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Alzheimer's disease (AD) is the most common type of dementia, and its pathogenesis is associated with accumulation of β-amyloid (Aβ) peptides. Aβ is produced from amyloid precursor protein (APP) that is sequentially cleaved by β- and γ-secretases. Therefore, APP processing has been a target in therapeutic strategies for managing AD; however, no effective treatment of AD patients is currently available. Here, to identify endogenous factors that modulate Aβ production, we performed a gene microarray–based transcriptome analysis of neuronal cells derived from human induced pluripotent stem cells, because Aβ production in these cells changes during neuronal differentiation. We found that expression of the glycophosphatidylinositol-specific phospholipase D1 (GPLD1) gene is associated with these changes in Aβ production. GPLD1 overexpression in HEK293 cells increased the secretion of galectin 3–binding protein (GAL3BP), which suppressed Aβ production in an AD model, neuroglioma H4 cells. Mechanistically, GAL3BP suppressed Aβ production by directly interacting with APP and thereby inhibiting APP processing by β-secretase. Furthermore, we show that cells take up extracellularly added GAL3BP via endocytosis and that GAL3BP is localized in close proximity to APP in endosomes where amyloidogenic APP processing takes place. Taken together, our results indicate that GAL3BP may be a suitable target of AD-modifying drugs in future therapeutic strategies for managing AD.

Neprilysin (NEP) and insulin-degrading enzyme (IDE) are considered the two major catabolic enzymes that degrade amyloid β peptide (Aβ), the primary cause of Alzheimer’s disease (AD). However, their roles in Aβ metabolism in vivo have never been compared in an impartial and side-by-side manner. Here, we crossbred single App knock-in mice with NEP (Mme) KO mice and with IDE (Ide) KO mice to generate double mutants that were analyzed for their biochemical and Aβ pathology properties. We found that NEP is responsible for the metabolism of amyloidogenic insoluble Aβ whereas IDE affects soluble Aβ. A deficiency of NEP, but not of IDE, augmented the formation of Aβ plaques, dystrophic neurites, and astrocytic and microglial activation, all of which are key pathological events in the development of AD. In addition, a deficiency of NEP had no significant impact on the levels of various neuropeptides (somatostatin, substance P, cholecystokinin, and neuropeptide Y), well known to be in vitro substrates for NEP, presumably because NEP is expressed in secretory vesicles and on the presynaptic membranes of excitatory neurons while most if not all neuropeptides are secreted from inhibitory neurons. This argues against the concern that NEP up-regulation for treatment of preclinical AD would reduce the levels of these neuropeptides. These findings indicate that NEP relatively selectively degrades Aβ in the brain. Whereas familial AD (FAD) is unambiguously caused by an increased anabolism of Aβ, and of Aβ 42 and Aβ 43 in particular, the anabolism of Aβ appears unaffected before its deposition in the brain that subsequently leads to the onset of sporadic AD (SAD). These observations thus suggest that NEP-sensitive amyloidogenic Aβ likely plays a primary pathogenic role in the etiology of SAD. Our findings are consistent with the aging-dependent decline of NEP expression in human brain and with recent genome-wide association studies (GWAS) indicating that variants of the gene encoding NEP (MME) are associated with the risk of SAD development. Taken together, our results imply that the aging-associated decrease in NEP expression is a primary cause of SAD and could thus be a target for the treatment of preclinical AD once other factors such as apolipoprotein E genotypes have also been considered.

Mutations of PRRT2 (proline-rich transmembrane protein 2) cause several neurological disorders, represented by paroxysmal kinesigenic dyskinesia (PKD), which is characterized by attacks of involuntary movements triggered by sudden voluntary movements. PRRT2 is reported to suppress neuronal excitation, but it is unclear how the function of PRRT2 is modulated during neuronal excitation. We found that PRRT2 is processed to a 12 kDa carboxy-terminal fragment (12K-CTF) by calpain, a calcium-activated cysteine protease, in a neuronal activity-dependent manner, predominantly via NMDA receptors or voltage-gated calcium channels. Furthermore, we clarified that 12K-CTF is generated by sequential cleavages at Q220 and S244. The amino-terminal fragment (NTF) of PRRT2, which corresponds to PKD-related truncated mutants, is not detected, probably due to rapid cleavage at multiple positions. Given that 12K-CTF lacks most of the proline-rich domain, this cleavage might be involved in the activity-dependent enhancement of neuronal excitation perhaps through transient retraction of PRRT2's function. Therefore, PRRT2 might serve as a buffer for neuronal excitation, and lack of this function in PKD patients might cause neuronal hyperexcitability in their motor circuits., FASEB journal, 34(1), pp.180-191; 2020

NEP (Neprilysin) degrades natriuretic peptides, and its inhibition is a clinically accepted target for heart failure treatment. NEP is widely expressed in various organs, including the heart. However, the pathophysiological significance of local cardiac NEP is not fully understood. To study the local function of NEP in the heart, we generated transgenic mice overexpressing NEP, specifically in cardiomyocytes (CM-NEP Tg). At baseline, CM-NEP Tg mice showed significantly lower levels of plasma ANP (atrial natriuretic peptide), plasma cGMP, and cardiac tissue cGMP versus wild-type (WT) mice. Blood pressure, heart weight, and cardiomyocyte diameter were greater in CM-NEP Tg than WT mice. There were no significant differences in interstitial fibrosis or ejection fraction. Transverse aortic constriction (TAC) surgery significantly increased left ventricular weight in WT and CM-NEP Tg mice 3 weeks post-op versus sham surgery; however, the cardiac hypertrophic response to TAC was higher in CM-NEP Tg than WT mice. Cardiac interstitial fibrosis was induced in TAC CM-NEP Tg mice, whereas TAC WT mice had none. TAC CM-NEP Tg, but not TAC WT, mice developed cardiac dysfunction secondary to TAC with echocardiography. Furthermore, administration of human ANP to raise plasma ANP levels comparable to those in WT mice neither improved the exacerbated cardiac hypertrophy and fibrosis nor recovered impaired cardiac function in CM-NEP Tg mice after TAC. In conclusion, overexpression of NEP in cardiomyocytes promoted degradation of natriuretic peptides in the heart and led to an exaggerated response of hypertrophy and fibrosis to pressure overload.

