[Show abstract][Hide abstract] ABSTRACT: The Duchenne muscular dystrophy (DMD) gene produces multiple dystrophin (Dp) products due to the presence of several promoters. We previously reported the existence of a novel short isoform of Dp, Dp40, in adult mouse brain. However, the exact biochemical expression profile and cytological distribution of the Dp40 protein remain unknown. In this study, we generated a polyclonal antibody against the NH2-terminal region of the Dp40 and identified the expression profile of Dp40 in the mouse brain. Through an analysis using embryonic and postnatal mouse cerebrums, we found that Dp40 emerged from the early neonatal stages until adulthood, whereas Dp71, an another Dp short isoform, was highly detected in both prenatal and postnatal cerebrums. Intriguingly, relative expressions of Dp40 and Dp71 were prominent in cultured dissociated neurons and non-neuronal cells derived from mouse hippocampus, respectively. Furthermore, the immunocytological distribution of Dp40 was analyzed in dissociated cultured neurons, revealing that Dp40 is detected in the soma and its dendrites, but not in the axon. It is worthy to note that Dp40 is localized along the subplasmalemmal region of the dendritic shafts, as well as at excitatory postsynaptic sites. Thus, Dp40 was identified as a neuron-type Dp possibly involving dendritic and synaptic functions.
Biochemical and Biophysical Research Communications 08/2014; 452(1). DOI:10.1016/j.bbrc.2014.08.064 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It has been reported that bisphenol A (BPA), a widespread xenoestrogen employed in the production of polycarbonate plastics, affects brain development in both humans and rodents. In the present study employing mice, we examined the effects of exposure to BPA (500 μg/kg/day) during fetal and lactational periods on the development of the locus coeruleus (LC) at the age of embryonic day 18 (E18), postnatal 3 weeks (P3W), P8W and P16W. The number of tyrosine hydroxylase-immunoreactive cells (TH-IR cells) in females exposed to BPA was decreased, compared with the control females at P3W. At P8W, the number of TH-IR cells in females exposed to BPA was significantly decreased, compared with the control females, whereas the number of TH-IR cells in males exposed to BPA was significantly increased, compared with the control males, which resulted in reversed transient sexual differences in the numbers of TH-IR cells observed in the controls at P8W. However, no significant changes were demonstrated at E18 or P16W. Next, we examined the density of the fibers containing norepinephrine transporter (NET) in the anterior cingulate cortex (ACC) and prefrontal cortex, at P3W, P8W and P16W, because NET would be beneficial in identifying the targets of the LC noradrenergic neurons. There were no significant differences shown in the density of the NET-positive fibers, between the control and the groups exposed to BPA. These results suggested that BPA might disrupt the development of physiological sexual differences in the LC-noradrenergic system in mice, although further studies are necessary to clarify the underlying mechanisms.
[Show abstract][Hide abstract] ABSTRACT: L1cam (L1) is a cell adhesion molecule associated with a spectrum of human neurological diseases, the most well-known being X-linked hydrocephalus. Although we recently demonstrated that L1 plays an important role in neuronal migration during cortical histogenesis, the mechanisms of delayed migration have still not been clarified. In this study, we found that cell locomotion in the intermediate zone and terminal translocation in the primitive cortical zone (PCZ) were affected by L1-knockdown (L1-KD). Time-lapse analyses revealed that L1-KD neurons produced by in utero electroporation of shRNA targeting L1 (L1-shRNAs) molecules showed decreased locomotion velocity in the intermediate zone, compared with control neurons. Furthermore, L1-KD neurons showed longer and more undulated leading processes during translocation through the primitive cortical zone. The curvature index, a quantitative index for curvilinearity, as well as the length of the leading process, were increased, whereas the somal movement was decreased in L1-KD neurons during terminal translocation in the PCZ. These results suggest that L1 has a role in radial migration of cortical neurons.
