Publications – Page 2

2004

Targeted introduction of V642I mutation in amyloid precursor protein gene causes functional abnormality resembling early stage of Alzheimer’s disease in aged mice

Kawasumi M, Chiba T, Yamada M, Miyamae-Kaneko M, Matsuoka M, Nakahara J, Tomita T, Iwatsubo T, Kato S, Aiso S, Nishimoto I, Kouyama K

Eur J Neurosci 2004;19(10):2826-2838

PMID: 15147316

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Targeted introduction of V642I mutation in amyloid precursor protein gene causes functional abnormality resembling early stage of Alzheimer’s disease in aged mice

V642I mutation in amyloid precursor protein (APP) conferred functional abnormality, but not organic abnormality, resembling early stage of Alzheimer's disease. Water-finding task (WFT) revealed that latent memory of 27-month-old V642I-APP heterozygous mutants was impaired.

Highlights

We generated V642I-APP knock-in mice to precisely examine the pathogenesis of familial Alzheimer's disease (FAD) by the exact genetic alteration that causes human FAD cases.
The V642I-APP knock-in heterozygous mutants were apparently indistinguishable from their wild-type littermates throughout the observation period of up to 29 months.
Long-term memory (latent learning performance) was deteriorated in 27-month-old V642I-APP mutants as assessed by water-finding task (WFT).
Brain sections of 29-month-old V642I-APP mutants were negative for formation of neuritic plaques or neurofibrillary tangles.

Abstract

While the exact aetiology of Alzheimer’s disease (AD) is unknown, distinct genetic mutations have been identified for the rare cases of familial AD (FAD). V642I mutation in amyloid precursor protein (APP) co-segregates with FAD with perfect penetration, and the clinicopathological characteristics of patients with this mutation resemble that of sporadic AD. To examine the pathogenic process of this FAD-linked trait in vivo, we produced a mouse with the corresponding point mutation in the APP gene using homologous recombination and Cre-loxP site-specific recombination (‘knock-in’ technique). Mice with the heterozygous V642I-APP allele most precisely reflected the genotype of humans bearing this mutation. For the observation period of 2.5 years the mutants stayed apparently indistinguishable from the wild-type littermates. However, behavioural analysis revealed significantly deteriorated long-term memory in mutants when examined for the retention of spatial attention. Interestingly, acquisition of spatial memory was slightly affected but short-term working memory was not deteriorated at all. Histological examination was negative for formation of neuritic plaques or neurofibrillary tangles, whereas the relative amount of longer form of β-amyloid species Aβ42(43) was significantly increased against that of the shorter form (Aβ40) in the mutant brain homogenates. We conclude that a V642I-APP mutant allele in aged mice confers functional components, but not organic components, of the AD-related phenotype that are observed in the early stage of AD. This V642I-APP knock-in mutant line may serve as a model to study the early pathogenic processes of AD in vivo and to develop therapeutics for this stage.

2003

Two serine residues distinctly regulate the rescue function of Humanin, an inhibiting factor of Alzheimer’s disease-related neurotoxicity: functional potentiation by isomerization and dimerization

Terashita K, Hashimoto Y, Niikura T, Tajima H, Yamagishi Y, Ishizaka M, Kawasumi M, Chiba T, Kanekura K, Yamada M, Nawa M, Kita Y, Aiso S, Nishimoto I

J Neurochem 2003;85(6):1521-1538

PMID: 12787071

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Abstract

The 24-residue peptide Humanin (HN), containing two Ser residues at positions 7 and 14, protects neuronal cells from insults of various Alzheimer’s disease (AD) genes and Aβ. It was not known why the rescue function of (S14G)HN is more potent than HN by two to three orders of magnitude. Investigating the possibility that the post-translational modification of Ser14 might play a role, we found that HN with D-Ser at position 14 exerts neuroprotection more potently than HN by two to three orders of magnitude, whereas D-Ser7 substitution does not affect the rescue function of HN. On the other hand, S7A substitution nullified the HN function. Multiple series of experiments indicated that Ser7 is necessary for self-dimerization of HN, which is essential for neuroprotection by this factor. These findings indicate that the rescue function of HN is quantitatively modulated by D-isomerization of Ser14 and Ser7-relevant dimerization, allowing for the construction of a very potent HN derivative that was fully neuroprotective at 10 pM against 25 μM Aβ1-43. This study provides important clues to the understanding of the neuroprotective mechanism of HN, as well as to the development of novel AD therapeutics.

