Seiichi Haga

Demonstration of microglial cells in and around senile (neuritic) plaques in the Alzheimer brain

SummaryA monoclonal antibody, termed AD11/8, reactive to microglial cells, was produced by immunization of mice with partially purified amyloid fibrils of senile (neuritic) plaques. With immunoperoxidase staining on human tissues, AD11/8 also recognized macrophages in the red pulp of the spleen, Kupffer cells in the liver, and macrophages in the bone marrow. The results show that AD11/8 recognizes the antigens associated with mononuclear phagocytes lineage. In normal brains a few resting microglial cells were stained in gray matter, and less frequently in white matter. In senile dementia of the Alzheimer type numerous microglial cells were stained intensively and they often formed clusters in gray matter. By double immunostaining with AD11/8 and a polyclonal antibody against synthetic amyloid β-protein, clustered microglial cells were observed in and around senile plaques with amyloid deposits. Some amyloid plaque cores were surrounded by microglial cell processes. These results indicate that microglial cells may play an important role in senile plaque formation.

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.

Enhanced carbonyl stress in a subpopulation of schizophrenia.

CONTEXT Various factors are involved in the pathogenesis of schizophrenia. Accumulation of advanced glycation end products, including pentosidine, results from carbonyl stress, a state featuring an increase in reactive carbonyl compounds (RCOs) and their attendant protein modifications. Vitamin B(6) is known to detoxify RCOs, including advanced glycation end products. Glyoxalase I (GLO1) is one of the enzymes required for the cellular detoxification of RCOs. OBJECTIVES To examine whether plasma levels of pentosidine and serum vitamin B(6) are altered in patients with schizophrenia and to evaluate the functionality of GLO1 variations linked to concomitant carbonyl stress. DESIGN An observational biochemical and genetic analysis study. SETTING Multiple centers in Japan. PARTICIPANTS One hundred six individuals (45 schizophrenic patients and 61 control subjects) were recruited for biochemical measurements. Deep resequencing of GLO1 derived from peripheral blood or postmortem brain tissue was performed in 1761 patients with schizophrenia and 1921 control subjects. MAIN OUTCOME MEASURES Pentosidine and vitamin B(6) concentrations were determined by high-performance liquid chromatographic assay. Protein expression and enzymatic activity were quantified in red blood cells and lymphoblastoid cells using Western blot and spectrophotometric techniques. RESULTS We found that a subpopulation of individuals with schizophrenia exhibit high plasma pentosidine and low serum pyridoxal (vitamin B(6)) levels. We also detected genetic and functional alterations in GLO1. Marked reductions in enzymatic activity were associated with pentosidine accumulation and vitamin B(6) depletion, except in some healthy subjects. Most patients with schizophrenia who carried the genetic defects exhibited high pentosidine and low vitamin B(6) levels in contrast with control subjects with the genetic defects, suggesting the existence of compensatory mechanisms. CONCLUSIONS Our findings suggest that GLO1 deficits and carbonyl stress are linked to the development of a certain subtype of schizophrenia. Elevated plasma pentosidine and concomitant low vitamin B(6) levels could be the most cogent and easily measurable biomarkers in schizophrenia and should be helpful for classifying heterogeneous types of schizophrenia on the basis of their biological causes.

Association between polymorphisms in the promoter region of the sialyltransferase 8b (SIAT8B) gene and schizophrenia

BACKGROUND Sialyltransferase 8B (SIAT8B) and 8D (SIAT8D) are two polysialyltransferases that catalyze the transfer of polysialic acid (PSA) to the neural cell adhesion molecule 1 (NCAM1). PSA modification of NCAM1 plays an important role in neurodevelopment of the brain and disruption of this process is postulated as an etiologic factor in psychiatric disorders. Altered levels of the PSA-NCAM1 in the brain of schizophrenics have been reported, suggesting a role for this molecule in the disorder. METHODS We performed an association study of single nucleotide polymorphisms (SNPs) within SIAT8B and SIAT8D, using 188 schizophrenics and 156 age and gender matched controls. All genotypes were determined by polymerase chain reaction (PCR) amplification and direct sequencing. RESULTS Two polymorphisms, -1126T > C and -851T > C, located in the promoter region of SIAT8B showed nominally significant association with schizophrenia (allelic associations, p = .014 and p = .007, respectively), and haplotypes constructed from three additional SNPs located in the same linkage disequilibrium block were associated with schizophrenia. Furthermore an in vitro promoter assay revealed that a reporter construct containing a risk haplotype for SIAT8B had significantly higher transcriptional activity compared with one containing a protective haplotype (p = .021). In contrast, no significant association was observed between any variations in SIAT8D and schizophrenia. CONCLUSIONS The present study suggests that functional promoter SNPs of SIAT8B could confer a risk for schizophrenia in the Japanese population.

