Increased expression of BIN1 mediates Alzheimer genetic risk by modulating tau pathology.

Genome-wide association studies (GWAS) have identified a region upstream the BIN1 gene as the most important genetic susceptibility locus in Alzheimer's disease (AD) after APOE. We report that BIN1 transcript levels were increased in AD brains and identified a novel 3 bp insertion allele ∼28 kb upstream of BIN1, which increased (i) transcriptional activity in vitro, (ii) BIN1 expression levels in human brain and (iii) AD risk in three independent case-control cohorts (Meta-analysed Odds ratio of 1.20 (1.14-1.26) (P=3.8 × 10(-11))). Interestingly, decreased expression of the Drosophila BIN1 ortholog Amph suppressed Tau-mediated neurotoxicity in three different assays. Accordingly, Tau and BIN1 colocalized and interacted in human neuroblastoma cells and in mouse brain. Finally, the 3 bp insertion was associated with Tau but not Amyloid loads in AD brains. We propose that BIN1 mediates AD risk by modulating Tau pathology.

Chronic treatment with a novel γ-secretase modulator, JNJ-40418677, inhibits amyloid plaque formation in a mouse model of Alzheimer's disease.

BACKGROUND AND PURPOSE: γ-Secretase modulators represent a promising therapeutic approach for Alzheimer's disease (AD) because they selectively decrease amyloid ß 42 (Aß42), a particularly neurotoxic Aß species that accumulates in plaques in the brains of patients with AD. In the present study, we describe the in vitro and in vivo pharmacological properties of a potent novel γ-secretase modulator, 2-(S)-(3,5-bis(4-(trifluoromethyl)phenyl)phenyl)-4-methylpentanoic acid (JNJ-40418677). EXPERIMENTAL APPROACH: The potency and selectivity of JNJ-40418677 for Aß reduction was investigated in human neuroblastoma cells, rat primary neurones and after treatment with single oral doses in non-transgenic mouse brains. To evaluate the effect of JNJ-40418677 on plaque formation, Tg2576 mice were treated from 6 until 13 months of age via the diet. KEY RESULTS: JNJ-40418677 selectively reduced Aß42 secretion in human neuroblastoma cells and rat primary neurones, but it did not inhibit Notch processing or formation of other amyloid precursor protein cleavage products. Oral treatment of non-transgenic mice with JNJ-40418677 resulted in an excellent brain penetration of the compound and a dose- and time-dependent decrease of brain Aß42 levels. Chronic treatment of Tg2576 mice with JNJ-40418677 reduced brain Aß levels, the area occupied by plaques and plaque number in a dose-dependent manner compared with transgenic vehicle-treated mice. CONCLUSIONS AND IMPLICATIONS: JNJ-40418677 selectively decreased Aß42 production, showed an excellent brain penetration after oral administration in mice and lowered brain Aß burden in Tg2576 mice after chronic treatment. JNJ-40418677 therefore warrants further investigation as a potentially effective disease-modifying therapy for AD.

The functional gamma-secretase inhibitor prevents production of amyloid beta 1-34 in human and murine cell lines.

The amyloid precursor protein (APP) undergoes two consecutive cleavages by different proteases, beta-secretase and gamma-secretase, leading to the release of an amyloidogenic 4 kDa fragment called amyloid beta (Abeta). Combining immunoprecipitation and mass spectrometry, we characterized soluble Abeta in cultured cell media of mouse neuroblastoma N2a cells and double hAPP/hBACE-1 transfected HEK293. The major Abeta isoforms detected were Abeta11-34, Abeta1-34, Abeta11-40 and Abeta1-40. In this study, we demonstrate that overexpression of human beta-secretase (BACE-1) in HEK293 cells resulted in predominant Abeta cleavage at position Glu(11) rather than Asp(1), as well as increased production of Abeta(x)-34, but not Abeta(x)-40. Incubation of cells with a specific gamma-secretase inhibitor suggests that cleavage of APP at Leu(34) could be mediated by gamma-secretase itself or by a gamma-secretase dependent process.

A beta peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer's disease.

Much evidence indicates that abnormal processing and extracellular deposition of amyloid-beta peptide (A beta), a proteolytic derivative of the beta-amyloid precursor protein (betaAPP), is central to the pathogenesis of Alzheimer's disease (reviewed in ref. 1). In the PDAPP transgenic mouse model of Alzheimer's disease, immunization with A beta causes a marked reduction in burden of the brain amyloid. Evidence that A beta immunization also reduces cognitive dysfunction in murine models of Alzheimer's disease would support the hypothesis that abnormal A beta processing is essential to the pathogenesis of Alzheimer's disease, and would encourage the development of other strategies directed at the 'amyloid cascade'. Here we show that A beta immunization reduces both deposition of cerebral fibrillar A beta and cognitive dysfunction in the TgCRND8 murine model of Alzheimer's disease without, however, altering total levels of A beta in the brain. This implies that either a approximately 50% reduction in dense-cored A beta plaques is sufficient to affect cognition, or that vaccination may modulate the activity/abundance of a small subpopulation of especially toxic A beta species.

