Hiroshi Tanahashi

Transgenic mice with Alzheimer presenilin 1 mutations show accelerated neurodegeneration without amyloid plaque formation.

Familial Alzheimer disease mutations of presenilin 1 (PS-1) enhance the generation of Aβ1–42, indicating that PS-1 is involved in amyloidogenesis. However, PS-1 transgenic mice have failed to show amyloid plaques in their brains. Because PS-1 mutations facilitate apoptotic neuronal death in vitro, we did careful quantitative studies in PS-1 transgenic mice and found that neurodegeneration was significantly accelerated in mice older than 13 months (aged mice) with familial Alzheimer disease mutant PS-1, without amyloid plaque formation. However, there were significantly more neurons containing intracellularly deposited Aβ42 in aged mutant transgenic mice. Our data indicate that the pathogenic role of the PS-1 mutation is upstream of the amyloid cascade.

Three novel alternatively spliced isoforms of the human beta-site amyloid precursor protein cleaving enzyme (BACE) and their effect on amyloid beta-peptide production.

Three novel alternatively spliced transcripts of the beta-site amyloid precursor protein cleaving enzyme (BACE) were cloned from human brain. Alternative splicing of the RNA occurs at an internal donor in exon 3 and/or an internal acceptor in exon 4. The splicing events lead to a deletion of 25 (BACE-I-476), 44 (BACE-I-457) and 69 (BACE-I-432) amino acids and the latter two caused the loss of two of four N-linked glycosylation sites. Although the mature form of BACE-501 was resistant to endoglycosidase H treatment, glycosylated forms of BACE-I-457 and BACE-I-476 were sensitive. This result suggests that BACE-I-457 and BACE-I-476 underwent different post-translational modifications. Moreover, the beta-secretase activity of BACE-I-457 and BACE-I-476 was significantly weaker than that of BACE-501. Thus, these isoforms may contribute to a physiological function of BACE.

Isolation of human delta-catenin and its binding specificity with presenilin 1

We screened proteins for interaction with presenilin (PS) 1, and cloned the full-length cDNA of human delta-catenin, which encoded 1225 amino acids. Yeast two-hybrid assay, GST binding assay and immunoprecipitation demonstrated that delta-catenin interacted with a hydrophilic loop region in the endoproteolytic C-terminal fragment of PS1, but not with that of PS-2. These results suggest that PS1 and PS2 partly differ in function. PS1 loop fragment containing the pathogenic mutation retained the binding ability. We also found another armadillo-protein, p0071, interacted with PS1.

Serial analysis of gene expression in a microglial cell line

We used the serial analysis of gene expression (SAGE) method to systematically analyze transcripts present in a microglial cell line. Over 10,000 SAGE tags were sequenced, and shown to represent 6,013 unique transcripts. Among the diverse transcripts that had not been previously detected in microglia were those for cytokines such as endothelial monocyte‐activating polypeptide I (EMAP I), and for cell surface antigens, including adhesion molecules such as CD9, CD53, CD107a, CD147, CD162 and mast cell high affinity IgE receptor. In addition, we detected transcripts that were characteristic of hematopoietic cells or mesodermal structures, such as E3 protein, A1, EN‐7, B94, and ufo. Furthermore, the profile contained a transcript, Hn1, that is important in hematopoietic cells and neurological development (Tang et al. Mamm Genome 8:695–696, 1997), suggesting the probable neural differentiation of microglia from the hematopoietic system in development. Messenger RNA expression of these genes was confirmed by RT‐PCR in primary cultures of microglia. Significantly, this is the first systematic profiling of the genes expressed in a microglial cell line. The identification and further characterization of the genes described here should provide potential new targets for the study of microglial biology. GLIA 28:265–271, 1999.

Sequence analysis of presenilin-1 gene mutation in Japanese Alzheimer's disease patients

The mutations of presenilins (PSs) gene and their clinicopathological correlations to Alzheimers disease (AD) have lately attracted considerable attention. In this report we analyzed fifteen Japanese familial Alzheimers disease (FAD) including 12 early-onset FAD and 13 sporadic AD patients for the mutation of PS-1 gene by direct sequence analysis. We found the mutations, G384A, E280A in two FAD and H163R in one sporadic AD patient, and no N1411 or M239V mutation in PS-2 gene, and no mutation in exons 16 and 17 in amyloid precursor protein (APP) gene. Families in which we failed to find the mutation by this screening may have mutations elsewhere in PSs or in APP gene, or yet unidentified other AD loci may exist. This is the first report to find a sporadic AD patient having PS-1 mutation.

