Etsuro Uemura

Microglial release of nitric oxide by the synergistic action of β-amyloid and IFN-γ

Abstract Alzheimers disease (AD) is a progressive neurodegenerative disorder characterized hisotpathologically by a loss of neurons and an accumulation of β-amyloid plaques, neurofibrillary tangles, dystrophic neurites, and reactive glial cells. While most previous studies on the neurodegeneration of AD have focused on neuronal cells and direct β-amyloid-mediated neurotoxicity, few have focused on the role of reactive glial cells in β-amyloid-mediated neurotoxicity. In the present study nitric oxide release from cultured rat microglia was examined by exposing the cells to synthetic β-amyloid peptides (β25–35 and β1–40) alone and in combination with the cytokines IFN-α/β (100 U/ml), IL-1β (100 U/ml), TNF-α (100 U/ml), TNF-β (100 U/ml), or IFN-γ (10, 100, 500, or 1000 U/ml). Assessment of microglial release of nitric oxide was based on the colorimetric assay for nitrite in the culture medium and histochemistry for nitric oxide synthase. Of the cytokines tested, only IFN-γ (1000 U/ml) induced nitric oxide release from microglia. β25–35 did not stimulate nitric oxide release by itself, but it did induce nitric oxide release when co-exposed with IFN-γ (100, 500, and 1000 U/ml). In contrast, β1–40 did induce microglial release of nitric oxide by itself, and this effect was enhanced significantly by co-exposure with IFN-γ (100 U/ml). These findings warrant a further investigation into the role of microglia in the neurodegeneration of Alzheimers disease via nitric oxide toxicity induced by the synergistic action of β-amyloid and a costimulatory factor.

Age-related changes in prefrontal cortex of Macaca mulatta: quantitative analysis of dendritic branching patterns.

Abstract Age-related changes in the dendritic branching pattern were observed in nine Macaca mulattas from 7 to 28 years of age. Morphometric measurements made with the computerized graphic digitizer revealed signficant age-related differences at various designated branch orders in both the centrifugal and centripetal ordering methods. It appeared that there was continued branching and growth of the apical dendrites in adulthood. Basal dendrites of adult Macaca mulattas did not show any added complexity, but rather growth of existing terminal branches continued. Data obtained from the three oldest animals suggested a preferential loss of whole dendritic branches on the apical portion of the dendritic tree, whereas basal dendrites seemed to manifest more of the distoproximal-type degeneration process.

Morphometric studies of the rat hippocampus following chronic delta-9-tetrahydrocannabinol (THC)

Persistent behavioral effects resembling those of hippocampal brain lesions have been reported following chronic administration of marijuana or its major psychoactive constituent, delta-9-tetrahydrocannabinol (THC) to rats. We used morphometric techniques to investigate the effects of chronic THC on the anatomical integrity of the hippocampus. Rats dosed orally for 90 days with 10 to 60 mg/kg THC or vehicle were evaluated by light and electron microscopy up to 7 months after their last dose of drug. Electron micrographs revealed a striking ultrastructural appearance and statistically significant decreases in mean volume of neurons and their nuclei sampled from the hippocampal CA3 region of rats treated with the highest doses of THC. A 44% reduction in the number of synapses per unit volume was demonstrated in these same rats. Golgi impregnation studies of additional groups of rats treated with 10 or 20 mg/kg/day THC and sacrificed 2 months after their last treatment with THC revealed a reduction in the dendritic length of CA3 pyramidal neurons, despite normal appearing ultrastructure and no changes in synaptic density. The hippocampal changes reported here may constitute a morphological basis for behavioral effects after chronic exposure to marijuana.

