Haruyasu Yamaguchi

Activation of tau protein kinase I/glycogen synthase kinase-3β by amyloid β peptide (25–35) enhances phosphorylation of tau in hippocampal neurons

According to the amyloid hypothesis for the pathogenesis of Alzheimer’s disease (AD), amyloid β peptide (Aβ) directly affects neurons, leading to neurodegeneration and tau phosphorylation, followed by the production of paired helical filaments (PHF) in neurofibrillary tangles (NFT). To analyze the relationship between the phosphorylation sites of tau and the activation of kinases in response to Aβ, we treated cultured rat hippocampal neurons with a peptide fragment of Aβ, Aβ(25–35). Aβ(25–35) treatment activated tau protein kinase I/glycogen synthase kinase-3β (TPK I/GSK-3β) but not glycogen synthase kinase-3α (GSK-3α) or mitogen activated protein kinase (MAP kinase) in primary culture of hippocampal neurons. Using antibodies that recognize phosphorylated sites of tau, we showed that tau phosphorylation was enhanced in at least five sites (Ser199, Ser202, Ser396, Ser404, and Ser413 numbered according to the human tau isoform containing 441 amino acid residues), to an extent that depended on the level of TPK I/GSK-3β. Treatment with TPK I/GSK-3β antisense oligonucleotide inhibited the enhancement of tau phosphorylation induced by Aβ(25–35) exposure. Thus, TPK I/GSK-3β activation by Aβ(25–35) may lead to extensive tau phosphorylation.

Immunohistochemical and immunoelectron microscopic localization of S-100 protein in the interdigitating reticulum cells of the human lymph node.

SummaryLymph nodes of 5 cases with non-specific lymphadenitis and of 4 cases with dermatopathic lymphadenitis were examined by the peroxidase anti-peroxidase (PAP) method and by immunoelectron microscopy using monospecific antibody against nervous system specific S-100 protein. S-100 protein was detected in dendritic shaped cells of the thymus-dependent area of non-specific lymphadenitis. The paracortical area of dermatopathic lymphadenitis showed a marked accumulation of S-100 protein-containing dendritic shaped cells. Immunoelectron micrographs revealed that S-100 proteincontaining d endritic shaped cells of both non-specific and dermatopathic lymphadenitis corresponded to interdigitating reticulum cells (IRC). The detection of S-100 protein in the nucleus and on the cytoplasmic and nuclear membrane suggested that IRCs themselves synthesized S-100 protein. S-100 protein is a very useful cell marker for the identification of IRC in the lymphoid tissue.

S100 protein in folliculostellate cells of the rat pituitary anterior lobe.

The anterior lobe of the rat pituitary was investigated immunohistochemically as to nervous tissue-specifis S100 protein. Granulated cells, both acidophils and basophils, were not stained with peroxidase-labelled anti-S100 rabbit serum (Fab) by the direct staining method. Only stellate-shaped cells with long slender cytoplasmic processes contained nervous tissue-specific S100 protein in the nucleus and cytoplasm, the presence of which was verified by both light and electron microscopic immunohistochemistry. Folliculostellate cells were devoid of specific secretory granules and in areas formed follicles with numerous microvilli and junctional complexes. They often extended long cytoplasmic processes between granulated cells. Ouchterlony double diffusion tests confirmed the presence of S100 protein in the pars distalis of rat pituitary glands. These findings lead to the hypothesis that folliculostellate cells may belong to the neuroectodermal cells and may be specialized cells playing some unknown role in rat adenohypophysis.

