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. 2023 Mar 16:16:1139118.
doi: 10.3389/fnmol.2023.1139118. eCollection 2023.

Excessive self-grooming, gene dysregulation and imbalance between the striosome and matrix compartments in the striatum of Shank3 mutant mice

Allain-Thibeault Ferhat  1   2   3 , Elisabeth Verpy  1 , Anne Biton  4 , Benoît Forget  1 , Fabrice De Chaumont  1 , Florian Mueller  5 , Anne-Marie Le Sourd  1 , Sabrina Coqueran  1 , Julien Schmitt  6 , Christelle Rochefort  6 , Laure Rondi-Reig  6 , Aziliz Leboucher  1 , Anne Boland  7 , Bertrand Fin  7 , Jean-François Deleuze  7   8 , Tobias M Boeckers  9   10 , Elodie Ey  1   11 , Thomas Bourgeron  1
Affiliations

Excessive self-grooming, gene dysregulation and imbalance between the striosome and matrix compartments in the striatum of Shank3 mutant mice

Allain-Thibeault Ferhat et al. Front Mol Neurosci. .

Abstract

Autism is characterized by atypical social communication and stereotyped behaviors. Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are detected in 1-2% of patients with autism and intellectual disability, but the mechanisms underpinning the symptoms remain largely unknown. Here, we characterized the behavior of Shank3 Δ11/Δ11 mice from 3 to 12 months of age. We observed decreased locomotor activity, increased stereotyped self-grooming and modification of socio-sexual interaction compared to wild-type littermates. We then used RNAseq on four brain regions of the same animals to identify differentially expressed genes (DEGs). DEGs were identified mainly in the striatum and were associated with synaptic transmission (e.g., Grm2, Dlgap1), G-protein-signaling pathways (e.g., Gnal, Prkcg1, and Camk2g), as well as excitation/inhibition balance (e.g., Gad2). Downregulated and upregulated genes were enriched in the gene clusters of medium-sized spiny neurons expressing the dopamine 1 (D1-MSN) and the dopamine 2 receptor (D2-MSN), respectively. Several DEGs (Cnr1, Gnal, Gad2, and Drd4) were reported as striosome markers. By studying the distribution of the glutamate decarboxylase GAD65, encoded by Gad2, we showed that the striosome compartment of Shank3 Δ11/Δ11 mice was enlarged and displayed much higher expression of GAD65 compared to wild-type mice. Altogether, these results indicate altered gene expression in the striatum of Shank3-deficient mice and strongly suggest, for the first time, that the excessive self-grooming of these mice is related to an imbalance in the striatal striosome and matrix compartments.

Keywords: GAD65; Shank3; autism; matrix; mouse model; stereotyped behavior; striatum compartmentation; striosomes.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MY declared a shared affiliation with the author ATF to the handling editor at the time of review.

