Tsuyoshi Ishii

Synthetic Alzheimer amyloid β/A4 peptides enhance production of complement C3 component by cultured microglial cells

Primary microglial cultures prepared from newborn mice showed the production and release of the third component of complement (C3). Newly synthesized [35S]methionine-labelled C3 was purified by immunoprecipitation using anti-C3-antibody. C3 was detected by SDS-PAGE and fluoroaraphy of the immunoprecipitated protein from cell lysates as a 195 kDa band, and from the supernatants of cultures as two major bands corresponding to the C3 alpha-chain (125 kDa) and beta-chain (75 kDa), consistent with known C3 characteristics. Increased biosynthesis of C3 was elicited by endotoxin lipopolysaccharide (LPS). Further, the synthesis of C3 was increased 5-10-fold in response to various synthetic peptides corresponding to the amyloid beta/A4 protein, which is the main constituent of extracellular amyloid deposits in Alzheimers disease (AD). The increased synthesis of C3 was shown to be dose dependent at concentrations of beta/A4 peptide ranging from 10 micrograms/ml to 50 micrograms/ml. These results suggest that complement components found previously in amyloid deposits may be partly derived from reactive microglia preferentially associated with senile plaques in AD brain.

Immuno-electron microscopic localization of immunoglobulins in amyloid fibrils of senile plaques.

SummaryAmyloid fibrils in senile plaques of the brain of patients with senile dementia or Alzheimers disease combined specifically with horseradish peroxidase (HRPO)-labeled rabbit anti-human IgG. Light and electron-microscopic immunoperoxidase technique was used to prove this. The fact may mean that immunological factors were involved in the pathogenesis of amyloid fibrils in the senile plaques, and probably also in that of senile dementia or Alzheimers disease.

The immunohistochemical demonstration of subsequences of the precursor of the amyloid A4 protein in senile plaques in Alzheimer's disease.

The actual presence of the predicted precursor of Alzheimers disease amyloid A4 protein, reported by Kang et al. (1987) in the Alzheimer brain, has yet to be verified. To identify the various regions of this precursor, antibodies were raised against three synthetic polypeptides, R35 (residues 274–286), R36 (residues 527–540), and R37 (residues 681–695), subsequences of the precursor protein; the specificity of these antibodies was ascertained by ELISA. Upon immunohistochemical examination, the antibody to R35 failed to react, but the antibody to R36 (the extracellular part) stained the amyloid of senile plaques and the staining pattern was identical to that of anti–A4 antibody. The antibody to R37 (the C–terminal intracellular part) stained what may be degenerating neurites in senile plaques whereas the amyloid remained unstained. An anti–neurofilament (NF) antibody reacted with some of the R37–positive grains, but R37–negative grains also were seen. Further, some R37–positive grains were not stained by the anti–NF antibody. The anti–GFAP antibody and the anti–macrophage antibody did not stain the R37–positive grains. These findings indicate that the amyloid protein in senile plaques actually contains a larger polypeptide than the A4 protein, and suggest that the intracellular C–terminal part of the precursor may exist in the degenerated neurites seen in senile plaques.

Presence of neurofilament protein in Alzheimer's neurofibrillary tangles (ANT)

SummaryLocalization of neurofilament (NF) protein in the neurons of rats brain, spinal cord, and peripheral nerves was studied by the indirect immunofluorescent method using goat-antirabbit plus rabbit anti-NF antibody. The NF protein was purified from the calf brain by the method of Yen et al. (1976). Control sections were treated with nonspecific rabbit serum and with the experimental rabbit serum absorbed with the NF protein. Fluorescence in the axons of the neurons, such as the motor neurons of the spinal cord, peripheral nerves, pontine nuclei, or those of the cerebellar cortex including Purkinje cells, was strong, while that of the dendrites and perikarya was weaker. Glial processes also showed intense fluorescence. Fluorescence in glia cells was interpreted as representing an insufficient separation of NF protein as the antigen from the glial fibrillary acidic protein (GFA). Localization of NF protein in the Alzheimers neurofibrillary tangles (ANT) in the hippocampus of the brain of a patient with Alzheimers disease, was examined by the same way as described above. ANT showed the whole gamut of the color of fluorescence from completely positive green to mixed green with increasingly bluer tints. Astroglial processes also showed intense green fluorescence. The results suggest that ANT comprises NF protein as the structural component and that the increasingly blue fluorescence may represent fewer antigenic sites due to increasing degrees of structural insolubilization or aging of ANT. Possibility of crosslinkage between NF protein and contribution of pathological factors as the cause of ANT formation were discussed.

