Rab13 Small G Protein and Junctional Rab13-binding Protein (JRAB) Orchestrate Actin Cytoskeletal Organization during Epithelial Junctional Development

During epithelial junctional development, both vesicle transport and reorganization of the actin cytoskeleton must be spatiotemporally regulated. Coordination of these cellular functions is especially important, but the precise mechanism remains elusive. Previously, we identified junctional Rab13-binding protein (JRAB)/molecules interacting with CasL-like 2 (MICAL-L2) as an effector of the Rab13 small G protein, and we found that the Rab13-JRAB system may be involved in the formation of cell-cell adhesions via transport of adhesion molecules. Here, we showed that JRAB interacts with two actin-binding proteins, actinin-1 and -4, and filamentous actin via different domains and regulates actin cross-linking and stabilization through these interactions. During epithelial junctional development, JRAB is prominently enriched in the actin bundle at the free border; subsequently, JRAB undergoes a Rab13-dependent conformational change that is required for maturation of cell-cell adhesion sites. These results suggest that Rab13 and JRAB regulate reorganization of the actin cytoskeleton throughout epithelial junctional development from establishment to maturation of cell-cell adhesion.     

Synthesis and evaluation of water-soluble resveratrol and piceatannol via BGLation

Synthesis of four water-soluble resveratrol and piceatannol derivatives bearing symmetrically branched glyceryl trimer (BGL003) with a non-biocleavable linkage, and their biological evaluation as a mitochondrial fusion-inducing agent with cellular fat-reducing effect from cells, is described. The effect of Piceatannol-BGL003 conjugate was as high as that of original stilbenoids.     

Rhabdom evolution in butterflies: insights from the uniquely tiered and heterogeneous ommatidia of the Glacial Apollo butterfly, Parnassius glacialis

The eye of the Glacial Apollo butterfly, Parnassius glacialis, a 'living fossil' species of the family Papilionidae, contains three types of spectrally heterogeneous ommatidia. Electron microscopy reveals that the Apollo rhabdom is tiered. The distal tier is composed exclusively of photoreceptors expressing opsins of ultraviolet or blue-absorbing visual pigments, and the proximal tier consists of photoreceptors expressing opsins of green or red-absorbing visual pigments. This organization is unique because the distal tier of other known butterflies contains two green-sensitive photoreceptors, which probably function in improving spatial and/or motion vision. Interspecific comparison suggests that the Apollo rhabdom retains an ancestral tiered pattern with some modification to enhance its colour vision towards the long-wavelength region of the spectrum.     

Calcium-induced synergistic inhibition of a translational factor eEF2 in nerve growth cones

Local protein synthesis in nerve growth cones has been suggested, but how it is controlled remains largely unknown. We found eukaryotic elongation factor-2 (eEF2), a key component of mRNA translation, in growth cones by immunocytochemistry. While phosphorylated eEF2 was weakly distributed in advancing growth cones, eEF2 phosphorylation was increased by high potassium-evoked calcium influx. In the growth cone, calcium elevation increased eEF2 kinase (EF2K), a calcim-calmodulin-dependent enzyme. Calcium also decreased the level of phosphorylated p70-S6 kinase (S6K), a kinase known to inhibit EF2K. Moreover, calcium elevation decreased total eEF2 in growth cones. Since phosphorylated eEF2 inhibits mRNA translation, calcium elevation appears to inhibit mRNA translation in growth cones by a synergistic mechanism involving regulation of EF2K, S6K, and eEF2 itself. Time-lapse imaging showed that calcium elevation induced growth arrest of neurites. The inhibitory effect on mRNA translation may thus be involved in the regulation of neurite outgrowth.     

Local intra-articular injection of rapamycin delays articular cartilage degeneration in a murine model of osteoarthritis

Introduction

Recent studies have revealed that rapamycin activates autophagy in human chondrocytes preventing the development of osteoarthritis (OA) like changes in vitro, while the systemic injection of rapamycin reduces the severity of experimental osteoarthritis in a murine model of OA in vivo. Since the systemic use of rapamycin is associated with numerous side effects, the goal of the current study was to examine the beneficial effect of local intra-articular injection of rapamycin in a murine model of OA and to elucidate the mechanism of action of rapamycin on articular cartilage.

