Yasuji Matsuoka

Females exhibit more extensive amyloid, but not tau, pathology in an Alzheimer transgenic model

Epidemiological studies indicate that women have a higher risk of Alzheimers disease (AD) even after adjustment for age. Though transgenic mouse models of AD develop AD-related amyloid beta (Abeta) and/or tau pathology, gender differences have not been well documented in these models. In this study, we found that female 3xTg-AD transgenic mice expressing mutant APP, presenilin-1 and tau have significantly more aggressive Abeta pathology. We also found an increase in beta-secretase activity and a reduction of neprilysin in female mice compared to males; this suggests that a combination of increased Abeta production and decreased Abeta degradation may contribute to higher risk of AD in females. In contrast to significantly more aggressive Abeta pathology in females, gender did not affect the levels of phosphorylated tau in 3xTg-AD mice. These results point to the involvement of Abeta pathways in the higher risk of AD in women. In addition to comparison of pathology between genders at 9, 16 and 23 months of age, we examined the progression of Abeta pathology at additional age points; i.e., brain Abeta load, intraneuronal oligomeric Abeta distribution and plaque load, in male 3xTg-AD mice at 3, 6, 9, 12, 16, 20 and 23 months of age. These findings confirm progressive Abeta pathology in 3xTg-AD transgenic mice, and provide guidance for their use in therapeutic research.

Pyridazine-derived γ-secretase modulators

SAR of a novel series of pyridine-derived γ-secretase modulators is described. Compound 5 was found to be a potent modulator in vitro, which on further profiling, was found to decrease Aβ42 and Aβ40, and maintain (or increase) the levels of total Aβ. Furthermore, representative compounds 1 and 5 demonstrated in vivo efficacy to lower Aβ42 in the brain without altering Notch processing in the peripheral.

Discovery of GSK2795039, a Novel Small Molecule NADPH Oxidase 2 Inhibitor.

AIMS The NADPH oxidase (NOX) family of enzymes catalyzes the formation of reactive oxygen species (ROS). NOX enzymes not only have a key role in a variety of physiological processes but also contribute to oxidative stress in certain disease states. To date, while numerous small molecule inhibitors have been reported (in particular for NOX2), none have demonstrated inhibitory activity in vivo. As such, there is a need for the identification of improved NOX inhibitors to enable further evaluation of the biological functions of NOX enzymes in vivo as well as the therapeutic potential of NOX inhibition. In this study, both the in vitro and in vivo pharmacological profiles of GSK2795039, a novel NOX2 inhibitor, were characterized in comparison with other published NOX inhibitors. RESULTS GSK2795039 inhibited both the formation of ROS and the utilization of the enzyme substrates, NADPH and oxygen, in a variety of semirecombinant cell-free and cell-based NOX2 assays. It inhibited NOX2 in an NADPH competitive manner and was selective over other NOX isoforms, xanthine oxidase, and endothelial nitric oxide synthase enzymes. Following systemic administration in mice, GSK2795039 abolished the production of ROS by activated NOX2 enzyme in a paw inflammation model. Furthermore, GSK2795039 showed activity in a murine model of acute pancreatitis, reducing the levels of serum amylase triggered by systemic injection of cerulein. INNOVATION AND CONCLUSIONS GSK2795039 is a novel NOX2 inhibitor that is the first small molecule to demonstrate inhibition of the NOX2 enzyme in vivo.

γ-Secretase modulators do not induce Aβ-rebound and accumulation of β-C-terminal fragment

J. Neurochem. (2012) 121, 277–286.

Orally bioavailable and brain-penetrant pyridazine and pyridine-derived γ-secretase modulators reduced amyloidogenic Aβ peptides in vivo

Accumulation of amyloid β (Aβ) in brain is a pathological hallmark of Alzheimers disease (AD). Aβ is generated after sequential cleavage of its parental molecule, amyloid precursor protein (APP), by β- and γ-secretases. Inhibition of γ-secretase activity is an effective approach for the reduction of Aβ levels. Since γ-secretase targets many different substrates, selective inhibition of its cleavage of APP is believed to be critical in order to avoid undesirable side effects. γ-Secretase modulator (GSM) shifts the cleavage site on APP and production of amyloidogenic to non-amyloidogenic Aβ fragments. Since GSMs only modulate and do not block cleavage of γ-secretase substrates, they are believed less likely to produce untoward adverse reactions. Here, we report in vivo Aβ-lowering profiles of a pyridazine and a pyridine-derived GSM: GSM-C (Wan et al., 2011a) and GSM-D (Wan et al., 2011b). Both compounds reduced Aβ40 and Aβ42 productions, increased shorter Aβ fragments, and had little effect on Notch signaling (∼100-fold selective). They had excellent oral bioavailability (97.8% for GSM-C, ∼100% for GSM-D) and good brain permeability (free brain to free blood AUC ratio of 0.41 and 1.10 for GSM-C and GSM-D, respectively). Oral administration of these compounds in both acute and sub-chronic conditions reduced Aβ levels in plasma and brain in rats in a dose- and time-dependent manner. Therefore, GSM-C and GSM-D represent two GSMs that are orally bioavailable and brain-permeable. They could serve as excellent tools in the investigation of the role of Aβ peptides in AD pathogenesis.

