A prochelator activated by beta-secretase inhibits Abeta aggregation and suppresses copper-induced reactive oxygen species formation.

The intersection of the amyloid cascade hypothesis and the implication of metal ions in Alzheimer's disease progression has sparked an interest in using metal-binding compounds as potential therapeutic agents. In the present work, we describe a prochelator SWH that is enzymatically activated by beta-secretase to produce a high affinity copper chelator CP. Because beta-secretase is responsible for the amyloidogenic processing of the amyloid precursor protein, this prochelator strategy imparts disease specificity toward copper chelation not possible with general metal chelators. Furthermore, once activated, CP efficiently sequesters copper from amyloid-beta, prevents and disassembles copper-induced amyloid-beta aggregation, and diminishes copper-promoted reactive oxygen species formation.

Functional Variants in Notch Pathway Genes NCOR2, NCSTN, and MAML2 Predict Survival of Patients with Cutaneous Melanoma.

BACKGROUND: The Notch signaling pathway is constitutively activated in human cutaneous melanoma to promote growth and aggressive metastatic potential of primary melanoma cells. Therefore, genetic variants in Notch pathway genes may affect the prognosis of cutaneous melanoma patients. METHODS: We identified 6,256 SNPs in 48 Notch genes in 858 cutaneous melanoma patients included in a previously published cutaneous melanoma genome-wide association study dataset. Multivariate and stepwise Cox proportional hazards regression and false-positive report probability corrections were performed to evaluate associations between putative functional SNPs and cutaneous melanoma disease-specific survival. Receiver operating characteristic curve was constructed, and area under the curve was used to assess the classification performance of the model. RESULTS: Four putative functional SNPs of Notch pathway genes had independent and joint predictive roles in survival of cutaneous melanoma patients. The most significant variant was NCOR2 rs2342924 T>C (adjusted HR, 2.71; 95% confidence interval, 1.73-4.23; Ptrend = 9.62 × 10(-7)), followed by NCSTN rs1124379 G>A, NCOR2 rs10846684 G>A, and MAML2 rs7953425 G>A (Ptrend = 0.005, 0.005, and 0.013, respectively). The receiver operating characteristic analysis revealed that area under the curve was significantly increased after adding the combined unfavorable genotype score to the model containing the known clinicopathologic factors. CONCLUSIONS: Our results suggest that SNPs in Notch pathway genes may be predictors of cutaneous melanoma disease-specific survival. IMPACT: Our discovery offers a translational potential for using genetic variants in Notch pathway genes as a genotype score of biomarkers for developing an improved prognostic assessment and personalized management of cutaneous melanoma patients.

Docetaxel Resistance in Prostate Cancer: Taking It Up a Notch.

Notch signaling is implicated in prostate cancer progression and docetaxel resistance. Cui and colleagues describe the additive efficacy and mechanisms of a γ-secretase inhibitor, PF-03084014, and docetaxel in preclinical models of prostate cancer, suggesting the need for further clinical development of Notch pathway modulators in men with metastatic prostate cancer.

Regulation of the Gene Encoding Thrombin-Activable Fibrinolysis Inhibitor in Non-Hepatic Cells

Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like pro-enzyme that, once activated, attenuates fibrinolysis. TAFIa also possesses anti-inflammatory properties. Although liver is the main source of plasma TAFI, platelet-derived TAFI has also been reported. An alternatively spliced TAFI variant resulted from the skipping of exon 6 and a 52-base deletion in exon 10 of CPB2 mRNA (∆6+10) was described to be brain specific. This TAFI variant is reputed to possess a secretase-like activity that cleaves β-amyloid precursor protein to form β-amyloid, a process involved in the onset of Alzheimer's disease. In this thesis, we report the identification of CPB2 mRNA and TAFI protein in various vascular and inflammatory cells. Specifically, we describe the expression of CPB2 mRNA in the megakaryocytic cell lines MEG-01 and Dami, the monocytic cell line THP-1, and peripheral blood mononuclear cells. TAFI protein was detected in differentiated Dami and THP-1 cells. We next describe the effect of external stimuli such as phorbol myristate acetate (PMA) on CPB2 expression in Dami and THP-1 cells. We found that PMA treatment increases both CPB2 mRNA abundance and promoter activity in Dami cells, and decreases both CPB2 mRNA abundance and promoter activity in THP-1 cells. Deletion analysis of the CPB2 promoter indicated cell-type specific regulation of CPB2 gene expression. Finally, we evaluated the expression of alternatively spliced CPB2 mRNA variants in hepatic and non hepatic cells. We found that exon 6 skipping variants are expressed in all cell types of interest. The variant previously reported to be brain specific was also found to be expressed in platelets. We found that the alternatively spliced TAFI variants accumulated inside the cells in a non-secretable, hypoglycosylated form and showed no carboxypeptidase activity. Taken together, this thesis provides further evidence supporting the hypothesis that platelet-derived TAFI is originated from CPB2 gene expression in megakaryocytes. Moreover, our data imply a potential for site-specific anti-inflammatory control provided by macrophage-derived TAFI. Alternative splicing of the CPB2 mRNA may give rise to variants with an intracellular role, perhaps as a peptidase chaperone, and may modulate the synthesis of secretable TAFI.

BACE1 Polymorphisms Do Not Influence Platelet Membrane β-secretase Activity or Genetic Susceptibility for Alzheimer’s Disease in the Northern Irish Population

ß-Site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is a biological and positional candidate gene for Alzheimer’s disease (AD). BACE1 is a protease that catalyses APP cleavage at the ß-secretase site. We evaluated all common and putatively functional polymorphisms in the genomic region encompassing BACE1 for an association with AD, and for functional effects on platelet ß-secretase activity. Tag SNPs (n = 10) derived from phase II of the International HapMap Project, and a nonsynonymous variant, were successfully genotyped in 901 Caucasian individuals from Northern Ireland using Sequenom iPLEX and TaqMan technologies. APOE genotyping was performed by PCR-RFLP. Platelet membrane ß-secretase activity was assayed in a subset of individuals (n = 311). Hardy–Weinberg equilibrium was observed for all variants. Evidence for an association with AD was observed with multi-marker haplotype analyses (P = 0.01), and with rs676134 when stratified for APOE genotype (P = 0.02), however adjusting for multiple testing negated the evidence for association of this variant with AD. ?2 analysis of genotype and allele frequencies in cases versus controls for individual SNPs revealed no evidence for association (5% level). No genetic factors were observed that significantly influenced platelet membrane ß-secretase activity. We have selected an appropriate subset of variants suitable for comprehensive genetic investigation of the BACE1 gene. Our results suggest that common BACE1 polymorphisms and putatively functional variants have no significant influence on genetic susceptibility to AD, or platelet ß-secretase activity, in this Caucasian Northern Irish population.

Discovery and Evaluation of BMS-708163, a Potent, Selective and Orally Bioavailable gamma-Secretase Inhibitor

During the course of our research efforts to develop a potent and selective gamma-secretase inhibitor for the treatment of Alzheimer's disease, we investigated a series of carboxamide-substituted sulfonamides. Optimization based on potency, Notch/amyloid-beta precursor protein selectivity, and brain efficacy after oral dosing led to the discovery of 4 (BMS-708163). Compound 4 is a potent inhibitor of gamma-secretase (A beta 40 IC50 = 0.30 nM), demonstrating a 193-fold selectivity against Notch. Oral administration of 4 significantly reduced A beta 40 levels for sustained periods in brain, plasma, and cerebrospinal fluid in rats and dogs.

A novel reciprocal and biphasic relationship between membrane cholesterol and beta-secretase activity in SH-SY5Y cells and in human platelets.

