Induction of the human CDC45 gene promoter activity by natural compound trans­resveratrol.

GGAA motifs in the human TP53 and HELB gene promoters play a part in responding to trans­resveratrol (Rsv) in HeLa S3 cells. This sequence is also present in the 5'­upstream region of the human CDC45 gene, which encodes a component of CMG DNA helicase protein complex. The cells were treated with Rsv (20 µM), then transcripts and the translated protein were analyzed by quantitative RT­PCR and western blotting, respectively. The results showed that the CDC45 gene and protein expression levels were induced after the treatment. To examine whether they were due to the activation of transcription, a 5'­upstream 556­bp of the CDC45 gene was cloned and inserted into a multi­cloning site of the Luciferase (Luc) expression vector. In the present study, various deletion/point mutation­introduced Luc expression plasmids were constructed and they were used for the transient transfection assay. The results showed that the GGAA motif, which is included in a putative RELB protein recognizing sequence, plays a part in the promoter activity with response to Rsv in HeLa S3 cells.

Long term administration of loquat leaves and their major component, ursolic acid, attenuated endogenous amyloid-ß burden and memory impairment.

Loquat (Eriobotrya japonica) leaves contain many bioactive components such as ursolic acid (UA) and amygdalin. We investigated the effects of loquat leaf powder and methanol extract in human neuroglioma H4 cells stably expressing the Swedish-type APP695 (APPNL-H4 cells) and C57BL/6 J mice. Surprisingly, the extract greatly enhanced cellular amyloid-beta peptide (Aß) 42 productions in APPNL-H4 cells. Administration of leaf powder increased Aß42 levels after 3 months and decreased levels after 12 months compared to control mice. Leaf powder had no effect on working memory after 3 months, but improved working memory after 12 months. Administration of UA decreased Aß42 and P-tau levels and improved working memory after 12 months, similar to the administration of leave powder for 12 months. Amygdalin enhanced cellular Aß42 production in APPNL-H4 cells, which was the same as the extract. Three-month administration of amygdalin increased Aß42 levels slightly but did not significantly increase them, which is similar to the trend observed with the administration of leaf powder for 3 months. UA was likely the main compound contained in loquat leaves responsible for the decrease in intracerebral Aß42 and P-tau levels. Also, amygdalin might be one of the compounds responsible for the transiently increased intracerebral Aß42 levels.

A New Species of Acoela Possessing a Middorsal Appendage with a Possible Sensory Function.

Acoels, belonging to Xenacoelomorpha, are small worms with a relatively simple body plan and are considered a critical clade for understanding the evolution of bilaterians. Despite acoels' importance, however, many undiscovered species are predicted to be present worldwide. Here, we describe a new marine acoel species, Amphiscolops oni sp. nov., based on materials collected from the intertidal and subtidal zones of rocky shores at several localities along the Japanese Pacific coast. The new species is approximately 3 mm long and shows typical characteristics of the family Convolutidae, such as the presence of eyespots, symbiosis with algae, position of the gonopores, morphology of the bursal nozzles, lack of central singlet microtubules in the axonemes of spermatozoa, and funnel-like posture of the anterior end. Based on morphology and the results of molecular phylogenetic analyses, we assign this species to the genus Amphiscolops. Interestingly, these worms show unique behaviors such as swimming by flapping the lateral sides and actively capturing prey by swinging the anterior funnel. Furthermore, they possess a dorsal appendage-a characteristic previously unreported in Xenacoelomorpha-representing an evolutionary novelty acquired by this species.

Molecular profiling of ginsenoside metabolites to identify estrogen receptor alpha activity.