PLoS ONE 01/2014; 9(1):e86186. DOI:10.1371/journal.pone.0086186 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Duchenne muscular dystrophy (DMD) causes cognitive impairment in one third of the patients, although the underlying mechanisms remain to be elucidated. Recent studies showed that mutations in the distal part of the dystrophin gene correlate well with the cognitive impairment in DMD patients, which is attributed to Dp71. The study on the expression of the shortest isoform, Dp40, has not been possible due to the lack of an isoform specific antibody. Dp40 has the same promoter as that found in Dp71 and lacks the normal C-terminal end of Dp427. In the present study, we have raised polyclonal antibody against the N-terminal sequence common to short isoforms of dystrophin, including Dp40, and investigated the expression pattern of Dp40 in the mouse brain. Affinity chromatography with this antibody and the consecutive LC-MS/MS analysis on the interacting proteins revealed that Dp40 was abundantly expressed in synaptic vesicles and interacted with a group of presynaptic proteins, including syntaxin1A and SNAP25, which are involved in exocytosis of synaptic vesicles in neurons. We thus suggest that Dp40 may form a novel protein complex and play a crucial role in presynaptic function. Further studies on these aspects of Dp40 function might provide more insight into the molecular mechanisms of cognitive impairment found in patients with DMD.
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The online version of this article (doi:10.1007/s12035-012-8233-5) contains supplementary material, which is available to authorized users.
[Show abstract][Hide abstract] ABSTRACT: Bisphenol A (BPA) is an endocrine-disrupting chemical, widely used in various industries and the field of dentistry. The consequent increase in BPA exposure among humans has led us to some concerns regarding the potential deleterious effects on reproduction and brain development. The emphasis of this review is on the effects of prenatal and lactational exposure to low doses of BPA on brain development in mice. We demonstrated that prenatal exposure to BPA affected fetal murine neocortical development by accelerating neuronal differentiation/migration during the early embryonic stage, which was associated with up- and down-regulation of the genes critical for brain development, including the basic helix-loop-helix transcription factors. In the adult mice brains, both abnormal neocortical architecture and abnormal corticothalamic projections persisted in the group exposed to the BPA. Functionally, BPA exposure disturbed murine behavior, accompanied with a disrupted neurotransmitter system, including monoamines, in the postnatal development period and in adult mice. We also demonstrated that epigenetic alterations in promoter-associated CpG islands might underlie some of the effects on brain development after exposure to BPA.
[Show abstract][Hide abstract] ABSTRACT: In order to clarify the mechanisms of resistance to paclitaxel in lung cancer, three human lung cancer cell lines which exhibit different sensitivity to paclitaxel were investigated from the following viewpoints: overexpression of ATP-binding cassette, sub-family B, member 1 (ABCB1), mutations on paclitaxel binding site of β-tubulin genes, quantity of polymerized tubulin and the intracellular localization of paclitaxel. ABCB1 expression was evaluated by real-time RT-PCR. No correlations were noted between the ABCB1 expression in the sensitive and resistant cell lines at the mRNA level. No mutations on the paclitaxel binding site of the β-tubulin genes were detected in either the resistant or sensitive cells. Live cell images obtained by confocal laser microscopy revealed that the resistant cell line, RERF-LC-KJ, had more accumulation of Oregon Green® 488 conjugated paclitaxel in the lysosomal and extra-lysosomal compartments of cytoplasm than other cell lines. The results obtained in this study indicated that the changes in the subcellular localization could contribute to the production of paclitaxel resistance in lung cancer cell lines. Further studies should be conducted to elucidate the molecular mechanisms that differentiate the intracellular localization of paclitaxel.
International Journal of Oncology 12/2011; 40(4):995-1004. DOI:10.3892/ijo.2011.1297 · 3.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It has been reported that premature infants in neonatal intensive care units are exposed to a high rate of bisphenol A (BPA), an endocrine disrupting chemical. Our previous studies demonstrated that corticothalamic projection was disrupted by prenatal exposure to BPA, which persisted even in adult mice. We therefore analyzed whether prenatal and lactational exposure to low doses of BPA affected the formation of the cortical barrel, the barreloid of the thalamus, and the barrelette of the brainstem in terms of the histology and the expression of genes involved in the barrel development. Pregnant mice were injected subcutaneously with 20 µg/kg of BPA daily from embryonic day 0 (E0) to postnatal 3 weeks (P3W), while the control mice received a vehicle alone. The barrel, barreloid and barrelette of the adult mice were examined by cytochrome C oxidase (COX) staining. There were no significant differences in the total and septal areas and the patterning of the posterior medial barrel subfield (PMBSF), barreloid and barrelette, between the BPA-exposure and control groups in the adult mice. The developmental study at postnatal day 1 (PD1), PD4 and PD8 revealed that the cortical barrel vaguely appeared at PD4 and completely formed at PD8 in both groups. The expression pattern of some genes was spatiotemporally altered depending on the sex and the treatment. These results suggest that the trigeminal projection and the thalamic relay to the cortical barrel were spared after prenatal and lactational exposure to low doses of BPA, although prenatal exposure to BPA was previously shown to disrupt the corticothalamic projection.