A tripartite motif protein TRIM11 binds and destabilizes Humanin, a neuroprotective peptide against Alzheimer’s disease-relevant insults

Niikura T, Hashimoto Y, Tajima H, Ishizaka M, Yamagishi Y, Kawasumi M, Nawa M, Terashita K, Aiso S, Nishimoto I

Eur J Neurosci 2003;17(6):1150-1158

PMID: 12670303

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Abstract

Humanin (HN) is a newly identified neuroprotective peptide that specifically suppresses Alzheimer’s disease (AD)-related neurotoxicity. HN peptide has been detected in the human AD brain as well as in mouse testis and colon by immunoblot and immunohistochemical analyses. By means of yeast two-hybrid screening, we identified TRIM11 as a novel HN-interacting protein. TRIM11, which is a member of protein family containing a tripartite motif (TRIM), is composed of a RING finger domain, which is a putative E3 ubiquitin ligase, a B-box domain, a coiled-coil domain and a B30.2 domain. Deletion of the B30.2 domain in TRIM11 abolished the interaction with HN, whereas the B30.2 domain alone did not interact with HN. For their interaction, at least the coiled-coil domain was indispensable together with the B30.2 domain. The intracellular level of glutathione S-transferase-fused or EGFP-fused HN peptides or plain HN was drastically reduced by the coexpression of TRIM11. Disruption of the RING finger domain by deleting the first consensus cysteine or proteasome inhibitor treatment significantly diminished the effect of TRIM11 on the intracellular level of HN. These results suggest that TRIM11 plays a role in the regulation of intracellular HN level through ubiquitin-mediated protein degradation pathways.

Involvement of c-Jun N-terminal kinase in amyloid precursor protein-mediated neuronal cell death

Hashimoto Y, Tsuji O, Niikura T, Yamagishi Y, Ishizaka M, Kawasumi M, Chiba T, Kanekura K, Yamada M, Tsukamoto E, Kouyama K, Terashita K, Aiso S, Lin A, Nishimoto I

J Neurochem 2003;84(4):864-877

PMID: 12562529

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Abstract

Amyloid precursor protein (APP), the precursor of Aβ, has been shown to function as a cell surface receptor that mediates neuronal cell death by anti-APP antibody. The c-Jun N-terminal kinase (JNK) can mediate various neurotoxic signals, including Aβ neurotoxicity. However, the relationship of APP-mediated neurotoxicity to JNK is not clear, partly because APP cytotoxicity is Aβ independent. Here we examined whether JNK is involved in APP-mediated neuronal cell death and found that: (i) neuronal cell death by antibody-bound APP was inhibited by dominant-negative JNK, JIP-1b and SP600125, the specific inhibitor of JNK, but not by SB203580 or PD98059; (ii) constitutively active (ca) JNK caused neuronal cell death and (iii) the pharmacological profile of caJNK-mediated cell death closely coincided with that of APP-mediated cell death. Pertussis toxin (PTX) suppressed APP-mediated cell death but not caJNK-induced cell death, which was suppressed by Humanin, a newly identified neuroprotective factor which inhibits APP-mediated cytotoxicity. In the presence of PTX, the PTX-resistant mutant of Gα_o, but not that of Gα_i, recovered the cytotoxic action of APP. These findings demonstrate that JNK is involved in APP-mediated neuronal cell death as a downstream signal transducer of G_o.

2002

Molecular mechanisms for neuronal cell death by Alzheimer’s amyloid precursor protein-relevant insults

Kawasumi M, Hashimoto Y, Chiba T, Kanekura K, Yamagishi Y, Ishizaka M, Tajima H, Niikura T, Nishimoto I

Neurosignals 2002;11(5):236-250 [Review]

PMID: 12566925

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Abstract

To develop a therapeutic intervention for Alzheimer’s disease (AD), it is necessary to clarify the mechanisms underlying the pathogenesis of AD, in which senile plaques, neurofibrillary tangles and neuronal loss in the cerebrum are the central abnormalities. A number of studies have focused on the major component of the senile plaques, which is amyloid-β (Aβ) and its precursor protein APP, and have investigated the roles of these molecules in the onset, progression and inhibition of AD. For multiple reasons, however, their roles in AD, especially in neuronal death, remain elusive and a unified concept for their roles has not yet been established. Recently, it has been found that APP functions normally as a neuronal surface transmembrane protein. In this article, we review the molecular mechanisms of neuronal cell death by these APP-relevant insults and discuss the functions of APP in regard to intracellular signal transducers, including c-Jun N-terminal kinase. We also revise the roles of Aβ in neuronal death and survival.