20(S)-Protopanaxadiol (PPD) and 20(S)-Protopanaxatriol (PPT) are major metabolites of ginseng in humans and are considered to have estrogenic activity in cellular bioassays. In this study, we conducted in silico analyses to determine whether PPD and PPT interact with estrogen receptor alpha (ERα) and compared them with ERα agonists, partial agonists, and antagonists to identify their ERα activity. The transcriptome profile of 17ß-estradiol (E2), PPD, and PPT in MCF-7 cells expressing ERα was further compared to understand the ERα activity of ginsenoside metabolites. The results showed that PPD and PPT interacted with the 1ERE, 1GWR, and 3UUD ERα proteins in the E2 interaction model, the 3ERD protein in the diethylstilbestrol (DES) interaction model, and the 1X7R protein in the genistein (GEN) interaction model. Conversely, neither the 4PP6 protein of the interaction model with the antagonist resveratrol (RES) nor the 1ERR protein of the interaction model with the antagonist raloxifene (RAL) showed the conformation of amino acid residues. When E2, PPD, and PPT were exposed to MCF-7 cells, cell proliferation and gene expression were observed. The transcriptomic profiles of E2, PPD, and PPT were compared using a knowledge-based pathway. PPD-induced transcription profiling was similar to that of E2, and the neural transmission pathway was detected in both compounds. In contrast, PPT-induced transcription profiling displayed characteristics of gene expression associated with systemic lupus erythematosus. These results suggest that ginsenoside metabolites have ERα agonist activity and exhibit neuroprotective effects and anti-inflammatory actions. However, a meta-analysis using public microarray data showed that the mother compounds GRb1 and GRg1 of PPD and PPT showed metabolic functions in insulin signaling pathways, condensed DNA repair and cell cycle pathways, and immune response and synaptogenesis. These results suggest that the ginsenoside metabolites have potent ERα agonist activity; however, their gene expression profiles may differ from those of E2.

Effect of the natural compound trans­resveratrol on human MCM4 gene transcription.

trans­Resveratrol (Rsv) is a natural compound contained in red wine and grape skins that has various beneficial effects for organisms such as lengthening of their life span. Rsv induces expression of the human TP53 and HELB genes, which are involved in the regulation of DNA maintenance. In the present study, a luciferase expression vector containing 309 bp of the 5' upstream end of the human MCM4 gene was transfected into HeLa S3 cells. A luciferase assay revealed that Rsv treatment increased the minichromosome maintenance 4 (MCM4) gene promoter activity by GC­box and GGAA (TTCC) motifs. Electro phoretic mobility shift assay revealed that the specific binding factor (complex) contains PU.1 (SPI1). Quantitative reverse transcription­polymerase chain reaction analysis indicated that MCM4 gene expression was transiently induced by Rsv. Moreover, western blotting revealed that the SP1/PU.1 ratio markedly increased after Rsv treatment, indicating that a balance or profile of these transcription factors may control Rsv­inducible initiation of transcription. These observations indicated that the beneficial effects of Rsv can be attributed to induction of the chromosomal DNA maintenance factor encoding gene expression.

Characterization of the human zinc finger nfx­1­type containing 1 encoding ZNFX1 gene and its response to 12­O­tetradecanoyl­13­acetate in HL­60 cells.

Human promyelocytic HL­60 cells can be differentiated into macrophage­like cells by treatment with 12­O­tetra decanoylphorbol­13­acetate (TPA). Certain 5' upstream regions of the zinc finger protein (ZNF)­encoding genes contain duplicated GGAA motifs, which are frequently found in the TPA­responding gene promoter regions. To examine transcriptional responses to TPA, 5'flanking regions of human zinc finger CCCH­type containing, antiviral, ZNF252, ZNF343, ZNF555, ZNF782 and zinc finger nfx­1­type containing 1 (ZNFX1) genes were isolated by polymerase chain reaction (PCR) and ligated into a multiple­cloning site of the pGL4.10[luc2] vector. Transient transfection and a luciferase assay revealed that the ZNFX1 promoter most prominently responded to the TPA treatment. Deletion and point mutation experiments indicated that the duplicated GGAA motif in the 100­bp region positively responded to TPA. In addition, reverse transcription­quantitative PCR and western blotting showed that the mRNA and protein of ZNFX1 accumulate during the differentiation of HL­60 cells. These results indicated that expression of the TPA­inducible ZNFX1 gene, which belongs to the group of interferon­responsive genes, is regulated by the cis­action of the duplicated GGAA motif.

Characterization of the human E2F4 promoter region and its response to 12-O-tetradecanoylphorbol-13-acetate.

The E2F transcription factors (TFs), which control the progression of the cell cycle in response to DNA-damage and various stresses, are known to interact with a tumour suppressor, Retinoblastoma 1 (RB1). We previously showed that the response of the human RB1 promoter to a 12-O-tetradecanoylphorbol-13-acetate (TPA) in HL-60 cells is mediated by a duplicated GGAA motif, which is also present in the 5'-upstream of the E2F family genes. The motifs are especially rich in the 5'-upstream of the E2F4 gene. In the present study, we constructed luciferase (Luc) expression vectors containing a 466 bp of the 5'-upstream of the human E2F4 gene. The transfection of this plasmid and deletion/mutation-introduced derivatives into HL-60 cells and a Luc reporter assay showed that duplicated and triplicated GGAA (TTCC) motifs in the E2F4 promoter respond to TPA. As expected, electrophoretic mobility shift assay indicated that SPI1 (PU.1) binds to the GGAA motif-containing element. A quantitative RT-PCR and western blotting showed that the E2F4 transcripts and its encoding proteins accumulate during the differentiation of HL-60 into macrophage-like cells. In contrast, the expression of the E2F1 gene and the protein, which possibly acts as a cell cycle accelerator, was greatly diminished.