Amyloid-β peptide (Aβ) accumulation is a triggering event leading to the Alzheimer's 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 DYRK1Amediated phosphorylation. DYRK1A inhibition may be a promising disease-modifying therapy for AD via neprilysin upregulation., Biological & Pharmaceutical Bulletin, 40(3), pp.327-333; 2017

Variants of triggering receptor expressed on myeloid cells 2 (TREM2) are associated with an increased incidence of Alzheimer’s disease, as well as other neurodegenerative disorders. Using a newly developed,highly sensitive reporter cell model, consisting of Jurkat T cells stably overexpressing a reporter gene and a gene encoding TREM2DAP12 fusion protein, we show here that TREM2-dependent signal transduction in response to apoptotic Neuro2a cells is mediated by aminophospholipid ligands,phosphatidylserine and phosphatidylethanolamine, which are not exposed on the intact cell surface, but become exposed upon apoptosis. We also show that signal-transducing TREM2 ligands different from aminophospholipids, which appear to be derived from neurons, might be present in membrane fractions of mouse cerebral cortex. These results may suggest that TREM2 regulates microglial function by transducing intracellular signals from aminophospholipids on apoptotic cells, as well as unidentified ligands in the membranes of the cerebral cortex., Scientific Reports, 9(1), art.no.7508; 2019

Calpain enzymes proteolytically modulate cellular function and have been implicated in inflammatory diseases. In this study, we found that calpain levels did not differ between intestinal tissues from inflammatory bowel disease (IBD) patients and healthy controls, but IBD tissues showed increased levels of the endogenous calpain inhibitor, calpastatin (CAST). To investigate the role of CAST in the immune system during IBD, mice were x-ray irradiated, reconstituted with either CAST-knockout (KO) or wild-type (WT) bone marrow, and subjected to dextran sulfate sodium–induced colitis. CAST-KO recipients with induced colitis exhibited more severe weight loss, bloody diarrhea, and anemia compared with WT controls. Histological evaluation of colons from KO recipients with colitis revealed increased inflammatory pathology. Macrophages purified from the colons of KO recipients had higher IL-6, TNF-α, and IFN-γ mRNA levels compared with WT controls. Mechanistic investigations using small interfering RNA and KO bone marrow to generate CAST-deficient macrophages showed that CAST deficiency during activation with bacterial pathogen associated molecular patterns, including heat-killed Enterococcus faecalis or CpG DNA, led to increased IκB cleavage, NF-κB nuclear localization, and IL-6 and TNF-α secretion. Thus, CAST plays a central role in regulating macrophage activation and limiting pathology during inflammatory disorders like IBD.

Accumulation of intracellular tau fibrils has been the focus of research on the mechanisms of neurodegeneration in Alzheimer's disease (AD) and related tauopathies. Here, we have developed a class of tau ligands, phenyl/pyridinyl-butadienyl-benzothiazoles/benzothiazoliums (PBBs), for visualizing diverse tau inclusions in brains of living patients with AD or non-AD tauopathies and animal models of these disorders. In vivo optical and positron emission tomographic (PET) imaging of a transgenic mouse model demonstrated sensitive detection of tau inclusions by PBBs. A pyridinated PBB, [(11)C]PBB3, was next applied in a clinical PET study, and its robust signal in the AD hippocampus wherein tau pathology is enriched contrasted strikingly with that of a senile plaque radioligand, [(11)C]Pittsburgh Compound-B ([(11)C]PIB). [(11)C]PBB3-PET data were also consistent with the spreading of tau pathology with AD progression. Furthermore, increased [(11)C]PBB3 signals were found in a corticobasal syndrome patient negative for [(11)C]PIB-PET.

Oligopeptidase B (OPB; EC 3.4.21.83) from 2 Gram-negative bacteria, Stenotrophomonas maltophilia (Stm) and Serratia marcescens (Sem), and the Gram-positive bacterium Rhodococcus erythropolis (Re) were cloned and characterized to clarify their activities and substrate specificities using peptidyl-MCA substrates containing Arg or Lys. The cloned enzymes, Stm, Sem and ReOPBs, in addition to Escherichia coli OPB (EcOPB) were expressed using a pET expression system. Although the Stm and SemOPBs share 45% sequence identity to each other and up to 60% identity with respect to their catalytic domains, their activities towards MCA substrates were quite different. StmOPB is approximately 100-500 times more active than SemOPB and 3-30 times more active than EcOPB. The activity of ReOPB is comparable to that of StmOPB and it shares 40% and 36% identity to StmOPB and SemOPB, respectively. Some features of Stm, Re and EcOPBs are similar to those of previously cloned OPBs, which were also strongly inhibited by substrates, but SemOPB differs from all other OPBs in that it is not inhibited by substrates; even substrates containing double arginine at 35μM did not inhibit SemOPB. On the other hand, the same substrates at only 5μM inhibited the activity of the Stm, Re, and EcOPB. This phenomenon was not observed with substrates containing single or double lysine., Biological and Pharmaceutical Bulletin, 35(11), pp.2010-2016; 2012

