Yasuo Ihara

The growth inhibitory factor that is deficient in the Alzheimer's disease brain is a 68 amino acid metallothionein-like protein

We have purified and characterized the growth inhibitory factor (GIF) that is abundant in the normal human brain, but greatly reduced in the Alzheimers disease (AD) brain. GIF inhibited survival and neurite formation of cortical neurons in vitro. Purified GIF is a 68 amino acid small protein, and its amino acid sequence is 70% identical to that of human metallothionein II with a 1 amino acid insert and a unique 6 amino acid insert in the NH2-terminal and the COOH-terminal portions, respectively. The antibodies to the unique sequence of GIF revealed a distinct subset of astrocytes in the gray matter that appears to be closely associated with neuronal perikarya and dendrites. In the AD cortex, the number of GIF-positive astrocytes was drastically reduced, suggesting that GIF is down-regulated in the subset of astrocytes during AD.

Ubiquitin is conjugated with amino-terminally processed tau in paired helical filaments

We have investigated ubiquitinated paired helical filaments, which produce a proteinaceous smear in SDS-polyacrylamide gel electrophoresis and immunoblotting. The smear consisted largely of the carboxy-terminal portion of tau and ubiquitin. The ubiquitin-targeted protein was identified as tau in paired helical filaments, and the conjugation sites were localized to the microtubule-binding region. Most ubiquitin in paired helical filaments occurred as a monoubiquitinated form, and only a small proportion of ubiquitin formed multiubiquitin chains. There was a ubiquitin-negative smear, in which tau was much less processed in the amino-terminal portion. This strongly suggests that the amino-terminal processing of tau in paired helical filaments precedes its ubiquitination.

Molecular cloning of human growth inhibitory factor cDNA and its down-regulation in Alzheimer's disease

In previous studies, we discovered a growth inhibitory factor (GIF) that was abundant in normal human brain, but greatly reduced in Alzheimers disease (AD) brain. Molecular cloning of a full-length cDNA for human GIF revealed that the GIF had striking homology to metallothioneins. Furthermore, it was determined that the GIF gene was on chromosome 16, as are the metallothionein genes. GIF, in contrast to metallothioneins, was found to be expressed exclusively in the nervous system. The GIF protein produced by Escherichia coli harboring the GIF cDNA in a prokaryotic expression vector inhibited the growth of neonatal rat cortical neurons. These results indicate that GIF is a new member of the metallothionein family with distinct tissue-specific expression and functions. Northern blot analysis revealed that expression of the GIF mRNA is drastically decreased in AD brains. The result raises the possibility that down-regulation of the GIF gene in AD brain plays an important role in the pathogenesis of AD.

Re-examination of ex-boxers' brains using immunohistochemistry with antibodies to amyloid beta-protein and tau protein.

SummaryA histopathological study was carried out on the brains of eight ex-boxers (ages 56 to 83) using conventional histological staining methods and immunocytochemistry with antibodies to amyloid β-protein and the PHF-related tau protein. All cases showed a large number of tau-immunoreactive neurofibrillary tangles and also β-protein immunoreactive senile plaques in the cortex. In the areas with many neurofibrillary tangles, neuropil threads with tau-immunoreactivity were also observed, and some of the senile plaque lesions were surrounded by abnormal neurites with tau-immunoreactivity. Moreover, three cases revealed β-protein-type cerebrovascular amyloid deposits on both leptomeningeal and cortical blood vessels. The present observations indicate that the cerebral pathology of dementia pugilistica is very similar to that of Alzheimers disease and suggest that these two disorders share some common etiological and pathogenic mechanisms.

Molecular cloning of rat growth inhibitory factor cDNA and the expression in the central nervous system.

Human growth inhibitory factor (GIF) is a new metallothionein-related molecule whose expression is markedly reduced in Alzheimers disease (AD) brain. We have recently isolated a full-length cDNA for human GIF that has a striking homology to metallothioneins (MTs). As an initial step to better understanding of the biological functions of GIF, we have isolated a full-length cDNA for rat GIF. Comparison of the predicted amino acid sequence with that of human GIF revealed a strikingly high homology (84% identity). Rat GIF cDNA also showed a striking homology to rat MT-1 and MT-II (66% and 62% identities on amino acid sequences, respectively). All cysteine residues were conserved among rat GIF, human GIF and MTs, indicating that cysteine residues play an important function in these molecules. Compared to mammalian MTs, there were 3 bp and 12 bp insertions near the amino-terminal and carboxy-terminal regions in the rat GIF. The rat GIF mRNA was found to be expressed exclusively in the central nervous system, being expressed predominantly in rat cortical astrocytes in primary culture. Although the rat GIF mRNA expression was low during the fetal stage, a dramatic increase of the rat GIF mRNA expression was demonstrated during the postnatal period of 10-17 days. These results indicate that transcriptional regulation of the rat GIF gene is quite different from that of MTs despite its strikingly high homology with MTs.

