Mitsunari Nakajima

Synthesis, aggregation, and neurotoxicity of the alzheimer's Aß1-42 amyloid peptide and its isoaspartyl isomers

Amyloid Abeta1-42 peptide (Abeta1-42) and its isomers with an isoaspartyl residue at position 7 or 23 [Abeta1-42(isoAsp7) and Abeta1-42(isoAsp23)] were synthesized in high purity by the Fmoc-solid phase technique, followed by HPLC on a silica-based reversed-phase column under the basic conditions. Importantly, Abeta1-42(isoAsp23) aggregated more strongly than native Abeta1-42 and showed significant neurotoxicity, while the aggregation ability and neurotoxicity of Abeta1-42(isoAsp7) was weak. This suggests that the isomerization of the aspartyl residues plays an important role in fibril formation in Alzheimers disease.

Abnormal blood vessel development in mice lacking presenilin-1.

Presenilin-1 (PS1) is a gene responsible for the development of early-onset familial Alzheimers disease. To explore the potential roles of PS1 in vascular development, we examined the vascular system of mouse embryos lacking PS1. PS1-deficient embryos exhibited cerebral hemorrhages and subcutaneous edema by mid gestation. Immunohistochemical analysis revealed vascular remodeling failure in the stomach and trunk dorsal median region of the skin and insufficient formation of the perineural plexus around the spinal cord of the PS1 mutant embryos. The number of capillary sprouting sites reduced and the capillary diameter increased in the mutant brains, especially at the amygdaloid and striatal regions. Endothelial cells in the sprouting capillaries of the mutant mice showed abnormal morphologies such as multiplication, apoptotic and necrotic images, in contrast to pericytes showing a normal appearance. An in vitro assay using para-aortic splanchnopleural mesoderm (P-Sp) revealed aberrant angiogenesis in the explant culture from the mutant. These findings suggest the essential roles of PS1 in angiogenesis.

EXT gene family member rib-2 is essential for embryonic development and heparan sulfate biosynthesis in Caenorhabditis elegans

EXT gene family members including EXT1, EXT2, and EXTL2 are glycosyltransferases required for heparan sulfate biosynthesis. To examine the biological functions of rib-2, a member of the Caenorhabditis elegans EXT gene family, we generated a mutant worm lacking the rib-2 gene using the UV-TMP method followed by sib-selection. Inactivation of rib-2 alleles induced developmental abnormalities in F2 and F3 homozygous worms, while F1 heterozygotes showed a normal morphology. The F2 homozygous progeny generated from the F1 heterozygous hermaphrodites somehow developed to adult stage but exhibited abnormal characteristics such as developmental delay and egg-laying defects. The F3 homozygous progeny from the F2 homozygous hermaphrodites showed early developmental defects and most of the F3 worms stopped developing during the gastrulation stage. Whole-mount staining analysis for heparan sulfate using Toluidine blue (pH 2.5) revealed a defect of heparan sulfate biosynthesis in the F2 homozygotes. The analysis using fluorometric post-column high-performance liquid chromatography also uncovered reduced production of heparan sulfate in the rib-2 mutant. These results indicate that rib-2 is essential for embryonic development and heparan sulfate biosynthesis in C. elegans.

Presenilin 1 is essential for cardiac morphogenesis

Presenilin 1 (PS1) is the gene responsible for the development of early‐onset familial Alzheimers disease. PS1‐deficient mice have been reported to show defects in neurogenesis, somitogenesis and angiogenesis. Here, we report cardiac anomaly in PS1‐deficient mice: the mutant hearts exhibited ventricular septal defect, double outlet right ventricle, and stenosis in the pulmonary artery. Immunohistochemistry using anti‐PS1 antibody revealed the prominent expression of PS1 in mesenchymal cells at the septal area of the wild‐type heart. These results suggest that PS1 may play an essential role in heart development. Developmental Dynamics 230:795–799, 2004.

Impaired cell cycle control of neuronal precursor cells in the neocortical primordium of presenilin-1-deficient mice

Recent studies have implicated presenilin‐1 (PS‐1) in the processing of the amyloid precursor protein and Notch‐1. We show that PS‐1 has biological effects on differentiation and cell cycle control of neuronal precursor cells in vivo using PS‐1‐deficient mice. The expression of Class III β‐tubulin was upregulated throughout the neocortical primordia of PS‐1‐deficient E14 embryos, especially on the ventricular surface. The increased speed of migration of the immature neurons from the ventricular zone outward in the PS‐1‐deficient neocortical primordia was indicated by an in vivo bromodeoxyuridine (BrdU)‐labeling assay and a DiI‐labeling assay in slice culture. Furthermore, we investigated the cell cycle of neuronal precursor cells in the neocortical ventricular zone using an in vivo cumulative BrdU‐labeling assay. The length of the cell cycle in the neocortical precursor cells of wild‐type mice was 11.4 hr whereas that of the PS‐1‐deficient mice was 15.4 hr. Among all phases of the cell cycle, S‐phase exhibited the most prominent change in length, increasing from 2.4 hr in the wild‐type mice to 7.4 hr in the mutant mice. The distribution of β–catenin was specifically affected in the ventricular zone of the PS‐1‐deficient mice. These findings suggest that PS‐1 is involved in the differentiation and the cell cycle control of neuronal precursor cells in the ventricular proliferating zone of the neocortical primordium.

