Takemi Kimura

Identification of monocyte chemoattractant protein-1 in senile plaques and reactive microglia of Alzheimer's disease

Abstract It has been shown that human monocytes express monocyte chemoattractant protein‐1 (MCP‐1), an inflammatry factor, in response to non‐fibrillar β‐amyloid protein. Reactive microglia and inflammatory factors were reported to be present in β‐amyloid deposits (senile plaques) in Alzheimers disease, suggesting the presence of MCP‐1 in senile plaques. To address this issue, we examined MCP‐1‐immunoreactivity in senile plaques using a mouse monoclonal anti‐MCP‐1 antibody. Monocyte chemoattractant protein‐1 was found immunohistochemically in mature senile plaques and reactive microglia but not in immature senile plaques of brain tissues from five patients with Alzheimers disease. These findings suggest that MCP‐1‐related inflammatory events induced by reactive microglia contribute to the maturation of senile plaques.

Sequential Changes of Tau-Site-Specific Phosphorylation during Development of Paired Helical Filaments

It has been reported that many tau sites in neurofibrillary tangles (NFT) are abnormally phosphorylated. We investigated the phosphorylation of tau in the hippocampus of nondemented patients and Alzheimers disease patients by immunostaining with five site-specific antibodies against phosphorylated tau. In the pretangle stage, tau in neuropil threads was phosphorylated at serines 199, 202 and 409, numbered according to the longest human tau isoform, whereas tau in some neuronal soma was phosphorylated at serines 199, 202, 409 and 422. Tau at the stage of NFT was phosphorylated at serine 396 and threonine 231 in addition to serines 199, 202, 409 and 422. In the advanced stage, tau in ghost tangles was phosphorylated mainly at serine 396. These results suggest that the phosphorylation of each site in tau differs among the maturing stages of neurofibrillary change and that abnormal phosphorylation of tau in the neuronal soma occurs at 199, 202, 409 and 422 earlier than at threonine 231 and serine 396.

Accumulation of advanced glycation end products of the Maillard reaction with age in human hippocampal neurons.

The recent immunological demonstration of advanced glycation end products (AGE) of the Maillard reaction in several human tissues suggests a possible involvement of AGE in the aging process. We previously prepared a monoclonal anti-AGE antibody (6D12) which recognized N epsilon-(carboxymethyl)lysine. We examined, immunohistochemically, the effect of aging on AGE-immunoreactivity in hippocampal pyramidal neurons in ten brain tissue samples obtained at autopsy from subjects aged 20-85 years old. Using 6D12 antibody, our results demonstrated a positive correlation between AGE-immunoreactivity in hippocampal pyramidal neurons and age. A more intense immunoreaction was observed in the CA3-4 pyramidal neurons compared with that of the CA1 neurons, known to be vulnerable to the formation of neurofibrillary tangles. Our results suggest that AGE are probably involved in the aging process affecting the human central nervous system, and that AGE do not mainly contribute to the formation of neurofibrillary tangles, at least in the CA1 neurons.

Role of Blood Vessels in Producing Pathological Changes in the Brain with Alzheimer's Disease

Abstract: Vascular factors have been shown to be highly involved in the deposition of the amyloid β‐protein (Aβ) in the brain of Alzheimers disease (AD). However, the detailed mechanism remains unknown. Here, we showed that more numerous deposits of Aβ40 and Aβ42 in the brain were found in AD patients than in controls. Together with evidence of no difference in the level of Aβ40 and Aβ42 in sera between sporadic AD and conrols, a certain dysfunction of the blood‐brain barrier could induce an abnormal transport of Aβ from sera to the parenchyma in AD. In addition, vascular Aβ deposits and mature Aβ plaques stained by Congo red in AD brains contained more Aβ40 than Aβ42, whereas Congo red‐negative immature plaques mainly consisted of Aβ42. Our confocal laser scanning microscopy demonstrated an intimate relationship between Aβ40 and the vascular network. The amount of mature plaques but not that of immature plaques was reportedly correlated with the severity of dementia in AD patients. These results suggest that serum‐derived Aβ40 and/or Aβ42 cause Aβ40 deposition in and around blood vessels through unknown but possible mechanisms such as (1) endocytosis of Aβ40, (2) selective transport Aβ40 and Aβ42 into blood vessels and the parenchyma, respectively, and (3) proteolysis of Aβ42 into Aβ40 induced by a putative carboxyl dipeptidase in blood vessels including vascular feet, which is involved in Aβ fibrillation and cognitive deterioration in the patients. Therefore, the accumulation of Aβ40 associated with blood vessels may play a critical role in the development of AD.

Are advanced glycation end-products associated with amyloidosis in Alzheimer's disease?

Recent studies have suggested that advanced glycation end-products (AGE) of the Maillard reaction are associated with amyloidosis in Alzheimer’s disease. To evaluate this possibility, the present immunohistochemical study was undertaken to locate AGE in cerebral cortices of Alzheimer’s disease, using a monoclonal antibody specific for AGE-proteins. Deposits of β-amyloid protein within cores of classic senile plaques and vascular walls in amyloid angiopathy showed no AGE-positive reaction, while primitive plaques, coronas of classic plaques and some glial cells were positive for AGE. These findings are inconsistent with the suggestion that AGE may be involved primarily in amyloidosis in Alzheimer’s disease.

