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Ephrin-B2 Deletion in GABAergic Neurons Induces Cognitive Deficits Associated with Single-Nucleus Transcriptomic Differences in the Prefrontal Cortex

ABSTRACT: A total of 11,308 single nuclei were sequenced. After quality filtering (Methods, Supplementary Fig. 3A, 4A-4C), 11,090 high-quality nuclei were obtained, including 5,336 cells from EB2-flox mouse and 5,754 from EB2-vGATCre mouse. These nuclei were grouped into 17 cell clusters (Supplementary Fig. 4D-4H), which were further assigned to 9 major cell types based on cell-type-specific markers: excitatory neurons (Slc17a7+), inhibitory neurons (Gad2+), astrocytes (Gja1+), microglia (C1qa+), oligodendrocytes (Aspa+), oligodendrocyte precursors (Pdgfra+), smooth muscle cell (Uaca+), endothelial (Flt1+) and ependymal (Dynlrb2+) cells (Fig. 4A, 4C, supplementary Fig. 3B).

ORGANISM(S): Mus musculus

PROVIDER: GSE297542 | GEO | 2025/05/31

REPOSITORIES: GEO

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Project description:The SRC inhibitor PP2 can block CASK nucleus translocation in macrophage under H5N1 infection. Since PP2 is a general inhibitor for Src family kinases (SFKs), which includes nine members (Csk, Yes, Fyn, Fgr, Lck, Hck, Blk, Lyn, and Frk), we aimed to identify the specific member responsible for the nuclear translocation of CASK. First, we examined the expression of SFKs in macrophages (Csk, Fyn, Fgr, and Hck) following H5N1-IAV infection (Fig. 4A). This observation suggests that Hck is the most abundant SFK and may contribute to virus-induced nuclear translocation of CASK. To test this, we generated constitutively active forms of HCK: HCK△513-524 (deletion mutant) and HCK Y499F mutant (YF mutation) [24], then co-transfected them with an EGFP-CASK construct into 293T cells. Immunoprecipitation with anti-GFP mAb, followed by trypsin digestion and LC-MS/MS analysis, revealed that compared to the control group, the phospho/nonphospho ratio at residue Serine 395 (S395) of CASK increased approximately 10-fold (0.008 to 0.072 by HCK△513-524, 0.008 to 0.108 by HCK Y499F), while the other 8 potential phosphorylation sites did not show significant alterations (Table 1). To confirm the phosphorylation of S395 of CASK is required for nuclear translocation, we generated an EGFP-CASK S395A mutant (SA mutation) to prevent CASK phosphorylation. In the absence of HCK Y499F, both the wild type (CASK S395) and mutant (CASK S395A) remained in the cytosol (left two columns, Fig. 4B). In contrast, transfection of HCK Y499F (red color) induced the translocation of wild type CASK (green color) into the nucleus (3rd column from left, Fig. 4B), an effect not observed with the CASK S395A mutant (4th column from left, Fig. 4B). The percentage of nuclear GFP-CASK (60% and 90% in WT and 35% in S395A) resulting from the overexpression of wild type HCK and HCK Y499F is shown in Figure 4C. Thus, we concluded that activated HCK induces the phosphorylation of CASK at Ser395, facilitating its nuclear translocation.

Project description:Since the intronic mutation in SLC2A2 did not influence the activity of GLUT2, we undertook more investigation to understand the underlying molecular mechanisms of dysglycemia in FBS. We ran Nanostring miRNA panel v3b covering ~800 miRNAs in the patient, mother, and aged- and gender-matching healthy controls with no family history of dysglycemia. We noticed a higher degree of correlation between the patient and the gender and age-matched healthy control, rather than between the patient and the mother, suggesting that the miRNA expression profile might be more influenced by the age and gender rather than relatedness (Supplementary Fig. S5). The unsupervised hierarchical clustering revealed 123 miRs expressed specifically in the patient sample (Supplementary Table S2). The function of these miRNAs was interrogated by using Ingenuity Pathway (IPA) analysis software which returned 118 mapped miRs (Fig. 10). Here we report 30 miRNAs with the highest number of counts difference in the patient in comparison to controls (Fig. 11). We found that 14 of them were correlated with T1DM: 10 miRNAs (miR-199a, miR-25-3p, miR-93-5p, miR-19b-3p, miR-107, miR-24-3p. miR-18a-5p, miR-125b-5p, miR-324-5p, miR-331-3p, and hsa-miR-143-3p) were overexpressed in the control as compared to the patient, and 3 miRNAs (miR-144-3p , let-7e-5p, hsa-miR-29a-3p) were significantly overexpressed in the patient in comparison to the control. Molecular networks, including molecules inferred from previous studies, were generated by IPA functional analysis software (Supplementary Fig. S6). The molecular networks were given a score based on the number of molecules represented in the study dataset as compared to the literature. Network 1 (score 33) includes the genes and miRNAs implicated in organismal injury and abnormalities, skeletal and muscular system development and function, and tissue morphology. The miR-144 family integrated into the network 1. Network 2 (score 31) includes insulin and other genes and miRNAs implicated in glomerular injury, inflammatory disease, inflammatory response, and included miR-29 and let-7 families. These results suggest that dysglycemia in the patient with intronic mutation might be associated with the deregulation of miRNAs involved in insulin production and secretion in beta cells.

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Ephrin-B2 Deletion in GABAergic Neurons Induces Cognitive Deficits Associated with Single-Nucleus Transcriptomic Differences in the Prefrontal Cortex

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