A variety of cerebral amyloid deposits in the brains of the Alzheimer-type dementia demonstrated by β protein immunostaining

SummaryWe studied cerebral amyloid deposits in the hippocampal area immunohistochemically, using antiserum to syntheticβ peptide (1–28) in 66 patients with or without dementia and aged 17 to 91 years old. Senile plaques (SP) and amyloid angiopathy (AA) were detected in 36 (55%) and 19 (29%) patients, respectively. Also, cerebral amyloid deposits from the brains of seven patients with dementia and five patients without were studied in serial sections stained with Bodian, modified Bielschowsky, Congo red, andβ protein immunostain. In the patients with Alzheimer-type dementia (ATD) diffuse plaques, typical of this group, were stained withβ protein antiserum but not with Bodian stain, because the plaques were devoid of abnormally swollen neuritic processes. The diffuse plaques often contained one or more neuronal cell bodies. As well as primitive and classic plaques and AA, theβ protein immunostain demonstrated small deposits among the SP, small stellate deposits of layer 1, subpial fibrillar deposits, and focal cribriform deposits of parasubiculum, which may be new types of amyloid deposits. Amyloid plaques within the subcortical white matter were only found in ATD brains. In the non-demented patients various kinds of SP, including diffuse and compact ones, were immunostained. They tended to be small and few.β protein immunostain with formic acid pretreatment is a useful method for the identification of a variety of senile cerebral amyloid deposits.

Preferential labeling of Alzheimer neurofibrillary tangles with antisera for tau protein kinase (TPK) I/glycogen synthase kinase-3β and cyclin-dependent kinase 5, a component of TPK II

Abstract Using immunohistochemistry, we examined the localization of four types of proline-directed kinases in the brains of control rats and in the brains of non-demented aged human subjects, subjects with Alzheimer’s disease and those with Down’s syndrome. The four kinases were: cyclin-dependent kinase (cdk) 5, a component of tau protein kinase (TPK) II; TPK I/glycogen synthase kinase (GSK)-3β; GSK-3α; and mitogen-activated protein kinase (MAPK/ERK2). Each of these kinases has been reported to promote the hyperphosphorylation of tau protein in vitro. The kinases were located essentially in neurons, although the intensity and distribution of labeling varied. Antiserum for cdk5 showed the most preferential and consistent labeling of intraneuronal neurofibrillary tangles (NFT). Antiserum for TPK I/GSK-3β also labeled intraneuronal NFT. Double immunolabeling for TPK I/GSK-3β and tau1 showed that TPK I/GSK-3β was closely associated with NFT. Antiserum for GSK-3α labeled neurons weakly, and the intensity of labeling did not differ between neurons with and without NFT. Antiserum for MAPK labeled neurons in superficial cortical layers, but NFT appeared in both superficial and deep cortical layers. These findings suggest that cdk5 and TPK I/GSK-3β are the critically important kinases for the generation in vivo of hyperphosphorylated tau, the main component of the paired helical filaments in NFT.

Intraneuronal Aβ42 accumulation in Down syndrome brain

Alzheimers disease (AD) brains display A beta (Aβ) plaques, inflammatory changes and neurofibrillary tangles (NFTs). Converging evidence suggests a neuronal origin of Aβ. We performed a temporal study of intraneuronal Aβ accumulation in Down syndrome (DS) brains. Sections from temporal cortex of 70 DS cases aged 3 to 73 years were examined immunohistochemically for immunoreactivity (IR) for the Aβ N-terminal, the Aβ40 C-terminus and the Aβ42 C-terminus. N-terminal antibodies did not detect intracellular Aβ Aβ40 antibodies did not detect significant intracellular Aβ, but older cases showed Aβ40 IR in mature plaques. in contrast, Aβ42 antibodies revealed clear-cut intraneuronal IR. All Aβ42 antibodies tested showed strong intraneuronal Aβ42 IR in very young DS patients, especially in the youngest cases studied (e.g., 3 or 4 yr. old), but this IR declined as extracellular Applaques gradually accumulated and matured. No inflammatory changes were associated with intraneuronal Aβ. We also studied the temporal development of gliosis and NFT formation, revealing that in DS temporal cortex, inflammation and NFT follow A β deposition. We conclude that Aβ42 accumulates intracellulary prior to extracellular Aβ deposition in Down syndrome, and that subsequent maturation of extracellular Aβ deposits elicits inflammatory responses and precedes NFTs.