Reduced neuropeptide Y mRNA levels in the frontal cortex of people with schizophrenia and bipolar disorder.

To study the change of gene expression in the brain tissues of schizophrenia, we used the gene expression monitoring technology and compared two sets of pools each containing four RNA samples of frontal cortex that were randomly selected from the control or schizophrenia group. We found that the expression of two genes were commonly altered in four pairwise comparisons; the expression of DEAD-box protein p72 (p72) gene was increased and neuropeptide Y (NPY) gene expression was decreased in the schizophrenia group compared with the control group. To substantiate these results, we estimated their mRNA levels by the real time TaqMan method in the 15 samples of each frontal or temporal cortex of four matched groups of schizophrenia, bipolar disorder, major depression and normal controls. A statistically significant decrease was observed for NPY in the frontal, but not in the temporal cortex, in the schizophrenia group (P=0.003). A decrease was also observed in the frontal cortex of the bipolar disorder group (P=0.031). In contrast, p72 gene expression showed no significant difference among the four groups. In conclusion, by novel technology of DNA array and TaqMan PCR analyses, we found that neuropeptide Y mRNA levels were significantly reduced in the frontal cortex in both schizophrenia and bipolar disorder.

Association between a novel polymorphism in the promoter region of the neuropeptide Y gene and schizophrenia in humans.

Hypoactivity of neuropeptide Y (NPY) is thought to be involved in the pathophysiology of schizophrenia, because post-mortem brain studies revealed a decrease of the NPY in schizophrenia, and antipsychotic treatments increase the NPY in animal brains and in cerebrospinal fluid of patients. We performed genetic analysis of the NPY gene in schizophrenia. Mutation screening of the gene detected nine single nucleotide polymorphisms, of which we typed a -485C>T variation from potential functional relevance. The -485T allele was overly represented in the disease group (P=0.0043). An in vitro promoter assay revealed that a C to T change at nt -485 significantly reduced transcriptional activity. These results suggest that the -485T allele in NPY may confer susceptibility to schizophrenia by decreasing the neuropeptide in brains.

Early senile plaques in Down's syndrome brains show a close relationship with cell bodies of neurons

A sensitive methenamine silver/Nissl stain was used to study the morphology and relationship of pre–plaques (presumed early senile plaques) in Downs syndrome brains to glial nuclei, capillaries and neuronal perikarya. The larger pre–plaques (> 50 μm) usually encompassed all of these tissue elements. However, the smaller pre–plaques (< 50 μm) were almost always found immediately adjacent to, or around the cell bodies of neurons (often with associated satellite cells), and they failed to show any consistent, close spatial relationship to the other tissue components. Thus we consider an early stage of pre–plaque formation to be the deposition of amyloid adjacent to the cell body of a morphologically normal neuron. Based on the study of transitional forms, we suggest that the amyloid progressively accumulates around the cell body until the enclosed neuron degenerates. How these pre–plaque lesions might eventually develop into the typical plaque structure is uncertain. Our observations support the theory of a neuronal origin for plaque amyloid.

Association of neural cell adhesion molecule 1 gene polymorphisms with bipolar affective disorder in Japanese individuals

BACKGROUND Although the pathogenesis of mood disorders remains unclear, heritable factors have been shown to be involved. Neural cell adhesion molecule 1 (NCAM1) is known to play important roles in cell migration, neurite growth, axonal guidance, and synaptic plasticity. Disturbance of these neurodevelopmental processes is proposed as one etiology for mood disorder. We therefore undertook genetic analysis of NCAM1 in mood disorders. METHODS We determined the complete genomic organization of human NCAM1 gene by comparing complementary deoxyribonucleic acid and genomic sequences; mutation screening detected 11 polymorphisms. The genotypic, allelic, and haplotype distributions of these variants were analyzed in unrelated control individuals (n = 357) and patients with bipolar disorder (n = 151) and unipolar disorder (n = 78), all from central Japan. RESULTS Three single nucleotide polymorphisms, IVS6+32T>C, IVS7+11G>C and IVS12+21C>A, displayed significant associations with bipolar disorder (for allelic associations, nominal p =.04, p =.02, and p =.004, respectively, all p >.05 after Bonferroni corrections). Furthermore, the haplotype located in a linkage disequilibrium block was strongly associated with bipolar disorder (the p value of the most significant three-marker haplotype is .005). CONCLUSIONS Our results suggest that genetic variations in NCAM1 or nearby genes could confer risks associated with bipolar affective disorder in Japanese individuals.