Dual roles of proteasome in the metabolism of presenilin 1.

Presenilin 1 (PS1) has been identified as a causative gene for most early-onset familial Alzheimer's 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 Alzheimer's 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.

Cloning of cDNA and expression of the gene encoding rat presenilin-2.

We have cloned the rat homologue of the presenilin-2 (PS-2) cDNA. PS-2 is responsible for chromosome 1-linked familial Alzheimer's disease. Sequence analysis predicted that the rat PS-2 encodes a 448 amino acid (aa) protein, and there was a very high degree of amino acid identity between rat and human PS-2 (95%). All the mutated codons in PS-2 and PS-1 in chromosome 1- or 14-linked familial Alzheimer's disease patients were conserved in rat PS-2. The expression of PS-2 was weaker than that of PS-1. The alternatively spliced short form of PS-2 mRNA, which was detected in human tissues was not detected in various rat tissues. During brain development, the expression level of both PS-2 and PS-1 increased but decreased in the adult. No remarkable change was observed in neural differentiation of PC12 cells.

Different effects of Alzheimer-associated mutations of presenilin 1 on its processing.

Presenilin 1 (PS 1) shows missense mutations in most early-onset familial Alzheimer's disease (FAD). Transfection of cDNA for wild type PS 1 into rat pheochromocytoma PC12 cells generated a 47 kDa full-size PS 1 protein, which was processed into a 28 kDa N-terminal fragment and a 19 kDa C-terminal fragment. We prepared selected Alzheimer-associated mutations (Gly384Ala, Leu392Val, and Cys410Tyr) of PS 1, which localized after a possible cleavage site. By transient expression in PC12 cells and rat glioma cell line, C6, we examined their influence on the processing of PS 1. Cys410Tyr inhibited proteolytic processing of PS 1, while Gly384Ala and Leu392Val did not. Thus, the Alzheimer related mutations can be divided into two groups in terms of their effect on the proteolytic cleavage of PS 1.

Immunocytochemistry of tau phosphoserine 413 and tau protein kinase I in Alzheimer pathology.

One unique phosphorylation site consistently found in paired helical filament tau, serine 413, is modified by tau protein kinase I/glycogen synthase kinase-3 beta but no other known tau kinase. Here we present immunocytochemistry from Alzheimer's disease brains showing that focal subpopulations of hippocampal CA1 pyramidal neurons and neuritic plaques are strongly reactive for tau protein kinase I/glycogen synthase kinase-3 beta and tau phosphoserine 413 in early stages of pathology. Colocalization of these epitopes suggests that tau protein kinase I/glycogen synthase kinase-3 beta abnormally phosphorylates tau and is in a position to disrupt neuronal metabolism in anatomical areas vulnerable to Alzheimer's disease.

Characterization of human presenilin 1 using N-terminal specific monoclonal antibodies: Evidence that Alzheimer mutations affect proteolytic processing.

The majority of cases of early-onset familial Alzheimer disease are caused by mutations in the recently identified presenilin 1 (PS1) gene, located on chromosome 14. PS1, a 467 amino acid protein, is predicted to be an integral membrane protein containing seven putative transmembrane domains and a large hydrophilic loop between the sixth and seventh membrane-spanning domain. We produced 7 monoclonal antibodies that react with 3 non-overlapping epitopes on the N-terminal hydrophilic tail of PS1. The monoclonal antibodies can detect the full-size PS1 at Mr 47000 and a more abundant Mr 28000 product in membrane extracts from human brain and human cell lines. PC12 cells transiently transfected with PS1 constructs containing two different Alzheimer mutations fail to generate the 28 kDa degradation product in contrast to PC12 cells transfected with wild-type PS1. Our results indicate that missense mutations in this form of familial Alzheimer disease may act via a mechanism of impaired proteolytic processing of PS1.

Isolation of IgG1-secreting switch variants from IgM hybridomas produced after in vitro immunization.

IgG1-secreting variants have been isolated from three different IgM-secreting hybridomas, in two instances following in vitro immunization. The method used was based on sequential sublining in combination with selection by an IgG1-specific two-site ELISA system employing two different IgG1-specific polyclonal antisera. Idiotypic identity between the IgG1 variants and their respective IgM parent was demonstrated using syngeneic anti-idiotypic antisera. The antigen binding specificity in the IgG1 variants was also conserved. Isolation of naturally occurring IgG1 switch variants from IgM-secreting hybridomas that are produced after in vivo immunization offers a solution to the major disadvantages associated with the generation of IgM hybridomas.