Possible involvement of Hcn1 ion channel in learning and memory dysfunction in SAMP8 mice

The senescence-accelerated mouse prone 8 (SAMP8) strain exhibits age-related learning and memory deficits (LMD) at 2 months of age. Combined linkage analysis of 264 F2 intercross SAMP8 × JF1 mice and RNA-seq analysis identified Hcn1 gene out of 29 genes in the LMD region on chromosome 13. Hcn1 in SAMP8 strain showed 15 times less polyglutamine repetition compared to Japanese fancy mouse 1 (JF1). Whole cell patch clamp analysis showed that Hcn1 ion conductivity was significantly lower in SAMP8 compared to that of JF1, which may be associated with learning and memory deficiency.     

C. elegans pharyngeal morphogenesis requires both de novo synthesis of pyrimidines and synthesis of heparan sulfate proteoglycans

The C. elegans pharynx undergoes elongation and morphogenesis to its characteristic bi-lobed shape between the 2- and 3-fold stages of embryogenesis. During this period, the pharyngeal muscles and marginal cells forming the isthmus between the anterior and posterior pharyngeal bulbs elongate and narrow. We have identified the spontaneous mutant pyr-1(cu8) exhibiting defective pharyngeal isthmus elongation, cytoskeletal organization defects, and maternal effect lethality. pyr-1 encodes CAD, a trifunctional enzyme required for de novo pyrimidine synthesis, and pyr-1(cu8) mutants are rescued by supplying exogenous pyrimidines. Similar pharyngeal defects and maternal effect lethality were found in sqv-1, sqv-8, rib-1 and rib-2 mutants, which affect enzymes involved in heparan sulfate proteoglycan (HSPG) synthesis. rib-1 mutant lethality was enhanced in a pyr-1 mutant background, indicating that HSPG synthesis is very sensitive to decreased pyrimidine pools, and HS disaccharides are moderately decreased in both rib-1 and pyr-1 mutants. We hypothesize that HSPGs are necessary for pharyngeal isthmus elongation, and pyr-1 functions upstream of proteoglycan synthesizing enzymes by providing precursors of UDP-sugars essential for HSPG synthesis.     

HuC:Kaede, a useful tool to label neural morphologies in networks in vivo

A simple and efficient procedure for labeling neurons is a prerequisite for investigating the development of neural networks in zebrafish. To label neurons we used Kaede, a fluorescent protein with a photoconversion property allowing conversion from green to red fluorescence following irradiation with UV or violet light. We established a zebrafish stable transgenic line, Tg(HuC:Kaede), expressing Kaede in neurons under the control of the HuC promoter. This transgenic line was used to label a small number of neurons in the trigeminal ganglion. Also, using embryos injected with the transgene, we labeled peripheral axon arbors of a Rohon-Beard neuron at 4 days postfertilization and observed the dendrite development of a tectal neuron for 3 days. These data indicate that Kaede is a useful tool to selectively label neural networks in zebrafish.     

Dynamical systems analysis of arterial blood pressure signals in relation to heart rate fluctuations in chick embryos

We attempted a new approach based on a modern dynamical system theory to reconstruct the arterial blood pressure signals in relation to heart rate fluctuations of developing chick embryos. The dynamical systems approach in general is to model a phenomenon that is presented by a single time series record and approximate the dynamical property (e.g. heart rate fluctuations) of a system based only on information contained in a single-variable (arterial blood pressure) of the system. The time-series data of the arterial blood pressure was reconstructed in 3-dimensional space to draw characteristic orbits. Since the reconstructed orbits of the blood pressure should retain information contained in the pressure signals, we attempted to derive instantaneous heart rate (IHR) from the reconstructed orbits. The derived IHR presenting HR fluctuations coincided well with the IHR obtained conventionally from the peak-to-peak time intervals of the maximum blood pressure. Movements of the reconstructed orbits of the arterial blood pressure in 3-dimensional space reflected HR fluctuations (i.e. transient decelerations and accelerations).     

Interaction with IQGAP1 links APC to Rac1, Cdc42, and actin filaments during cell polarization and migration

Rho family GTPases, particularly Rac1 and Cdc42, are key regulators of cell polarization and directional migration. Adenomatous polyposis coli (APC) is also thought to play a pivotal role in polarized cell migration. We have found that IQGAP1, an effector of Rac1 and Cdc42, interacts directly with APC. IQGAP1 and APC localize interdependently to the leading edge in migrating Vero cells, and activated Rac1/Cdc42 form a ternary complex with IQGAP1 and APC. Depletion of either IQGAP1 or APC inhibits actin meshwork formation and polarized migration. Depletion of IQGAP1 or APC also disrupts localization of CLIP-170, a microtubule-stabilizing protein that interacts with IQGAP1. Taken together, these results suggest a model in which activation of Rac1 and Cdc42 in response to migration signals leads to recruitment of IQGAP1 and APC which, together with CLIP-170, form a complex that links the actin cytoskeleton and microtubule dynamics during cell polarization and directional migration.     

Cthrc1 is a positive regulator of osteoblastic bone formation

Background

Bone mass is maintained by continuous remodeling through repeated cycles of bone resorption by osteoclasts and bone formation by osteoblasts. This remodeling process is regulated by many systemic and local factors.