Familial Alzheimer's disease genes in Japanese

More than 40 missense mutations and a splice-site mutation in the presenilin 1 (PS-1) gene, two missense mutations of presenilin 2 (PS-2), and more than three missense mutations of amyloid precursor protein (APP) cosegregate with early onset familial Alzheimers disease (FAD). In order to determine the incidence of mutations of these genes in Japanese patients, we screened 25 early onset FAD families, one late-onset FAD case, 33 early onset AD cases and five late-onset AD cases for mutations in the coding regions of the genes using SSCP analysis. Four different missense mutations of the PS-1 gene, including a novel mutation, Glu273Ala, were identified in five early onset FAD families and one missense mutation of PS-1 in one isolated AD patient. While no missense mutations of PS-2 were detected, four silent nucleotide substitutions were observed. Our data indicate that PS-1 mutations account for 20.0% of early onset FAD cases in Japan. Since mutations in PS-2 and APP genes were not found in the remaining cases, which could be explained only partially by apolipoprotein E epsilon4, important FAD genes or risk-factor genes remain to be identified.

Both N-terminal and C-terminal fragments of Presenilin 1 colocalize with neurofibrillary tangles in neurons and dystrophic neurites of senile plaques in Alzheimer's disease

Presenilin 1 (PS1) is a causative gene for chromosome 14‐linked familial Alzheimers disease. The gene product is known to be cleaved into N‐terminal fragments (PS1‐N) and C‐terminal fragments (PS1‐C). To understand the pathophysiological role of PS1, we conducted immunohistochemical studies using antibodies specific for PS1‐N and PS1‐C in sporadic Alzheimers disease (AD). Both antibodies showed punctuate staining exclusively in neurons and their processes in both control and AD brains. PS1‐N immunolabeling colocalized with neurofibrillary tangles (NFTs) in 36% of NFT‐bearing neurons and with dystrophic neurites in 28% of senile plaques (SPs). PS1‐C immunolabeling colocalized with dystrophic neurites in 70% of NFT‐bearing SPs and with intraneuronal NFTs in 32% of NFT‐bearing neurons. Both antibodies did not detect PHF‐tau‐positive neuropil threads and Aβ amyloid fibrils. The colocalization was also found in 33–38% of NFT‐bearing neurons in progressive supranuclear palsy. These results indicate that both PS1‐N and PS1‐C fragments are deposited in part of NFT‐bearing neurons and dystrophic neurites in SPs; both are the pathologic hallmarks of AD. J. Neurosci. Res. 53:99–106, 1998.

Molecular cloning of human Fe65L2 and its interaction with the Alzheimer's β-amyloid precursor protein

We report the cDNA sequence of human Fe65L2. The human Fe65L2 encoded 486 amino acids; the deduced amino acid sequence was shorter by 18 amino acids than the rat protein and had 86% identity to the rat protein Three protein-protein interaction domains, a WW and two PID/PTB elements, were conserved among the Fe65 protein family. Human Fe65L2 mRNA was expressed in various tissues; a transcript of about 2.2 kb was mainly expressed in the brain. A splicing variant lacking two amino acids in the first PID/PTB element was detected. We also confirmed that the carboxyl-terminal region of PID/PTB of the Fe65L2 interacted with the intracellular domain of the Alzheimers β-amyloid precursor protein (APP) and APP-like proteins.

Characterization of an amyloid precursor protein-binding protein Fe65L2 and its novel isoforms lacking phosphotyrosine-interaction domains.

Human Fe65L2 is a member of the Fe65 protein family, which interacts with amyloid precursor protein (APP). Fe65L2 contains an N-terminal WW (Trp-Trp) domain followed by two phosphotyrosine-interaction domains, and consists of 486 amino acids. In the present study, we cloned and characterized two novel isoforms of Fe65L2, designated I-214 and I-245, which are produced by alternative splicing of the RNA. The splicing events disrupt the ability to bind with APP and low-density-lipoprotein-receptor-related protein. Fe65L2 was highly expressed in the brain, whereas I-214 and I-245 were expressed in various tissues. In HEK-293 cells, Fe65L2 was expressed in the nucleus and cytosol, whereas I-245 and I-214 were localized exclusively to the nucleus. The ratio of I-214 to Fe65L2 mRNA was increased by apoptotic stimuli. Although the overexpression of either Fe65L2 or I-214 did not significantly affect the half-life and maturation of APP, or the secretion of secreted APP, the secretion of beta-amyloid peptide (Abeta)40 and Abeta42 was increased by overexpression of Fe65L2, but not by that of I-214. These results suggest that Fe65L2 affects Abeta production and a possible regulation of Fe65L2 function by alternative splicing.

Association between tau polymorphism and male early-onset Alzheimer's disease.

Neurofibrillary tangles, containing hyperphosphorylated microtubule-associated protein tau, are one of the major pathological hallmarks of Alzheimers disease. To investigate a possible association between tau genotypes and the risk of Alzheimers disease, we screened for polymorphisms in the tau gene and found a novel polymorphism IVS11+90G→A. A case-control study (874 patients and 678 controls) showed a significant association between possession of the A allele and male Alzheimers disease with early-onset (age of onset before 65, odds ratio = 2.65; 95% confidence interval 1.30–5.42), suggesting that age and gender modify the risk effect. However, we failed to replicate the reported association between the Saitohin gene located in the tau intron 9 and Japanese Alzheimers disease.