Ohoshi Murayama

Presenilin-1 mutations downregulate the signalling pathway of the unfolded-protein response.

Missense mutations in the human presenilin-1 (PS1) gene, which is found on chromosome 14, cause early-onset familial Alzheimer’s disease (FAD). FAD-linked PS1 variants alter proteolytic processing of the amyloid precursor protein and cause an increase in vulnerability to apoptosis induced by various cell stresses. However, the mechanisms responsible for these phenomena are not clear. Here we report that mutations in PS1 affect the unfolded-protein response (UPR), which responds to the increased amount of unfolded proteins that accumulate in the endoplasmic reticulum (ER) under conditions that cause ER stress. PS1 mutations also lead to decreased expression of GRP78/Bip, a molecular chaperone, present in the ER, that can enable protein folding. Interestingly, GRP78 levels are reduced in the brains of Alzheimer’s disease patients. The downregulation of UPR signalling by PS1 mutations is caused by disturbed function of IRE1, which is the proximal sensor of conditions in the ER lumen. Overexpression of GRP78 in neuroblastoma cells bearing PS1 mutants almost completely restores resistance to ER stress to the level of cells expressing wild-type PS1. These results show that mutations in PS1 may increase vulnerability to ER stress by altering the UPR signalling pathway.

Alterations in Glucose Metabolism Induce Hypothermia Leading to Tau Hyperphosphorylation through Differential Inhibition of Kinase and Phosphatase Activities: Implications for Alzheimer's Disease

Alzheimers disease (AD) brains contain neurofibrillary tangles (NFTs) composed of abnormally hyperphosphorylated tau protein. Regional reductions in cerebral glucose metabolism correlating to NFT densities have been reported in AD brains. Assuming that reduced glucose metabolism might cause abnormal tau hyperphosphorylation, we induced in vivo alterations of glucose metabolism in mice by starvation or intraperitoneal injections of either insulin or deoxyglucose. We found that the treatments led to abnormal tau hyperphosphorylation with patterns resembling those in early AD brains and also resulted in hypothermia. Surprisingly, tau hyperphosphorylation could be traced down to a differential effect of low temperatures on kinase and phosphatase activities. These data indicate that abnormal tau hyperphosphorylation is associated with altered glucose metabolism through hypothermia. Our results imply that serine-threonine protein phosphatase 2A plays a major role in regulating tau phosphorylation in the adult brain and provide in vivo evidence for its crucial role in abnormal tau hyperphosphorylation in AD.

Direct association of presenilin-1 with β-catenin

Families bearing mutations in the presenilin‐1 (PS1) gene develop Alzheimers disease (AD). However, the mechanism through which PS1 causes AD is unclear. The co‐immunoprecipitation with PS1 in transfected COS‐7 cells indicates that PS1 directly interacts with endogenous β‐catenin, and the interaction requires residues 322–450 of PS1 and 445–676 of β‐catenin. Both proteins are co‐localized in the endoplasmic reticulum. Over‐expression of PS1 reduces the level of cytoplasmic β‐catenin, and inhibits β‐catenin‐T cell factor‐regulated transcription. These results indicate that PS1 plays a role as inhibitor of the β‐catenin signal, which may be connected with the AD dysfunction.

Lithium inhibits amyloid secretion in COS7 cells transfected with amyloid precursor protein C100

To examine the regulation of amyloid secretion in more detail, Abeta sandwich ELISAs with high sensitivity and specificity were developed. Using this technique, we measured Abeta secreted from COS7 cells transiently transfected with APP C100 in the presence of LiCl, a potent glycogen synthase kinase (GSK)-3beta inhibitor. We found that both Abetax-40 and Abetax-42 secretion were reduced by LiCl treatment in a dose-dependent manner. Diminished amyloid secretion was associated with GSK-3beta activity. These results suggest that GSK-3beta might function as a possible mediator for regulating both amyloid deposition and tau pathology in Alzheimers disease (AD), and that lithium should be re-evaluated as a candidate reagent for preventing AD pathology.

Formation of filamentous tau aggregations in transgenic mice expressing V337M human tau

Formation of neurofibrillary tangles (NFTs) is the most common feature in several neurodegenerative diseases, including Alzheimers disease (AD). Here we report the formation of filamentous tau aggregations having a beta-sheet structure in transgenic mice expressing mutant human tau. These mice contain a tau gene with a mutation of the frontotemporal dementia parkinsonism (FTDP-17) type, in which valine is substituted with methionine residue 337. The aggregation of tau in these transgenic mice satisfies all histological criteria used to identify NFTs common to human neurodegenerative diseases. These mice, therefore, provide a preclinical model for the testing of therapeutic drugs for the treatment of neurodegenerative disorders that exhibit NFTs.

Enhancement of amyloid β 42 secretion by 28 different presenilin 1 mutations of familial Alzheimer's disease

Families bearing mutations in the presenilin 1 (PS1) gene develop early onset familial Alzheimers disease (FAD). Further, some PS1 mutants enhance secretion of the longer form of amyloid β protein (Aβ42). We constructed cDNAs encoding human PS1 harboring 28 FAD-linked mutations, and examined the effects of the expressed PS1 mutants on Aβ42 secretion in β amyloid precursor producing COS-1 cells. All the mutants significantly enhanced the ratio of Aβ42 to total Aβ compared with wild-type PS1. However, the increase in Aβ42 ratio in cells with each PS1 mutation did not correlate with the reported age of onset of FAD caused by that mutation. These results suggest that increased Aβ42 secretion is important for the development of Alzheimers disease (AD), but may not be the only factor contributing to the onset of AD.

