Masashi Asai

Putative function of ADAM9, ADAM10, and ADAM17 as APP -secretase

Abstract The putative α-secretase cleaves the amyloid precursor protein (APP) of Alzheimer’s disease in the middle of the amyloid β peptide (Aβ) domain. It is generally thought that the α-secretase pathway mitigates Aβ formation in the normal brain. Several studies have suggested that ADAM9, ADAM10, and ADAM17 are candidate α-secretases belonging to the ADAM (a disintegrin and metalloprotease) family, which are membrane-anchored cell surface proteins. In this comparative study of ADAM9, ADAM10, and ADAM17, we examined the physiological role of ADAMs by expressing these ADAMs in COS-7 cells, and both “constitutive” and “regulated” α-secretase activities of these ADAMs were determined. We tried to suppress the expression of these ADAMs in human glioblastoma A172 cells, which contain large amounts of endogenous α-secretase, by lipofection of the double-stranded RNA (dsRNA) encoding each of these ADAMs. The results indicate that ADAM9, ADAM10, and ADAM17 catalyze α-secretory cleavage and therefore act as α-secretases in A172 cells. This is the first report that to suggest the endogenous α-secretase is composed of several ADAM enzymes.

Anti-Aβ Drug Screening Platform Using Human iPS Cell-Derived Neurons for the Treatment of Alzheimer's Disease

Background Alzheimers disease (AD) is a neurodegenerative disorder that causes progressive memory and cognitive decline during middle to late adult life. The AD brain is characterized by deposition of amyloid β peptide (Aβ), which is produced from amyloid precursor protein by β- and γ-secretase (presenilin complex)-mediated sequential cleavage. Induced pluripotent stem (iPS) cells potentially provide an opportunity to generate a human cell-based model of AD that would be crucial for drug discovery as well as for investigating mechanisms of the disease. Methodology/Principal Findings We differentiated human iPS (hiPS) cells into neuronal cells expressing the forebrain marker, Foxg1, and the neocortical markers, Cux1, Satb2, Ctip2, and Tbr1. The iPS cell-derived neuronal cells also expressed amyloid precursor protein, β-secretase, and γ-secretase components, and were capable of secreting Aβ into the conditioned media. Aβ production was inhibited by β-secretase inhibitor, γ-secretase inhibitor (GSI), and an NSAID; however, there were different susceptibilities to all three drugs between early and late differentiation stages. At the early differentiation stage, GSI treatment caused a fast increase at lower dose (Aβ surge) and drastic decline of Aβ production. Conclusions/Significance These results indicate that the hiPS cell-derived neuronal cells express functional β- and γ-secretases involved in Aβ production; however, anti-Aβ drug screening using these hiPS cell-derived neuronal cells requires sufficient neuronal differentiation.

BACE1 interacts with lipid raft proteins

A neuropathological hallmark of Alzheimers disease is the presence of amyloid plaques in the brain. Amyloid‐β peptide (Aβ) is the major constituent of the plaques and is generated by proteolytic cleavages of amyloid precursor protein (APP) by β‐ and γ‐secretases. Growing evidence shows that lipid rafts are critically involved in regulating the Aβ generation. In support of this, APP, Aβ, and presenilins have been found in lipid rafts. Although cholesterol plays a crucial role in maintaining lipid rafts, functions of other components in the generation of Aβ are unknown. Caveolins (CAVs) and flotillins (FLOTs) are principal proteins related to lipid rafts and have been suggested to be involved in APP processing. Here, we report that FLOT‐1 binds to BACE1 (beta‐site APP cleaving enzyme 1) and that overexpression of CAV‐1 or FLOT‐1 results in recruiting BACE1 into lipid rafts and influence on β‐secretase activity in cultured cells. Our results show that both CAV‐1 and FLOT‐1 may modulate β‐secretase activity by interacting with BACE1.

