31 research outputs found
Cilia in the choroid plexus: their roles in hydrocephalus and beyond
Cilia are whip-like projections that are widely conserved in eukaryotes and function as a motile propeller and/or sensory platform to detect various extracellular stimuli. In vertebrates, cilia are ubiquitously found in most cells, showing structural and functional diversities depending on the cell type. In this review, we focus on the structure and function of cilia in choroid plexus epithelial cells (CPECs). CPECs form one or two dozen non-motile 9+0 cilia, which display transient acquisition of motility during development. Genetic malfunction of cilia can lead to failure of multiple organs including the brain. Especially, several groups have demonstrated that the defects in CPEC cilia cause the communicating form of hydrocephalus. In order to elucidate the molecular mechanisms underlying the hydrocephalus, we have previously demonstrated that the cilia possess an NPFF receptor for autocrine signaling to regulate transepithelial fluid transport. In this perspective, we also discuss the potential involvement of cilia in the other aspects of choroid plexus functions, such as the regulation of brain development and neuroinflammation
Odontoblast differentiation is regulated by an interplay between primary cilia and the canonical Wnt pathway
Primary cilium is a protruding cellular organelle that has various physiological functions, especially in sensory reception. While an avalanche of reports on primary cilia have been published, the function of primary cilia in dental cells remains to be investigated. In this study, we focused on the function of primary cilia in dentin-producing odontoblasts. Odontoblasts, like most other cell types, possess primary cilia, which disappear upon the knockdown of intraflagellar transport-88. In cilia-depleted cells, the expression of dentin sialoprotein, an odontoblastic marker, was elevated, while the deposition of minerals was slowed. This was recapitulated by the activation of canonical Wnt pathway, also decreased the ratio of ciliated cells. In dental pulp cells, as they differentiated into odontoblasts, the ratio of ciliated cells was increased, whereas the canonical Wnt signaling activity was repressed. Our results collectively underscore the roles of primary cilia in regulating odontoblastic differentiation through canonical Wnt signaling. This study implies the existence of a feedback loop between primary cilia and the canonical Wnt pathway
Dynamic Changes in Ultrastructure of the Primary Cilium in Migrating Neuroblasts in the Postnatal Brain
New neurons, referred to as neuroblasts, are continuously generated in the ventricular-subventricular zone of the brain throughout an animal's life. These neuroblasts are characterized by their unique potential for proliferation, formation of chain-like cell aggregates, and long-distance and high-speed migration through the rostral migratory stream (RMS) toward the olfactory bulb (OB), where they decelerate and differentiate into mature interneurons. The dynamic changes of ultrastructural features in postnatal-born neuroblasts during migration are not yet fully understood. Here we report the presence of a primary cilium, and its ultrastructural morphology and spatiotemporal dynamics, in migrating neuroblasts in the postnatal RMS and OB. The primary cilium was observed in migrating neuroblasts in the postnatal RMS and OB in male and female mice and zebrafish, and a male rhesus monkey. Inhibition of intraflagellar transport molecules in migrating neuroblasts impaired their ciliogenesis and rostral migration toward the OB. Serial section transmission electron microscopy revealed that each migrating neuroblast possesses either a pair of centrioles or a basal body with an immature or mature primary cilium. Using immunohistochemistry, live imaging, and serial block-face scanning electron microscopy, we demonstrate that the localization and orientation of the primary cilium are altered depending on the mitotic state, saltatory migration, and deceleration of neuroblasts. Together, our results highlight a close mutual relationship between spatiotemporal regulation of the primary cilium and efficient chain migration of neuroblasts in the postnatal brain
Study of ceramide glucosyltransferase
Ceramide glucosyltransferase (CGT) is a key enzyme in glycosphingolipid (GSL) biosynthesis in eukaryotic cells. Inhibition of enzyme activity by an N-alkylated imino sugar, N-butyl-deoxynojirimycin (NB-DNJ), has been evaluated for the therapeutic treatment of inherited glycosphingolipid lysosomal storage diseases. To develop more selective drugs for potential clinical use, further investigation of possible side effects and the design of a more selective inhibitor is required. One concern for clinical use of NB-DNJ is the potential activation of CGT in vivo. When rats were treated with various concentrations of NB-DNJ for 13 weeks to assess the depletion of glycosphingolipids and up-regulation of CGT activity, the reduction of ganglioside levels was observed following an increase in NB-DNJ dose level up to 180 mg/kg/day. However, CGT activity levels were not significantly affected by NB-DNJ treatment. The lack of CGT up-regulation while reducing GSLs by NB-DNJ would be desirable in the clinic to avoid a rapid accumulation of GSLs if patient treatment was concluded. To aid in design of highly selective inhibitors for CGT, enzyme kinetic studies were performed using recombinant human CGT and five different imino sugars. The recombinant enzyme showed similar enzyme kinetics to a native enzyme from HL-60 cells. All the tested imino sugars showed a mixed-type inhibition for ceramide, and an increase in N-alkyl chain provided an improved uncompetitive inhibition. These data suggest that CGT may have two different sites for binding of imino sugars, and the N-alkyl chain length may affect the preference for binding site. When the protein sequence of CGT was analysed using www server programs to predict protein structure, a Rossman fold was predicted in the nucleotide-binding domain as observed in other nucleotide-sugar glycosyltransferase structures. Also, a significant folding similarity to bacterial glycosyltransferase SpsA was predicted. Based on these observations, a possible inhibitor-binding mechanism is discussed that may aid the design of highly selective inhibitors for CGT.</p
Study of ceramide glucosyltransferase
Ceramide glucosyltransferase (CGT) is a key enzyme in glycosphingolipid (GSL)
biosynthesis in eukaryotic cells. Inhibition of enzyme activity by an N-alkylated imino sugar,
N-butyl-deoxynojirimycin (NB-DNJ), has been evaluated for the therapeutic treatment of
inherited glycosphingolipid lysosomal storage diseases. To develop more selective drugs for
potential clinical use, further investigation of possible side effects and the design of a more
selective inhibitor is required.