Fyn kinase regulates phosphorylation and localization of APP and Dab1 to lipid rafts

Background: We have recently shown that Fyn, a Src family tyrosine kinase, phosphorylates APP, increases cell surface APP, and increases alpha-cleavage of APP. These effects were mediated through Dab1, an adaptor protein whose interaction with APP alters its trafficking and decreases its amyloidogenic processing. However, it is unknown where and how these interactions between APP, Fyn, and Dab1 take place. Fyn is highly enriched in lipid rafts, suggesting that its effect on APP and Dab1 may be compartmentally regulated. Methods: We isolated lipid rafts from wild-type and Fyn knock-out mouse brains in 1% Triton-X using a sucrose gradient and ultracentrifuged for 12 hrs at 4 C at 33,000rpm (Beckman S4w40Ti). Co-immunoprecipitations were conducted with 200mg lysate, 20mL Sepharose beads, and 1mL antibody. Primary neurons (DIV14) were treated with 100x concentrated Reelin or control media for 20min, 1hr, or 4hrs. Results: A large fraction of Dab1 and APP was found in lipid rafts of wild-type mice, but significantly lower fractions were found in lipid rafts of knock-out mice. Co-immunoprecipitation revealed similar levels of interaction between APP and Dab1 in and out of lipid rafts in wild-type mice. Surprisingly, despite the decreased fraction of APP and Dab1 in lipid rafts of Fyn knock-out mice, interaction between APP and Dab1 occurred predominantly in lipid rafts. We found that tyrosine-phosphorylated Dab1 and APP were found only in lipid rafts of wild-type mice; however, pDab1 was distributed equally in and out of lipid rafts isolated from Fyn knock-out mice. To determine whether Dab1 phosphorylation by Fyn regulated the APP-Dab1 interaction, we treated primary neurons with Reelin, an extracellular protein that activates Dab1 through Fyn, and measured co-precipitation of APP and Dab1 at various time points. We found that the APPDab1 interaction decreased as Dab1 phosphorylation increasedan effect that recovered over time as levels of p-Dab1 returned to baseline levels. Conclusions: These data suggest that Fyn localizes APP and Dab1 to lipid rafts, and that phosphorylation of Dab1 by Fyn in lipid rafts reduces the interaction between APP and Dab1. Fyn regulation of APP and Dab1 phosphorylation and localization may have significant effects on the trafficking and processing of APP.

γ-secretase inhibitor, but not modulator, causes Aβ overshoot and accumulation of β-CTF

peptides mainly generate two isoform, Aß40 and Aß42 by enzymatic proteolysis of amyloid precursor protein (APP). In particular, the Aß42 is believed to be the major etiologic agent in pathogenesis of AD due to its higher fibrillation or oligomerization properties than that of Aß40. Recently we have established conformation dependent human antibody, B6, which binds to Aß42 fibril, but not to soluble form of Aß42, inhibiting Aß42 fibril formation. Concurrently, we have identified a mimotope of B6, B6-C15, using the PhD.C7C phage library. We chemically synthesized TAT-conjugated B6-C15 peptide, TATB6-C15. This synthetic peptide has inhibitory activity on Aß42 fibrillation. Furthermore, TAT-B6-C15 specifically binds to the oligomer form of Aß42, but not to freshly prepared monomer Aß42 nor its fibril form. In this study, we investigated the effect of this TAT-B6-C15 peptide on Aß40 assembly. Methods: Aß42 or Aß40 was incubated at 37 C in the presence or absence of TAT-B6-C15 peptide. Aß fibrillation was monitored by amyloid specific fluorescence dye, Thioflavin T. To identify the Aß conformers which specifically bound to TAT-B6-C15 peptide, we performed dot blot analysis. Aß conformers were periodically sampled after the onset of Aß40 or 42 fibrillation assay and hand-spotted onto nitrocellulose membrane, followed by incubation with detection probes such as TAT-B6-C15 or anti-Aß antibody. Results: The TAT-B6-C15 peptide exhibited inhibitory effect on Aß42, but not Aß40 fibrillation. Furthermore, the TAT-B6-C15 showed binding activity to the Aß42 prefibrillar oligomer, but not any Aß40 conformers. Conclusions: The mimotope peptide which identified as conformation dependent antibody epitope, specifically binds to prefibrillar oligomers of Aß42, inhibiting Aß42 but not Aß40 fibril formation.