Figures

Figure 1
Figure 1
Hypoactivity, reduced exploration and increased stereotyped behaviors in Shank3Δ11/Δ11 mice at 3, 8, and 12 months of age. (A) Total distance traveled during 30-min free exploration of an open field in wild-type (green), heterozygous (blue) and Shank3Δ11/Δ11 (orange) for males (left panel) and females (right panel). (B) Total time spent digging in fresh bedding during 10 min observation in a new test cage, after 10 min habituation. (C) Total time spent in contact during male/female and female/female social interaction. (D) Total time spent self-grooming during 10 min observation in a new test cage, after 10 min habituation. (E) Z-score for the time spent self-grooming. (F) Proportion and number of individuals displaying different levels of severity in self-grooming. “Dead” corresponds to animals that had to be euthanized due to severe self-inflicted injuries. (A–D) Black points represent mean with standard error of the mean (s.e.m). Mann–Whitney U test with Bonferroni correction for multiple testing (two tests): * Corrected value of p < 0.05; ** corrected value of p < 0.01.
Figure 2
Figure 2
The striatum displays the largest gene expression differences between Shank3+/+ and Shank3Δ11/Δ11 mice and the largest number of impacted pathways. (A) Upset plot displaying the intersection between brain structures for the genes differentially expressed between Shank3Δ11/Δ11 and Shank3+/+ mice (adjusted value of p < 0.05; determined by limma-voom using observation quality weights). Genes located around the Shank3 region on chromosome 15 are shown in blue. The horizontal bar plots on the left show the number of differentially expressed genes in the different brain structures according to the value of their log-fold changes (logFC). (B) GO (Gene Ontology), GeneSetDB Pathway (gsdbpath), and KEGG (Kyoto Encyclopaedia of Genes and Genomes) gene sets found to be over-represented (top 20 gene sets with adjusted value of p < 0.05) in the genes detected as differentially expressed in the striatum. (C) Protein–protein interaction (PPI) network for the differentially expressed genes (DEGs). Nodes, colored ovals, represent DEGs; edges, black lines, represent direct protein–protein interactions (from BioGrid) between DEGs products. The color of the node represents the Log2FC of DEGs: red means up-regulation and blue means down-regulation in Shank3Δ11/Δ11 mice compared to wild-type mice.
Figure 3
Figure 3
Altered transcriptome of D1- and D2-MSN, over-expression of GAD65, and modification of the striosome/matrix balance in the striatum of Shank3Δ11/Δ11 mice. (A) Enrichment of RNAseq DEGs in the striatum of Shank3Δ11/Δ11 mice compared to cell-specific gene clusters suggested by Gokce et al. (2016) and Montalban et al. (2020). Genes under-expressed in Shank3Δ11/Δ11 striatum are enriched in D1-MSN clusters while genes over-expressed in Shank3Δ11/Δ11 striatum are enriched in D2-MSN clusters. Ratio genes correspond to the proportion of DEGs enriched in the cluster. Over-representation analyses were performed with Fisher’s hypergeometric tests and p-values were adjusted for multiple testing with the Benjamini–Hochberg procedure within each category of DEGs. (B) Comparison of the log of the fold change of gene expression of selected genes in Shank3Δ11/Δ11 vs. Shank3+/+ mice (LogFC RNAseq, Y axis) and the β values (slope of the linear regression) for low self-grooming vs. high self-grooming (β value grooming, X axis). (C,D) Confocal images of striatum coronal sections of one-year old Shank3+/+ male mice. (C) GAD65 immunoreactivity (red) is enriched in striatal microzones identified as striosomes by immunostaining for μ-opioid receptor (MOR, in green), a canonical marker of striosomes. (D) Increased GAD65 immunoreactivity in striosomes (arrows) and subcallosal streak (two-headed arrows) compared to surrounding matrix is observed with two different antibodies: the rabbit polyclonal antibody (Invitrogen PA5-77983 in red) used in (C,E,F) and a mouse monoclonal antibody (Millipore MAB351, in green). (E,F) Distribution and quantification of GAD65 immunoreactivity in dorsal striatum sections of a Shank3+/+ (E) and a Shank3Δ11/Δ11 (F) mouse (images generated by stitching multiple maximum-intensity projected z-stacks). The arrows point to striosomes and the arrowheads to subcallosal streak. In the dorsal striatum ROI (surrounded in green), the unlabeled myelinated fibers are colored in red, the regions of higher expression of GAD65 (striosomes) are colored in green, and the regions of lower expression of GAD65 (matrix) are uncolored. The cortex ROI used as a reference is surrounded in red. Note that the acquisition parameters were adjusted for each brain hemi-section in order to have no saturating signal for GAD65 in the striatal ROI. Increased intensity in the striatum thus leads to a staining which appears weaker in the cortex. (G–I) Comparison of GAD65 immunoreactivity in the striosome and matrix compartments of the dorsal striatum in 5 Shank3+/+ (green) and 9 Shank3Δ11/Δ11 (orange) 1-year old male mice. (G) Relative surface of the GAD65-enriched striosome compartment (surface of striosomes/surface of (striosomes + matrix). (H) Relative GAD65 labeling intensity in the striosomal compartment of the striatum compared to cortex. (I) Relative GAD65 labeling intensity in the matrix compartment of the striatum compared to cortex. Data, generated from analysis of 9–14 images per animal, are presented as box-plots (median, first, and third quartiles).
Figure 4
Figure 4
Proposed model linking increased activity in the striosomal pathway and imbalance between the direct and indirect pathways in the Shank3Δ11/Δ11 mice. The modifications of connection strength (line thickness) induced by striosomal compartment alterations in Shank3Δ11/Δ11 mice are only represented between the striatum and its output target areas (colored boxes). Increased activity of the striosomal pathway in Shank3Δ11/Δ11 mice, due to both larger size and increased GAD65 activity of the striosomal compartment (light/dark orange) enhances GABAergic inhibition of the dopamine-producing neurons of SNpc (green). This inhibition of the SNpc neurons (light green) results in an imbalance between the direct and indirect pathways by decreasing both the activation of D1-MSN (D1) and the inhibition of D2-MSN (D2). GPI, internal globus pallidus; GPE, external globus pallidus; SNpr, substantia nigra pars reticulata; SNpc, substantia nigra pars compacta; STN, subthalamic nucleus.
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