A simplified and sensitive method to identify Alzheimer's disease biomarker candidates using patient-derived induced pluripotent stem cells (iPSCs).

We developed a simplified and sensitive method to identify Alzheimer's disease (AD) biomarker candidates by a quantitative and targeted proteomic analysis (combination of liquid chromatography tandem mass spectrometry and multiplexed-multiple reaction monitoring/selected reaction monitoring analysis) of culture media from neurons differentiated from induced pluripotent stem cells (iPSCs) established from AD patients. We found that alpha-1-acid glycoprotein (ORM1) was decreased in the culture media of AD-iPSC-derived neurons, consistent with previous observations for AD patient cerebrospinal fluid, thus validating our new strategy. Moreover, our method is applicable for identifying biomarker candidates for other neurodegenerative disorders using patient-derived iPSCs.

Elucidating Pathogenic Mechanisms of Early-onset Alzheimer's Disease in Down Syndrome Patients.

Down syndrome (DS) patients demonstrate the neuropathology of Alzheimer's disease (AD) characterized by the formation of senile plaques and neurofibrillary tangles by age 40-50 years. It has been considered for a number of years that 1.5-fold expression of the gene for the amyloid precursor protein (APP) located on chromosome 21 leading to overproduction of amyloid-ß peptide (Aß) results in the early onset of AD in adults with DS. However, the mean age of onset of familial AD with the Swedish mutation on APP which has high affinity for ß-secretase associated with a dramatic increase in Aß production is about 55 years. This paradox indicates that there is a poor correlation between average ages of AD onset and the theoretical amount of Aß production and that there are factors exacerbating AD on chromosome 21. We therefore focused on dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), since overexpressing transgenic mice show AD-like brain pathology. The overexpression of DYRK1A caused suppression of the activity of neprilysin (NEP), which is a major Aß-degrading enzyme in the brain, and phosphorylation at the NEP cytoplasmic domain. NEP activity was markedly reduced in fibroblasts derived from DS patients compared with that in fibroblasts derived from healthy controls. This impaired activity of NEP was rescued by DYRK1A inhibition. These results show that DYRK1A overexpression causes suppression of NEP activity through its phosphorylation in DS patients. Our results suggest that DYRK1A inhibitors could be effective against AD not only in adults with DS but also in sporadic AD patients.

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

Amyloid-ß peptide (Aß) accumulation is a triggering event leading to the Alzheimer's disease (AD) pathological cascade. Almost all familial AD-linked gene mutations increase Aß production and accelerate the onset of AD. The Swedish mutation of amyloid precursor protein (APP) affects ß-secretase activity and increases Aß 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 Aß production. However, when comparing the causal relationship of Aß accumulation with the onset age between the above two populations, early DS pathogenesis does not appear to be accounted for by the increased Aß production alone. In this study, we found that neprilysin, a major Aß-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 Aß 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.

Alzheimer's 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-ß 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.

Perturbed Calcineurin-NFAT Signaling Is Associated with the Development of Alzheimer's Disease.

Down syndrome (DS), the most common genetic disorder, is caused by trisomy 21. DS is accompanied by heart defects, hearing and vision problems, obesity, leukemia, and other conditions, including Alzheimer's disease (AD). In comparison, most cancers are rare in people with DS. Overexpression of dual specificity tyrosine-phosphorylation-regulated kinase 1A and a regulator of calcineurin 1 located on chromosome 21 leads to excessive suppression of the calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway, resulting in reduced expression of a critical angiogenic factor. However, it is unclear whether the calcineurin-NFAT signaling pathway is involved in AD pathology in DS patients. Here, we investigated the association between the calcineurin-NFAT signaling pathway and AD using neuronal cells. Short-term pharmacological stimulation decreased gene expression of tau and neprilysin, and long-term inhibition of the signaling pathway decreased that of amyloid precursor protein. Moreover, a calcineurin inhibitor, cyclosporine A, also decreased neprilysin activity, leading to increases in amyloid-ß peptide levels. Taken together, our results suggest that a dysregulation in calcineurin-NFAT signaling may contribute to the early onset of AD in people with DS.