Research into the cause of Alzheimer's disease (AD) has identified strong connections to cholesterol. Cholesterol and cholesterol esters can modulate amyloid precursor protein (APP) processing, thus altering production of the A beta peptides that deposit in cortical amyloid plaques. Processing depends on the encounter between APP and cellular secretases, and is thus subject to the influence of cholesterol-dependent factors including protein trafficking, and distribution between membrane subdomains. We have directly investigated endogenous membrane beta-secretase activity in the presence of a range of membrane cholesterol levels in SH-SY5Y human neuroblastoma cells and human platelets. Membrane cholesterol significantly influenced membrane beta-secretase activity in a biphasic manner, with positive correlations at higher membrane cholesterol levels, and negative correlations at lower membrane cholesterol levels. Platelets from individuals with AD or mild cognitive impairment (n = 172) were significantly more likely to lie within the negative correlation zone than control platelets (n = 171). Pharmacological inhibition of SH-SY5Y beta-secretase activity resulted in increased membrane cholesterol levels. Our findings are consistent with the existence of a homeostatic feedback loop between membrane cholesterol level and membrane beta-secretase activity, and suggest that this regulatory mechanism is disrupted in platelets from individuals with cognitive impairment.

Variation in RTN3 and PPIL2 Genes Does not Influence Platelet Membrane β-Secretase Activity or Susceptibility to Alzheimer’s Disease in the Northern Irish Population

beta-site amyloid precursor protein cleaving enzyme (BACE1) is the rate-limiting enzyme for production of beta-amyloid peptides (A beta), which are proposed to drive the pathological changes found in Alzheimer's disease (AD). Reticulon 3 (RTN3) is a negative modulator of BACE1 (beta-secretase) proteolytic activity, while peptidylprolyl isomerase (cyclophilin)-like 2 (PPIL2) positively regulates BACE1 expression. The present study investigated whether there was any association between genetic variation in RTN3 and PPIL2, and either risk for AD, or levels of platelet beta-secretase activity, in a large Northern Irish case-control sample. Four hundred and sixty-nine patients with a diagnosis of probable AD (NINCDS-ADRDA criteria) and 347 control individuals (MMSE > 28/30) were genotyped. SNPs in both genes were selected by downloading genotype data from the International HapMap Project (Phase II) and tags selected using multimarker approach in Haploview, where r (2) > 0.8 and LOD > 3.0. Non-synonymous SNPs of interest were also included. Genotyping was performed by Sequenom iPLEX and TaqMan technologies. Alleles, genotypes and multi-marker haplotypes were tested for association with AD, and platelet beta-secretase activities were measured for a subset of individuals (n = 231). Eight SNPs in RTN3 and 7 in PPIL2 were genotyped. We found no significant associations between allele, genotype or haplotype frequencies and risk of AD. Further, there was no effect of genotype on platelet membrane beta-secretase activity. We conclude that common or potentially functional genetic variation in these BACE1 interacting proteins does not affect platelet membrane beta-secretase activity or contribute to risk of AD in this population.

A Family of Helminth Molecules that Modulate Innate Cell Responses via Molecular Mimicry of Host Antimicrobial Peptides

Over the last decade a significant number of studies have highlighted the central role of host antimicrobial (or defence) peptides in modulating the response of innate immune cells to pathogen-associated ligands. In humans, the most widely studied antimicrobial peptide is LL-37, a 37-residue peptide containing an amphipathic helix that is released via proteolytic cleavage of the precursor protein CAP18. Owing to its ability to protect against lethal endotoxaemia and clinically-relevant bacterial infections, LL-37 and its derivatives are seen as attractive candidates for anti-sepsis therapies. We have identified a novel family of molecules secreted by parasitic helminths (helminth defence molecules; HDMs) that exhibit similar biochemical and functional characteristics to human defence peptides, particularly CAP18. The HDM secreted by Fasciola hepatica (FhHDM-1) adopts a predominantly alpha-helical structure in solution. Processing of FhHDM-1 by F. hepatica cathepsin L1 releases a 34-residue C-terminal fragment containing a conserved amphipathic helix. This is analogous to the proteolytic processing of CAP18 to release LL-37, which modulates innate cell activation by classical toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). We show that full-length recombinant FhHDM-1 and a peptide analogue of the amphipathic C-terminus bind directly to LPS in a concentration-dependent manner, reducing its interaction with both LPS-binding protein (LBP) and the surface of macrophages. Furthermore, FhHDM-1 and the amphipathic C-terminal peptide protect mice against LPS-induced inflammation by significantly reducing the release of inflammatory mediators from macrophages. We propose that HDMs, by mimicking the function of host defence peptides, represent a novel family of innate cell modulators with therapeutic potential in anti-sepsis treatments and prevention of inflammation.

