Kayo Tsuzuki

Advanced Glycation End Products in Alzheimer’s Disease and Other Neurodegenerative Diseases

Advanced glycation end products (AGEs) have been implicated in the chronic complications of diabetes mellitus and have been reported to play an important role in the pathogenesis of Alzheimers disease. In this study, we examined the immunohistochemical localization of AGEs, amyloid beta protein (A beta), apolipoprotein E (ApoE), and tau protein in senile plaques, neurofibrillary tangles (NFTs), and cerebral amyloid angiopathy (CAA) in Alzheimers disease and other neurodegenerative diseases (progressive supranuclear palsy, Picks disease, and Guamanian amyotrophic lateral sclerosis/Parkinsonism-dementia complex). In most senile plaques (including diffuse plaques) and CAA from Alzheimers brains, AGE and ApoE were observed together. However, approximately 5% of plaques were AGE positive but A beta negative, and the vessels without CAA often showed AGE immunoreactivity. In Alzheimers disease, AGEs were mainly present in intracellular NFTs, whereas ApoE was mainly present in extracellular NFTs. Picks bodies in Picks disease and granulovacuolar degeneration in various neurodegenerative diseases were also AGE positive. In non-Alzheimer neurodegenerative diseases, senile plaques and NFTs showed similar findings to those in Alzheimers disease. These results suggest that AGE may contribute to eventual neuronal dysfunction and death as an important factor in the progression of various neurodegenerative diseases, including Alzheimers disease.

Transthyretin binds amyloid β peptides, Aβ1-42 and Aβ1-40 to form complex in the autopsied human kidney: possible role of transthyretin for Aβ sequestration

Abstract The deposition of amyloid β protein (A β ), a proteolytic cleavage product of amyloid precursor protein (APP), is an invariable pathological feature of the Alzheimers disease brain, while APP gene is widely expressed in all neuronal and non-neuronal tissues with the highest levels of expression in the brain, and kidney. To understand the role transthyretin (TTR) plays in the sequestration mechanism of A β in the kidney, we have investigated interactions of TTR with A β 1–40 and A β 1–42 molecules by an immunoprecipitation method, in vitro binding studies, and overlay assay. These in vivo and in vitro biochemical experiments showed that TTR bound A β 1–42 preferentially, and A β 1–40 only to a limited extent, to form TTR-monomer and -dimer-A β complexes in the normal human kidney. We provide new evidence supporting the hypothesis that TTR, an A β binding protein, plays an important role in the sequestration of A β and prevents amyloid formation in the kidney.

The complete nucleotide sequence of the gene encoding the nontoxic component of Clostridium botulinum type E progenitor toxin

We have analysed the genes borne on a 6.0 kb HindIII fragment cloned from the chromosome of Clostridium botulinum type E strain Mashike. This fragment, cloned within plasmid pU9EMH, contains part of the structural gene for botulinum toxin type E neurotoxin as well as the entire structural gene for a nontoxic component of botulinum type E progenitor neurotoxin gene, ent-120. ent-120 is transcribed in the same direction as the neurotoxin gene and consists of one open reading frame encoding 1162 amino acid residues. Western blotting with anti-nontoxic component sera demonstrates that ent-120 encodes a protein of 120 kDa which forms part of the nontoxic component. ent-120 is homologous to an analogous gene found in botulinum type C strains (69.3% identity at the nucleotide level and 56.1% at the amino acid level). Two stretches of amino acids at the N-terminus of the ent-120 protein are highly homologous to amino acid sequences within the type E neurotoxin. The stop codon of the ent-120 gene is situated 27 nucleotides upstream from the start codon of the neurotoxin gene.

Amino-terminus truncated apolipoprotein E is the major species in amyloid deposits in Alzheimer's disease-affected brains: a possible role for apolipoprotein E in Alzheimer's disease

Amyloid deposits in Alzheimers disease (AD) are composed of amyloid beta protein (A beta) and many other components called amyloid-associated proteins. Apolipoprotein E (apoE) is one of the most important amyloid-associated proteins. The role apoE plays in AD, however, is yet to be determined. In this study, we present the biochemical and histochemical nature of apoE in AD-affected brains using four monoclonal antibodies (mAbs) against apoE and newly established antibodies against the amino-terminal (anti-apoE-N), and carboxyl-terminal regions (anti-apoE-C) of apoE. Competitive ELISA and Western-blot analysis combined with thrombolytic digestion of apoE indicated that our four mAbs recognized at least two different epitopes within a 22-kDa amino-terminal domain of apoE. Using these mAbs and an anti-A beta mAb, double immunostaining showed that the majority of amyloid deposits were stained by both anti-apoE and anti-A beta mAbs, but the minority of them were detected only by either anti-apoE or anti-A beta mAbs. Differences in staining properties between anti-apoE-N and anti-apoE-C were that anti-apoE-C recognized both amyloid deposits and astrocytes similar to anti-apoE mAbs, but anti-apoE-N strongly stained only astrocytes. Preliminary semi-quantitative determinations of apoE in CSF and brain homogenate showed that the amount of apoE increased in AD and Creutzfeldt-Jakob disease brains compared to normal samples. Our immunological data, using antibodies specific for the amino and carboxyl termini of apoE, suggest that apoE may, in some circumstances, initiate plaque formation, and that apoE in amyloid deposits has at least part of its amino termini cleaved out.