Cathepsin K-mediated Notch1 activation contributes to neovascularization in response to hypoxia.

Cysteine proteases play important roles in pathobiology. Here we reveal that cathepsin K (CatK) has a role in ischaemia-induced neovascularization. Femoral artery ligation-induced ischaemia in mice increases CatK expression and activity, and CatK-deficient mice show impaired functional recovery following hindlimb ischaemia. CatK deficiency reduces the levels of cleaved Notch1 (c-Notch1), Hes1 Hey1, Hey2, vascular endothelial growth factor, Flt-1 and phospho-Akt proteins of the ischaemic muscles. In endothelial cells, silencing of CatK mimicked, whereas CatK overexpression enhanced, the levels of c-Notch1 and the expression of Notch downstream signalling molecules, suggesting CatK contributes to Notch1 processing and activates downstream signalling. Moreover, CatK knockdown leads to defective endothelial cell invasion, proliferation and tube formation, and CatK deficiency is associated with decreased circulating endothelial progenitor cells-like CD31(+)/c-Kit(+) cells in mice following hindlimb ischaemia. Transplantation of bone marrow-derived mononuclear cells from CatK(+/+) mice restores the impairment of neovascularization in CatK(-/-) mice. We conclude that CatK may be a potential therapeutic target for ischaemic disease.

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

Accumulation of amyloid-ß peptide (Aß) in the brain is closely associated with cognitive decline in Alzheimer's disease (AD). Stereotaxic infusion of neprilysin-encoding viral vectors into the hippocampus has been shown to decrease Aß 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 Aß 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 Aß 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.

Modeling Alzheimer's disease with iPSCs reveals stress phenotypes associated with intracellular Aß and differential drug responsiveness.

Oligomeric forms of amyloid-ß peptide (Aß) are thought to play a pivotal role in the pathogenesis of Alzheimer's disease (AD), but the mechanism involved is still unclear. Here, we generated induced pluripotent stem cells (iPSCs) from familial and sporadic AD patients and differentiated them into neural cells. Aß oligomers accumulated in iPSC-derived neurons and astrocytes in cells from patients with a familial amyloid precursor protein (APP)-E693Δ mutation and sporadic AD, leading to endoplasmic reticulum (ER) and oxidative stress. The accumulated Aß oligomers were not proteolytically resistant, and docosahexaenoic acid (DHA) treatment alleviated the stress responses in the AD neural cells. Differential manifestation of ER stress and DHA responsiveness may help explain variable clinical results obtained with the use of DHA treatment and suggests that DHA may in fact be effective for a subset of patients. It also illustrates how patient-specific iPSCs can be useful for analyzing AD pathogenesis and evaluating drugs.

[A pathogenic mechanism of N-terminally pyroglutamylated Aß and possible application of Alzheimer's disease by inhibition of the pyroglutamylation].

Aggregation and accumulation of amyloid-ß peptide (Aß) in the brain are triggering events leading to the pathological cascade of Alzheimer's disease (AD). Aß accumulates in AD brains and forms amyloid plaques, which consist mostly of amino-terminally truncated and/or modified Aßs, among which Aß3pyroglutamate (Aß3pE) is a major product. Thus, the N-terminal structures of accumulated species of Aß are different from those secreted from neurons. Aß3pE-42 is more hydrophobic, more easily self-aggregated (250-fold), and is more resistant to proteolytic degradation (4-fold) than Aß1-42. Therefore, Aß3pE appears to act as a seed for the formation of oligomers and amyloid plaques. Aß 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 Aß3pE. 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 Aß3pE production and also alleviated impaired cognitive function. Thus, inhibition of Aß3pE formation appears to be a novel target for therapy and prevention of AD.

Anti-Aß drug screening platform using human iPS cell-derived neurons for the treatment of Alzheimer's disease.

BACKGROUND: Alzheimer's 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 ß peptide (Aß), which is produced from amyloid precursor protein by ß- 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, ß-secretase, and γ-secretase components, and were capable of secreting Aß into the conditioned media. Aß production was inhibited by ß-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 (Aß surge) and drastic decline of Aß production. CONCLUSIONS/SIGNIFICANCE: These results indicate that the hiPS cell-derived neuronal cells express functional ß- and γ-secretases involved in Aß production; however, anti-Aß drug screening using these hiPS cell-derived neuronal cells requires sufficient neuronal differentiation.

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-ß peptide (Aß), 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 ß and to decrease levels of p3 or Aß 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 Aß, 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.