Wound healing responses of urinary extravasation after urethral injury.

The post-surgical fluid leakage from the tubular tissues is a critical symptom after gastrointestinal or urinary tract surgeries. Elucidating the mechanism for such abnormalities is vital in surgical and medical science. The exposure of the fluid such as peritonitis due to urinary or gastrointestinal perforation has been reported to induce severe inflammation to the surrounding tissue. However, there have been no reports for the tissue responses by fluid extravasation and assessment of post-surgical and injury complication processes is therefore vital. The current model mouse study aims to investigate the effect of the urinary extravasation of the urethral injuries. Analyses on the urinary extravasation affecting both urethral mesenchyme and epithelium and the resultant spongio-fibrosis/urethral stricture were performed. The urine was injected from the lumen of urethra exposing the surrounding mesenchyme after the injury. The wound healing responses with urinary extravasation were shown as severe edematous mesenchymal lesions with the narrow urethral lumen. The epithelial cell proliferation was significantly increased in the wide layers. The mesenchymal spongio-fibrosis was induced by urethral injury with subsequent extravasation. The current report thus offers a novel research tool for surgical sciences on the urinary tract.

Functional analysis of PAX8 variants identified in patients with congenital hypothyroidism in situ.

Paired box transcription factor 8 (PAX8) is essential for thyroid organogenesis and development. Heterozygous pathogenic variants of PAX8 typically cause congenital hypothyroidism (CH) due to thyroid hypoplasia. Additionally, pathogenic PAX8 variants have been identified in patients with gland in situ (GIS). This study was conducted to analyze the in vitro functional consequences of four PAX8 variants (p.D94N, p.E90del, p.V58I, and p.L186Hfs*22) previously identified in patients with CH and GIS. The transcriptional activity of PAX8 variants on the thyroglobulin (TG) promoter was assessed in a luciferase reporter assay. The levels of transcriptional activity on the TG promoter of p.E90del and p.L186Hfs*22 were significantly reduced, whereas p.D94N and p.V58I showed residual activation. In addition, a dominant negative effect on the wild-type (WT) was not detected in any PAX8 variant using a luciferase reporter assay. Two PAX8 variants (p.E90del and p.L186Hfs*22) may be pathogenic causes of CH with GIS.

Negative feedback loop of bone resorption by NFATc1-dependent induction of Cadm1.

Trimethylation of histone H3 lysine 4 and lysine 27 (H3K4me3 and H3K27me3) at gene promoter regions critically regulates gene expression. Key developmental genes tend to exhibit changes in histone modification patterns from the H3K4me3/H3K27me3 bivalent pattern to the H3K4me3 monovalent pattern. Using comprehensive chromatin immunoprecipitation followed by sequencing in bone marrow-derived macrophages (BMMs) and mature osteoclasts, we found that cell surface adhesion molecule 1 (Cadm1) is a direct target of nuclear factor of activated T cells 1 (NFATc1) and exhibits a bivalent histone pattern in BMMs and a monovalent pattern in osteoclasts. Cadm1 expression was upregulated in BMMs by receptor activator of nuclear factor kappa B ligand (RANKL), and blocked by a calcineurin/NFATc1 inhibitor, FK506. Cadm1-deficient mice exhibited significantly reduced bone mass compared with wild-type mice, which was due to the increased osteoclast differentiation, survival and bone-resorbing activity in Cadm1-deficient osteoclasts. These results suggest that Cadm1 is a direct target of NFATc1, which is induced by RANKL through epigenetic modification, and regulates osteoclastic bone resorption in a negative feedback manner.

Foxp2 Regulates Identities and Projection Patterns of Thalamic Nuclei During Development.

The molecular mechanisms underlying the formation of the thalamus during development have been investigated intensively. Although transcription factors distinguishing the thalamic primordium from adjacent brain structures have been uncovered, those involved in patterning inside the thalamus are largely unclear. Here, we show that Foxp2, a member of the forkhead transcription factor family, regulates thalamic patterning during development. We found a graded expression pattern of Foxp2 in the thalamic primordium of the mouse embryo. The expression levels of Foxp2 were high in the posterior region and low in the anterior region of the thalamic primordium. In Foxp2 (R552H) knockin mice, which have a missense loss-of-function mutation in the forkhead domain of Foxp2, thalamic nuclei of the posterior region of the thalamus were shrunken, while those of the intermediate region were expanded. Consistently, Foxp2 (R552H) knockin mice showed changes in thalamocortical projection patterns. Our results uncovered important roles of Foxp2 in thalamic patterning and thalamocortical projections during development.

