The novel beta-secretase inhibitor KMI-429 reduces amyloid beta peptide production in amyloid precursor protein transgenic and wild-type mice

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid plaques and neurofibrillary tangles in the brain. The major component of the plaques, amyloid beta peptide (Abeta), is generated from amyloid precursor protein (APP) by beta- and gamma-secretase-mediated cleavage. Because beta-secretase/beta-site APP cleaving enzyme 1 (BACE1) knockout mice produce much less Abeta and grow normally, a beta-secretase inhibitor is thought to be one of the most attractive targets for the development of therapeutic interventions for AD without apparent side-effects. Here, we report the in vivo inhibitory effects of a novel beta-secretase inhibitor, KMI-429, a transition-state mimic, which effectively inhibits beta-secretase activity in cultured cells in a dose-dependent manner. We injected KMI-429 into the hippocampus of APP transgenic mice. KMI-429 significantly reduced Abeta production in vivo in the soluble fraction compared with vehicle, but the level of Abeta in the insoluble fraction was unaffected. In contrast, an intrahippocampal injection of KMI-429 in wild-type mice remarkably reduced Abeta production in both the soluble and insoluble fractions. Our results indicate that the beta-secretase inhibitor KMI-429 is a promising candidate for the treatment of AD.     

Structural Stabilities of Different Regions of the Titin I27 Domain Contribute Differently to Unfolding upon Mitochondrial Protein Import

Protein import into mitochondria requires unfolding of the folded mature domain of precursor proteins. Here we compared the effects of amino-acid replacement between the core region and the N-terminal region of the titin I27 domain (the 27th Ig domain of human titin) on its import into isolated mitochondria when attached to a short presequence (pb₂(35)). We found that several mutations in the core region around Trp34 of the I27 domain enhanced the import rates of the fusion proteins, while the N-terminal K6P mutation, which increases mechanical stability around the N-terminal region, decreases the import rate. When the K6P mutation is combined with core-destabilizing mutations, the import rates of the fusion proteins still decrease, unless a long segment is deleted. These results suggest that mutations in the core region could destabilize the transition state for unfolding from the intermediate with the detached N-terminal segment during import, leading to enhanced unfolding rates, although stabilization of the N-terminal region masks these effects. In other words, the rate-limiting step of the global unfolding upon import into mitochondria switches, depending on the balance between the stability of the N-terminal structure and the stability of the core region of the I27 domain.     

Cell Surface Expression of the Major Amyloid-β Peptide (Aβ)-degrading Enzyme, Neprilysin, Depends on Phosphorylation by Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (MEK) and Dephosphorylation by Protein Phosphatase 1a

Neprilysin is one of the major amyloid-β peptide (Aβ)-degrading enzymes, the expression of which declines in the brain during aging. The decrease in neprilysin leads to a metabolic Aβ imbalance, which can induce the amyloidosis underlying Alzheimer disease. Pharmacological activation of neprilysin during aging therefore represents a potential strategy to prevent the development of Alzheimer disease. However, the regulatory mechanisms mediating neprilysin activity in the brain remain unclear. To address this issue, we screened for pharmacological regulators of neprilysin activity and found that the neurotrophic factors brain-derived neurotrophic factor, nerve growth factor, and neurotrophins 3 and 4 reduce cell surface neprilysin activity. This decrease was mediated by MEK/ERK signaling, which enhanced phosphorylation at serine 6 in the neprilysin intracellular domain (S6-NEP-ICD). Increased phosphorylation of S6-NEP-ICD in primary neurons reduced the levels of cell surface neprilysin and led to a subsequent increase in extracellular Aβ levels. Furthermore, a specific inhibitor of protein phosphatase-1a, tautomycetin, induced extensive phosphorylation of the S6-NEP-ICD, resulting in reduced cell surface neprilysin activity. In contrast, activation of protein phosphatase-1a increased cell surface neprilysin activity and lowered Aβ levels. Taken together, these results indicate that the phosphorylation status of S6-NEP-ICD influences the localization of neprilysin and affects extracellular Aβ levels. Therefore, maintaining S6-NEP-ICD in a dephosphorylated state, either by inhibition of protein kinases involved in its phosphorylation or by activation of phosphatases catalyzing its dephosphorylation, may represent a new approach to prevent reduction of cell surface neprilysin activity during aging and to maintain physiological levels of Aβ in the brain.     

Morphology of filamentous thalli of Analipus japonicus (Ralfsiales), temperature and irradiance effects on their formation and growth,and erect thallus formation in the field

Journal of Applied Phycology - Free-living filamentous thalli derived from an Analipus japonicus erect thallus were established in laboratory culture. The filamentous thalli were composed of...     