Global brain delivery of neprilysin gene by intravascular administration of AAV vector in mice

Accumulation of amyloid-β peptide (Aβ) in the brain is closely associated with cognitive decline in Alzheimers disease (AD). Stereotaxic infusion of neprilysin-encoding viral vectors into the hippocampus has been shown to decrease Aβ in AD-model mice, but more efficient and global delivery is necessary to treat the broadly distributed burden in AD. Here we developed an adeno-associated virus (AAV) vector capable of providing neuronal gene expression throughout the brains after peripheral administration. A single intracardiac administration of the vector carrying neprilysin gene in AD-model mice elevated neprilysin activity broadly in the brain, and reduced Aβ oligomers, with concurrent alleviation of abnormal learning and memory function and improvement of amyloid burden. The exogenous neprilysin was localized mainly in endosomes, thereby effectively excluding Aβ oligomers from the brain. AAV vector-mediated gene transfer may provide a therapeutic strategy for neurodegenerative diseases, where global transduction of a therapeutic gene into the brain is necessary.

BACE1 interacts with nicastrin.

Beta-amyloid peptide (Abeta) is generated through the proteolytic cleavage of beta-amyloid precursor protein (APP) by beta- and gamma-secretases. The beta-secretase, BACE1, initiates Abeta formation followed by gamma-cleavage within the APP transmembrane domain. Although BACE1 localizes in the transGolgi network (TGN), its physiological substrates and modulators are not known. In addition, the relationship to other secretase(s) also remains unidentified. Here, we demonstrate that BACE1 binds to nicastrin, a component of gamma-secretase complexes, in vitro, and that nicastrin activates beta-secretase activity in COS-7 cells.

An alternative metabolic pathway of amyloid precursor protein C-terminal fragments via cathepsin B in a human neuroglioma model

γ‐Secretase catalyzes the cleavage of the intramembrane region of the Alzheimer amyloid precursor protein (APP), generating p3, amyloid‐β peptide (Aβ), and the APP intracellular domain (AICD). Although a γ‐secretase inhibitor has been shown to cause an accumulation of the APP C‐terminal fragments (CTFs) α and β and to decrease levels of p3 or Aβ and AICD, we found that treatment with a lysosomotropic weak base, such as chloroquine or ammonium chloride, caused simultaneous accumulation of both CTFs and AICD, suggesting that lysosomal proteases are also involved in processing of APP. This observation was reinforced by the results that cysteine protease inhibitor E‐64d and cathepsin B specific inhibitor CA‐074Me caused the accumulation of both CTFs and AICD with no change in known secretase activities. γ‐Secretase preferentially cleaved phosphorylated CTFs to produce Aβ, but cathepsin B degraded CTFs regardless of phosphorylation. Our results suggest that cathepsin B plays novel roles in the metabolism of APP and that an inhibition of APP phosphorylation is an attractive therapeutic target for Alzheimers disease.—Asai, M., Yagishita, S., Iwata, N., Saido, T. C., Ishiura, S., Maruyama, K. An alternative metabolic pathway of amyloid precursor protein C‐terminal fragments via cathepsin B in a human neuroglioma model. FASEB J. 25, 3720–3730 (2011). www.fasebj.org

Efficient four-drug cocktail therapy targeting amyloid-β peptide for Alzheimer's disease

Cocktail treatment is an effective multidrug medication therapy for some diseases, such as cancer and AIDS, because of the additive or synergistic effect of each medicine and relief from adverse effects. Amyloid‐β peptide (Aβ), which is now recognized as central to the development of Alzheimers disease (AD), is derived from the sequential proteolysis of amyloid precursor protein (APP) by β‐ and γ‐secretases. Secretase inhibitors are one of most attractive targets for therapeutic intervention in AD. However, because β‐ and γ‐secretases cleave not only APP but also other substrate proteins, strong inhibition of these secretases leads to severe adverse effects. Some nonsteroidal antiinflammatory drugs (NSAIDs) and cholesterol‐lowering drugs (statins) can modify the production of Aβ. Here, we report that a cocktail treatment with four drugs (NSAID, statin, and β‐ and γ‐secretase inhibitors) had additive effects on the reduction of Aβ levels in cultured cells without competing with each other. Moreover, the four‐drug cocktail treatment caused no changes in processing of the γ‐secretase substrate Notch. This is suggests that this cocktail treatment could be a new therapeutic approach for AD.