CASPR2 forms a complex with GPR37 via MUPP1 but not with GPR37(R558Q), an autism spectrum disorder-related mutation.

Autism spectrum disorder (ASD) is a developmental brain disorder. Mutations in synaptic components including synaptic adhesion molecules have been found in ASD patients. Contactin-associated protein-like 2 (CASPR2) is one of the synaptic adhesion molecules associated with ASD. CASPR2 forms a complex with receptors via interaction with multiple PDZ domain protein 1 (MUPP1). Little is known about the relationship between impaired CASPR2-MUPP1-receptor complex and the pathogenesis of ASD. GPR37 is a receptor for survival factors. We recently identified mutations including R558Q in the G-protein-coupled receptor 37 (GPR37) gene in ASD patients. The mutated GPR37s accumulate in the endoplasmic reticulum. In this study, we show that GPR37 is a component of the CASPR2-MUPP1 receptor complex in the mouse brain. CASPR2 and GPR37 mainly interacted with the PDZ3 and PDZ11 domains of MUPP1, respectively. Compared to GPR37, GPR37(R558Q) slightly interacted with MUPP1 and caused dendritic alteration. GPR37, but not GPR37(R558Q) nor GPR37-deltaC which lacks its PDZ binding domain, was transported to the cell surface by MUPP1. In primary hippocampal neurons, GPR37 co-localized with MUPP1 and CASPR2 at the synapse, but not GPR37(R558Q). Thus, ASD-related mutation of GPR37 may cause the impaired CASPR2-MUPP1-GPR37 complex on the dendrites associated with one of the pathogenesis of ASD. In this study, we identified that GPR37 is a component of the MUPP1 and CASPR2 receptor complex. Autism deleterious mutated GPR37(R558Q) slightly interacts with MUPP1 and retains in ER, resulting in dendritic alteration. In neuron, GPR37, but not GPR37(R558Q), is transported to the dendrite and synapse by MUPP1. Thus, ASD-related mutation of GPR37 may cause the impaired CASPR2-MUPP1-GPR37 complex on the dendrites associated with one of the pathogenesis of ASD.

The association of GPR85 with PSD-95-neuroligin complex and autism spectrum disorder: a molecular analysis.

BACKGROUND: Autism spectrum disorder (ASD) has a complex genetic etiology. Some symptoms and mutated genes, including neuroligin (NLGN), neurexin (NRXN), and SH3 and multiple ankyrin repeat domains protein (SHANK), are shared by schizophrenia and ASD. Little is known about the molecular pathogenesis of ASD. One of the possible molecular pathogenesis is an imbalance of excitatory and inhibitory receptors linked with the NLGN-PSD-95-SHANK complex via postsynaptic density protein/Drosophila disc large tumor suppressor/zonula occludens-1 protein (PDZ) binding. In the present study, we focused on GPR85 as a candidate gene for ASD because the C-terminal amino acid sequence of GPR85 [Thr-Cys-Val-Ile (YCVI)] is classified as a type II PDZ-binding motif, and GPR85 is a risk factor for schizophrenia. GPR85 is an orphan receptor that regulates neural and synaptic plasticity and modulates diverse behaviors, including learning and memory. While searching for molecules that associate with GPR85, we found that GPR85 was associated with postsynaptic density protein (PSD)-95 linked with NLGN in the brain. METHODS: We examined the proteins that associate with the C-terminal sequence of GPR85 by pull-down assay and immunoblot analysis and searched for a mutation of the GPR85 gene in patients with ASD. We used immunostaining to examine the intracellular localization of mutated GPR85 and its influence on the morphology of cells and neurons. RESULTS: The C-terminal sequence of GPR85 interacted with PSD-95 at PDZ1, while NLGN interacted with PSD-95 at PDZ3. Two male patients with ASD from independent Japanese families possessed inherited missense mutations at conserved sites in GPR85: one had T1033C (M152T) and the other had G1239T (V221L). These mutations were located in a domain related to G protein interaction and signal transduction. In contrast to wild-type GPR85, mutated GPR85 was more preferentially accumulated, causing endoplasmic reticulum stress, and disturbed the dendrite formation of hippocampal neurons. CONCLUSIONS: GPR85 associated with the PSD-95 linked with NLGN, which is related to ASD. GPR85 carrying the mutations detected in ASD patients disturbed dendrite formation that could be the candidate for molecular pathogenesis of ASD through the associated NLGN-PSD-95 receptor complex.