[Show abstract][Hide abstract] ABSTRACT: Quantum dots (QDs) are well known for their potential application in biosensing, ex vivo live-cell imaging and in vivo animal targeting. The brain is a challenging organ for drug delivery, because the blood brain barrier (BBB) functions as a gatekeeper guarding the body from exogenous substances. Here, we evaluated the distribution of bioconjugated QDs, i.e., captopril-conjugated QDs (QDs-cap) following intraperitoneal injection into male ICR mice as a model system for determining the tissue localization of QDs, employing ICP-MS and confocal microscopy coupled with spectrometric analysis. We have demonstrated that intraperitoneally administered QDs-cap were delivered via systemic blood circulation into liver, spleen, kidney and brain at 6 h after injection. QDs-cap were located predominantly inside the blood vessels in the liver, kidney and brain, but a few were distributed in the parenchyma, especially noteworthy in the brain. Careful studies on acute as well as chronic toxicity of QDs in the brain are required prior to clinical application to humans.
[Show abstract][Hide abstract] ABSTRACT: Bisphenol A (BPA) is one of endocrine disrupting chemicals, being distributed widely in the environment. We have been studying the low dose effects of BPA on murine forebrain development. Here, we have investigated the genome-wide effect of maternal exposure to BPA on the epigenome in mouse forebrain at E12.5 and at E14.5. We scanned CpG methylation status in 2500 NotI loci, representing 48 (de)methylated unique loci. Methylation status in most of them was primarily developmental stage-dependent. Each of almost all cloned NotI loci was located in a CpG island (CGI) adjacent to 5' end of the transcriptional unit. The mRNA expression of two functionally related genes changed with development as well as the exposure to BPA. In both genes, changes at the transcriptional level correlated well with the changes in NotI methylation status. Taken together, epigenetic alterations in promoter-associated CGIs after exposure to BPA may underlie some effects on brain development.
Biochemical and Biophysical Research Communications 10/2008; 376(3):563-7. DOI:10.1016/j.bbrc.2008.09.028 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Microcephaly is a malformation associated with in utero exposed atomic bomb survivors and can be induced in mice by fetal exposure to ionizing radiation (IR). The pathogenesis of IR-induced microcephaly, however, has not been fully understood. Our analyses of high-coverage expression profiling (HiCEP) demonstrated that the abnormal spindle-like microcephaly associated gene (ASPM) was down-regulated in irradiated human diploid fibroblasts. ASPM was recently reported as the causative gene for MCPH-5, the most common type of congenital microcephaly in humans. Here, we show that the expression of the Aspm gene was significantly reduced by IR in various human and murine cells. Additionally, Aspm was found downregulated in the irradiated fetal mouse brain, particularly in the ventricular zones. A similar suppression was observed in the irradiated neurosphere cultures. This is the first report suggesting that the suppression of Aspm by IR could be the initial molecular target leading to the future microcephaly formation.
Biochemical and Biophysical Research Communications 06/2008; 369(3):953-7. DOI:10.1016/j.bbrc.2008.02.149 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Most previously reported mutations in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) result in an odd number of cysteine residues within the epidermal growth factor (EGF)-like repeats in Notch3. We report here R75P mutation in two Japanese CADASIL families not directly involving cysteine residues located within the first EGF-like repeats. Probands in both families had repeated episodes of stroke, depression, dementia as well as T2 high-intensity lesions in the basal ganglia and periventricular white matter, but fewer white matter lesions in the temporal pole on MRI. These families provide new insights into the diagnosis and pathomechanisms of CADASIL.
