Ryo Fukatsu

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.

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.

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.

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.

Immunohistochemical evidence for amyloid β in rat soleus muscle in chloroquine-induced myopathy

Deposition of amyloid beta (A beta) is one of the pathological hallmarks of brains affected with Alzheimers disease (AD). The accumulation of A beta have been observed in human myopathies with rimmed vacuoles (RVs) which might involve lysosomal function. Chloroquine, a potent lysosomotropic agent, induces muscle pathology in experimental animals similar to myopathy with RV. In this study, we demonstrate, for the first time, immunohistochemical evidence that A beta and cathepsin D, a lysosomal enzyme, accumulate in vacuolated rat soleus muscle due to chloroquine-induced myopathy. These data indicate that lysosomes are important in the metabolism of amyloid precursor protein to generate A beta. This experimental system seems to be useful not only to study basic mechanisms underlying RV myopathy but also to understand processing of amyloid precursor protein to A beta in AD.

Evidence for presenilin-1 involvement in amyloid angiopathy in the Alzheimer's disease-affected brain

Presenilin-1 (PS-1) has been identified as the protein encoded by the chromosome 14 locus that, when mutated, leads to familial Alzheimers disease (FAD). The role PS-1 plays in the pathogenesis of Alzheimers disease (AD) remains unclear. Using a set of antibodies raised against PS-1 synthetic peptides, polyclonal antibody to amyloid beta protein (Abeta) and end-specific antibodies against Abeta40, and Abeta42, immunohistochemical studies were performed on brain sections obtained from AD cases and controls. The PS-1 antibodies clearly stained amyloid angiopathies in AD-affected brains, but no recognizable immunoreactions were observed in any other vessels free from amyloid involvement in either AD-affected brains or controls. Abeta antibodies and the end-specific antibody against Abeta40 also decorated amyloid angiopathies, showing localization similar to that of PS-1. Western blot analyses predominantly detected protein band polypeptide species of a 50 kDa, band, presumably full-length PS-1 protein with N-terminus antisera, since these antibodies turned out to recognize a 50-kDa full-length band in cell lysate of transfected HeLa cell overexpressing PS-1. In addition, we recognized 30, 27 and 25 kDa proteins in both AD and control brain homogenate with these antibodies. In microvessel fractions extracted from brain homogenates, the 50, and 27 kDa fragments were observed in AD-affected brains but not in those of controls. C-terminus rabbit antisera reacted strongly with the 33 and 27 kDa bands, and additionally detected a small amount of full-length PS-1 protein in extracts from AD and control brains. Our present data indicate that PS-1 might be involved in the pathogenesis of amyloid angiopathy in the AD brain.

Co-localization of amyloid-associated proteins with amyloid β in rat soleus muscle in chloroquine-induced myopathy: a possible model for amyloid β formation in Alzheimer's disease

Chloroquine, a potent lysosomotropic agent, induces myopathy in experimental animals similar to rimmed vacuole (RV) myopathy in humans. The abnormal accumulation of amyloid beta protein (A beta), which is the invariable pathological alterations in the brains affected by Alzheimers disease (AD), has been demonstrated in denervated soleus muscle fibers in chloroquine-induced myopathy in rats. In AD affected brains, a variety of additional proteins are associated with the extracellular deposition of A beta, which leads to the intracellular accumulation of neurofibrillary tangles and finally to neuronal death. In this study, we demonstrate that amyloid-associated proteins, alpha 1-antichymotrypsin, apolipoprotein E, SP-40,40 and ubiquitin co-localize with A beta in vacuolated muscle fibers in chloroquine-induced myopathy. There are striking similarities in immunopathology between experimental RV myopathy and AD. Chloroquine-induced myopathy in rats provides a suitable model not only to obtain insight into the basic mechanisms underlying RV formation in muscle, but also to understand amyloid precursor protein processing into A beta, and the role of amyloid-associated proteins in terms of the pathogenesis of AD.

Pathologic processes leading to cerebral hemorrhage in amyloid angiopathy

This report concerns a study undertaken to eluidate the pathogenesis of arterial rupture related to amyloid deposition. For this purpose, histochemical and immunohistochemical analyses were carried out on brain tissue specimens of a case of cerebral amyloid angiopathy (CAA) with multiple cortical hemorrhages. A small amount of amyloid β‐protein was found in the tunica media vasorum in which the smooth muscle cells were well preserved. with increases in amyloiod deposition, the entire arteriolar wall became concenmtrically thick, with amyloid largely occupying the tunica media; the number of smooth muscle cells was decreased and these cells were located in the intimal side of the vessel wall. Under these conditions, the elastic fibers and endothhelial cells were relatively well preserved. In the advanced stage of amyloid deposition, the arterioles became devoid of smooth muscle cells and underwent wither fibrous luminal occlusionor aneurysmal dilatation with fibrinoid necrosis and loss of elastic fibers; the latterwould eventually rupture causing the hemorrhage. These findings suggest that amyloid β‐protein‐related loss of arteriolar smooth muscle cells could be the initial event, with the subsequent damage of the vascular wall leading to cerebral hemorrhages in CAA patients.