The inhibitory effects of β-amyloid on glutamate and glucose uptakes by cultured astrocytes

beta-Amyloid is the primary protein component of neuritic plaques, which are degenerative foci in brains of patients with Alzheimers disease (AD). The effects of this naturally occurring beta-amyloid on the cells of the central nervous system have not been completely understood. beta-Amyloid increases the vulnerability of cultured neurons to glutamate-induced excitotoxic damage. Because astrocytes play a key role in uptake of extracellular glutamate and glutamate uptake is ATP-dependent, we studied the effect of beta25-35 on glutamate and glucose uptake in cultured hippocampal astrocytes following 7 days of exposure to beta25-35. Astrocytic glutamate uptake was studied at 1, 5, 10, 15, 20, and 60 min following the addition of [3H]glutamate (5 nM) to the culture media, and astrocytic glucose uptake was assessed at 60 min after the addition of [14C]glucose (600 and 640 nM) to the media. Glutamate uptake by control astrocytes was time-dependent. Astrocytes exposed to beta25-35, however, showed significantly lower glutamate uptake at all sampling times. Similarly, [14C]glucose uptake by astrocytes was inhibited by beta25-35. When glucose uptake was blocked by phloretin (10 mM), astrocytic [3H]glutamate uptake was also blocked, suggesting that the inhibitory effect of beta-amyloid on glutamate uptake is caused by diminished glucose uptake. Thus, our present study suggests a possible link between two proposed mechanisms of pathogenesis of the Alzheimers disease: glutamate neurotoxicity and global defect in cerebral energy metabolism.

Effects of halothane on synaptogenesis and learning behavior in rats

Synaptic density was quantitated in the entorhinal cortex and subiculum of rats at 5, 21, 34, and 95 postnatal days. These rats were offspring of mothers that had been subjected to four different concentrations of halothane during gestation and for 60 days after birth. The exposure conditions were control, intermittent halothane (25 +/- 5 ppm or 100 +/- 5 ppm, 8 h/day, 5 days/week) and continuous halothane (25 +/- 5 ppm, 24 h/day, 7 days/week). Synaptic density in rats exposed to halothane was significantly less than in control rats. Animals exposed intermittently to 25 +/- 5 ppm halothane had higher synaptic density than animals exposed continuously to 25 +/- 5 ppm halothane or intermittently to 100 +/- 5 ppm halothane. The latter two exposure conditions exerted similar effects. The lag in synaptic development was established at 5 days postnatal and remained the same throughout the first 95 postnatal days in both the entorhinal cortex and subiculum. Delayed synaptogenesis caused by halothane was indicated by the presence of growth cones in halothane-exposed rats to 34 days compared with 21 days in the control rats. The spontaneous alternation test indicated that the delayed synaptogenesis by halothane was sufficient to suppress behavioral development. Thus, the delay in the initial synaptic maturation caused by halothane exposure in utero may result in permanent morphologic and functional deficits of the brain.

Age-related changes in the subiculum of Macaca mulatta: dendritic branching pattern.

The dendritic branching pattern was studied in the subiculum of nine Macaca mulatta from 7 to 28 years of age. Morphometric analysis of pyramidal neurons revealed significant age-related differences at various designated branch orders in both the centrifugal and centripetal ordering methods. There was continued branching and growth of the apical dendrites in adulthood. Basal dendrites did not show any added complexity, but rather showed continued growth of existing terminal branches. The three oldest animals showed a preferential loss of whole terminal branches on the apical portion of the dendritic tree, whereas shortening of existing terminal branches was the characteristic feature of the basal dendrites. Data obtained from the subiculum provide quantitative evidence indicating the considerable potential for dendritic plasticity beyond the early developmental stages and eventual loss of dendritic complexity in the old M. mulatta.

Insulin regulates neuronal glucose uptake by promoting translocation of glucose transporter GLUT3.

Neurons have been classically considered insulin-insensitive cells. In this in vitro study, the effect of insulin on neuronal glucose uptake was studied by assaying glucose uptake, translocation of glucose transporter isoform GLUT3, and fusion of GLUT3 vesicles with the plasma membrane. Insulin (50 nM) promoted translocation of GLUT3 to the plasma membrane. However, insulin neither promoted fusion of GLUT3 with the plasma membrane nor increased neuronal glucose uptake. In cells pre-exposed to insulin, depolarization with 40 mM KCl markedly increased fusion of GLUT3 with plasma membrane and neuronal uptake of glucose. Based on these data, we propose that insulin regulates neuronal glucose uptake by promoting translocation of GLUT3 to the plasma membrane, and that insulin enables neurons to respond to demand for energy induced by increased neuronal activity.