Methods

Destabilization of the medial meniscus (DMM) was performed on 10-week-old male mice to induce OA. Intra-articular injections of 10 μl of rapamycin (10 μM) were administered twice weekly for 8 weeks. Articular cartilage damage was analyzed by histology using a semi-quantitative scoring system at 8 and 12 weeks after surgery. Mammalian target of rapamycin (mTOR), light chain 3 (LC3), vascular endothelial growth factor (VEGF), collagen, type X alpha 1 (COL10A1), and matrix metallopeptidase 13 (MMP13) expressions were analyzed by immunohistochemistry. VEGF, COL10A1, and MMP13 expressions were further examined via quantitative RT-PCR (qPCR).

Results

Intra-articular injection of rapamycin significantly reduced the severity of articular cartilage degradation at 8 and 12 weeks after DMM surgery. A reduction in mTOR expression and the activation of LC3 (an autophagy marker) in the chondrocytes was observed in the rapamycin treated mice. Rapamycin treatment also reduced VEGF, COL10A1, and MMP13 expressions at 8 and 12 weeks after DMM surgery.

Conclusion

These results demonstrate that the intra-articular injection of rapamycin could reduce mTOR expression, leading to a delay in articular cartilage degradation in our OA murine model. Our observations suggest that local intra-articular injection of rapamycin could represent a potential therapeutic approach to prevent OA.     

Disruption of the temporally regulated cloaca endodermal β-catenin signaling causes anorectal malformations

