Katherine Quesnel

Mutations in the ATRX gene are a primary cause of alpha‐thalassemia intellectual disability X‐linked (ATRX) syndrome, which is characterized by intellectual disability, autism, and a range of brain structural abnormalities, including microcephaly. We previously showed that mice with conditional ATRX ablation in forebrain excitatory neurons display deficits in fear memory and autism‐related behaviors, with some effects exhibiting sexual dimorphism. In this study, we used high‐resolution magnetic resonance imaging (MRI) to systematically characterize brain structural changes associated with these behavioral abnormalities. Whole‐brain analysis revealed male‐specific microcephaly, while subregional analysis identified significant reductions in hippocampal structures and increased volume of the caudal cortex in mutant animals of both sexes. We also identified structural alterations in regions retaining ATRX expression, such as the thalamus, midbrain, cerebellum, and several fiber tracts. These findings suggest that ATRX loss disrupts the coordinated development of interconnected brain regions. Overall, our results implicate impaired cortico‐thalamic‐cerebellar connectivity as a potential neural substrate underlying the autistic‐like behaviors observed in this mouse model, providing new insights into the neurobiological basis of ATR‐X syndrome.

Mutations in the ATRX gene are a primary cause of ATR-X syndrome, which is characterized by intellectual disability, autism, and a range of brain structural abnormalities, including microcephaly. We previously showed that mice with conditional ATRX ablation in forebrain excitatory neurons display deficits in fear memory and autism-related behaviors, with some effects exhibiting sexual dimorphism. In this study, we used high-resolution magnetic resonance imaging (MRI) to systematically characterize brain structural changes associated with these behavioral abnormalities. Whole-brain analysis revealed male-specific microcephaly, while subregional analysis identified significant reductions in hippocampal structures and increased volume of the caudal cortex in mutant animals of both sexes. We also identified structural alterations in regions retaining ATRX expression, such as the thalamus, midbrain, cerebellum, and several fiber tracts. These findings suggest that ATRX loss disrupts the coordinated development of interconnected brain regions. Overall, our results implicate impaired cortico-thalamic-cerebellar connectivity as a potential neural substrate underlying the autistic-like behaviors observed in this mouse model, providing new insights into the neurobiological basis of ATR-X syndrome. LAY SUMMARY: Changes in a gene called ATRX are known to affect brain development and are linked to intellectual disability and autism. In our previous work, we found that removing this gene early in brain development caused mice to show behaviors like those seen in people with autism. In this study, we used detailed brain scans to see if these behavioral changes were linked to differences in brain structure. We found that male mice without ATRX had smaller brains and bodies, while female mice did not show the same brain size reduction. However, both male and female mice had smaller areas of the brain important for memory and movement, and larger areas involved in thinking and sensing. We also saw changes in parts of the brain where ATRX was still present, suggesting that early changes in one area can affect how the whole brain develops. These findings help us understand how early disruptions in brain development might lead to autism-related behaviors.

CCN1 +/+ stroma vasculature microct

CCN1 deficient stroma vasculature microct

CCN1 is expressed by a subset of CAFs and correlates with expression of angiogenic markers. A,CCN1 expression correlates with bulk tumor stromal scores. Scatterplot representation of stromal score (ESTIMATE algorithm) to CCN1 expression in bulk primary melanoma tumors from TCGA (n = 82). The linear regression line is shown in blue, and the 95% confidence interval is shown as a gray area around the regression line. Spearman correlation coefficient and corresponding P value are displayed in the bottom right corner. B,CCN1 gene expression (TPM/10) in melanoma tumor single cells [malignant cells (n = 2,018), T cells (n = 3,321), B cells (n = 818), macrophages (n = 420), natural killer cells (n = 92), endothelial cells (n = 104), and CAFs (n = 106)]. Boxes represent interquartile ranges, and points represent individual sample values. C, Heat map representation of the 50 genes with the highest SD in CAF single cells from human melanoma tumors. CCN1 expression is higher in a particular subset of CAFs (GSE115978). Columns represent single CAF cells, and rows represent Z-score mRNA expression (TPM/10). The horizontal bar denotes different melanoma samples. Scatterplots show CAF-specific CCN1 scores versus gene set enrichment of angiogenesis (D), or EMT gene sets (E; GAGE t statistics) of melanoma bulk primary melanoma tumors from TCGA samples (n = 82). The linear regression line is shown in blue, and the 95% confidence interval is shown as a gray area around the regression line. Spearman correlation coefficient and associated P values are displayed in the bottom right corner.