A zinc-protease specific domain in botulinum and tetanus neurotoxins

Neurotoxins produced by Clostridium botulinum are classified into groups (A to G) based on their serological nature. They consist of two subunits, heavy and light chains, linked by one or more disulphide bridges. The light chain is responsible for the blocking of acetylcholine release. Amino acid sequences of light chains have already been reported for botulinum toxins types A, C, D and E. Five highly homologous regions are found between these four toxins. One of these homologous regions, sequence HELIHSL, shows strong similarity with the active site of zinc-proteases. We suggest that inhibition of acetylcholine release might be associated with this protease activity.

Potentially amyloidogenic fragment of 50 kDa and intracellular processing of amyloid precursor protein in cells cultured under leupeptin

The principal neuropathological feature of Alzheimers disease is extracellular deposition of approximately 4-kDa proteinous fragment, designated as beta-amyloid peptides (beta/A4 peptides) derived by proteolytic cleavage from amyloid precursor protein (APP), a large cell-surface receptor-like protein. There has been evidence that APP is proteolytically degraded in the secretory and endosomal/lysosomal pathways. The pathway in which APP is cleaved to generate beta/A4 peptides is still not identified. To clarify the intracellular processing of APP into the generation of beta/A4 peptides, we detected and characterized potentially amyloidogenic or non-amyloidogenic fragments using newly established monoclonal and polyclonal antibodies in the cultured cells with or without leupeptin, potent lysosomal protease inhibitor of lysosome. APP fragments of 50 and 20 kDa containing full-length beta/A4 peptides were identified in the cultured cells. Immunoblot analysis, biochemical study for specific marker enzyme activity of the fractions obtained from subcellular fractionation, sucrose density gradient centrifugation indicated that the 50-kDa APP fragment was produced in the compartment closely related to endosomal/lysosomal system. Our data suggest that the endosomal/lysosomal pathway is involved in the processing and generation of beta/A4 peptides.

Amyloid β protein and transthyretin, sequestrating protein colocalize in normal human kidney

The localization of amyloid beta protein (A beta), A beta 40, A beta 42, and transthyretin (TTR) was investigated immunohistochemically in the autopsied human kidney, using polyclonal antibodies against TTR, A beta and C-terminal end-specific antibodies against A beta 40 and 42. Immunoreactivities of A beta and A beta 40 were found both in the proximal and distal tubular epithelial cells. But the immunolocalization of A beta 40 was observed predominantly in the distal tubules whereas that of A beta 42 was predominantly recognized in the proximal tubules. TTR, sequestrating protein for A beta, was present in the proximal tubules. The mechanism by which A beta does not form amyloid in Alzheimers disease outside the brain remains unknown. The tubular epithelial cells in the kidney may provide a useful system to shed light on this issue.

Amyloid β protein in rat soleus muscle in chloroquine-induced myopathy using end-specific antibodies for Aβ40 and Aβ42: immunohistochemical evidence for amyloid β protein

Abstract Previous immunohistochemical studies from this laboratory demonstrated that monoclonal antibodies raised against various regions of amyloid precursor protein (APP) (i.e., N-terminus, amyloid β protein (Aβ), and C-terminus) strongly labeled vacuoles in chloroquine-induced myopathy-affected muscle in rats. In this study, we used antibodies end specific for the Aβ40 and Aβ42 species, and a monoclonal antibody to A01-9 which reacts with APP and Aβ. Most vacuoles clearly reacted with anti-Aβ1–9, while about half reacted with anti-Aβ42, and only a few reacted with anti-Aβ40. These results demonstrate that vacuoles in chloroquine-induced myopathy-affected muscle contain cleaved Aβ, and that distribution of the two major Aβ species is similar to what is observed in Aβ deposition in Alzheimers disease (AD)-affected brain. This provides further evidence that chloroquine-induced myopathy in rats provides a suitable model to understand APP processing into Aβ, and the role of APP in terms of the pathogenesis of AD.

Binding of botulinum type C1, D and E neurotoxins to neuronal cell lines and synaptosomes

Clostridium botulinum 125I-labeled Cl neurotoxin bound to NG108 hybridoma cell line. Unlabeled type Cl neurotoxin inhibited the binding of the labeled Cl toxin but neither types D nor E toxin. 125I-labeled type D neurotoxin bound to rat brain synaptosomes but did not bind to NG108 cells. It is suggested that receptors for types C and D or E toxins on neuronal cell membranes are different.

Amyloid precursor protein, Aβ and amyloid-associated proteins involved in chloroquine retinopathy in rats – immunopathological studies

To understand the retinal changes in Alzheimer disease (AD) patients, pathological and immunocytochemical studies were performed on retinal cells in the chloroquine-treated rats at 0, 4, 8, 12, 16, 20, and 24 weeks after the initial injection, using anti-amyloid precursor protein (APP), -amyloid beta protein (A beta), -apolipoprotein E (apoE), -ubiquitin, and -cathepsin D antibodies. Pathological alterations consistent with chloroquine retinopathy were recognized in the ganglion cells of the ganglion cell layer (GCL) and the inner plexiform layer (IPL) 4 weeks after initial chloroquine injection. Rat retinal changes appear to have a direct relationship to the duration of chloroquine administration. Intense immunoreactivities for anti-APP, A beta, apoE (an associated protein), and ubiquitin co-localized in the swollen ganglion cells and Muller cells by 20-24 weeks together with the lysosomal enzyme cathepsin D. The present data indicate that the endosomal/lysosomal pathway plays an important role in the processing of APP in rat retina. This experimental model is considered to be a suitable neural model to understand retinal pathology and the processing of APP in terms of the pathogenesis of AD, whereas chloroquine-induced myopathy is a useful extra neuronal model.