The mechanism by which amyloid-β peptide (Aβ) accumulation causes neurodegeneration in Alzheimer's disease (AD) remains unresolved. Given that Aβ perturbs calcium homeostasis in neurons, we investigated the possible involvement of calpain, a calcium-activated neutral protease. We first demonstrated close postsynaptic association of calpain activation with Aβ plaque formation in brains from both patients with AD and transgenic (Tg) mice overexpressing amyloid precursor protein (APP). Using a viral vector-based tracer, we then showed that axonal termini were dynamically misdirected to calpain activation-positive Aβ plaques. Consistently, cerebrospinal fluid from patients with AD contained a higher level of calpain-cleaved spectrin than that of controls. Genetic deficiency of calpastatin (CS), a calpain-specific inhibitor protein, augmented Aβ amyloidosis, tau phosphorylation, microgliosis, and somatodendritic dystrophy, and increased mortality in APP-Tg mice. In contrast, brain-specific CS overexpression had the opposite effect. These findings implicate that calpain activation plays a pivotal role in the Aβ-triggered pathological cascade, highlighting a target for pharmacological intervention in the treatment of AD.

Core pathologies of Alzheimer's disease (AD) are aggregated amyloid-β peptides (Aβ) and tau, and the latter is also characteristic of diverse neurodegenerative tauopathies. These amyloid lesions provoke microglial activation, and recent neuroimaging technologies have enabled visualization of this response in living brains using radioligands for the peripheral benzodiazepine receptor also known as the 18 kDa translocator protein (TSPO). Here, we elucidated contributions of Aβ and tau deposits toin vivoTSPO signals in pursuit of mechanistic and diagnostic significance of TSPO imaging in AD and other tauopathies. A new antibody to human TSPO revealed induction of TSPO-positive microgliosis by tau fibrils in tauopathy brains. Emergence of TSPO signals before occurrence of brain atrophy and thioflavin-S-positive tau amyloidosis was also demonstrated in living mice transgenic for mutant tau by positron emission tomography (PET) with two classes of TSPO radioligands, [11C]AC-5216 and [18F]fluoroethoxy-DAA1106. Meanwhile, only modest TSPO elevation was observed in aged mice modeling Aβ plaque deposition, despite the notably enhancedin vivobinding of amyloid radiotracer, [11C]Pittsburgh Compound-B, to plaques. In these animals, [11C]AC-5216 yielded better TSPO contrasts than [18F]fluoroethoxy-DAA1106, supporting the possibility of capturing early neurotoxicity with high-performance TSPO probes. Furthermore, an additional line of mice modeling intraneuronal Aβ accumulation displayed elevated TSPO signals following noticeable neuronal loss, unlike TSPO upregulation heralding massive neuronal death in tauopathy model mice. Our data corroborate the utility of TSPO-PET imaging as a biomarker for tau-triggered toxicity, and as a complement to amyloid scans for diagnostic assessment of tauopathies with and without Aβ pathologies.

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 Alzheimer's 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.

Cerebral deposition of beta-amyloid (Abeta) peptides is a pathological hallmark of Alzheimer disease. Intramembranous proteolysis of amyloid precursor protein by a multiprotein gamma-secretase complex generates Abeta. Previously, it was reported that CD147, a glycoprotein that stimulates production of matrix metalloproteinases (MMPs), is a subunit of gamma-secretase and that the levels of secreted Abeta inversely correlate with CD147 expression. Here, we show that the levels and localization of CD147 in fibroblasts, as well as postnatal expression and distribution in brain, are distinct from those of integral gamma-secretase subunits. Notably, we show that although depletion of CD147 increased extracellular Abeta levels in intact cells, membranes isolated from CD147-depleted cells failed to elevate Abeta production in an in vitro gamma-secretase assay. Consistent with an extracellular source that modulates Abeta metabolism, synthetic Abeta was degraded more rapidly in the conditioned medium of cells overexpressing CD147. Moreover, modulation of CD147 expression had no effect on epsilon-site cleavage of amyloid precursor protein and Notch1 receptor. Collectively, our results demonstrate that CD147 modulates Abeta levels not by regulating gamma-secretase activity, but by stimulating extracellular degradation of Abeta. In view of the known function of CD147 in MMP production, we postulate that CD147 expression influences Abeta levels by an indirect mechanism involving MMPs that can degrade extracellular Abeta.

A subtle but chronic alteration in metabolic balance between amyloid-beta peptide (Abeta) anabolic and catabolic activities is thought to cause Abeta accumulation, leading to a decade-long pathological cascade of Alzheimer disease. However, it is still unclear whether a reduction of the catabolic activity of Abeta in the brain causes neuronal dysfunction in vivo. In the present study, to clarify a possible connection between a reduction in neprilysin activity and impairment of synaptic and cognitive functions, we cross-bred amyloid precursor protein (APP) transgenic mice (APP23) with neprilysin-deficient mice and biochemically and immunoelectron-microscopically analyzed Abeta accumulation in the brain. We also examined hippocampal synaptic plasticity using an in vivo recording technique and cognitive function using a battery of learning and memory behavior tests, including Y-maze, novel-object recognition, Morris water maze, and contextual fear conditioning tests at the age of 13-16 weeks. We present direct experimental evidence that reduced activity of neprilysin, the major Abeta-degrading enzyme, in the brain elevates oligomeric forms of Abeta at the synapses and leads to impaired hippocampal synaptic plasticity and cognitive function before the appearance of amyloid plaque load. Thus, reduced neprilysin activity appears to be a causative event that is at least partly responsible for the memory-associated symptoms of Alzheimer disease. This supports the idea that a strategy to reduce Abeta oligomers in the brain by up-regulating neprilysin activity would contribute to alleviation of these symptoms.