Localization of identified advanced glycation end-product structures, Nε=(carboxymethyl)lysine and pentosidine, in age-related inclusions in human brains

The recent identification of age‐related accumulation of advanced glycation end‐products (AGE) of the Maillard reaction In neurons and vessels of the human brain suggests the involvement of AGE in the aging process. A variety of inclusions such as lipofuscin granules, corpora amylacea, Hirano bodies, granulovacuolar degenerations and ubiquitin‐positive granular structures are found in the aged human brain. These age‐related Inclusions contain insoluble and non‐degradable proteins. Advanced glycation end‐product‐modified proteins are also known to be insoluble and protease resistant. The similarlty between proteins in such inclusions and AGE‐modified proteins suggests the presence of AGE in inclusions. To investigate this possiblllty, the presence of two known AGE structures, N‐(carboxymethyl)lysine (CML) and pentosidine, was exarnined in age‐related inclusions. immunohistochemical examination of the medial temporal area of brain tissues obtained at autopsy from seven nondemented elderly individuals demonstrated positive reactions in lipofuscin granules and corpora amylacea but not in other inclusions for anti‐CML and anti‐pentosldine antibodies. As CML and pentosidine are glycoxidation products among AGE, the results suggest that glycation andor Oxidation may be involved In the formation of lipofuscin granules and corpora amylacea.

Accumulation of imidazolone, pentosidine and Nε-(carboxymethyl)lysine in hippocampal CA4 pyramidal neurons of aged human brain

Previous studies from our laboratory demonstrated that Nɛ‐(carboxymethyl)lysine (CML), one of the major advanced glycation end products (AGE), was accumulated in human pyramidal neurons in the hippocampus in an age‐dependent manner. This suggests a potential link between AGE‐accumulation and the aging process in neurons. The purpose of the present study was to examine whether this notion could be extended to other AGE structures, such as imidazolone and pentosidine. This was done using 19 human brains that were not affected by dementia. The immunohistochemical survey on distribution in brain tissues of imidazolone and pentosidine was carried out with monoclonal antibodies specific for imidazolone and pentosidine. A parallel control experiment was carried out with anti‐CML antibody. The results showed that pentosidine and imidazolone were localized in neurons in different areas of human brain tissue, especially in neurons of CA4 in the hippocampus. The characteristic distribution of pentosidine and imidazolone is very similar to that of CML. Furthermore, when the accumulation of these AGE structures was compared with the age of individual brains it was found that accumulation of imidazolone, pentosidine and CML in the CA4 region increased with age. These findings taken together support the notion that the accumulation of AGE structures in the CA4 region might be closely related to the aging process in neurons.

Identification of advanced glycation end products of the Maillard reaction in Pick's disease

The recent identification of advanced glycation end products (AGEs) of the Maillard reaction in the central nervous system suggests their potential involvement in the aging process. Other studies have also suggested a possible link between AGEs and Alzheimers disease. To investigate the role of AGEs in Picks disease, another neurodegenerative disease associated with dementia, we examined the AGE-immunoreaction in Picks bodies and ballooned neurons, two characteristic neuropathological features of Picks disease, using antibodies against identified AGE-structures, N epsilon-(carboxymethyl)lysine and pentosidine. These two structures were observed immunohistochemically in Picks bodies and ballooned neurons of brain tissues obtained at autopsy from three elderly patients diagnosed with Picks disease. These immunoreactions were further confirmed by absorption experiments and double-immunostaining. The present findings suggest that AGEs are involved in the pathology of Picks disease.

Nuclear Localization of the δ Subunit of Ca2+/Calmodulin-Dependent Protein Kinase II in Rat Cerebellar Granule Cells

Abstract : To examine the physiological roles of the δ subunit of Ca2+/calmodulin‐dependent protein kinase ∥ (CaM kinase ∥δ) in brain, we examined the localization of CaM kinase ∥δ in the rat brain. A specific antibody to CaM kinase ∥δ1‐δ4 isoforms was prepared by immunizing rabbits with a synthesized peptide corresponding to the unique carboxyl‐terminal end of these isoforms. The prepared antibody did not recognize the α, β, and γ subunits, which were each overexpressed in NG108‐15 cells. Immunoblot analysis on various regions and the nuclear fractions from rat brains suggested that some isoforms of CaM kinase ∥δ1‐δ4 were abundant in the nucleus in the cerebellum. Total RNA from the cerebellum was analyzed by RT‐PCR with a primer pair from variable domain 1 to variable domain 2. We detected the three PCR products δ3.1, δ3.4, and δ3 that contained the nuclear localization signal. These CaM kinase ∥δ3 isoforms were localized in the nuclei in transfected NG108‐15 cells. Immunohistochemical study suggested the existence of these isoforms in the nuclei in cerebellar granule cells. These results suggest that CaM kinase ∥δ3 isoforms are involved in nuclear Ca2+ signaling in cerebellar granule cells.

Observations of Microvessels in the Brain with Alzheimer's Disease by the Scanning Electron Microscopy

Abstract: Microvessels in the brain with Alzheimers disease and those of a control group were observed by means of the scanning electron microscopy. There were no morphological differences between the relatively large arteries in Alzheimers disease and those of the control. Remarkable differences existed in terminal arterioles and capillaries. In Alzheimers brain, the terminal arterioles frequently had focal constrictions and smooth muscle cells with an irregular shape and arrangement, and the capillaries also showed an irregular abluminal surface and irregular constriction and dilatation along their paths. In the control brain, there were no changes such as those observed in Alzheimers disease. The findings suggest that diffuse neuronal loss in Alzheimers cerebral cortex might be induced by circulatory disturbance through the changed micro‐vessels.