Estrogen Stimulates Proliferation and Differentiation of Neural Stem/Progenitor Cells through Different Signal Transduction Pathways

Our previous study indicated that both 17β-estradiol (E2), known to be an endogenous estrogen, and bisphenol A (BPA), known to be a xenoestrogen, could positively influence the proliferation or differentiation of neural stem/progenitor cells (NS/PCs). The aim of the present study was to identify the signal transduction pathways for estrogenic activities promoting proliferation and differentiation of NS/PCs via well known nuclear estrogen receptors (ERs) or putative membrane-associated ERs. NS/PCs were cultured from the telencephalon of 15-day-old rat embryos. In order to confirm the involvement of nuclear ERs for estrogenic activities, their specific antagonist, ICI-182,780, was used. The presence of putative membrane-associated ER was functionally examined as to whether E2 can activate rapid intracellular signaling mechanism. In order to confirm the involvement of membrane-associated ERs for estrogenic activities, a cell-impermeable E2, bovine serum albumin-conjugated E2 (E2-BSA) was used. We showed that E2 could rapidly activate extracellular signal-regulated kinases 1/2 (ERK 1/2), which was not inhibited by ICI-182,780. ICI-182,780 abrogated the stimulatory effect of these estrogens (E2 and BPA) on the proliferation of NS/PCs, but not their effect on the differentiation of the NS/PCs into oligodendroglia. Furthermore, E2-BSA mimicked the activity of differentiation from NS/PCs into oligodendroglia, but not the activity of proliferation. Our study suggests that (1) the estrogen induced proliferation of NS/PCs is mediated via nuclear ERs; (2) the oligodendroglial generation from NS/PCs is likely to be stimulated via putative membrane-associated ERs.

Etoposide induces programmed death in neurons cultured from the fetal rat central nervous system

The effects of etoposide on the death of neurons cultured from the central nervous system (CNS) of fetal rats were examined. The cultured neurons died in the presence of 1-40 micrograms/ml of etoposide, which is known to induce programmed death in some kinds of cells, and this cytotoxic effect was prevented by inhibition of protein synthesis and/or RNA synthesis. Furthermore, DNA degradation, including a ladder-like pattern, became evident in these neurons 3 h after incubation with etoposide (10 micrograms/ml), whereas cell death commenced after about 6 h. These results indicate that etoposide-treated CNS neurons require new protein and RNA synthesis to undergo an active death programme, and that internucleosomal fragmentation of DNA mediates the etoposide-induced programmed cell death. This culture system of etoposide-treated CNS neurons is thought to be a useful model for the study of programmed neuronal cell death.

Anti-inflammatory and neuroprotective effects of auraptene, a citrus coumarin, following cerebral global ischemia in mice

Cerebral ischemia causes delayed neuronal cell death in the hippocampus resulting in sequential cognitive impairments. Hyper-activated inflammation following ischemia is one of the etiologies for delayed neuronal cell death. In the present study, using a transient global ischemia mouse model, we showed that auraptene (AUR), a citrus coumarin, effectively inhibited microglia activation, cyclooxygenase-2 expression by astrocytes, and neuronal cell death in the hippocampus following ischemic insults. These results suggest that AUR acts as a neuroprotective agent in the ischemic brain, which may be mediated by suppression of the inflammatory response.

Notch‐1 activation by familial Alzheimer's disease (FAD)‐linked mutant forms of presenilin‐1

We prepared a cleavage site‐directed antibody against Notch‐1, that specifically recognized the cleaved Notch‐1 intracellular domain (NICD). To assess Notch‐1 processing and its nuclear localization in familial Alzheimers disease (FAD)‐linked presenilin‐1 (PS‐1) mutants, we overexpressed wild type, M146V, A246E, C410Y, or δE9 PS‐1 mutant with a membrane‐bound Notch‐1 in a PS‐1‐deficient cell line. On Western blot and immunocytochemical analyses using the NICD specific antibody, M146V and A246E mutants showed the comparable levels of Notch‐1 processing and nuclear localizing activities to wild type PS‐1 whereas C410Y and δE9 mutants failed to show these activities. These results suggest that the loss or partial loss of PS‐1 activities in Notch‐1 proteolysis and its nuclear translocation may be irrelevant for the neuropathology of Alzheimers disease. J. Neurosci. Res. 62:311–317, 2000.

Deficiency of presenilin-1 increases calcium-dependent vulnerability of neurons to oxidative stress in vitro.

We examined the function of presenilin‐1 (PS1) on neuronal resistance to oxidative stress. CNS neurons cultured from PS1‐deficient mice exhibited increased vulnerability to H2O2 treatment compared with those from wild‐type mice. Antioxidants protected the cultured neurons against the oxidative stress. An intracellular calcium chelator, BAPTA AM, as well as an L‐type voltage‐dependent calcium channel blocker, nifedipine, rescued the neurons from H2O2‐induced death, while an N‐type voltage‐dependent calcium channel blocker, ω‐conotoxin, or calcium release blockers from ER stores, dantrolene and xestospongin C, failed to rescue them. Wild‐type and PS1‐deficient neurons showed comparable increases of cytoplasmic free calcium levels after exposure to H2O2. Taken together with the data that PS1‐deficient neurons exhibited increased vulnerability to glutamate, these findings imply that PS1 confers resistance to oxidative stress on neurons in calcium‐dependent manners.