Decrease in p3-Alcß37 and p3-Alcß40, products of Alcadein ß generated by γ-secretase cleavages, in aged monkeys and patients with Alzheimer's disease.

INTRODUCTION: Neuronal p3-Alcß peptides are generated from the precursor protein Alcadein ß (Alcß) through cleavage by α- and γ-secretases of the amyloid ß (Aß) protein precursor (APP). To reveal whether p3-Alcß is involved in Alzheimer's disease (AD) contributes for the development of novel therapy and/or drug targets. METHODS: We developed new sandwich enzyme-linked immunosorbent assay (sELISA) systems to quantitate levels of p3-Alcß in the cerebrospinal fluid (CSF). RESULTS: In monkeys, CSF p3-Alcß decreases with age, and the aging is also accompanied by decreased brain expression of Alcß. In humans, CSF p3-Alcß levels decrease to a greater extent in those with AD than in age-matched controls. Subjects carrying presenilin gene mutations show a significantly lower CSF p3-Alcß level. A cell study with an inverse modulator of γ-secretase remarkably reduces the generation of p3-Alcß37 while increasing the production of Aß42. DISCUSSION: Aging decreases the generation of p3-Alcß, and further significant decrease of p3-Alcß caused by aberrant γ-secretase activity may accelerate pathogenesis in AD.

Induction of Amyloid-ß42 Production by Fipronil and Other Pyrazole Insecticides.

Generation of amyloid-ß peptides (Aßs) by proteolytic cleavage of the amyloid-ß protein precursor (AßPP), especially increased production of Aß42/Aß43 over Aß40, and their aggregation as oligomers and plaques, represent a characteristic feature of Alzheimer's disease (AD). In familial AD (FAD), altered Aß production originates from specific mutations of AßPP or presenilins 1/2 (PS1/PS2), the catalytic subunits of γ-secretase. In sporadic AD, the origin of altered production of Aßs remains unknown. We hypothesize that the 'human chemical exposome' contains products able to favor the production of Aß42/Aß43 over Aß40 and shorter Aßs. To detect such products, we screened a library of 3500 + compounds in a cell-based assay for enhanced Aß42/Aß43 production. Nine pyrazole insecticides were found to induce a ß- and γ-secretase-dependent, 3-10-fold increase in the production of extracellular Aß42 in various cell lines and neurons differentiated from induced pluripotent stem cells derived from healthy and FAD patients. Immunoprecipitation/mass spectrometry analyses showed increased production of Aßs cleaved at positions 42/43, and reduced production of peptides cleaved at positions 38 and shorter. Strongly supporting a direct effect on γ-secretase activity, pyrazoles shifted the cleavage pattern of another γ-secretase substrate, alcadeinα, and shifted the cleavage of AßPP by highly purified γ-secretase toward Aß42/Aß43. Focusing on fipronil, we showed that some of its metabolites, in particular the persistent fipronil sulfone, also favor the production of Aß42/Aß43 in both cell-based and cell-free systems. Fipronil administered orally to mice and rats is known to be metabolized rapidly, mostly to fipronil sulfone, which stably accumulates in adipose tissue and brain. In conclusion, several widely used pyrazole insecticides enhance the production of toxic, aggregation prone Aß42/Aß43 peptides, suggesting the possible existence of environmental "Alzheimerogens" which may contribute to the initiation and propagation of the amyloidogenic process in sporadic AD.

Facilitation of brain mitochondrial activity by 5-aminolevulinic acid in a mouse model of Alzheimer's disease.