Development of the vitellaria of the liver fluke, Fasciola hepatica in the rat host

The development of the vitellaria of Fasciola hepatica within the liver of its rat host was studied by means of whole-mount stained preparations and transmission electron microscopy, together with light and electron immunocytochemistry using an antibody to vitelline protein B, an eggshell precursor protein synthesized by F. hepatica. No vitelline cells could be identified in flukes recovered from the liver parenchyma, by any of the methods used. In contrast, follicles were present in flukes at the earliest time of recovery from the bile duct, namely, 5 weeks 3 days post-infection. The vitellaria in these flukes formed a row of small follicles on either side of the body. Development of the follicles was rapid: by 6 weeks 3 days, the vitellaria resembled those in the adult fluke and eggs were present in the uterus. Immunolabelling was confined to the shell protein globules in the vitelline cells, confirming the packaging of the eggshell protein within the shell globule clusters.

Ornithokinin (avian bradykinin) from the skin of the Chinese bamboo odorous frog, Odorrana versabilis

One of the most widespread and abundant families of pharmacologically active peptides in amphibian defensive skin secretions is the bradykinins and related peptides. Despite retaining certain primary structural attributes that assign them to this peptide family, bradykinins and related peptides are unique among amphibian skin peptides in that they exhibit a wide range of primary structural variations, post-translational modifications and/or N-terminal or C-terminal extensions. Initially it was believed that their high degree of primary structural heterogeneity was reflective of random gene mutations within species, but latterly, there is an increasing body of evidence that the spectrum of structural modifications found within this peptide family is reflective of the vertebrate predator spectrum of individual species. Here we report the discovery of ornithokinin (avian bradykinin – Thr6, Leu8-bradykinin) in the skin secretion of the Chinese bamboo odorous frog, Odorrana versabilis. Molecular cloning of its biosynthetic precursor-encoding cDNA from a skin secretion-derived cDNA library revealed a deduced open-reading frame of 86 amino acid residues, encoding a single copy of ornithokinin towards its C-terminus. The domain architecture of this ornithokinin precursor protein was consistent with that of a typical amphibian skin peptide and quite different to that of the ornithokininogen from chicken plasma. Ornithokinin was reported to induce hypotension in the chicken and to contract the chicken oviduct but to have no obvious effect on the rat uterus. However, in this study, synthetic ornithokinin was found to contract the rat ileum (EC50 = 539 nM) and to increase contraction frequency in the rat uterus (EC50 = 1.87 μM).

mRNA Levels of BACE1 and its interacting proteins, RTN3 and PPIL2, correlate in human post mortem brain tissue