Spatial and temporal expression of RA70/Scap2 in the developing neural tube.

Src kinase-associated phosphoprotein 2 (Ra70/scap2), which was originally isolated as a retinoic acid (RA)-induced gene, associates with molecules that modulate integrin-survival signals. Although RA is essential for vertebrate organogenesis in the posterior region, little is known about the biological role of RA70/Scap2 during development. In the present study, we demonstrate that Ra70/scap2 mRNA is temporally expressed during the RA-induced neuronal differentiation of P19 embryonic carcinoma cells. Homozygous knockout mice in which the Ra70/scap2 gene was replaced with LacZ exhibited embryonic lethality, while heterozygous mice displayed preferential expression of LacZ in posterior neural tissues, including the neural tube and hindbrain during development (E7.5-11.5), but not the forebrain. Ra70/scap2 was expressed in the ependymal layer and ventricular zone in the neural tube, where neuroepithelial cells and neuroblasts with proliferation capacity are localized, respectively. Thus, RA70/Scap2 may be necessary for RA-induced neuronal differentiation from the posterior neuroectoderm.

Specific expression of FOXP2 in cerebellum improves ultrasonic vocalization in heterozygous but not in homozygous Foxp2 (R552H) knock-in pups.

The R553H mutation has been found in the FOXP2 gene of patients with speech-language disorder. Foxp2(R552H) knock-in (KI) mice exhibit poor dendritic development of Purkinje cells in the cerebellum and impaired ultrasonic vocalization (USV), which is related to human speech and language; compared with wild-type mice, heterozygous Foxp2(R552H)-KI pups exhibit the reduced number of whistle-type USVs and the increased short-type ones, while homozygous pups exhibit only click-type USVs but no whistle-type or short-type ones. To make clear the relationship between the role of Foxp2 in the cerebellum and whistle-type USVs activity, we prepared transgenic (Tg) mice specifically expressing human FOXP2-myc in cerebellum (Pcp2-FOXP2-myc-Tg mice) by using purkinje cell protein-2 (Pcp2) promoter. FOXP2-myc expression in the cerebellum increased the relative numbers of whistle-type USVs in the heterozygous Foxp2(R552H)-KI pups and recovered their USVs but did not in the homozygous ones. Foxp2 in the cerebellum may pertain to the brain network engaged in whistle-type USVs activities including modification, but not their production. There may be common molecular contribution of Purkinje cells to human FOXP2-mediated speech-language and mouse Foxp2-mediated USVs.

Mutation in Parkinson disease-associated, G-protein-coupled receptor 37 (GPR37/PaelR) is related to autism spectrum disorder.

Little is known about the molecular pathogenesis of Autism spectrum disorder (ASD), a neurodevelopmental disorder. Here we identified two mutations in the G-protein-coupled receptor 37 gene (GPR37) localized on chromosome 7q31-33, called the AUTS1 region, of ASD patients; 1585-1587 ttc del (Del312F) in one Japanese patient and G2324A (R558Q) in one Caucasian patient. The Del312F was located in the conserved transmembrane domain, and the R558Q was located in a conserved region just distal to the last transmembrane domain. In addition, a potential ASD-related GPR37 variant, T589M, was found in 7 affected Caucasian men from five different families. Our results suggested that some alleles in GPR37 were related to the deleterious effect of ASD. GPR37 is associated with the dopamine transporter to modulate dopamine uptake, and regulates behavioral responses to dopaminergic drugs. Thus, dopaminergic neurons may be involved in the ASD. However, we also detected the Del321F mutation in the patient's unaffected father and R558Q in not only an affected brother but also an unaffected mother. The identification of unaffected parents that carried the mutated alleles suggested that the manifestation of ASD was also influenced by factors other than these mutations, including endoplasmic reticulum stress of the mutated proteins or gender. Our study will provide the new insight into the molecular pathogenesis of ASD.