One concern for clinical use of NB-DNJ is the potential activation of CGT in vivo.
When rats were treated with various concentrations of NB-DNJ for 13 weeks to assess the
depletion of glycosphingolipids and up-regulation of CGT activity, the reduction of
ganglioside levels was observed following an increase in NB-DNJ dose level up to 180
mg/kg/day. However, CGT activity levels were not significantly affected by NB-DNJ treatment.
The lack of CGT up-regulation while reducing GSLs by NB-DNJ would be desirable in the
clinic to avoid a rapid accumulation of GSLs if patient treatment was concluded.
To aid in design of highly selective inhibitors for CGT, enzyme kinetic studies were
performed using recombinant human CGT and five different imino sugars. The recombinant
enzyme showed similar enzyme kinetics to a native enzyme from HL-60 cells. All the tested
imino sugars showed a mixed-type inhibition for ceramide, and an increase in N-alkyl chain
provided an improved uncompetitive inhibition. These data suggest that CGT may have two
different sites for binding of imino sugars, and the N-alkyl chain length may affect the
preference for binding site.
When the protein sequence of CGT was analysed using www server programs to predict
protein structure, a Rossman fold was predicted in the nucleotide-binding domain as observed
in other nucleotide-sugar glycosyltransferase structures. Also, a significant folding similarity to
bacterial glycosyltransferase SpsA was predicted. Based on these observations, a possible
inhibitor-binding mechanism is discussed that may aid the design of highly selective inhibitors
for CGT.</p
CFAP70 Is a Novel Axoneme-Binding Protein That Localizes at the Base of the Outer Dynein Arm and Regulates Ciliary Motility
In the present study, we characterized CFAP70, a candidate of cilia-related protein in mice. As this protein has a cluster of tetratricopeptide repeat (TPR) domains like many components of the intraflagellar transport (IFT) complex, we investigated the domain functions of particular interest in ciliary targeting and/or localization. RT-PCR and immunohistochemistry of various mouse tissues demonstrated the association of CFAP70 with motile cilia and flagella. A stepwise extraction of proteins from swine tracheal cilia showed that CFAP70 bound tightly to the ciliary axoneme. Fluorescence microscopy of the cultured ependyma expressing fragments of CFAP70 demonstrated that the N-terminus rather than the C-terminus with the TPR domains was more important for the ciliary localization. When CFAP70 was knocked down in cultured mouse ependyma, reductions in cilia beating frequency were observed. Consistent with these observations, a Chlamydomonas mutant lacking the CFAP70 homolog, FAP70, showed defects in outer dynein arm (ODA) activity and a reduction in flagellar motility. Cryo-electron tomography revealed that the N-terminus of FAP70 resided stably at the base of the ODA. These results demonstrated that CFAP70 is a novel regulatory component of the ODA in motile cilia and flagella, and that the N-terminus is important for its ciliary localization
Discovery of Vertebrate-Specific Factor that Processes Flagellar Glycolytic Enolase During Motile Ciliogenesis
Koshu GSE augments cell survival of cultured hippocampal neurons after excitotoxic treatment.
<p>Hippocampal neurons plated at 2.0×10<sup>5</sup> cells/cm<sup>2</sup> were treated with 50 µM glutamate in the presence or absence of 1.0 ng/ml KOS GSE for 10 min and allowed to recover in conditioned medium for 24 hr. (A) Representative images of hippocampal neurons after treatment. Bar, 100 µm. (B) The mean density of MAP2-positive neurons surviving 24 hr after glutamate treatment (<i>n</i> = 8). ***, <i>P</i><0.001; *, <i>P</i><0.05. (C) Quantification of the mean dendrite length by morphometric analysis (<i>n</i> = 8). ***, <i>P</i><0.001; *, <i>P</i><0.05.</p