Methodology/Principal Findings

We identified collagen triple helix repeat containing-1 (Cthrc1) as a downstream target of bone morphogenetic protein-2 (BMP2) in osteochondroprogenitor-like cells by PCR-based suppression subtractive hybridization followed by differential hybridization, and found that Cthrc1 was expressed in bone tissues in vivo. To investigate the role of Cthrc1 in bone, we generated Cthrc1-null mice and transgenic mice which overexpress Cthrc1 in osteoblasts (Cthrc1 transgenic mice). Microcomputed tomography (micro-CT) and bone histomorphometry analyses showed that Cthrc1-null mice displayed low bone mass as a result of decreased osteoblastic bone formation, whereas Cthrc1 transgenic mice displayed high bone mass by increase in osteoblastic bone formation. Osteoblast number was decreased in Cthrc1-null mice, and increased in Cthrc1 transgenic mice, respectively, while osteoclast number had no change in both mutant mice. In vitro, colony-forming unit (CFU) assays in bone marrow cells harvested from Cthrc1-null mice or Cthrc1 transgenic mice revealed that Cthrc1 stimulated differentiation and mineralization of osteoprogenitor cells. Expression levels of osteoblast specific genes, ALP, Col1a1, and Osteocalcin, in primary osteoblasts were decreased in Cthrc1-null mice and increased in Cthrc1 transgenic mice, respectively. Furthermore, BrdU incorporation assays showed that Cthrc1 accelerated osteoblast proliferation in vitro and in vivo. In addition, overexpression of Cthrc1 in the transgenic mice attenuated ovariectomy-induced bone loss.

Conclusions/Significance

Our results indicate that Cthrc1 increases bone mass as a positive regulator of osteoblastic bone formation and offers an anabolic approach for the treatment of osteoporosis.     

Crystal structure of the rac activator, Asef, reveals its autoinhibitory mechanism

The Rac-specific guanine nucleotide exchange factor (GEF) Asef is activated by binding to the tumor suppressor adenomatous polyposis coli mutant, which is found in sporadic and familial colorectal tumors. This activated Asef is involved in the migration of colorectal tumor cells. The GEFs for Rho family GTPases contain the Dbl homology (DH) domain and the pleckstrin homology (PH) domain. When Asef is in the resting state, the GEF activity of the DH-PH module is intramolecularly inhibited by an unidentified mechanism. Asef has a Src homology 3 (SH3) domain in addition to the DH-PH module. In the present study, the three-dimensional structure of Asef was solved in its autoinhibited state. The crystal structure revealed that the SH3 domain binds intramolecularly to the DH domain, thus blocking the Rac-binding site. Furthermore, the RT-loop and the C-terminal region of the SH3 domain interact with the DH domain in a manner completely different from those for the canonical binding to a polyproline-peptide motif. These results demonstrate that the blocking of the Rac-binding site by the SH3 domain is essential for Asef autoinhibition. This may be a common mechanism in other proteins that possess an SH3 domain adjacent to a DH-PH module.     

Epidemiologic study of clinically amyopathic dermatomyositis and anti-melanoma differentiation-associated gene 5 antibodies in central Japan

Introduction

Several reports have found the onset or activity of inflammatory myopathies to show spatial clustering and seasonal association. We recently detected autoantibodies against melanoma differentiation-associated gene 5 (MDA-5) in more than 20% of patients with dermatomyositis. Anti-MDA-5 antibodies were associated with the presence of rapidly progressive interstitial lung disease in clinically amyopathic dermatomyositis (CADM). The present study aims to assess the growing prevalence of CADM and the geographical incidence of anti-MDA-5-positive patients.

Methods

We reviewed medical charts and examined the presence of anti-MDA-5 antibodies in 95 patients, including 36 CADM patients. Sera were obtained from 1994 through 2011. Statistical analyses were performed to assess whether CADM development and the presence of anti-MDA-5 antibodies were associated with various parameters, including age at disease onset, season of onset, annual positivity, and population of resident city.

Results

Tertiles based on the year when the sera were collected showed increasing tendencies of CADM and anti-MDA-5-positive patients among all of the dermatomyositis patients. From 1994 to 2010, the relative prevalence of CADM and anti-MDA-5 antibody-positive patients significantly increased. Interestingly, the presence of anti-MDA-5 antibodies in 26 patients was inversely associated with the population of their city of residence.

Conclusions

This is the first study to examine the distribution of anti-MDA-5-positive dermatomyositis phenotypes in Japan. Regional differences in the incidences of these phenotypes would suggest that environmental factors contribute to the production of antibodies against MDA-5, which triggers innate antiviral responses.     

Interferon-Inducible Mechanism of Dendritic Cell-Mediated HIV-1 Dissemination Is Dependent on Siglec-1/CD169

Human immunodeficiency virus type 1 (HIV-1) interactions with myeloid dendritic cells (DCs) can result in virus dissemination to CD4⁺ T cells via a trans infection pathway dependent on virion incorporation of the host cell derived glycosphingolipid (GSL), GM3. The mechanism of DC-mediated trans infection is extremely efficacious and can result in infection of multiple CD4⁺ T cells as these cells make exploratory contacts on the DC surface. While it has long been appreciated that activation of DCs with ligands that induce type I IFN signaling pathway dramatically enhances DC-mediated T cell trans infection, the mechanism by which this occurs has remained unclear until now. Here, we demonstrate that the type I IFN-inducible Siglec-1, CD169, is the DC receptor that captures HIV in a GM3-dependent manner. Selective downregulation of CD169 expression, neutralizing CD169 function, or depletion of GSLs from virions, abrogated DC-mediated HIV-1 capture and trans infection, while exogenous expression of CD169 in receptor-naïve cells rescued GSL-dependent capture and trans infection. HIV-1 particles co-localized with CD169 on DC surface immediately following capture and subsequently within non-lysosomal compartments that redistributed to the DC – T cell infectious synapses upon initiation of T cell contact. Together, these findings describe a novel mechanism of pathogen parasitization of host encoded cellular recognition machinery (GM3 – CD169 interaction) for DC-dependent HIV dissemination.