2001

c-Jun N-terminal kinase (JNK)-interacting protein-1b/islet-brain-1 scaffolds Alzheimer’s amyloid precursor protein with JNK

Matsuda S, Yasukawa T, Homma Y, Ito Y, Niikura T, Hiraki T, Hirai S, Ohno S, Kita Y, Kawasumi M, Kouyama K, Yamamoto T, Kyriakis JM, Nishimoto I

J Neurosci 2001;21(17):6597-6607

PMID: 11517249

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Abstract

Using a yeast two-hybrid method, we searched for amyloid precursor protein (APP)-interacting molecules by screening mouse and human brain libraries. In addition to known interacting proteins containing a phosphotyrosine-interaction-domain (PID)-Fe65, Fe65L, Fe65L2, X11, and mDab1, we identified, as a novel APP-interacting molecule, a PID-containing isoform of mouse JNK-interacting protein-1 (JIP-1b) and its human homolog IB1, the established scaffold proteins for JNK. The APP amino acids Tyr⁶⁸², Asn⁶⁸⁴, and Tyr⁶⁸⁷ in the G⁶⁸¹YENPTY⁶⁸⁷ region were all essential for APP/JIP-1b interaction, but neither Tyr⁶⁵³ nor Thr⁶⁶⁸ was necessary. APP-interacting ability was specific for this additional isoform containing PID and was shared by both human and mouse homologs. JIP-1b expressed by mammalian cells was efficiently precipitated by the cytoplasmic domain of APP in the extreme Gly⁶⁸¹-Asn⁶⁹⁵ domain-dependent manner. Reciprocally, both full-length wild-type and familial Alzheimer’s disease mutant APPs were precipitated by PID-containing JIP constructs. Antibodies raised against the N and C termini of JIP-1b coprecipitated JIP-1b and wild-type or mutant APP in non-neuronal and neuronal cells. Moreover, human JNK1β1 formed a complex with APP in a JIP-1b-dependent manner. Confocal microscopic examination demonstrated that APP and JIP-1b share similar subcellular localization in transfected cells. These data indicate that JIP-1b/IB1 scaffolds APP with JNK, providing a novel insight into the role of the JNK scaffold protein as an interface of APP with intracellular functional molecules.

A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer’s disease genes and Aβ

Hashimoto Y, Niikura T, Tajima H, Yasukawa T, Sudo H, Ito Y, Kita Y, Kawasumi M, Kouyama K, Doyu M, Sobue G, Koide T, Tsuji S, Lang J, Kurokawa K, Nishimoto I

Proc Natl Acad Sci U S A 2001;98(11):6336-6341 Erratum in: Proc Natl Acad Sci U S A 2001;98(22):12854

PMID: 11371646

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A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer’s disease genes and Aβ

Humanin (HN) is a unique secretory polypeptide and suppresses Aβ-induced cell death in primary neurons. Also, HN protects neuronal cells from death by three types of familial Alzheimer's disease (FAD) genes [amyloid precursor protein (APP), presenilin 1 (PS1), and presenilin 2 (PS2)].

Highlights

To discover molecules that suppress familial Alzheimer's disease (FAD) gene-induced death of neuronal cells, we performed a functional screening for antagonistic genes against death insults, termed a death-trap screening.
We transfected V642I-APP-inducible neuronal cells with a cDNA library constructed from the occipital cortex of an Alzheimer's disease patient brain and recovered the plasmids from surviving cells.
We discovered a cDNA, encoding a 24-residue peptide (MAPRGFSCLLLLTSEIDLPVKRRA), that suppresses neuronal cell death induced by three different types of FAD genes and by Aβ, designated Humanin (HN).
The rescue action of HN cDNA requires extracellular secretion of the corresponding polypeptide, not its presence inside the cell, indicating that HN acts on cells from the outside.

Abstract

Through functional expression screening, we identified a gene, designated Humanin (HN) cDNA, which encodes a short polypeptide and abolishes death of neuronal cells caused by multiple different types of familial Alzheimer’s disease genes and by Aβ amyloid, without effect on death by Q79 or superoxide dismutase-1 mutants. Transfected HN cDNA was transcribed to the corresponding polypeptide and then was secreted into the cultured medium. The rescue action clearly depended on the primary structure of HN. This polypeptide would serve as a molecular clue for the development of new therapeutics for Alzheimer’s disease targeting neuroprotection.

Secreted Aβ does not mediate neurotoxicity by antibody-stimulated amyloid precursor protein

Sudo H, Hashimoto Y, Niikura T, Shao Z, Yasukawa T, Ito Y, Yamada M, Hata M, Hiraki T, Kawasumi M, Kouyama K, Nishimoto I

Biochem Biophys Res Commun 2001;282(2):548-556

PMID: 11401495

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Abstract

Antibodies against APP, a precursor of Aβ deposited in Alzheimer’s disease brain, have been shown to cause neuronal death. Therefore, it is important to determine whether Aβ mediates antibody-induced neurotoxicity. When primary neurons were treated with anti-APP antibodies, Aβ40 and Aβ42 in the cultured media were undetectable by an assay capable of detecting 100 nM Aβ peptides. However, exogenously treated Aβ1-42 or Aβ1-43 required >3 µM to exert neurotoxicity, and 25 µM Aβ1-40 was not neurotoxic. Glutathione-ethyl-ester inhibited neuronal death by anti-APP antibody, but not death by Aβ1-42, whereas serum attenuated toxicity by Aβ1-42, but not by anti-APP antibody. Using immortalized neuronal cells, we specified the domain responsible for toxicity to be cytoplasmic His⁶⁵⁷-Lys⁶⁷⁶, but not the Aβ1-42 region, of APP. This indicates that neuronal cell death by anti-APP antibody is not mediated by secreted Aβ.