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.

Neprilysin (NEP) is a rate-limiting amyloid beta peptide (Abeta)-degrading enzyme in the brain. We demonstrated previously that overexpression of neprilysin in primary cortical neurons remarkably decreased not only extracellular but also intracellular Abeta levels. To investigate the subcellular compartments where neprilysin degrades Abeta most efficiently, we expressed neprilysin chimeric proteins containing various subcellular compartment-targeting domains in neurons. Sec12-NEP, beta-galactoside alpha2,6-sialyltransferase-NEP, transferrin receptor-NEP, and growth-associated protein 43-NEP were successfully sorted to the endoplasmic reticulum, trans-Golgi network, early/recycling endosomes, and lipid rafts, respectively. We found that intracellularly, wild-type neprilysin and all the chimeras showed equivalent Abeta40-degrading activities. Abeta40 was more effectively cleared than Abeta42, and this tendency was greater for intracellular Abeta than for extracellular Abeta. Wild-type and trans-Golgi network-targeted ST-NEP cleared more intracellular Abeta42 than the other chimeras. Wild-type neprilysin cleared extracellular Abeta more effectively than any of the chimeras, among which endoplasmic reticulum-targeted Sec12-NEP was the least effective. These observations indicate that different intracellular compartments may be involved in the metabolism of distinct pools of Abeta (Abeta40 and Abeta42) to be retained or recycled intracellularly and to be secreted extracellularly, and that the endogenous targeting signal in wild-type neprilysin is well optimized for the overall neuronal clearance of Abeta.

Aggregation and accumulation of amyloid-β peptide (Aβ) in the brain are triggering events leading to the pathological cascade of Alzheimer's disease (AD). Aβ accumulates in AD brains and forms amyloid plaques, which consist mostly of amino-terminally truncated and/or modified Aβs, among which Aβ3pyroglutamate (Aβ3pE) is a major product. Thus, the N-terminal structures of accumulated species of Aβ are different from those secreted from neurons. Aβ3pE-42 is more hydrophobic, more easily self-aggregated (250-fold), and is more resistant to proteolytic degradation (4-fold) than Aβ1-42. Therefore, Aβ3pE appears to act as a seed for the formation of oligomers and amyloid plaques. Aβ is physiologically degraded via the neprilysin-mediated pathway in the brain. However, if neprilysin activity is low, a compensatory metabolic pathway is up-regulated, in which exopeptidases, such as aminopeptidase or dipeptidyl peptidase, and glutaminyl cyclase (QC) may be involved, generating Aβ3pE. It is reported that QC is up-regulated with AD development. Recent study revealed that administration of synthetic QC inhibitor reduced total amyloid burden in the brains of APP transgenic mice (Tg2576) via inhibition of Aβ3pE production and also alleviated impaired cognitive function. Thus, inhibition of Aβ3pE formation appears to be a novel target for therapy and prevention of AD.

Deposition of amyloid-b peptide (Ab), a pathological hallmark of Alzheimer's disease (AD), locates upstream of the AD cascade. Ab is constantly anabolized and catabolized in the brain, and the steady-state Ab level is physiologically determined by the metabolic balance between the anabolic and catabolic activities. Even subtle alterations in this metabolic balance over a long period of time could turn Ab into a pathogenic agent that influences both the pathological progression and incidence of the disease. Therefore, AD may be a consequence of proteolytic disorder. In order to overcome AD, lowering the Ab levels in the brain is necessary. There are many strategies to address this, such as b-, g-secretase inhibitors or Ab vaccination. Recently, we demonstrated that neprilysin is the most potent Ab-degrading enzyme in vivo and hypothesized that neprilysin deficiency would influence the steady-state Ab levels in the brain by alternating the Ab metabolism. Therefore, to maintain or upregulate neprilysin activity in the brain will be a relevant strategy for prevention and therapy of AD through reduction of the Ab levels. In this review, we describe several therapeutic strategies for manipulating the Ab level and our current understanding of Ab metabolism, focusing on the Ab-degrading enzyme, neprilysin. Drug Dev. Res. 56:171-183, 2002. � c 2002 Wiley-Liss, Inc.

To identify the amyloid beta peptide (Abeta) 1-42-degrading enzyme whose activity is inhibited by thiorphan and phosphoramidon in vivo, we searched for neprilysin (NEP) homologues and cloned neprilysin-like peptidase (NEPLP) alpha, NEPLP beta, and NEPLP gamma cDNAs. We expressed NEP, phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PEX), NEPLPs, and damage-induced neuronal endopeptidase (DINE) in 293 cells as 95- to 125-kDa proteins and found that the enzymatic activities of PEX, NEPLP alpha, and NEPLP beta, as well as those of NEP and DINE, were sensitive to thiorphan and phosphoramidon. Among the peptidases tested, NEP degraded both synthetic and cell-secreted Abeta1-40 and Abeta1-42 most rapidly and efficiently. PEX degraded cold Abeta1-40 and NEPLP alpha degraded both cold Abeta1-40 and Abeta1-42, although the rates and the extents of the digestion were slower and less efficient than those exhibited by NEP. These data suggest that, among the endopeptidases whose activities are sensitive to thiorphan and phosphoramidon, NEP is the most potent Abeta-degrading enzyme in vivo. Therefore, manipulating the activity of NEP would be a useful approach in regulating Abeta levels in the brain.

