Ichiro Isobe

Apolipoprotein E Exhibits Isoform‐Specific Promotion of Lipid Efflux from Astrocytes and Neurons in Culture

Abstract: Many studies have shown that apolipoprotein E (apoE) plays important roles in maintaining intracellular lipid homeostasis in nonneuronal cells. However, little is known about the extracellular transport of lipids in the CNS. In this study, we determined whether and to what degree lipid efflux from astrocytes and neurons depended on apoE. Our results showed that exogenously added apoE promoted the efflux of cholesterol and phosphatidylcholine from both astrocytes and neurons in culture, resulting in the generation of high‐density lipoprotein‐like particles. The order of potency of the apoE isoforms as lipid acceptors was apoE2 > apoE3 = apoE4 in astrocytes and apoE2 > apoE3 > apoE4 in neurons. Treatment with brefeldin A, monensin, and a protein kinase C inhibitor, H7, abolished the ability of apoE to promote cholesterol efflux from cultured astrocytes, without altering apoE‐mediated phosphatidylcholine efflux. In contrast, the efflux of both cholesterol and phosphatidylcholine promoted by apoE was abolished following treatment with heparinase or lactoferrin, which block the interaction of apoE with heparan sulfate proteoglycans (HSPGs) or low‐density lipoprotein receptor‐related protein (LRP), respectively. This study suggests that apoE promotes lipid efflux from astrocytes and neurons in an isoform‐specific manner and that cell surface HSPGs and/or HSPG‐LRP pathway may mediate this apoE‐promoted lipid efflux.

Enhancement of MTT, a tetrazolium salt, exocytosis by amyloid β-protein and chloroquine in cultured rat astrocytes

The effect of amyloid beta-protein (Abeta) on the cellular reducing activity has been a controversial issue. We determined the cellular reducing activity in cultured astrocytes using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl )-2H-tetrazolium (WST-8) reduction assays following Abeta treatment. MTT reduction was inhibited whereas WST-8 reduction was unaffected by the Abeta treatment. Furthermore, the early extracellular appearance of MTT formazan, a reduced product of MTT, was observed in association with the rapid disappearance of intracellular formazan granules. Notably, similar results were obtained in cultures treated with chloroquine, a perturbant of membrane trafficking. Our results suggest that MTT formazan exocytosis is enhanced in a similar manner by Abeta and chloroquine and that this biological activity of Abeta may underlie the pathogenesis of Alzheimers disease.

Astrocytic contributions to blood-brain barrier (BBB) formation by endothelial cells: a possible use of aortic endothelial cell for in vitro BBB model.

Astrocytic contribution of endothelial cell monolayer permeability was examined in two blood-brain barrier (BBB) models, using the coculture in a double chamber system: rat astrocytes and bovine aortic endothelial cells (BAECs) or bovine brain endothelial cells (BBECs). In system 1, where astrocytes were separated from endothelial cells, a 40% reduction in L-glucose permeability of the BBEC monolayer, but not the BAEC monolayer, was observed by cocultivation with astrocytes. Although several passages of BBEC in culture elicited morphological transformation from spindle-shapes to cobblestone-like features, the passaged BBECs remained responsive to astrocytes in coculture in system 1 (37% reduction of the L-glucose permeability). By contrast, in system 2, where respective endothelial cells and astrocytes layered on the upper and lower surfaces of a membrane, the permeability of both BAEC and BBEC monolayers was reduced by cocultivation with astrocytes (75% reduction for BAEC and 40% reduction for BBEC). BAECs in this contiguous coculture (system 2) with astrocytes showed numerous tight junction-like structures characteristic of the BBB in vivo. These results suggest that primary cultured BBECs, which had been primed by astrocytes in vivo, retain a higher sensitivity to astrocytes possibly through an astrocytic soluble factor (s) to exhibit BBB-specific phenotypes, and that even BAEC from extra-neural tissues, when cultured with astrocytes in close proximity in vitro, may acquire the similar phenotypes and serve for an extensive use of BBB model in vitro.

A Novel Glial Growth Inhibitory Factor, Gliostatin, Derived from Neurofibroma

Neurofibroma tissue was investigated for the presence of glial growth modulators that would suppress the proliferation of glial cells. A novel endogenous polypeptide inhibitor of proliferation and DNA synthesis in glial cells, gliostatin, was purified from the extracts of neurofibroma by a procedure comprising dye and anion‐exchange column chromatography, and HPLC. A monoclonal antibody raised against partially purified gliostatin showed no cross‐reactivity with known cytokines, but adsorbed the growth inhibitory activity of gliostatin and immunochemi‐cally visualized the putative gliostatin bands on western blot analyses. Although the product showed an apparent Mr of 100,000 accompanied by an inhibitory activity on gel filtration column chromatography, it migrated at a lower apparent Mr of 50,000 under the reducing conditions on western blotting, indicating that a homodimeric structure of native gliostatin consisted of 50‐kDa subcomponents. Gliostatin was a potent growth inhibitor acting at nanomolar concentrations against all glial tumor cells and glia maturation factor‐stimulated astroblasts, but not neuronal cells.