Extracellular neurofibrillary tangles associated with degenerating neurites and neuropil threads in Alzheimer-type dementia

SummaryWe examined the cellular components of extracellular neurofibrillary tangles (E-NFT) in the hippocampal areas in cases with Alzheimer-type dementia. Immunohistochemically, the E-NFT were labeled for the C terminus of tau and glial fibrillary acidic protein. Moreover, the majority of the E-NFT was associated with intensely argyrophilic rods and with tau-and ubiquitin-immunoreactive dots. Ultrastructurally, the E-NFT consisted mainly of extracellular paired helical filaments (PHF) and astroglial processes. The extracellular PHF tended to be straighter and thinner. One third of the E-NFT was associated with small degenerating neurites containing many dense bodies and with neuropil threads containing PHF. These findings suggested that extracellular PHF promote both intense astroglial reaction and neuritic alteration, and that the E-NFT are continuously changing their morphology.

Tau 2: A Probe for a Ser Conformation in the Amino Terminus of τ

Abstract: We have determined the epitope for Tau 2, a monoclonal antibody that intensely stained tangles, plaque neurites, and curly fibers in the tissue section, and strongly labeled bovine τ, but only very weakly labeled human r on the blot. The epitope has been localized to Ala95 through Ala108 of bovine τ. Ser101 is critical for Tau 2 reactivities; the replacement of Ser by Pro, which is found in rat, mouse, and human T, brings about very weak Tau 2 reactivities. The strong Tau 2 staining of tangles and its effective absorption with a synthetic Ser peptide (Ala95 through Ala108) suggest that the τ in paired helical filaments takes a Ser conformation, rather than a Pro conformation, in its ammo‐terminal portion.

Amyloid β/A4 protein precursor is bound to neurofibrillary tangles in Alzheimer-type dementia

Abstract Localization of amyloid β/A4 protein precursor (APP) in Alzheimers neurofibrillary tangles (NFT) was examined immunohistochemically. Antiserum directed to N-terminal of APP intensely labeled intracellular NFT and some neuropil threads. The NFT, extracted from Alzheimer brains by detergent treatments, were also immunoreactive with this antiserum. Antisera to other parts of APP labeled NFT after the formic acid pretreatment. However, Western blot analysis of NFT demonstrated no immunoreactive bands with APP antiserum. These findings suggest that APP is a minor component of the NFT.

Differential expression of β amyloid protein precursor (APP) and tau mRNA in the aged human brain : individual variability and correlation between APP-751 and four-repeat tau

We investigated the relationship between the differential expression of β amyloid protein precursor (APP) and tau mRNA, and the extent of β and tau deposition in three regions from each of the 38 aged brains obtained from consecutive autopsied cases. Remarkable variabilities were noted in the ratios of APP-770/-751/ −695 and four-repeat tau among elderly individuals. There was no consistent alteration in the APP differential expression among β plaque (-), (+), and (+ +-+ + +) groups. Also, no differences in the four-repeat tau ratios were noted among tangle (-), (+), and (++) groups. Despite these great individual variabilities, APP-751 was found to be well-correlated with four-repeat tau. It is possible that APP-751 and four-repeat tau are increasing during aging, while APP-695 and three-repeat tau are decreasing.

The protease inhibitor leupeptin induces several signs of aging in brain, retina and internal organs of young rats☆

The protease inhibitor leupeptin was administered to brain, retina and internal organs of young rats for up to two weeks in order to determine if specifically decreased proteolysis could cause symptoms of cellular aging in a variety of tissues. Electron microscopy showed that leupeptin induced the formation of dense substances with fine morphologies similar to and, in many cases, apparently identical with those of natural lipofuscin from aged tissues. Leupeptin also caused increased immunoreactivity to ubiquitin in cerebellar Purkinje cells and presumed Bergmann glia perikarya of brain tissue as well as in hepatocytes of liver tissue. Both of these effects were found in aged tissues as well. Finally, both leupeptin treatment and normal aging led to the onset of immunoreactivity in Purkinje cells to antibodies to the abnormal tau molecule of paired helical filaments from Alzheimers disease brain. Together, these results indicate that inhibition of thiol (and possibly some serine) proteases by leupeptin is sufficient to cause obvious morphological manifestations of aging in several tissues, and are thus consistent with the hypothesis that lipofuscinogenesis as well as a build-up of ubiquilinated proteins with age is caused by decreased or defective proteolysis. These effects are likely secondary to the mechanism(s) interfering with proteolysis itself.