Amyloid beta peptide (Abeta) is a physiological peptide that is constantly catabolized in the brain. We previously demonstrated that an endopeptidase sensitive to phosphoramidon and thiorphan conducts the initial rate-limiting proteolysis of Abeta in vivo, but the exact molecular identity of the peptidase(s) has remained unknown because of the molecular redundancy of such activity. We analyzed the brain-derived enzyme by means of immuno-depletion and gene disruption, and demonstrate here that neprilysin accounts for the majority of the Abeta-degrading activity. Furthermore, kinetic analysis, giving a K(m) value of 2.8 +/- 0.76 microM, indicated that Abeta(1-42) is a relevant substrate for neprilysin.

The c- Cbl protooncogene product is a prominent substrate of protein tyrosine kinases and is rapidly tyrosine-phosphorylated upon stimulation of a wide variety of cell-surface receptors. We have identified a novel c-Cbl-interacting protein termed CIN85 with a molecular mass of 85 kDa which shows similarity to adaptor proteins, CMS and CD2AP. CIN85 mRNA is expressed ubiquitously in normal human tissues and cancer cell lines analyzed. CIN85 was basally associated with c-Cbl. For interaction of CIN85 with c-Cbl, the second SH3 domain of CIN85 was shown to serve as a central player. The CIN85–c-Cbl association was enhanced shortly after stimulation of 293 cells with epidermal growth factor (EGF) and gradually diminished to a basal level, which correlated with a tyrosine phosphorylation level of c-Cbl. Our results suggest that CIN85 may play a specific role in the EGF receptor-mediated signaling cascade via its interaction with c-Cbl.

The effects of C60, a fullerene, on the activities of glutathione 5-transferase (GST), glutathione peroxidase (GSH-Px) and glutathione reductase (GR) in rodent and human livers were investigated. the GST activity in rat liver towards frans-4-phenyl-3-buten-2-one and that in mouse liver towards ethacrynic acid were inhibited by C60- the GST activity towards 1,2-dichloro-4-nitrobenzene, isomerase activity of GST towards androst-5-ene-3,17-dione, GSH-Px activity and GR activity were not affected by C60 a kinetic study using purified mouse GST-π with ethacrynic acid (25-100 μM) as the substrate revealed that C60 was a non-competitive inhibitor of the enzyme with a Ki = 48.8 ± 0.25 μM and a Ki = 47.9 ±0.18 μM. However, C60 did not inhibit the activity of purified mouse GST-π towards l-chloro-2,4-dinitrobenzene. Thus, the inhibition by C60 appears to be substrate-specific. In human liver, C60 inhibited the GST activity towards ethacrynic acid, and moderately inhibited GSH-Px and GR activities.

A potent nitric oxide (NO) synthase inhibitor, Nω-nitro- l -arginine ( l -NA), suppressed tonic seizure elicited by pentylenetetrazol (PTZ; 100 mg/kg, SC) in a dose-related manner (25 to 100 mg/kg, IP), but had no effect on clonk seizure. The effect was most potent at 1 h after the administration of l -NA. l -NA (100 mg/kg, IP) suppressed clonic seizure as well as tonic seizure in bicuculline-treated (3.0 or 4.5 mg/kg, SC) mice. However, it did not affect seizures elicited by picrotoxin (2.0 to 6.0 mg/kg, SC). On the other hand, N -methyl-DL-aspartate (NMDLA; 300 mg/kg or 350 mg/kg, IP) induced clonic seizure, but tonic seizure was not always noted. All mice with clonic and tonic seizures died, and some mice with clonic seizure died without accompanying tonic seizure. l -NA did not influence NMDLA-induced seizures, but it appeared to enhance NMDLA lethality, though without statistical significance. These findings suggest distinct roles of NO in seizures induced by different drugs in mice.

We could prepare the microsomal fraction of mouse liver, without using an ultracentrifuge but with a low speed centrifuge. The procedure includes 1) lyophilization of post-mitochondrial fraction (9,000 x g supernatant) of mouse liver, 2) powdering of the lyophilized sample, 3) the addition of 1.15 per cent potassium chloride solution or distilled water, which afforded microsomal aggregates, 4) sedimentation of microsomal fraction by low-speed centrifugation (20,000 x g, 20 min). The sedimented microsomal fraction showed normal contents of cytochrome P-450 and cytochrome b5, and gave a normal pattern on SDS polyacrylamide gel electrophoresis and normal electron microscopic feature. This method should be convenient for rapid and large-scale preparation of microsomes, especially for the preparation of cytochrome b5 and cytochrome P-450.

Both mislocalization of TDP-43 and downregulation of RNA-editing enzyme ADAR2 co-localize in the motor neurons of amyotrophic lateral sclerosis patients, but how they are linked is not clear. Here we demonstrate that activation of calpain, a Ca2+-dependent cysteine protease, by upregulation of Ca2+-permeable AMPA receptors generates carboxy-terminal-cleaved TDP-43 fragments and causes mislocalization of TDP-43 in the motor neurons expressing glutamine/arginine site-unedited GluA2 of conditional ADAR2 knockout (AR2) mice that mimic the amyotrophic lateral sclerosis pathology. These abnormalities are inhibited in the AR2res mice that express Ca2+-impermeable AMPA receptors in the absence of ADAR2 and in the calpastatin transgenic mice, but are exaggerated in the calpastatin knockout mice. Additional demonstration of calpain-dependent TDP43 fragments in the spinal cord and brain of amyotrophic lateral sclerosis patients, and high vulnerability of amyotrophic lateral sclerosis-linked mutant TDP43 to cleavage by calpain support the crucial role of the calpain-dependent cleavage of TDP43 in the amyotrophic lateral sclerosis pathology.