Evidence of local short‐distance spawning migration of tropical freshwater eels,and implications for the evolution of freshwater eel migration

Freshwater eels have fascinated biologists for centuries due to the spectacular long‐distance migrations between the eels’ freshwater habitats and their spawning areas far out in the ocean and the mysteries of their ecology. The spawning areas of Atlantic eels and Japanese eel were located far offshore in the Atlantic Ocean and the Pacific Ocean, respectively, and their reproduction took place thousands of kilometers away from their growth habitats. Phylogenetic studies have revealed that freshwater eels originated in the Indonesian region. However, remarkably little is known about the life histories of tropical freshwater eels despite the fact that tropical eels are key to understanding the nature of primitive forms of catadromous migration. This study found spawning‐condition tropical freshwater eels in Lake Poso, central Sulawesi, Indonesia, with considerably high gonadosomatic index values and with histologically fully developed gonads. This study provides the first evidence that under certain conditions, freshwater eels have conditions that are immediately able to spawn even in river downstream. The results suggest that, in contrast to the migrations made by the Atlantic and Japanese eels, freshwater eels originally migrated only short distances of <100 kilometers to local spawning areas adjacent to their freshwater growth habitats. Ancestral eels most likely underwent a catadromous migration from local short‐distance movements in tropical coastal waters to the long‐distance migrations characteristic of present‐day temperate eels, which has been well established as occurring in subtropical gyres in both hemispheres.     

Interleukin-1 beta up-regulates TACE to enhance alpha-cleavage of APP in neurons: resulting decrease in Abeta production

The proinflammatory cytokine interleukin (IL)-1β is up-regulated in microglial cells surrounding amyloid plaques, leading to the hypothesis that IL-1β is a risk factor for Alzheimer's disease. However, we unexpectedly found that IL-1β significantly enhanced α-cleavage, indicated by increases in sAPPα and C83, but reduced β-cleavage, indicated by decreases in sAPPβ and Aβ40/42, in human neuroblastoma SK-N-SH cells. IL-1β did not significantly alter the mRNA levels of BACE1, ADAM-9, and ADAM-10, but up-regulated that of TACE by threefold. The proform and mature form of TACE protein were also significantly up-regulated. A TACE inhibitor (TAPI-2) concomitantly reversed the IL-1β-dependent increase in sAPPα and decrease in sAPPβ, suggesting that APP consumption in the α-cleavage pathway reduced its consumption in the β-cleavage pathway. IL-1Ra, a physiological antagonist for the IL-1 receptor, reversed the effects of IL-1β, suggesting that the IL-1β-dependent up-regulation of α-cleavage is mediated by the IL-1 receptor. IL-1β also induced this concomitant increase in α-cleavage and decrease in β-cleavage in mouse primary cultured neurons. Taken together we conclude that IL-1β is an anti-amyloidogenic factor, and that enhancement of its signaling or inhibition of IL-1Ra activity could represent potential therapeutic strategies against Alzheimer's disease.     

Migratory histories of three types of Cottus pollux (small-egg, middle-egg, and large-egg types) as revealed by otolith microchemistry

 Migratory histories of three types of Cottus pollux, the small-egg type (SE type), middle-egg type (ME type), and large-egg type (LE type), were studied by examining strontium (Sr) and calcium (Ca) in their otoliths with wavelength dispersive X-ray spectrometry on an electron microprobe. The Sr : Ca ratios in the otoliths changed both with ontogenetic development and with salinity of the habitat. Otolith Sr : Ca ratios of LE-type samples and the ME-type samples from the Honmyo River, Kyushu Island, showed consistently low ratios, averaging 1.8 × 10⁻³ and 2.4 × 10⁻³ from the core to the edge, respectively. In contrast, otolith Sr : Ca ratios of SE-type samples and the other four ME-type samples from Hokkaido and Honshu Islands fluctuated strongly along the life history transects in accordance with migration patterns from freshwater to the sea and vice versa. The otolith Sr : Ca ratios of SE-type samples showed low ratios from the core to a point around 15 μm, averaging 1.5 × 10⁻³, and subsequently increased sharply with a high Sr : Ca ratio phase to a point around 400 μm, averaging 5.5 × 10⁻³, and followed again a low ratio phase to the edge with averages of 3.1 × 10⁻³. Similar fluctuation patterns in otolith Sr : Ca ratios were found for the four ME-type samples. These findings clearly demonstrated that otolith Sr : Ca ratios reflected the sculpin's life histories, as being fluvial for the LE type and the Honmyo River ME type and amphidromous for the SE type and the other four populations of ME type. Received: August 1, 2002 / Revised: October 15, 2002 / Accepted: October 28, 2002 Acknowledgments We thank Dr. N. Miyazaki, University of Tokyo, for his kind guidance of our joint research. Thanks are also offered to Drs. H. Sakai, National Fisheries University, Y. Yamazaki, Toyama University, and R. Yokoyama, Hokkaido University, and Mrs. N. Okabe and Y. Suzuki of Yamagata Prefecture for their help in sample collection. This work was partly supported by a Grant-in-Aid (No. 13660171) from the Japan Ministry of Education, Science, Sports and Culture to A. Goto. Correspondence to:Akira Goto