Astrocytic contribution to functioning synapse formation estimated by spontaneous neuronal intracellular Ca2+ oscillations

Glial contribution to in vitro synaptic function was investigated in a neuron-glia co-culture system by monitoring spontaneous oscillations of intracellular Ca2+ in neurons. Rat cortical neurons, grown stably on a cortical astrocyte monolayer, extended neurites resulting in marked functional synapse formation. Little synapse formation was observed in neuronal co-culture with meningeal fibroblasts or endothelial cells. Aged astrocytes in vitro (C35) were found to attenuate synaptic development, while young astrocytes (C5) markedly promoted synaptic function. C5 and C35 astrocyte media conditioned yielded no significant synaptogenic effect, indicating diffusible factor(s) are not responsible for our observation. Modulation of astrocytic proliferation and differentiation by gliostatin, a glial growth inhibitor, or dibutyryl cAMP affected neuronal synaptic function on the co-cultures. Site-specific analysis in homologous and heterologous neuron-astrocyte co-cultures among cortex, hippocampus, septum, and striatum revealed that homologous combinations of neurons and astrocytes derived from identical brain regions elicited the largest number of synchronizing neurons. These results suggest that in vivo neuronal synaptic function essentially requires the participation of adjacent astrocytes, which is site-specific and age-dependent.

Mitochondrial sequence haplotype in the Japanese population

Sequence polymorphysms of the mitochondrial DNA (mtDNA) control region, hypervariable regions I and II, from 50 unrelated Japanese were determined by PCR amplification and cycle sequencing.

An autopsy case of poisoning by massive absorption of cresol a short time before death

A 65-year-old male patient who was hospitalized with schizophrenia died about 15 min later after ingestion of a large volume of saponated cresol solution in a mental hospital. Fatal levels of free p- and m-cresol in the heart blood were detected at 458.8 and 957.3 microg/ml, respectively, which far exceeded the fatal levels determined previously. The levels in the heart muscle, liver and spleen tissues were also extremely high, and there was 250 ml of cresol-odor-emitting fluid in the stomach. The levels of glucuronic-acid-conjugated p- and m-cresols in the heart blood were 38.2 and 85.6 microg/ml, respectively. Although the high levels of cresols in the heart blood may be due to diffusion from the stomach contents, it is surmised that the essential levels of free and conjugated forms in blood were at least 99 and 240 microg/ml, respectively, considering the results of postmortem examinations and some case reports. It was concluded that about 340 microg/ml of the total cresols was absorbed in a very short period following oral ingestion of saponated cresol solution in this case.

Neurotrophic action of gliostatin on cocultured neurons with glial cells

Gliostatin is a polypeptide factor (apparent M(r) = 100 k with a homodimeric structure comprising two 50 kDa subunits) acting on cortical neurons (neurotrophic action) as well as astrocytic cells (growth inhibition). Under the coculture system of cerebral cortical neurons and astrocytes from fetal rats (E15 or E16), the neurotrophic action of gliostatin was examined immunocytochemically. Immunostaining by an anti-neurofilament (NF) monoclonal antibody visualized a marked neurite-outgrowth and interconnecting bundles of neuritic processes induced by gliostatin in the coculture system. Neurons stimulated by gliostatin formed dense aggregates in clumps, while neurons in control coculture spread out. Gliostatin has also shown survival-promoting effects on neurons. Furthermore, it was shown that gliostatin induced the differentiation of protoplasmic astrocytes to fibrous astrocytes. These results further support our previous contention that gliostatin plays physiological roles on neuronal and glial development.

A Possible Model of Senile Plaques Using Synthetic Amyloid β-Protein and Rat Glial Culture

The senile plaque (SP) is one of the pathological hallmarks in the brains of patients with Alzheimers disease (AD), but the mechanism of its formation and its role in AD progression are not yet fully understood. Synthetic amyloid beta-protein (Abeta)1-40 is known to aggregate in vitro, and the aggregated Abeta has been widely used for in vitro experiments, in which its peculiar effects on neuronal and glial cells have been shown. To date, however, the formation of a SP-like structure in a culture system using synthetic Abeta has not been demonstrated. In this study, we established a possible SP model using synthetic Abeta1-40 and rat glial cultures as follows: (1) large spherical aggregates of synthetic Abeta (sAmys) were produced from synthetic Abeta1-40 (10-50 microm in diameter), (2) the sAmys were added to a glial culture, and (3) the characteristics of the sAmys and the reactions of glial cells (microglia and astrocytes) around the sAmys were analyzed. We found that the sAmys exhibited the same features as the dense amyloid core in SPs, including the intense green birefringence under polarized light with Congo red, and induced reactive features in glial cells, including induction of major histocompatibility complex class II antigen in the microglia and interleukin-1beta in the astrocytes, similar to those seen in SPs in the brain in AD. Given our findings, we consider that this glial culture system with the sAmys is a possible in vitro SP model and useful for investigating the effects of massive amyloid deposition on neuronal and glial cells.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) causes Akt phosphorylation and morphological changes in intracellular organellae in cultured rat astrocytes

3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) is widely used for cell viability and cytotoxicity assays, but cell biological effects of MTT itself have not been investigated. In this paper we show that MTT induces a morphological change in an intracellular membranous compartment labeled with anti‐Rab5 antibody, dissociation of early endosomal auto‐antigen (EEA1) from the membrane fraction, and phosphorylation of Akt probably through a phosphatidylinositol‐3‐OH kinase [PI(3)K] pathway in cultured rat astrocytes. These findings suggest that MTT affects cellular functions and conditions to some extent, and such effects of MTT may cause some discrepancies of measurement of cell viability using MTT assay and other assays. That is, the effects of MTT on cells could influence the results of cell viability assay. Moreover, MTT or other tetrazolium salts could be used as interesting activators of Akt to investigate the mechanism by which Akt or PI(3)K is activated.