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 Alzheimer's disease.

GABA is the major inhibitory neurotransmitter in the CNS and changes in GABAergic neurotransmission affect the overall activity of neuronal networks. The uptake of GABA into synaptic vesicles is mediated by the vesicular GABA transporter (VGAT), and changes in the expression of the transporter directly regulate neurotransmitter release. In this work we investigated the changes in VGAT protein levels during ischemia and in excitotoxic conditions, which may affect the demise process. We found that VGAT is cleaved by calpains following excitotoxic stimulation of hippocampal neurons with glutamate, giving rise to a stable truncated cleavage product (tVGAT). VGAT cleavage was also observed after transient middle cerebral artery occlusion in mice, a cerebral ischemia model, and following intrahippocampal injection of kainate, but no effect was observed in transgenic mice overexpressing calpastatin, a calpain inhibitor. Incubation of isolated cerebrocortical synaptic vesicles with recombinant calpain also induced the cleavage of VGAT and formation of stable tVGAT. Immunoblot experiments using antibodies targeting different regions of VGAT and N-terminal sequencing analysis showed that calpain cleaves the transporter in the N-terminal region, at amino acids 52 and 60. Immunocytochemistry of GABAergic striatal neurons expressing GFP fusion proteins with the full-length VGAT or tVGAT showed that cleavage of the transporter induces a loss of synaptic delivery, leading to a homogeneous distribution of the protein along neurites. Our results show that excitotoxicity downregulates full-length VGAT, with a concomitant generation of tVGAT, which is likely to affect GABAergic neurotransmission and may influence cell death during ischemia.

Paraquat (30-70 mg/kg intraperitoneally) caused typical shaking behaviour in rats in a dose-dependent manner. Myoclonus also appeared after the shaking behaviour in several rats treated with the highest dose of paraquat. Morphine (5 mg/kg intraperitoneally, 30 min. before paraquat) significantly reduced the frequency of shaking behaviour. The alleviation by morphine disappeared when naloxone (1.5 mg/kg intraperitoneally 15 min. after morphine) was coadministered. Although there was no histological change in brain slices of paraquat-treated rats (70 mg/kg intraperitoneally), the fluorescein uptake into brain was increased by the treatment. Morphine prevented the increase of fluorescein uptake, but naloxone failed to antagonize this effect. On the other hand, intracerebroventricularly administered paraquat (25.7 micrograms/rat) caused tremor in all rats, but not shaking behaviour nor myoclonus. These findings suggest that paraquat administered systemically as well as centrally may be toxic to the brain. Although the actions of paraquat on the brain seem to be complicated, opioid receptors may play a role in the actions.

The inducing effects of musk xylene, a non-mutagenic carcinogen, on hepatic glutathione S-transferase (GST) in mice were investigated. Musk xylene posessed a potency of inducing all isozymes of mouse GSTs, whereas the effects were different between male and female mice. Induction of both GST MI (α class) and GST MII (π class) by musk xylene was observed only ip female. GST MIII (μ class) was induced up to about 5-fold by musk xylene in both sexes. The induction of MIII was higher than those of the other isozymes. There were strain differences in the induction of MII and MIII by musk xylene in female mice. The induction observed in C57BL/6N female mice was higher than that in DBA/2N female mice. These results indicate that musk xylene possesses multifunctional effects on GST induction in male and female mice.

We investigated the effects of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) on ovarian carbonyl reductase activities towards 13, 14-dihydro-15-ketoprostaglandin F 2α (15KD-PGF 2α ), p -nitroacetophenone (PNAP) and p -nitrobenzaldehyde (PNBA) in mice and hamsters, and compared with their effects on those we observed previously in rats. The treatment with PMSG and hCG caused a significant increase in ovarian weights and superovulation in both mice and hamsters. Hamster ovary possessed appreciable carbonyl reductase activities towards all three substrates. whereas the activities were lower than those in rat ovary. The reductase activities were not increased by the treatment with gonadotropins, differing from rat ovarian carbonyl reductase. In untreated mice, carbonyl reductase activity towards 15KD-PGF 2α was not detected, whereas the activities towards PNAP and PNBA were detected, which activities were lower than those in rats and hamsters. The PNAP and PNBA reductase activities in mouse ovary were significantly increased up to 7.1- and 1.7-fold, respectively, by the treatment with gonadotropins. These results show that there are species differences in ovarian carbonyl reductase and response of the enzyme to gonadotropins.

The nitroreductase activities of rat liver microsomes and cytosol towards various nitrated naphthalenes (1-, 2-mononitro-, 1,3-, 1,5-, 1,7-, 1,8-dinitro-1,3,5- and 1,3,8-trinitronaphthalenes) were characterized as follows. (1) The rates of reduction of nitrated naphthalenes in either microsomal or cytosolic incubation were found to increase in the order of trinitro- > dinitro- > mono-nitronaphthalene, although, in the case of microsomal nitroreduction, trinitronaphthalenes were reduced more rapidly than in cytosol. (2) The effective cofactors, electron donors, in the nitroreduction of nitrated naphthalenes in cytosol were NADH and hypoxanthine, but not NADPH. (3) The nitrated naphthalenes with a nitro group at a beta-position appear to be more easily reduced among the various isomers. The cytosolic nitroreductase activities towards the nitrated naphthalenes were closely related to the single-electron reduction potentials measured by cyclic voltammetry and hence, there was a good relationship between the logarithm of nitroreductase activities and the electrochemical reduction potentials. In microsomes, nitroreductase activities were rather less well related to electrochemical reduction potentials.

