versus cDKO Saline P = 0.0495, CTL Saline versus cDKO Flx P = 0.0119). (d,e) qRT-PCR analysis showed that mRNA expression levels of Sapap3 were unchanged in the frontal cortex (FC), hippocampus (HC), striatum (STR), and cerebellum (CBL) of both HDAC1 cKO mice (d) and HDAC2 cKO mice (e) compared to their respective CTLs (HDAC1 FC CTL n=6, cKO n=6; HC CTL n=5, cKO n=6; STR CTL n=6, cKO n=6; CBL CTL n=5, cKO n=6; two-tailed t-test; t(10) = 0.4718; P = 0.6472 for FC CTL versus cKO; t(9) = 0.4535; P = 0.6609 for HC CTL versus cKO; t(10) = 0.918; P = 0.3802 for STR CTL versus cKO; t(9) = 1.084; P = 0.3064 for CBL CTL versus cKO; HDAC2 FC CTL n=6, cKO n=6; HC CTL n=5, cKO n=6; STR CTL n=5, cKO n=6; CBL CTL n=6, cKO n=6; two-tailed t-test; t(10) = 1.332; P = 0.2123 for FC CTL versus cKO; t(9) = 1.422; P = 0.1887 for HC CTL versus cKO; t(9) = 1.42; P = 0.1892 for STR CTL versus cKO; t(10) = 0.0277; P = 0.9784 for CBL CTL versus cKO.) (f) qRT-PCR analysis showed that mRNA levels of Slitrk5 are similar between cDKO and CTL mice in the frontal cortex (FC), striatum (STR), hippocampus (HC), and cerebellum (CBL) (FC CTL n=8, cDKO n=7; STR CTL n=7, cDKO n=7; HC CTL n=6, cDKO n=7; CBL CTL n=7, cDKO=4; two-tailed t-test; t(13) = 1.247; P =0.2344 for FC CTL versus cDKO; t(12) = 1.788; P = 0.0990 for STR CTL versus cDKO; t(11) = 0.897; P = 0.3889 for HC CTL versus cDKO; t(9) = 0.5751; P = 0.5793 for CBL CTL versus cDKO). Data are shown as median, 25th and 75th percentile, and min and max value (a-f). *P < 0.05.

Supplementary Figure 5 Characterization of striatal specific deletion of Hdac1/Hdac2.

(a) Locomotor activity of mice with a striatal specific deletion of HDAC1/2 was measured over a 2 hour testing period in 5 minute increments with (inset) representing the total number of beam breaks during the testing period. AAV-GFP-Cre mice show no deficits in locomotor activity during the 2 hour testing period compared to AAV-GFP control mice (AAV-GFP n=9; AAV-GFP-Cre n=8; two-tailed t-test; t(15) = 0.3065; P = 0.7634). (b) Motor coordination was assessed using the rotarod test. No significant differences were measured in the amount of time spent on the rod between AAV-GFP-Cre mice and AAV-GFP control mice, and AAV-GFP-Cre mice showed improvement over the 8 trials at a similar rate as the AAV-GFP control mice (AAV-GFP n=9; AAV-GFP-Cre n=8; two-way ANOVA F(1,15) = 0.2112 for genotype, Bonferroni post hoc analysis P = 0.6524). (c) Mice with a striatal specific loss of HDAC1/2 do not display any anxiety like behavior in the open field paradigm. There were no significant differences in the total time spent in the complete center or periphery of the arena between AAV-GFP-Cre mice and AAV-GFP (AAV-GFP n=9; AAV-GFP-Cre n=8; two-tailed t-test; t(15) = 0.5173; P = 0.6125 for complete center, P = 0.6122 for periphery). (d) TUNEL staining did not reveal any cell death in the striatum following the AAV-mediated deletion of HDAC1 and HDAC2. Both AAV-GFP-Cre and AAV-GFP groups show no TUNEL positive cells indicating an absence of apoptotic activity. Images were captured using an Olympus BX51 epifluorescence microscope and Olympus DP70 software. Scale bar represents 100µm. Data are shown as median, 25th and 75th percentile, and min and max value (a inset, c) and mean value ± s.e.m. (a,b). *P < 0.05.

Supplementary Figure 6 MeCP2 associates with Hdac2 in the striatum.

Representative western blot following immunoprecipitation (IP) of HDAC2 from striatum of Mecp2 cKO and CTL mice. Following IP using an antibody against HDAC2, western blot analysis with an antibody against MeCP2 indicates that MeCP2 and HDAC2 interact in vivo in CTL mice, and this interaction is diminished in Mecp2 cKO mice. Results were replicated in a second cohort of mice. Full length blots are presented in Supplementary Figure 8.

Supplementary Figure 7 Rescue of Sapap3 in Mecp2 cKO mice.

(a and inset) Mecp2 cKO mice injected with AAV-GFP or AAV-SAPAP3 display normal locomotor activity compared to AAV-GFP CTL mice (AAV-GFP CTL n=10; AAV-GFP cKO n=8; AAV-SAPAP3 CTL n=10; AAV-SAPAP3 cKO n=7; one-way ANOVA F(3,31)= 1.625 for treatment, P = 0.2036). (b) Mecp2 cKO mice injected with AAV-GFP have significant deficits in rotarod performance compared to AAV-GFP CTL mice. Injection of AAV-SAPAP3 in Mecp2 cKO mice does not rescue this deficit, and mice are unable to show improvement on the rotarod over the 8 day trial compared to AAV-GFP CTLs (AAV-GFP CTL n=10; AAV-GFP cKO n=8; AAV-SAPAP3 CTL n=10; AAV-SAPAP3 cKO n=7; two-way ANOVA F(3,248) = 16.74 for genotype, P<0.0001, Tukey’s post hoc analysis for AAV-GFP CTL versus AAV-GFP cKO P < 0.0001, AAV-GFP CTL versus AAV-SAPAP3 cKO P =.0023). Data are shown as median, 25th and 75th percentile, and min and max value (a inset) and mean value ± s.e.m. (a, b). *P < 0.05.

Supplementary Figure 8 Full length western blots.

(a) Full length western blots of HDAC1 and GAPDH for cropped images from Fig. 1c. (b) Full length western blots of HDAC2 and GAPDH for cropped images from Figure 1d. (c) Full length western blot of MeCP2 for cropped images from Supplementary Fig. 6. (d) Full length western blot of Actin and MeCP2 for cropped images from Supplementary Fig. 6.

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Mahgoub, M., Adachi, M., Suzuki, K. et al. MeCP2 and histone deacetylases 1 and 2 in dorsal striatum collectively suppress repetitive behaviors. Nat Neurosci 19, 1506–1512 (2016). https://doi.org/10.1038/nn.4395

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