β-Site amyloid precursor protein cleaving enzyme (BACE1) is the rate-limiting enzyme for production of Aβ peptides, proposed to drive the pathological changes found in Alzheimer’s disease (AD). Reticulon 3 (RTN3) is a negative modulator of BACE1 (β-secretase) proteolytic activity, while peptidylprolyl isomerase (cyclophilin)-like 2 (PPIL2) positively regulated BACE1 gene expression in a cell-based assay. This study aimed to analyze RTN3 and PPIL2 mRNA levels in four brain regions from individuals with AD and controls. BACE1 mRNA had been previously quantified in the samples, as had glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE), to track changing cell populations in the tissue. mRNA levels in the human post mortem brain tissue were assayed using quantitative real-time polymerase chain reaction (qPCR) and qbasePLUS, employing validated stably expressed reference genes. No differences in RTN3 or PPIL2 mRNA levels were found in individuals with AD, compared to controls. Both RTN3 and PPIL2 mRNA levels correlated significantly with BACE1 mRNA and all three showed similar disease stage-dependent changes with respect to NSE and GFAP. These findings indicated that the in vitro data demonstrating an effect of PPIL2 on BACE1 expression have functional relevance in vivo. Further research into BACE1-interacting proteins could provide a fruitful approach to the modulation of this protease and consequently Aβ production.

O sistema dois-hidrido de levedura para confirmação da sinalização de APP Tipo-Noth

Inúmeras potenciais funções foram sugeridas para a APP (Proteína Precursora de Amilóide de Alzheimer), contudo, fisiologicamente, a função precisa da APP não foi ainda desvendada. A APP tem características consistentes com a função de molécula-receptora, capaz de reconhecer sinais extracelulares. Também relevante para este trabalho, é o facto de que a sinalização através de RIP (Proteólise Intramembranar Regulada) tem consequências na expressão génica, como no caso da sinalização tipo-Notch. Tal como a proteína Notch, a APP é processada resultando num fragmento C-terminal designado por AICD (Domínio Intracelular da APP). Neste trabalho é focado o papel importante na sinalização nuclear desempenhado pelo fragmento AICD, especificamente através da interacção com proteínas adaptadoras, promovendo a transcrição. Com o objectivo de contribuir para uma melhor compreensão da base molecular da DA (Doença de Alzheimer), torna-se importante investigar as vias de localização nuclear do AICD e o seu envolvimento na transcrição génica, possivelmente afectando proteínas até agora não associadas à DA. Por estes motivos é fundamental a identificação de novas proteínas que interajam com a APP. Um rastreio foi efectuado, utilizando o sistema Dois-Híbrido em Levedura, para identificar interacções específicas do AICD no cérebro humano e, assim, caracterizar o interactoma do AICD. Foi feito o rastreio de aproximadamente 1.1x108 clones de uma biblioteca de cDNA de cérebro humano com o domínio C-terminal da APP com a mutação Y687E, que mimetiza o estado fosforilado. De experiências anteriores deste laboratório sabemos que a tirosina-687 afecta a localização subcelular da APP e é também consensual que a fosforilação é importante nos mecanismos de transdução de sinais, daí a utilização deste mutante parecer apropriada. O rastreio originou 55 clones positivos que foram analisados para identificar proteínas que interagem com a APP. Dois clones são particularmente importantes, a RanBPM e a Transportin-SR2, visto que estão associadas ao transporte de proteínas para o núcleo e confirmam a sinalização nuclear da APP. ABSTRACT: Many putative functions for APP (Alzheimer’s amyloid precursor protein) have been suggested, although the precise physiological function of APP remains to be elucidated. APP has characteristics consistent with it having a role as a receptor, capable of mediating extracellular signals. Also of relevance to the work described here is that RIP (Regulated Intramembrane Proteolysis) signalling can have consequences in gene expression, similar to Notch signalling. Like the latter, APP is processed by RIP resulting in a C-terminal fragment known as AICD. Here we test the hypothesis that the AICD fragment may play an important role in nuclear signalling, specifically by interacting with adaptor proteins potentiating transcription. Therefore, in order to contribute to our understanding of the molecular basis of AD (Alzheimer’s disease) it is important to investigate the pathways of AICD nuclear targeting and its involvement in gene transcription, possibly affecting other proteins hitherto not associated with AD. Thus, it is important to identify AICD binding proteins. A Yeast Two-Hybrid (YTH) screen was performed to identify human brainspecific AICD binding proteins, and thus characterize the AICD interactome. The screen of approximately 1.1 x108 clones from a human brain cDNA library was carried out using the AICD fragment with an Y687E mutation, which mimics phosphorylation on that residue. From previous work carried out in the laboratory we know that tyrosine-687 phosphorylation affects subcellular localization of APP, and it is also recognized that phosphorylation events are important in signal transduction mechanisms, hence the use of this mutant is appropriate. The YTH screen yielded 55 positive clones that were analysed and several novel brain-specific APP binding proteins were identified. Two clones were particularly important, RanBPM and Transportin-SR2, being that they are associated with the nuclear transport of proteins, and support the nuclear signalling for APP.