The effect of musk xylene on contents of both cytochrome P-450IA1 and cytochrome P-450IA2 in rat liver was investigated using Western blotting analysis. Rats were treated i.p. for five consecutive days with either 50, 100 or 200 mg musk xylene/kg body weight. Musk xylene increased both total cytochrome P-450 and cytochrome b5 contents in rat liver microsomes. Musk xylene induced cytochrome P-450IA2 (384 pmol/mg protein) strongly and preferentially and the ratio of cytochrome P450IA2/P-450IA1 was about 12 at the lowest dose tested. Musk xylene also induced the cytochrome P-450IA1 dose-dependently, but these extents were very small (32-174 pmol/mg protein). These results suggest that musk xylene may be a more specific inducer for cytochrome P-450IA2 than any other inducers reported.

Increased oxidative damage is a prominent and early feature in Alzheimer disease. We previously crossed Alzheimer disease transgenic (APPsw) model mice with alpha-tocopherol transfer protein knock-out (Ttpa(-/-)) mice in which lipid peroxidation in the brain was significantly increased. The resulting double-mutant (Ttpa(-/-)APPsw) mice showed increased amyloid beta (Abeta) deposits in the brain, which was ameliorated with alpha-tocopherol supplementation. To investigate the mechanism of the increased Abeta accumulation, we here studied generation, degradation, aggregation, and efflux of Abeta in the mice. The clearance of intracerebral-microinjected (125)I-Abeta(1-40) from brain was decreased in Ttpa(-/-) mice to be compared with wild-type mice, whereas the generation of Abeta was not increased in Ttpa(-/-)APPsw mice. The activity of an Abeta-degrading enzyme, neprilysin, did not decrease, but the expression level of insulin-degrading enzyme was markedly decreased in Ttpa(-/-) mouse brain. In contrast, Abeta aggregation was accelerated in Ttpa(-/-) mouse brains compared with wild-type brains, and well known molecules involved in Abeta transport from brain to blood, low density lipoprotein receptor-related protein-1 (LRP-1) and p-glycoprotein, were up-regulated in the small vascular fraction of Ttpa(-/-) mouse brains. Moreover, the disappearance of intravenously administered (125)I-Abeta(1-40) was decreased in Ttpa(-/-) mice with reduced translocation of LRP-1 in the hepatocytes. These results suggest that lipid peroxidation due to depletion of alpha-tocopherol impairs Abeta clearances from the brain and from the blood, possibly causing increased Abeta accumulation in Ttpa(-/-)APPsw mouse brain and plasma.

Three enzyme forms (T1, T2, T3) from rat testis and two from rat vas deferens (V1, V2) of carbonyl reductase have been highly purified to apparent homogeneity. These carbonyl reductases from rat reproductive organs have several similarities in terms of molecular mass (32-33 kDa), isoelectric point (pI 5.9-6.4), immunochemical properties, cofactor requirement (NADPH dependency) and sensitivity to sulfhydryl reagents. The isoenzymes from the vas deferens (V1, V2) have similar catalytic activities, whereas those from the testis (T1, T2, T3) showed different catalytic activities from each other. All enzymes, however, reduced quinones, aromatic aldehydes and ketones, while T3, V1 and V2 were characterized as possessing high affinity towards prostaglandins. An immunoinhibition study using a specific antibody indicated that these enzymes were solely responsible for the overall catalytic activities of 13, 14-dihydro-15-oxo-prostaglandin F2 alpha, 4-benzoylpyridine, and 4-nitroacetophenone reduction and prostaglandin F2 alpha oxidation in both testis and vas deferens cytosol. The immunohistochemical staining revealed a positive immunoreactivity to antibody only in the Leydig cells of the testis, but neither the germ cells nor Sertoli cells in the seminiferous tubule. The staining also showed that the enzymes in the vas deferens were primarily localized in mucosal epithelium cells.

The tissue distribution of carbonyl reductase in ovary and liver of various animal species was investigated by measuring the reduction of 13,14-dihydro-15-keto-prostaglandin F2a, a specific substrate for rat ovarian carbonyl reductases, and by means of Western blotting analysis using anti-rat ovarian carbonyl reductase antibody. The highest ovarian carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a was found in rat among ten animal species tested, followed by hamster and monkey. The immunoreactive protein was detected in hamster and monkey ovaries. Although carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a was not detectable in non-pregnant rabbit ovary, pregnant rabbit ovary showed not only moderate activity but also immunoreactivity with anti-rat ovarian carbonyl reductase antibody. On the other hand, carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a was detected in hepatic tissue of all the species tested, except for rat and left-eye flounder. Immunoreactive proteins were present in hepatic tissue of various species that exhibited measurable carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a.

Carbonyl reductase activity and content in the rat ovary were measured at various stages of the estrous cycle, and the enzyme protein in the ovary was localized by immunohistochemistry. The enzyme activity increased after the preovulatory surge of luteinizing hormone (LH) on proestrus, and the enzyme content began to increase prior to the LH surge. Although the enzyme content reached the highest level at 2000 h and remained at a plateau for 8 h, the enzyme activity increased linearly until it reached the highest level at 0800 h on the morning of estrus. At their maximum, enzyme activity and content were approximately 1.5-fold and 2-fold greater, respectively, then basal diestrus values. The enzyme protein amounted to 1-4% of the ovarian cytosolic protein. An immunohistochemical study revealed that the enzyme was primarily localized in interstitial gland cells and theca interna cells of secondary and Graafian follicles as well as atretic follicles.