Integrin Mac-1 and β-amyloid in microglial release of nitric oxide

The beta-amyloid protein associated with Alzheimers disease (AD) has been well characterized biochemically; however, its primary biological function and mode of action in AD has not been determined. We have shown previously that beta-amyloid (beta25-35), in combination with interferon-gamma (IFN-gamma), can induce nitric oxide release from cultured hippocampal microglial cells. In the present study, binding of beta-amyloid with the leukocyte integrin Mac-1, a cell surface receptor on microglia, was studied by observing (1) inhibition of beta-amyloid (beta25-35)-mediated release of nitric oxide from cultured microglial cells following exposure to monoclonal antibodies against Mac-1 (anti-CD18 and anti-CD11b) and (2) competitive binding of fluorochrome-labeled beta25-35 with anti-CD18 or anti-CD11b using fluorescent flow cytometry. Wt.3 (anti-CD18 antibody) and OX42 (anti-CD11b antibody) were as effective as opsonized zymosan at inducing the release of nitric oxide from microglia. Furthermore, Wt.3 and OX42 acted synergistically to induce maximum nitric oxide release. An interaction between beta-amyloid and CD18 of Mac-1 was evidenced by the suppressive action of beta25-35 on Wt.3-mediated release of nitric oxide and the synergistic action between OX42 and beta25-35 in inducing nitric oxide release from microglia. The tissue culture study was supported by competitive binding assays of fluorochrome-labeled beta25-35 and Mac-1 antibodies (Wt.3 or OX42). The majority of microglial cells (71%) did bind biotinylated beta-amyloid in the presence of cytochalasin B, suggesting that beta-amyloid binding to microglia is a receptor-mediated event. Furthermore, pre-exposure to Wt.3, but not OX42, significantly decreased binding of biotinylated beta25-35 to microglia. These findings suggest that CD18 of Mac-1 may play a role in beta-amyloid-mediated release of nitric oxide.

Intranuclear aluminum accumulation in chronic animals with experimental neurofibrillary changes

Young New Zealand white rabbits (1.5 kg body weight) subjected to daily subcutaneous injection of aluminum tartrate (7.7 mg/kg body weight) developed neurofibrillary tangles (NFTs) in specific areas of the central nervous system. The NFTs were initially observed in the spinal cord and brain stem. The hippocampus was the last region to show NFTs. Animals neither died nor showed any neurologic signs. Their growth pattern during and following the aluminum injection was comparable to that of controls. Energy-dispersive X-ray spectrometry detected aluminum within the nucleus of a high percentage of NFT-bearing neurons in the spinal cord and hippocampus. Aluminum was not detected in NFT-free neurons. These findings suggest the association of intranuclear aluminum with NFTs induced experimentally in the chronic animals.

RNA content and volume of nerve cell bodies in human brain. I. Prefrontal cortex in aging normal and demented patients.

The age-related change in the neuronal RNA content, volume, and the RNA concentration of 2,160 single cell bodies was examined from the prefrontal cortex. Human brains from 15 normal and 3 demented patients of ages ranging from 8 months to 94 years were obtained at post-mortem examination. The neuronal RNA showed an adult level at age 9 years and remained unchanged until age 66; the mean RNA content was 27.15 pg during this period of time. A decline in the RNA content followed with increasing ages, but it leveled off to an average of 17.97 pg after the age of 80 years. A comparative observation of morphological changes of normal and demented patients reveal the quantitative spectrum of senile plaques. In spite of the presence of significantly more senile plaques, patients with senile dementia showed the RNA content and the volume of the cell body like those of normal patients of similar age. There seems to be no criterion which is characteristic of senile dementia in terms of the RNA content in cortical cell bodies.