Aß biology and its contribution to Alzheimer's disease

A doença de Alzheimer (DA) é uma desordem neurodegenerativa progressiva patologicamente caracterizada pela presença de placas de amilóide (placas senis) insolúveis e também pela presença de tranças neurofibrilhares,formadas pela proteína Tau hiperfosforiladada. O principal constituinte das placas senis é o peptídeo beta-amilóide (Ab), que deriva do processamento proteolítico da proteína precursora de amilóide de Alzheimer (APP). Embora Ab exista como um agregado pouco solúvel nas placas senis, ele é secretado pelas células como uma molécula solúvel. O Ab “per se” pode afectar o metabolismo da APP. Alguns autores sugerem que o Ab exerce o seu efeito alterando o processamento ou catabolismo da APP, outros sugerem que ele também induz a transcrição da APP, onde aumentando os níveis da APP pode estar a contribuir para a sua própria produção (mecanismo de “feedback” positivo). Assim sendo, torna-se difícil consolidar todas estas observações e identificar as potenciais funções fisiológicas do Ab “in vivo”, ou as consequências da sua produção. Neste trabalho caracterizaram-se os efeitos do Ab no metabolismo da APP. Os nossos estudos revelaram que um dos mecanismos induzidos pelo Ab é a acumulação intracelular do fragmento neuroprotector sAPP (isAPPa) em estruturas com características vesiculares associadas ao citosqueleto. Estudos adicionais em culturas primárias revelaram que o Ab estava a exercer o seu efeito ao nível da secreção vesicular, provavelmente interferindo com o transporte de APP/sAPP ao longo da rede do citosqueleto. Esta hipótese é sustentada pelo facto do Ab estar a afectar a estabilidade e a polimerização de proteínas envolvidas na dinâmica do citosqueleto. Contrariamente a publicações anteriores o Ab não induziu a transcrição da APP, na verdade em culturas primárias neuronais foi observado uma diminuição nos níveis de expressão da APP. Isto foi acompanhado por um aumento nos fragmentos C-terminais da APP (CTFs) e uma diminuição na localização nuclear do seu domínio intracelular (AICD), sugerindo alterações na sinalização nuclear da APP. O Ab pode afectar outras vias de sinalização, particularmente alterando o balanço entre as actividades das proteínas cinases e fosfatases, o que pode ter consequências para o desenvolvimento da doença. Os dados obtidos indicam que o Ab é capaz de inibir a actividade da proteína fosfatase1, a sua importância numa perspectiva de futuras terapias é discutida. Devido à relevância da agregação do Ab para a sua toxicidade, a formação de complexos com proteínas que promovem a sua desagregação/degradação e o seu efeito no processamento da APP foi avaliado. Na presença destes complexos observou-se uma reversão da acumulação isAPP, demonstrando o potencial terapêutico destas proteínas como moduladores do metabolismo da APP. Este trabalho permitiu compreender melhor os mecanismos envolvidos nos efeitos do Ab no processamento da APP e descobrir algumas moléculas que podem ser relevantes numa perspectiva de diagnóstico e terapia na DA.

Identification of the cellular and molecular mechanism by which CD2AP, a regulator of neuronal intracellular transport contributes to the development of Alzheimer’s disease

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências,2014