Effects of three glucocorticoids on ovulation, and on content and activity of ovarian carbonyl reductase in rats were investigated. Glucocorticoid treatment caused both significant decrease in ovarian and uterine weights, blockade of ovulation and marked decrease in content and activity of ovarian carbonyl reductase. Furthermore, the weak immunoreactivity to antibody against ovarian carbonyl reductase was shown in the theca cells and the interstitial gland cells in the glucocorticoid-treated ovary. The treatment with hCG (LH activity specific) restored the ovarian weight, and both the content and activity of ovarian carbonyl reductase, which were inhibited by glucocorticoids, to control level as well as ovulation. These results indicate that the ovarian carbonyl reductase may be closely involved in ovarian function, especially ovulation, and may be an LH-dependent enzyme, because it is well known that glucocorticoids inhibit both LH surge and ovulation in rats.

Berberine is an isoquinoline alkaloid isolated from Coptidis rhizoma, a major herb widely used in Chinese herbal medicine. Berberine's biological activity includes antidiarrheal, antimicrobial, and anti-inflammatory effects. Recent findings show that berberine prevents neuronal damage due to ischemia or oxidative stress and that it might act as a novel cholesterol-lowering compound. The accumulation of amyloid-beta peptide (Abeta) derived from amyloid precursor protein (APP) is a triggering event leading to the pathological cascade of Alzheimer's disease (AD); therefore the inhibition of Abeta production should be a rational therapeutic strategy in the prevention and treatment of AD. Here, we report that berberine reduces Abeta levels by modulating APP processing in human neuroglioma H4 cells stably expressing Swedish-type of APP at the range of berberine concentration without cellular toxicity. Our results indicate that berberine would be a promising candidate for the treatment of AD.

An imbalance between anabolism and catabolism causes an accumulation of amyloid beta-peptide (Abeta), which is a proposed trigger of the onset of Alzheimer's disease. Neprilysin is a rate-limiting peptidase that participates in the catabolism of Abeta in the brain. We examined whether rats continuously infused with thiorphan, a specific neprilysin inhibitor, into the hippocampus develop cognitive impairments through accumulation of Abeta. Thiorphan infusion elevated hippocampal Abeta40 and Abeta42 levels in the insoluble but not the soluble fraction. Thiorphan-infused rats displayed cognitive impairments in the ability to discriminate in the object recognition test, associative learning in the conditioned fear learning test, and spatial memory in the water maze test, tasks that depend on the hippocampus. These cognitive abilities in the battery of behavioral tasks inversely correlated with insoluble Abeta contents in the hippocampus. The nicotine-stimulated release of acetylcholine in the hippocampus of thiorphan-infused rats was significantly lower than that in vehicle-infused rats. These results indicate that continuous infusion of thiorphan into the hippocampus causes cognitive dysfunction and reduces cholinergic activity by raising the level of Abeta in the hippocampus and suggest that a reduction of neprilysin activity contributes to the deposition of Abeta and development of Alzheimer's disease.

An accumulation of amyloid beta peptide (Abeta) due to an imbalance between anabolism and catabolism triggers Alzheimer's disease (AD). Neprilysin is a rate-limiting peptidase, which participates in the catabolism of Abeta in brain. We investigated whether rats continuously infused with thiorphan, a specific inhibitor for neprilysin, into the cerebral ventricle cause cognitive dysfunction, with an accumulation of Abeta in the brain. Thiorphan-infused rats displayed significant cognitive dysfunction in the ability to discriminate in the object recognition test and spatial memory in the water maze test, but not in other hippocampus-dependent learning and memory tasks. Thiorphan infusion also elevated the Abeta40 level in the insoluble fraction of the cerebral cortex, but not that of the hippocampus. There was no significant difference in the nicotine-stimulated release of acetylcholine in the hippocampus between vehicle- and thiorphan-infused rats. These results indicate that continuous infusion of thiorphan into the cerebral ventricle causes cognitive dysfunction by raising the level of Abeta in the cerebral cortex, and suggest that a reduction of neprilysin activity contribute to the deposition of Abeta and development of AD.

The accumulation of amyloid-beta peptide (Abeta), a physiological peptide, in the brain is a triggering event leading to the pathological cascade of Alzheimer's disease (AD) and appears to be caused by an increase in the anabolic activity, as seen in familial AD cases or by a decrease in catabolic activity. Neprilysin is a rate-limiting peptidase involved in the physiological degradation of Abeta in the brain. As demonstrated by reverse genetics studies, disruption of the neprilysin gene causes elevation of endogenous Abeta levels in mouse brain in a gene-dose-dependent manner. Thus, the reduction of neprilysin activity will contribute to Abeta accumulation and consequently to AD development. Evidence that neprilysin in the hippocampus and cerebral cortex is down-regulated with aging and from an early stage of AD development supports a close association of neprilysin with the etiology and pathogenesis of AD. Therefore, the up-regulation of neprilysin represents a promising strategy for therapy and prevention. Recently, somatostatin, which acts via a G-protein-coupled receptor (GPCR), has been identified as a modulator that increases brain neprilysin activity, resulting in a decrease of Abeta levels. Thus, it may be possible to pharmacologically control brain Abeta levels with somatostatin receptor agonists.