The activities of mitochondrial enzymes, which are essential for neural function, decline with age and in age-related disease. In particular, the activity of cytochrome c oxidase (COX/complex IV) decreases in patients with Alzheimer's disease (AD). COX, a mitochondrial inner membrane protein complex that contains heme, plays an essential role in the electron transport chain that generates ATP. Heme synthesis begins with 5-aminolevulinic acid (5-ALA) in mitochondria. 5-ALA synthetase is the rate-limiting enzyme in heme synthesis, suggesting that supplementation with 5-ALA might help preserve mitochondrial activity in the aged brain. We administered a diet containing 5-ALA to triple-transgenic AD (3xTg-AD) model mice for 6 months, starting at 3 months of age. COX activity and protein expression, as well as mitochondrial membrane potential, were significantly higher in brains of 5-ALA-fed mice than in controls. Synaptotagmin protein levels were also significantly higher in 5-ALA-fed mice, suggesting improved preservation of synapses. Although brain Aß levels tended to decrease in 5-ALA-fed mice, we observed no other significant changes in other biochemical and pathological hallmarks of AD. Nevertheless, our study suggests that daily oral administration of 5-ALA could preserve mitochondrial enzyme activities in the brains of aged individuals, thereby contributing to the preservation of neural activity.

Specific Triazine Herbicides Induce Amyloid-ß42 Production.

Proteolytic cleavage of the amyloid-ß protein precursor (AßPP) by secretases leads to extracellular release of amyloid-ß (Aß) peptides. Increased production of Aß42 over Aß40 and aggregation into oligomers and plaques constitute an Alzheimer's disease (AD) hallmark. Identifying products of the 'human chemical exposome' (HCE) able to induce Aß42 production may be a key to understanding some of the initiating causes of AD and to generate non-genetic, chemically-induced AD animal models. A cell model was used to screen HCE libraries for Aß42 inducers. Out of 3500+ compounds, six triazine herbicides were found that induced a ß- and γ-secretases-dependent, 2-10 fold increase in the production of extracellular Aß42 in various cell lines, primary neuronal cells, and neurons differentiated from human-induced pluripotent stem cells (iPSCs). Immunoprecipitation/mass spectrometry analyses show enhanced production of Aß peptides cleaved at positions 42/43, and reduced production of peptides cleaved at positions 38 and lower, a characteristic of AD. Neurons derived from iPSCs obtained from a familial AD (FAD) patient (AßPP K724N) produced more Aß42 versus Aß40 than neurons derived from healthy controls iPSCs (AßPP WT). Triazines enhanced Aß42 production in both control and AD iPSCs-derived neurons. Triazines also shifted the cleavage pattern of alcadeinα, another γ-secretase substrate, suggesting a direct effect of triazines on γ-secretase activity. In conclusion, several widely used triazines enhance the production of toxic, aggregation prone Aß42/Aß43 amyloids, suggesting the possible existence of environmental "Alzheimerogens" which may contribute to the initiation and propagation of the amyloidogenic process in late-onset AD.

Increased levels of plasma p3-alcα35, a major fragment of Alcadeinα by γ-secretase cleavage, in Alzheimer's disease.

p3-Alcα is a metabolic fragment of Alcadeinα (Alcα). Similar to the generation of the p3 fragment from amyloid-ß protein precursor (AßPP) processing, Alcα is cleaved by α- and γ-secretases, leading to the secretion of p3-Alcα peptides into cerebrospinal fluid (CSF). p3-Alcα is also detected in the plasma, similar to amyloid-ß (Aß), which is a metabolic fragment of AßPP cleaved by amyloidogenic ß- and γ-secretases. Because p3-Alcα is a non-aggregatable and stable peptide, unlike aggregatable Aß and metabolically labile p3 of AßPP, the changes of p3-Alcα in quality and/or quantity in CSF and plasma are expected to be a marker for assessing alteration of substrate cleavage by γ-secretase, such as Aß generation from AßPP. The present study describes a sandwich enzyme-linked immunosorbent assay for quantifying levels of p3-Alcα35, the major form of the p3-Alcα species, and examines levels of p3-Alcα35 in the plasma of three independent Japanese cohorts. In two of the three cohorts, the p3-Alcα35 levels were significantly increased with a concomitant decrease in the Mini-Mental State Examination score, or in clinically diagnosed Alzheimer's disease (AD) patients, when compared with age-matched non-demented subjects. The values were significantly lower in AD subjects who were administered donepezil, when compared to AD subjects without donepezil treatment. The increase in plasma p3-Alcα35 levels may indicate an endophenotype in subjects in whom AD is due to a progressing cognitive impairment in subjects with a γ-secretase malfunction, or a disorder of the clearance of peptides.