Yuji Takamaru

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.

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.

An adult-type metachromatic leukodystrophy caused by substitution of serine for glycine-122 in arylsulfatase A

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease with autosomal recessive inheritance caused by a deficiency of the enzyme arylsulfatase A (ASA). We have identified a new mutation in the ASA gene of a patient with adult-type MLD. In this mutation, the glycine at position 122, a highly conserved residue in the AS gene family, was replaced by serine. In a transient expression study, COS cells transfected with the mutant cDNA carrying 122Gly→Ser did not show an increase of ASA activity and produced little material immunoreactive to an anti-ASA antibody, despite normal mRNA levels.

Snake coiled fibres in rat soleus muscle in chloroquine induced myopathy share immunohistochemical characteristics with amyloid depositions in Alzheimer's disease brain tissue

Pathological and immunopathological studies were carried out on snake coiled fibres (SCF) which occurred in affected soleus muscle in chloroquine treated rats. The SCF began to appear in denervated soleus muscle by 8 days after chloroquine injection. By day 14, typical SCF were observed with an unusual swirling pattern of the myofibrils, presenting a bizarre appearance. By day 21 or later, the SCF became less remarkable, and were fragmented and broken apart to form large vacuoles. Immunopathological studies demonstrated that the amyloid β (Aβ ) and N and C‐terminal regions of amyloid precursor protein (APP), and the amyloid associated proteins tested, apolipoprotein E (apoE), SP‐40,40, α1‐antichymotrypsin (α1‐ACT), and ubiquitin, which are known to be components of amyloid depositions found in Alzheimers disease (AD) affected brains, were present in the SCF. ApoE, SP‐40,40, α1‐ACT, and ubiquitin are induced following certain cell challenges (e.g. heat shock, various drugs and injury). The significance of APP, Aβ, and amyloid associated proteins are discussed in respect to snake coiled fibre formations in chloroquine rat myopathy and in the amyloidogenesis of AD.

Amyloidogenic Fragments of Amyloid Precursor Protein in Cells Cultured under Leupeptin

The extracellular deposition of β/A4 protein is the most characteristic neuropathological change observed in the brain of patients suffering from Alzheimer’s disease. The accumulation of β/A4 protein is notable at an early stage of the disease, and increases to a high extent during the progression of the disease which, in turn, is thought to be responsible for generation of neurofibrillary tangles leading to neuronal cell death. The β/A4 protein is a proteolytically cleaved product of receptor-like integral transmembrane protein, the amyloid precursor protein (APP).1,2,3 The mechanism and subcellular compartment of β/A4 protein production are still unknown.