Insulin-like growth factor I (IGF-I) protects cells from apoptosis by Alzheimer’s V642I mutant amyloid precursor protein through IGF-I receptor in an IGF-binding protein-sensitive manner

Niikura T, Hashimoto Y, Okamoto T, Abe Y, Yasukawa T, Kawasumi M, Hiraki T, Kita Y, Terashita K, Kouyama K, Nishimoto I

J Neurosci 2001;21(6):1902-1910

PMID: 11245675

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Abstract

It has been found that insulin-like growth factor I (IGF-I) exerts cytoprotection against Aβ amyloid-induced neuronal cell death. Deposits of Aβ amyloid are one of the pathological hallmarks of Alzheimer’s disease (AD). Here, we examined whether IGF-I exerts protective activity against cell death induced by a familial AD (FAD)-linked mutant of amyloid precursor protein (APP), and we found that IGF-I protected cells from toxicity of FAD-associated V642I mutant of APP in multiple cell systems. IGFBP-3 blocked this action of IGF-I, but not of des(1-3)IGF-I, which was as active as IGF-I in the presence of IGFBP-3. The data also demonstrated that the IGF-I receptor (IGF-IR) mediates the protective activity of IGF-I. The antagonizing function of the IGF-I/IGF-IR system against V642I-APP, which is further antagonized by IGFBP-3, provides a molecular clue to the understanding of AD pathophysiology and to the establishment of potential therapy for AD.

2000

Antibody-regulated neurotoxic function of cell-surface β-amyloid precursor protein

Sudo H, Jiang H, Yasukawa T, Hashimoto Y, Niikura T, Kawasumi M, Matsuda S, Takeuchi Y, Aiso S, Matsuoka M, Murayama Y, Nishimoto I

Mol Cell Neurosci 2000;16(6):708-723

PMID: 11124892

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Abstract

APP is a transmembrane precursor of β-amyloid, and its mutations cause early-onset familial Alzheimer’s disease. We report a toxic function of normal wild-type APP (wtAPP). Treatment of neuronal F11 cells, immortalized embryonic day 13 neurons, overexpressing wtAPP with anti-APP antibodies caused death. Death was not induced by antibody in parental F11 cells. Death by antibody occurred through cell-surface APP, not through secreted APP, in a pertussis toxin-sensitive manner and was typical apoptosis, not observed in primary astrocytes or glioma cells overexpressing wtAPP, but observed in primary cortical neurons. Cell-surface APP thus performs a toxic function as an extracellularly controllable regulator of neuronal death. This study provides a novel insight into the normal and pathological functions of cell-surface wtAPP.

1998

Phosphorylation of Sic1, a cyclin-dependent kinase (Cdk) inhibitor, by Cdk including Pho85 kinase is required for its prompt degradation

Nishizawa M, Kawasumi M, Fujino M, Toh-e A

Mol Biol Cell 1998;9(9):2393-2405

PMID: 9725902

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Phosphorylation of Sic1, a cyclin-dependent kinase (Cdk) inhibitor, by Cdk including Pho85 kinase is required for its prompt degradation

Abstract

In the yeast Saccharomyces cerevisiae, Sic1, an inhibitor of Clb-Cdc28 kinases, must be phosphorylated and degraded in G₁ for cells to initiate DNA replication, and Cln-Cdc28 kinase appears to be primarily responsible for phosphorylation of Sic1. The Pho85 kinase is a yeast cyclin-dependent kinase (Cdk), which is not essential for cell growth unless both CLN1 and CLN2 are absent. We demonstrate that Pho85, when complexed with Pcl1, a G₁ cyclin homologue, can phosphorylate Sic1 in vitro, and that Sic1 appears to be more stable in pho85Δ cells. Three consensus Cdk phosphorylation sites present in Sic1 are phosphorylated in vivo, and two of them are required for prompt degradation of the inhibitor. Pho85 and other G₁ Cdks appear to phosphorylate Sic1 at different sites in vivo. Thus at least two distinct Cdks can participate in phosphorylation of Sic1 and may therefore regulate progression through G₁.

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