Takeshi Yaoi

Murine neocortical histogenesis is perturbed by prenatal exposure to low doses of Bisphenol A.

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 μg/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 THRα 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.

Effects of pre- and neonatal exposure to bisphenol A on murine brain development

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.

Bisphenol A, an endocrine-disrupting chemical, and brain development

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.

N‐copine: a novel two C2‐domain‐containing protein with neuronal activity‐regulated expression

Neuronal activity is often associated with changes in gene expression. By a two‐dimensional cDNA‐display system, restriction landmark cDNA scanning, we identified a novel gene whose expression in the hippocampus was up‐regulated by kainate stimulation. The mRNA expression was detected only in brain and up‐regulated by the stimulation evoking CA3‐CA1 long‐term potentiation. The encoded protein contains two copies of C2‐domain, known as the Ca2+‐binding domain of PKC‐γ, and shows 49% identity with human copine I. We designated this protein N‐copine (neuronal‐copine). N‐copine may have a role in synaptic plasticity.

Organ distribution of quantum dots after intraperitoneal administration, with special reference to area-specific distribution in the brain

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.

Localization and Subcellular Distribution of N-Copine in Mouse Brain

Abstract : N‐Copine is a novel protein with two C2 domains. Its expression is brain specific and up‐regulated by neuronal activity such as kainate stimulation and tetanus stimulation evoking hippocampal CA1 long‐term potentiation. We examined the localization and subcellular distribution of N‐copine in mouse brain. In situ hybridization analysis showed that N‐copine mRNA was expressed exclusively in neurons of the hippocampus and in the main and accessory olfactory bulb, where various forms of synaptic plasticity and memory formation are known to occur. In immunohistochemical analyses, N‐copine was detected mainly in the cell bodies and dendrites in the neurons, whereas presynaptic proteins such as synaptotagmin I and rab3A were detected in the regions where axons pass through. In fractionation experiments of brain homogenate, N‐copine was associated with the membrane fraction in the presence of Ca2+ but not in its absence. As a GST‐fusion protein with the second C2 domain of N‐copine showed Ca2+ ‐dependent binding to phosphatidylserine, this domain was considered to be responsible for the Ca2+ ‐dependent association of N‐copine with the membrane. Thus, N‐copine may have a role as a Ca2+ sensor in postsynaptic events, in contrast to the known roles of “double C2 domain‐containing proteins,” including synaptotagmin I, in presynaptic events.

Ca2(+)-dependent interaction of N-copine, a member of the two C2 domain protein family, with OS-9, the product of a gene frequently amplified in osteosarcoma.

N‐copine is a novel two C2 domain protein that shows Ca2+‐dependent phospholipid binding and membrane association. By using yeast two‐hybrid assays, we identified OS‐9 as a protein capable of interacting with N‐copine. We further revealed that the second C2 domain of N‐copine bound with the carboxy‐terminal region of OS‐9. Their interaction in vivo was also confirmed by co‐immunoprecipitation from 293E cells co‐expressing transfected N‐copine and OS‐9. In vitro binding assays showed that this interaction was Ca2+‐dependent. By Northern blot analysis, N‐copine and OS‐9 were co‐expressed in the same regions of human brain. These results reveal that OS‐9 is a potential target of N‐copine.

Ionizing radiation downregulates ASPM, a gene responsible for microcephaly in humans

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.

The shortest isoform of dystrophin (Dp40) interacts with a group of presynaptic proteins to form a presumptive novel complex in the mouse brain.

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.

Spatiotemporal patterns of Musashi1 expression during inner ear development.

Musashi1 (Msi 1) is an RNA binding protein associated with asymmetric cell divisions in neural progenitor cells. To investigate the involvement of Msi1 in the inner ear development, we studied the expression of Msi1 in mouse inner ears with RT-PCR and immunohistochemistry. Immunohistochemistry revealed that Msi1 was expressed in all otocyst cells at embryonic day (E) 10 and 12. Msi1 immunoreactivity became lost in hair cells after E14 in vestibule and after E16 in cochlea, whereas it persisted in supporting cells until adulthood. The subcellular localization of Msi1 changed from “cytoplasmic predominance” to “nuclear predominance” during the first 2 weeks after birth. The present data suggested that Msi may play a role in inner ear development.