The cloaca is temporally formed and eventually divided by the urorectal septum (URS) during urogenital and anorectal organ development. Although congenital malformations, such as anorectal malformations (ARMs), are frequently observed during this process, the underlying pathogenic mechanisms remain unclear. β-Catenin is a critical component of canonical Wnt signaling and is essential for the regulation of cell differentiation and morphogenesis during embryogenesis. The expression of β-catenin is observed in endodermal epithelia, including URS epithelia. We modulated the β-catenin gene conditionally in endodermal epithelia by utilizing tamoxifen-inducible Cre driver line (Shh^CreERT2). Both β-catenin loss- and gain-of-function (LOF and GOF) mutants displayed abnormal clefts in the perineal region and hypoplastic elongation of the URS. The mutants also displayed reduced cell proliferation in the URS mesenchyme. In addition, the β-catenin GOF mutants displayed reduced apoptosis and subsequently increased apoptosis in the URS epithelium. This instability possibly resulted in reduced expression levels of differentiation markers, such as keratin 1 and filaggrin, in the perineal epithelia. The expression of bone morphogenetic protein (Bmp) genes, such as Bmp4 and Bmp7, was also ectopically induced in the epithelia of the URS in the β-catenin GOF mutants. The expression of the Msx2 gene and phosphorylated-Smad1/5/8, possible readouts of Bmp signaling, was also increased in the mutants. Moreover, we introduced an additional mutation for a Bmp receptor gene: BmprIA. The Shh^CreERT2/+; β-catenin^flox(ex3)/+; BmprIA^flox/− mutants displayed partial restoration of URS elongation compared with the β-catenin GOF mutants. These results indicate that some ARM phenotypes in the β-catenin GOF mutants were caused by abnormal Bmp signaling. The current analysis revealed the close relation of endodermal β-catenin signaling to the ARM phenotypes. These results are considered to shed light on the pathogenic mechanisms of human ARMs.During embryonic development, the cloaca is a temporal structure that is subsequently divided into the urogenital sinus and rectum by the urorectal septum (hereafter referred to as the URS).^{1, 2, 3} The URS develops from the proximal umbilical mesenchyme (the rostral portion of the cloaca) around embryonic day 10.5 (E10.5) (Figure 1a, asterisk)^{3, 4, 5} and subsequently extends caudally along the cloaca reaching the cloacal membrane. Part of the cloacal membrane degrades with the approximation of the URS tip. As a result, the cloaca is divided into the urogenital sinus and rectum, and the tip of the URS (endodermal epithelia) contributes to forming the ectodermal epithelia of the perineum and external genitalia.⁶ The contribution of these cells and growth factors expressed in the endodermal epithelia are essential for the proper morphogenesis of the perineal region, based on the phenotypes of several mutants. For instance, Sonic hedgehog (Shh) is expressed in the endodermal epithelia and has an essential role in both URS formation and GT protrusion by affecting neighboring mesenchymal cells.^{7, 8, 9, 10, 11} Apoptotic cells are observed in the urogenital tract during URS formation, being distributed primarily in the epithelial layers of the URS, cloacal membrane and mesenchyme of the dorsal surface of the caudal hindgut at E11.5 and E11.75.^{5, 12} From E12.5, apoptotic cells are also observed in the URS mesenchyme and are thought to be involved in the transformation of the URS and disintegration of the cloacal membrane.⁵Open in a separate windowFigure 1Temporally labeled Shh-expressing URS endodermal cells contribute to the ectodermal epithelia. The expression of the Shh gene in sagittal sections of wild-type embryos at E10.5, E11.5, E12.5 and E13.5 (a–d). The asterisk in (a) indicates future URS derived from the proximal umbilical mesenchyme. Ventral view of the genital tubercle (e–j). Tissue labeling experiments of Shh-expressing cells were performed at E15.5. Shh^CreERT2/+; R26^LacZ/+ embryos subsequent to TM administration at E8.5, E9.5, E10.5, E11.5, E12.5 or E13.5 (e–j). The red arrows indicate the LacZ-positive ectodermal cells. β-Catenin was expressed in the endoderm, including the URS epithelia, at E11.5 in the wild-type embryos (k). b, Bladder; c, cloaca; gt, genital tubercle; hl, hindlimb; r, rectum; t, tail; uc, umbilical cord; urs, urorectal septumSeveral congenital anomalies are frequently observed during urogenital organ development. These abnormalities are usually accompanied by deficient excretory and copulatory functions, influencing the quality of life of the patient. In particular, the incidence of anorectal malformations (ARMs) is approximately 1 in 5000 human births;¹³ however, the underlying pathogenic mechanisms of this condition are currently unknown. ARM phenotypes are observed in several diseases, including Currarino syndrome, Townes Brocks syndrome and VACTERL complex.^{14, 15, 16, 17} Affected patients often display other malformations, such as anal fistulas, sacral malformations and renal malformations. Human and mouse genetic analyses have shed light on the possible genetic causes of some of these abnormalities.Several mouse mutants for hedgehog signaling genes, ephrin-Eph signaling genes, fibroblast growth factor (Fgf) signaling genes and Wnt signaling genes are reported to display ARM phenotypes.^{18, 19, 20, 21, 22} Another causative factor for the development of ARM is all-trans retinoic acid (RA), a teratogen and active form of vitamin A. RA treatment in pregnant mice results in imperforate anus in embryos.^{23, 24} Although these reports have identified causative factors for ARM phenotypes, the pathogenic mechanisms underlying the development of ARM and URS remain elusive.The Wnt signaling pathway is essential for embryonic development, and its dysregulation has been implicated in developmental disorders and human diseases. Wnt signaling is transmitted primarily via three divergent pathways: the canonical Wnt/β-catenin pathway, the planner cell polarity pathway and the Wnt/Ca²⁺ pathway.^{25, 26} β-Catenin is a key factor for the canonical Wnt pathway and also acts as a subunit of the cadherin protein complex, which controls cell–cell adhesion. Owing to the early lethality of β-catenin-deficient mice,²⁷ analyses of the function of β-catenin in organogenesis have been performed using conditional mutants. Previous reports have revealed that β-catenin induces the differentiation of hair follicles during hair/skin development according to loss- and gain-of-function (LOF and GOF) approaches.^{28, 29} The ectopic activation of canonical Wnt signaling results in the ectopic induction of bone morphogenetic protein (Bmp) signaling, which is essential for hair follicle formation.^{30, 31} With respect to genital tubercle (primordia of external genitalia: GT) development, region-specific (ectodermal, endodermal and mesenchymal) modulation of the β-catenin gene has revealed essential functions, such as regulation of cell proliferation, epithelial integrity and protrusion/elongation of GT.^{8, 32, 33} However, the regulatory functions of the β-catenin gene during the development of the URS have not been investigated.The current study aimed to investigate the function of the β-catenin gene in URS development. We modulated the β-catenin activity in the endodermal epithelia and studied the phenotypic consequences of dysregulated endodermal β-catenin signaling for urorectal development. Both β-catenin LOF and GOF mutations resulted in ARM phenotypes. The β-catenin GOF mutation led to the ectopic induction of Bmp signaling. Moreover, the ARM phenotypes in the β-catenin GOF mutants were restored by additionally introducing the BmprIA gene mutation. These results suggest that adequately controlled β-catenin signaling and its downstream growth factor signaling are essential for proper URS formation.     