Complement gene expression in mouse microglia and astrocytes in culture: comparisons with mouse peritoneal macrophages

We investigated the mRNA expression of the various complement components in cultured mouse microglia and astrocytes by the reverse transcription and polymerase chain reaction. C1q, C2, C3, and C4 mRNAs were detected in microglial cultures. C3 and C4 mRNAs were found in astrocyte cultures. Microglia showed enhanced expression of C2, C3, and C4 mRNAs when they were treated with lipopolysaccharide. Much higher expression of C1q, C2, C3, and C4 mRNAs was detected in microglia after stimulation with interferon-gamma. Our data suggest that microglia and astrocytes may produce some of the complement components also in vivo, which can be facilitated in certain infectious and inflammatory diseases in the central nervous system.

Characteristic residual neuropathological features of Japanese B encephalitis.

SummaryCharacteristic residual (12–67 years) neuropathological features of 4 verified or suspected cases of Japanese B encephalitis (JBE) are reported. These features are summarized as: 1. unique distribution pattern of the main lesions, i.e. combination of lesions in the thalamus, substantia nigra and Ammons horn. Lesions in the thalamus consistently involved, in a linear fashion, lamina medullaris medialis with nucleus intralaminalis and adjacent portions of the nucleus lateralis thalami. Lesions in the substantia nigra usually occupied the middle parts of zona compacta. These lesions were usually symmetrical, though unequal in extent. 2. Unique nature of the lesions, especially those in the thalamus and substantia nigra. Characteristic “light circumscribed foci (LCF)”, which consisted of small rarefied areas, with few cellular and fibrous elements, surrounded by dense gliomesenchymal scarring, were observed there and occasionally in cerebral cortices. Lesions were thought to be vestiges of “circumscribed necrotic foci” reported in the CNS of acute stage of JBE. Additional characteristic features in the thalamic lesions were calcified and binucleated nerve cells. Alzheimers neurofibrillary tangles were not found. Authors consider that the distribution and nature of the lesions are of diagnostic value.

The binding of basic fibroblast growth factor to Alzheimer's neurofibrillary tangles and senile plaques

Brain sections from Alzheimers disease (AD) patients and controls were treated with basic fibroblast growth factor (bFGF) and then immunostained with anti-bFGF. Additional sections were treated with biotinylated bFGF without using the anti-bFGF. Labelling was visualized by the ABC method. Both protocols above intensely labelled neurofibrillary tangles, senile plaques and amyloidotic vessels in AD brains. Omission of the bFGF treatment abolished the staining of the AD lesions. The pretreatment of sections with heparitinase also reduced their staining. These results indicate that AD lesions contain bFGF-binding sites and that the chemical substrate for bFGF binding to AD lesions was heparan sulfate.

Presence of Immunoglobulins and Complements in the Amyloid Plaques in the Brain of Patients with Alzheimer’s Disease

The presence of immunoglobulins and complement factors in the amyloid in senile plaques in the brain of six patients with Alzheimer’s disease has been demonstrated by means of immunofluorescence, immunoperoxidase, or the avitin-biotin complex immunoperoxidase (ABC) method.

Ultrastructure of 6-aminonicotinamide (6-AN)-induced lesions in the central nervous system of rats

SummaryFollowing a single i.p. injection of 6-AN (10 mg/kg), the anterior horn cells of 20- and 25-month-old rats increased more in size and recovered slower from chromatolytic changes than those of 3-month-old rats. Neurofilamentous hyperplasia of the perikarya was more prominent in aged rats; proliferated neurofilaments were arranged in thick parallel bundles.In the acute stage, reactive and degenerative changes of glial and mesenchymal elements were more conspicuous in 3-month-old rats; however, they disappeared by day 14 with prominent proliferation of hypertrophic astrocytes.The older rats showed less intensity and slower progression of these changes; sponginess and swelling of the astrocytic cytoplasm were still observed at day 14.Our results suggest that these age-dependent changes in the response to neurotoxins are not only induced on the neuron without mitotic phenomena after birth, but also on neuroglial cells. Furthermore, an alteration or reduction in the support of the neuron augments its intensified and delayed susceptibility to neurotoxins.

Alzheimer amyloid β/A4 peptide binding sites and a possible ‘APP-secretase’ activity associated with rat brain cortical membranes

We carried out ligand binding experiments on membranes from rat brain cortical grey matter using radioiodinated beta/A4 8-17, with non-specific binding determined by the addition of 10 microM unlabelled peptide. Specific, reversible binding amounted to 60-75% of total binding and showed a clear dependence on time, temperature, pH and membrane concentration. Kinetic analyses indicated a high-affinity binding site with an apparent KD of 440 pM. However, the ligand was partly degraded with loss of the Ser8, Lys16 and Leu17 residues. Excision of the two C-terminal amino acids was inhibited by EDTA, EGTA, dithiothreitol or Zn2+ but was stimulated by Ca2+ or Mn2+. These studies demonstrate high-affinity binding sites for beta/A4 8-17 (or its derivatives) in rat brain, suggesting that this region may contain a physiologically important amino acid sequence and identify a potential membrane-associated amyloid precursor protein (APP) secretase activity.