Internal Medicine 02/2008; 47(23):2067-72. DOI:10.2169/internalmedicine.47.1391 · 0.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bisphenol A (BPA), known as an environmental endocrine disrupter, is widely used in industry and dentistry. We investigated the effects of fetal and neonatal exposure to bisphenol A (BPA) on the brain development of mice. The density of tyrosine hydroxylase (TH)-immunoreactive (IR) neurons in substantia nigra was significantly decreased in BPA-exposed female mice (3 microg/g powder food), but not in the male mice, as compared with that of the control mice. The densities of calbindin D-28 K-, calretinin- and parvalbumin-IR neurons in the cerebral cortex were not different between BPA-exposed and the control mice. The present study indicates that chronic exposure of BPA during prenatal and neonatal periods causes a decrease of TH-positive neurons in substantia nigra only in female mice brain.
Brain and Development 08/2007; 29(6):352-6. DOI:10.1016/j.braindev.2006.10.003 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The objective of the study was to evaluate possible changes of the gene expression and localization of the enzymes, heme oxygenase and nitric oxide synthase (NOS), with reference to increase of collagen type III in response to the partial obstruction of the bladder. Following initial obstruction, whole rat bladders were removed for real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Real-time RT-PCR demonstrated significantly enhanced expression of HO (p < 0.01) and collagen type III (p < 0.001) gene on postoperative day 14. Enhanced expression of NOS gene was seen only on postoperative day 4 (p < 0.01). Immunohistochemistry revealed that immunoreactivity to HO-1 had much in common in neural cells and fibers, although immunoreactivity to HO-2 and iNOS was relatively weak. This study suggested gene expression of HO, especially HO-1, was more dramatically changed than NOS, and was upregulated simultaneously with increase of collagen type III after obstruction. HO systems could be involved in the pathogenesis of bladder dysfunction related to increase of collagen type III after obstruction.
Urologia Internationalis 04/2007; 78(3):270-7. DOI:10.1159/000099351 · 1.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bisphenol A (BPA) has been shown to disrupt thyroid hormone function. We therefore studied whether prenatal exposure to low-doses of BPA affects the morphology and the expression of some genes related to brain development in the murine fetal neocortex. Pregnant mice were injected subcutaneously with 20 microg/kg of BPA daily from embryonic day 0 (E0). Control animals received vehicle alone. For evaluating cell proliferation, neuronal differentiation and migration, bromodeoxyuridine (BrdU) was injected intraperitoneally into pregnant mice with various regimens and the brains were processed for immunohistochemistry. The total RNA was extracted from the embryonic telencephalon at various embryonic stages. The BrdU-labeled cells examined 1 hour after BrdU injection showed no differences between the BPA-treated and control groups (n = 10, each), which indicated that the proliferation of precursor cells was not affected. The BrdU-labeled cells, analysed 2 days after BrdU injection, were decreased in the ventricular zone of BPA-treated mice at E14.5 and E16.5, whereas they were increased in the cortical plate at E14.5 as compared with those in control mice (n = 10, each). Furthermore, the expression of Math3, Ngn2, Hes1, LICAM, and THRalpha was significantly upregulated at E14.5 in the BPA-treated group. These results suggested that BPA might disrupt normal neocortical development by accelerating neuronal differentiation/migration.
Journal of Neuroscience Research 11/2006; 84(6):1197-205. DOI:10.1002/jnr.21020 · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Terminal Schwann cells (TSCs) that cover motor neuron terminals, are known to play an important role in maintaining neuromuscular junctions, as well as in the repair process after nerve injury. However, the molecular characteristics of TSCs remain unknown, because of the difficulties in analyzing them due to their paucity. By using our previously reported method of selectively and efficiently collecting TSCs, we have analyzed the difference in expression patterns of lysophospholipid (LPL) receptor genes (LPA1, LPA2, LPA3, S1P1, S1P2, S1P3, S1P4, and S1P5) between TSCs and myelinating Schwann cells (MSCs). LPL, which includes lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), is the bioactive lipid that induces a myriad of cellular responses through specific members of G-protein coupled receptors for LPA. It turned out that LPA3 was expressed only in TSCs, whereas S1P1 was expressed in TSCs and skeletal muscle, but not in MSCs. Other types of LPL receptor genes, including LPA1, S1P2, S1P3, S1P4, were expressed in both types of Schwann cells. None of the LPL receptor gene family showed MSCs-specific expression.
Acta histochemica et cytochemica official journal of the Japan Society of Histochemistry and Cytochemistry 05/2006; 39(2):55-60. DOI:10.1267/ahc.06002 · 1.39 Impact Factor