Human Apolipoprotein E Resequencing by Proteomic Analysis and Its Application to Serotyping

Background

Apolipoprotein E (ApoE) typing is considered important because of the association between ApoE and Alzheimer’s disease and familial dyslipidemia and is currently performed by genetic testing (APOE genotyping). ApoE levels in plasma and serum are clinically determined by immunoassay.

Methods

Combining an ApoE immunoassay reagent with proteomic analysis using an Orbitrap mass spectrometer, we attempted to resequence ApoE from trace amounts of serum for typing (serotyping). Most (24 of 33) ApoE mutant proteins registered to date with Online Mendelian Inheritance in Man, such as ApoE2 and ApoE4, involve lysine and arginine mutations. Digestion of mutant ApoE with trypsin will thus result in fragments that differ substantially from wild-type ApoE3 in terms of mass, making serotyping ideally suited to mass spectrometry analysis.

Results

The mean coverage of the amino acid sequence of full-length ApoE was 91.6% in the protein resequence. Residues 112 and 158 (which are mutated in ApoE2 and ApoE4) were covered in all samples, and the protein sequences were used for serotyping. Serotypes including all heterozygous combinations (ApoE2/E3, E2/E4, E3/E4) corresponded exactly to the APOE genotyping results in each of the subjects.

Conclusion

Our novel ApoE serotyping method with protein resequencing requires no synthesis of stable isotope-labeled peptides or genome analysis. The method can use residual blood from samples collected for routine clinical tests, thus enabling retrospective studies with preserved body fluids. The test could be applied to samples from subjects whose DNA is unavailable. In future studies, we hope to demonstrate the capability of our method to detect rare ApoE mutations.     

Genetic and Infectious Profiles Influence Cerebrospinal Fluid IgG Abnormality in Japanese Multiple Sclerosis Patients

Background

Abnormal intrathecal synthesis of IgG, reflected by cerebrospinal fluid (CSF) oligoclonal IgG bands (OBs) and increased IgG index, is much less frequently observed in Japanese multiple sclerosis (MS) cohorts compared with Western cohorts. We aimed to clarify whether genetic and common infectious backgrounds influence CSF IgG abnormality in Japanese MS patients.

Methodology

We analyzed HLA-DRB1 alleles, and IgG antibodies against Chlamydia pneumoniae, Helicobacter pylori, Epstein-Barr virus nuclear antigen (EBNA), and varicella zoster virus (VZV) in 94 patients with MS and 367 unrelated healthy controls (HCs). We defined CSF IgG abnormality as the presence of CSF OBs and/or increased IgG index (>0.658).

Principal Findings

CSF IgG abnormality was found in 59 of 94 (62.8%) MS patients. CSF IgG abnormality-positive patients had a significantly higher frequency of brain MRI lesions meeting the Barkhof criteria compared with abnormality-negative patients. Compared with HCs, CSF IgG abnormality-positive MS patients showed a significantly higher frequency of DRB1*1501, whereas CSF IgG abnormality-negative patients had a significantly higher frequency of DRB1*0405. CSF IgG abnormality-positive MS patients had a significantly higher frequency of anti-C. pneumoniae IgG antibodies compared with CSF IgG abnormality-negative MS patients, although there was no difference in the frequency of anti-C. pneumoniae IgG antibodies between HCs and total MS patients. Compared with HCs, anti-H. pylori IgG antibodies were detected significantly less frequently in the total MS patients, especially in CSF IgG abnormality-negative MS patients. The frequencies of antibodies against EBNA and VZV did not differ significantly among the groups.

Conclusions

CSF IgG abnormality is associated with Western MS-like brain MRI features. DRB1*1501 and C. pneumoniae infection confer CSF IgG abnormality, while DRB1*0405 and H. pylori infection are positively and negatively associated with CSF IgG abnormality-negative MS, respectively, suggesting that genetic and environmental factors differentially contribute to MS susceptibility according to the CSF IgG abnormality status.