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Scribble (Scrib) is a conserved polarity protein acting as a scaffold involved in multiple cellular and developmental processes. Recent evidence from our group indicates that Scrib is also essential for brain development as early global deletion of Scrib in the dorsal telencephalon induced cortical thickness reduction and alteration of interhemispheric connectivity. In addition, Scrib conditional knockout (cKO) mice have behavioral deficits such as locomotor activity impairment and memory alterations. Given Scrib broad expression in multiple cell types in the brain, we decided to determine the neuronal contribution of Scrib for these phenotypes. In the present study, we further investigate the function of Scrib specifically in excitatory neurons on the forebrain formation and the control of locomotor behavior. To do so, we generated a novel neuronal glutamatergic specific Scrib cKO mouse line called Nex -Scrib −/− cKO. Remarkably, cortical layering and commissures were impaired in these mice and reproduced to some extent the previously described phenotype in global Scrib cKO. In addition and in contrast to our previous results using Emx1-Scrib −/− cKO, the Nex-Scrib −/− cKO mutant mice exhibited significantly reduced locomotion. Altogether, the novel cKO model described in this study further highlights an essential role for Scrib in forebrain development and locomotor behavior. [ABSTRACT FROM AUTHOR]

Chondroitin sulfate (CS) is a sulfated glycosaminoglycan (GAG) chain. In cartilage, CS plays important roles as the main component of the extracellular matrix (ECM), existing as side chains of the major cartilage proteoglycan, aggrecan. Six glycosyltransferases are known to coordinately synthesize the backbone structure of CS; however, their in vivo synthetic mechanism remains unknown. Previous studies have suggested that two glycosyltransferases, Csgalnact1 (t1) and Csgalnact2 (t2), are critical for initiation of CS synthesis in vitro. Indeed, t1 single knockout mice (t1 KO) exhibit slight dwarfism and a reduction in CS content in cartilage compared with wild-type (WT) mice. To reveal the synergetic roles of t1 and t2 in CS synthesis in vivo, we generated systemic single and double knockout (DKO) mice and cartilage-specific t1 and t2 double knockout (Col2-DKO) mice. DKO mice exhibited postnatal lethality, whereas t2 KO mice showed normal size and skeletal development. Col2-DKO mice survived to adulthood and showed severe dwarfism compared with t1 KO mice. Histological analysis of epiphyseal cartilage from Col2-DKO mice revealed disrupted endochondral ossification, characterized by drastic GAG reduction in the ECM. Moreover, DKO cartilage had reduced chondrocyte proliferation and an increased number of apoptotic chondrocytes compared with WT cartilage. Conversely, primary chondrocyte cultures from Col2-DKO knee cartilage had the same proliferation rate as WT chondrocytes and low GAG expression levels, indicating that the chondrocytes themselves had an intact proliferative ability. Quantitative RT-PCR analysis of E18.5 cartilage showed that the expression levels of Col2a1 and Ptch1 transcripts tended to decrease in DKO compared with those in WT mice. The CS content in DKO cartilage was decreased compared with that in t1 KO cartilage but was not completely absent. These results suggest that aberrant ECM caused by CS reduction disrupted endochondral ossification. Overall, we propose that both t1 and t2 are necessary for CS synthesis and normal chondrocyte differentiation but are not sufficient for all CS synthesis in cartilage. [ABSTRACT FROM AUTHOR]

Gravity change affects many immunological systems. We investigated the effects of hypergravity (2G) on murine thymic cells. Exposure of mice to 2G for three days reduced the frequency of CD4+CD8+ thymocytes (DP) and mature medullary thymic epithelial cells (mTECs), accompanied by an increment of keratin-5 and keratin-8 double-positive (K5+K8+) TECs that reportedly contain TEC progenitors. Whereas the reduction of DP was recovered by a 14-day exposure to 2G, the reduction of mature mTECs and the increment of K5+K8+ TEC persisted. Interestingly, a surgical lesion of the inner ear’s vestibular apparatus inhibited these hypergravity effects. Quantitative PCR analysis revealed that the gene expression of Aire and RANK that are critical for mTEC function and development were up-regulated by the 3-day exposure and subsequently down-regulated by the 14-day exposure to 2G. Unexpectedly, this dynamic change in mTEC gene expression was independent of the vestibular apparatus. Overall, data suggest that 2G causes a temporary reduction of DP and a persistent reduction of mature mTECs in a vestibular system-dependent manner, and also dysregulates mTEC gene expression without involving the vestibular system. These data might provide insight on the impact of gravity change on thymic functions during spaceflight and living. [ABSTRACT FROM AUTHOR]

Abstract: Chondroitin sulfate (CS), the carbohydrate chain of chondroitin sulfate proteoglycans, is involved in neuronal circuit formation during development. CS shows great structural diversity with combination of disaccharide units of different structure (A-, C-, D-, or E-unit). However, whether its structural diversity contributes to pathway formation remains unclear. We chemically coupled the reducing end of various types of CS to the amino group of phosphatidylethanolamine (lipid-derivatized CS, CS-PE) and established an in vitro time-lapse assay to observe the behaviors of growth cones of retinal ganglion cells from embryonic day 6 chick retina on exposure to beads coated with lipid-derivatized CS (CS-PE beads). Among CS-PEs with different content of the structural units, the beads coated with E-unit-containing CS-PE [E-unit: GlcAβ1-3GalNAc(4,6-O-disulfate)] (CSE-PE beads) significantly caused the growth cones to retract and to turn away from the beads, but the beads coated with CSA-, CSC- or CSD-PE beads did not. Importantly, not all the growth cones retracted equally from the CSE-PE beads, but they showed continuum of the repulsive behaviors; some behaved moderately and others remarkably. The growth cones distinguished different samples of CS: CSE and the others. Moreover, the continuum of the repulsive behaviors suggests that CS might be involved with the fine regulation of growth cones'' behavior through its characteristic structure. [Copyright &y& Elsevier]

Hindlimb unloading (HU) of rodents has been used as a ground-based model of spaceflight. In this study, we investigated the detailed impact of 14-day HU on the murine thymus. Thymic mass and cell number were significantly reduced after 14 days of hindlimb unloading, which was accompanied by an increment of plasma corticosterone. Although corticosterone reportedly causes selective apoptosis of CD4 + CD8 + thymocytes (CD4 + CD8 + DPs) in mice treated with short-term HU, the reduction of thymocyte cellularity after the 14-day HU was not selective for CD4 + CD8 + DPs. In addition to the thymocyte reduction, the cellularity of thymic epithelial cells (TECs) was also reduced by the 14-day HU. Flow cytometric and RNA-sequencing analysis suggested that medullary TECs (mTECs) were preferentially reduced after HU. Moreover, immunohistochemical staining suggested that the 14-day HU caused a reduction of the mTECs expressing autoimmune regulator (Aire). Our data suggested that HU impacts both thymocytes and TECs. Consequently, these data imply that thymic T cell repertoire formation could be disturbed during spaceflight-like stress. [ABSTRACT FROM AUTHOR]