CCN1-deficient stroma show disorganized collagen fibers. Digital pathology phenotypic quantification (PharmaNest, Inc) phenotypic fibrosis histologic heat chart (A) where each row represents a principal qFT, at the three phenotypic levels (collagen deposition, fibers morphometry, and fibrosis architecture). B, The Ph-FCS recapitulates all the qFTs for one sample, and quantifies the phenotype of fibrosis and its differences between CCN1-deficient (KO; n = 5, mean = 1.5, Std dev = 0.5) and wild-type (WT) mice (n = 6, mean = 4.6, Std dev = 0.9) groups (3.1 fold change, P < 0.001, Student t test). C, The collagen fiber morphometric scores recapitulates the differences at the morphometric level between KO (n = 5, mean = 1.6, Std dev = 0.5) and WT (n = 6, mean = 4.5, Std dev = 0.9) groups (2.9 fold change, P < 0.001, Student t test). D, Representative images with augmented digital pathology layers. “Entropy Texture” is one of the architectural phenotypes of fibrosis (39). KO, knockout.

CCN1 deficient stroma vasculature microct

In a syngeneic model of metastasis, CCN1-deficient stroma possesses reduced collagen deposition, whereas myofibroblast formation is unaffected. Skin tissue showed a significant decrease in collagen shown trichrome and Sirius red staining (A; arrow indicates stroma). Representative images shown; Graph shows mean area of collagen, detected using trichrome stain, present in stroma images ± SD; Student t test; n = 3). α-SMA–expressing myofibroblasts (B) and YAP-positive nuclei (C) are present in both wild-type and CCN1-deficient stroma (Representative images shown; Graphs represent mean α-SMA positive cells ± SD; one-way ANOVA; n = 5). Mice were examined after 14 days of tumor growth of B16F10 melanoma cells.

CCN1 deficient stroma vasculature microct

Mice harboring Col1A2-Cre-fibroblast–specific deletion for Ccn1 show impaired metastasis of B16F10 melanoma cells to the lung. A–C, Mice were examined after 14 days of tumor growth. A, Sections of tumor stroma were stained with anti-CCN1 antibody (N = 3, representative images are shown), verifying loss of CCN1 protein expression in mice deleted for CCN1 in fibroblasts. Note CCN1 staining remains in the tumor. Scale bar = 50 µm. B, Representative images of lung sections from wild-type or mice harboring a deletion for Ccn1 in Col1A2-Cre-fibroblasts. Hematoxylin and eosin was used to reveal detect dense metastatic foci (purple). Scale Bar = 300 µm. Total area of the lung section covered by metastases was quantified. Deletion of Ccn1 caused reduced metastasis (N = 6, ***, P < 0.001). C, Reduced CCN2 expression in the stroma did not significantly alter tumor growth. Kruskal–Wallis analysis (N = 8). D, To more accurately define the cells in which CCN1 was deleted, three-week old Col1A2-cre(ER)T/); mTmG mice were injected for 5 consecutive days with tamoxifen to activate Cre recombinase. Six weeks later, skin was isolated, digested with collagenase, and cells expressing GFP were isolated by FACS, and subjected to RNA-seq analysis. Col1A2 lineage fibroblasts were identified as universal fibroblasts based on the gene expression of Pi16, Col15a1, C3, and Cd84 genes, but not papillary or reticular fibroblasts based on the gene expression of Crabp1 and Defb8 or Nexn, Trim63, Actn2, and Hspb7, respectively.