Neprilysin Is Suppressed by Dual-Specificity Tyrosine-Phosphorylation Regulated Kinase 1A (DYRK1A) in Down-Syndrome-Derived Fibroblasts

Amyloid-β peptide (Aβ) accumulation is a triggering event leading to the Alzheimers disease (AD) pathological cascade. Almost all familial AD-linked gene mutations increase Aβ production and accelerate the onset of AD. The Swedish mutation of amyloid precursor protein (APP) affects β-secretase activity and increases Aβ production up to ca. 6-fold in cultured cells; the onset age is around 50. Down syndrome (DS) patients with chromosome 21 trisomy present AD-like pathologies at earlier ages (40s) compared with sporadic AD patients, because APP gene expression is 1.5-fold higher than that in healthy people, thus causing a 1.5-fold increase in Aβ production. However, when comparing the causal relationship of Aβ accumulation with the onset age between the above two populations, early DS pathogenesis does not appear to be accounted for by the increased Aβ production alone. In this study, we found that neprilysin, a major Aβ-degrading enzyme, was downregulated in DS patient-derived fibroblasts, compared with healthy people-derived fibroblasts. Treatment with harmine, an inhibitor of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), which is located in the DS critical region of chromosome 21, and gene knockdown of DYRK1A, upregulated neprilysin in fibroblasts. These results suggest that a decrease in the Aβ catabolic rate may be, at least in part, one of the causes for accelerated AD-like pathogenesis in DS patients if a similar event occurs in the brains, and that neprilysin activity may be regulated directly or indirectly by DYRK1A-mediated phosphorylation. DYRK1A inhibition may be a promising disease-modifying therapy for AD via neprilysin upregulation.

Paradigm shift from diagnosing patients based on common symptoms to categorizing patients into subtypes with different pathogenic mechanisms to guide treatment for Alzheimer’s disease

Alzheimers disease (AD) is a major cause of dementia in the elderly, and the number of AD patients is rapidly growing as life expectancy increases. However, disease-modifying drugs are not yet available. According to the amyloid hypothesis, disease onset is triggered by aggregation and accumulation of amyloid-β peptide, followed by the formation of neurofibrillary tangles composed of hyperphosphorylated tau, and synaptic loss/neuronal cell death leading to dementia. Based on this hypothesis, various clinical trials for treatment of AD have been conducted, but most were discontinued due to failure to achieve cognitive improvement or appearance of adverse effects. Here we discuss the reasons for the failure of these trials. We suggest that biomarkers of specific, distinct molecular mechanisms of amyloidogenesis should be developed concomitantly with disease-modifying drugs (the so-called companion diagnosis) to aid the proper design of clinical trials, as well as to enable personalized treatment of individual AD patients.

Modeling Alzheimer's disease using iPS cells reveals stress phenotypes associated with intracellular beta-amyloid oligomer and differential drug responsiveness

Background:Aberrant metabolism of amyloid b peptide (A b) results in the formation of amyloid plaques (APs), which are pathological hallmarks of Alzheimer’s disease (AD). Most cases of AD are also associated with cerebral amyloid angiopathy (CAA) which is characterized by A b deposition around brain blood microvessels. The molecular and cellular mechanism underlying the formation of APs and CAA remain unclear. In particular, the neural cell(s) responsible for the formation of these amyloid deposits has not yet been identified, although neurons are currently believed to be the major source of A b. However, this view lacks concrete evidence. In fact, reactive astrocytes have been shown to be very active for the production of A b, and brain vascular microvessel endothelial cells (ECs) and brain vascular smooth muscle cells (BVSMCs) can produce A b. In this study, we further characterized the A b production in ECs in terms of the pathogenesis of CAA. Methods: Human umbilical vein endothelial cells were used as a model of ECs. A b is adhesive and can bind to the extracellular matrix (EM). As the EM is more abundant at a high cell density, the level of free A b released from ECs may be affected by the cell density.To test this hypothesis, ECs were cultured at different cell densities, and the level of A b in the culture medium was analyzed by ELISA. Results: The Ab production in ECs was 2-4-fold higher at a low cell density than at a high cell density, where A b degrading activity was detected in the culture medium. Conclusions: The A b production in ECs was dependent on the cell density and it was suppressed at a high cell density, where physiologically-relevant cell-cell interactions in bloodmicrovessels may occur. Even under such conditions, the A b production of ECs was more than 100-fold higher than that of human BVSMCs cultured under similar conditions. These findings suggest that ECs, rather than the BVSMCs, play a major causative role in CAA, although the contribution of pericytes and astroglial cells, which are other components of the brain microvessels, remains unclear.