A Seed for Alzheimer Amyloid in the Brain

A fundamental question about the early pathogenesis of Alzheimers disease (AD) concerns how toxic aggregates of amyloid β protein (Aβ) are formed from its nontoxic soluble form. We hypothesized previously that GM1 ganglioside-bound Aβ (GAβ) is involved in the process. We now examined this possibility using a novel monoclonal antibody raised against GAβ purified from an AD brain. Here, we report that GAβ has a conformation distinct from that of soluble Aβ and initiates Aβ aggregation by acting as a seed. Furthermore, GAβ generation in the brain was validated by both immunohistochemical and immunoprecipitation studies. These results imply a mechanism underlying the onset of AD and suggest that an endogenous seed can be a target of therapeutic strategy.

Diffuse type of senile plaques in the brains of Alzheimer-type dementia

SummaryWe studied the nature of diffuse type of senile plaques (SP) in the brains of six autopsied subjects with Alzheimer-type dementia (ATD). The densities of SP in the entorhinal cortex were evaluated using serial sections stained by four different methods. Compared with β protein immunostaining (100% as a reference), the modified Bielschowsky stain (103%) and the periodic acid-methenamine silver (PAM) stain (109%) labeled similar numbers of SP, whereas the Bodian stain labeled only a minor proportion (42%) of these. The vast majority of Bodian-negative plaques were diffuse plaques, which were seen as ill-defined areas of fine fibrillar material after β protein immunostain with formic acid pretreatment, modified Bielschowsky stain, and PAM stain. They were not stained by Congo red or periodic acid-Schiff stains. Double staining using Bodian and β protein methods demonstrated that diffuse plaques were free of swollen neurites. Argyrophilia of the diffuse plaques shown by the modified Bielschowsky and PAM stains, became undetectable when sections were pretreated with formic acid. Such treatment made the diffuse plaques immunoreactive to β protein antiserum, suggesting that diffuse plaques consisted mainly of amyloid, but not neuritic components. The diffuse plaques were distributed in various cortical areas and in the amygdala, and comprised a considerable population of the SP in the ATD brains.

Phosphorylated tau in human cerebrospinal fluid is a diagnostic marker for Alzheimer's disease.

Microtubule-associated protein tau in cerebrospinal fluid (CSF) has been proposed as a diagnostic marker for Alzheimers disease (AD), but there is overlap between AD patients and non-AD controls. To improve the diagnostic accuracy, we measured phosphorylated tau in CSF, because phosphorylated tau accumulates as pathological paired helical filaments in neurons of the AD brain. Immunoblot showed that CSF contained a 32 kDa N-terminal fragment of tau that was partially phosphorylated on Ser199, Thr231 and Ser235. A sandwich enzyme immunoassay revealed that phosphorylated CSF-tau levels were significantly higher in AD patients than those in non-AD controls. Discrimination between the two groups was clearer in phosphorylated CSF-tau than in total CSF-tau. The data indicate that elevated phosphorylated CSF-tau level is a more specific diagnostic marker for AD.

Oligodendroglial Microtubular Tangles in Olivopontocerebellar Atrophy

Oligodendroglial microtubular tangles (OMT), a distinctive oligodendroglial change, was observed in seven of eight cases of olivopontocerebellar atrophy (OPCA). This change was a well-defined glassy cytoplasmic structure showing intense argyrophilia. OMT were distributed in the brain stem, cerebellum, and basal ganglia where severe neurodegenerative changes were consistently observed. In 45 control cases, no OMT were found regardless of the presence or absence of neurological disorders. The OMT were immunostained by anti-tubulin antibodies, but no other antibodies reacted with them. Each OMT consisted of a meshwork of randomly oriented fibrils studded with granular and fuzzy material. The fibrillary elements were between 20 and 30 nm in diameter. It is suggested that OMT are primarily composed of altered microtubules, and are related to the neurodegenerative process of OPCA.