Dual Roles of Proteasome in the Metabolism of Presenilin 1

Abstract : Presenilin 1 (PS1) has been identified as a causative gene for most early‐onset familial Alzheimers disease. Biochemical studies revealed that PS1 exists predominantly as two processed fragments in cells and brain tissues. We prepared stably transfected cells expressing the wild‐type and familial Alzheimers disease‐associated mutants of PS1 and investigated the enzyme that participates in the metabolism of PS1. After treatment of the cells with proteasome inhibitors, the full‐length PS1 was significantly accumulated. The levels of N‐ and C‐terminal fragments were also increased. The accumulation of PS1 with a deletion of exon 10, which is unable to be processed, on treatment of the transfected cells with lactacystin indicated that proteasome can degrade full‐length PS1. A synthetic peptide that includes the processing region of PS1 was cleaved by 20S proteasome at the putative processing sites after Met288 and Glu299. Metabolic labeling experiments showed that the appearance of the N‐terminal fragment was attenuated by the inhibitor. Finally, 28‐kDa N‐ and 20‐kDa C‐terminal fragments were generated by purified PS1 in vitro. These data indicated that the proteasome pathway is involved in PS1 processing. These results demonstrate that the proteasome pathway plays dual roles in processing and degradation of PS1.

Homogeneity of the 16S rDNA sequence among geographically disparate isolates of Taylorella equigenitalis

BackgroundAt present, six accessible sequences of 16S rDNA from Taylorella equigenitalis (T. equigenitalis) are available, whose sequence differences occur at a few nucleotide positions. Thus it is important to determine these sequences from additional strains in other countries, if possible, in order to clarify any anomalies regarding 16S rDNA sequence heterogeneity. Here, we clone and sequence the approximate full-length 16S rDNA from additional strains of T. equigenitalis isolated in Japan, Australia and France and compare these sequences to the existing published sequences.ResultsClarification of any anomalies regarding 16S rDNA sequence heterogeneity of T. equigenitalis was carried out. When cloning, sequencing and comparison of the approximate full-length 16S rDNA from 17 strains of T. equigenitalis isolated in Japan, Australia and France, nucleotide sequence differences were demonstrated at the six loci in the 1,469 nucleotide sequence. Moreover, 12 polymorphic sites occurred among 23 sequences of the 16S rDNA, including the six reference sequences.ConclusionHigh sequence similarity (99.5% or more) was observed throughout, except from nucleotide positions 138 to 501 where substitutions and deletions were noted.

Presenilin-1 Mutation L271V Results in Altered Exon 8 Splicing and Alzheimer's Disease with Non-cored Plaques and No Neuritic Dystrophy

The mutation L271V in exon 8 of the presenilin-1 (PS-1) gene was detected in an Alzheimers disease pedigree. Neuropathological examination of affected individuals identified variant, large, non-cored plaques without neuritic dystrophy, reminiscent of cotton wool plaques. Biochemical analysis of L271V mutation showed that it increased secretion of the 42-amino acid amyloid-β peptide, suggesting a pathogenic mutation. Analysis of PS-1 transcripts from the brains of two mutation carriers revealed a 17–50% increase in PS-1 transcripts with deletion of exon 8 (PS-1Δexon8) compared with unrelated Alzheimers disease brains. Exon trapping analysis confirmed that L271V mutation enhanced the deletion of exon 8. Western blots of brain lysates indicated that PS-1Δexon8 was overexpressed in an affected individual. Biochemical analysis of PS-1Δexon8 in COS and BD8 cells indicate the splice isoform is not intrinsically active but interacts with wild-type PS-1 to generate amyloid-β. Western blots of cell lysates immunoprecipitated with anti-Tau or anti-GSK-3β antibodies indicated that PS-1Δexon8, unlike wild-type PS-1, does not interact directly with Tau or GSK-3β, potential modifiers of neuritic dystrophy. We postulate that variant plaques observed in this family are due in part to the effects of PS-1Δexon8 and that interaction between PS-1 and various protein complexes are necessary for neuritic plaque formation.

Twenty-nine missense mutations linked with familial Alzheimer's disease alter the processing of presenilin 1

1. Full-length form of human presenilin 1 (PS1) is processed and an N-terminal fragment (28 KD) and C-terminal fragment (19 KD) are generated. To elucidate the possible role of presenilin mutations in Alzheimers disease (AD), the authors analyze the effects of AD-linked mutations on PS1 processing in cultured cells. 2. Complementary DNAs encoding genes for human PS1 harboring twenty-nine missense mutations linked with familial Alzheimers disease (FAD) were introduced into PC12 cells. Human PS1 exogenously expressed in the cells was detected by immunoblotting using a monoclonal antibody that recognized the N-terminal region of human PS1. The amounts of full-length form (48 KD) and N-terminal fragment (28 KD) of PS1 was quantified by densitometrical analysis. 3. The ratio of the N-terminal fragment to total PS1 was reduced by twenty-nine mutations. The specific effects on PS1 processing varied according to mutation. 4. These results suggest that AD-linked missense mutations of PS1 are involved in neurodegeneration via inhibition of PS1 processing.