Synthesis, radiolabeling, and evaluation of a potent ß-site APP cleaving enzyme (BACE1) inhibitor for PET imaging of BACE1 in vivo.

The ß-site APP-cleaving enzyme 1 (BACE1) plays important roles in the proteolytic processing of amyloid precursor protein, and can be regarded as an important target for the diagnosis and treatment of AD. This study aimed to report the synthesis and evaluation of an 18F-labeled 2-amino-3,4-dihydroquinazoline analog as a potential BACE1 radioligand. A fluoropropyl side chain was introduced to the phenyl of this 3,4-dihydroquinazoline scaffold to generate the radioligand. Our preliminary data indicated that although the 2-amino-3,4-dihydroquinazoline scaffold possessed favorable in-vitro properties as a PET ligand, its poor brain uptake hindered the in-vivo imaging of BACE1. Further investigation would be required to optimize the scaffold for the development of a blood-brain-barrier-permeable BACE1-targeted PET ligand.

The metalloproteinase ADAM10 requires its activity to sustain surface expression.

The metalloproteinase ADAM10 critically contributes to development, inflammation, and cancer and can be controlled by endogenous or synthetic inhibitors. Here, we demonstrate for the first time that loss of proteolytic activity of ADAM10 by either inhibition or loss of function mutations induces removal of the protease from the cell surface and the whole cell. This process is temperature dependent, restricted to mature ADAM10, and associated with an increased internalization, lysosomal degradation, and release of mature ADAM10 in extracellular vesicles. Recovery from this depletion requires de novo synthesis. Functionally, this is reflected by loss and recovery of ADAM10 substrate shedding. Finally, ADAM10 inhibition in mice reduces systemic ADAM10 levels in different tissues. Thus, ADAM10 activity is critically required for its surface expression in vitro and in vivo. These findings are crucial for development of therapeutic ADAM10 inhibition strategies and may showcase a novel, physiologically relevant mechanism of protease removal due to activity loss.

Brain functions and unusual ß-amyloid accumulation in the hypertensive white matter lesions of rats.

This study used Sprague Dawley (SD) rats with stroke-prone renovascular hypertension (RHRSP) to establish an animal model of hypertensive white matter lesions (WML), so as to explore the brain functions and unusual ß-amyloid (Aß) accumulation in WML. Hypertensive WML and brain dysfunctions were evaluated by measuring the caudal arterial pressure of model rats, and by observing the histomorphological deformations o f the prefrontal lobe, temporal lobe, hippocampus and corpus callosum, as well as by counting of the number of neurons using Hematoxylin and Eosin (H and E) staining, and by evaluating the changes in rat brain functions, including memory and the ability of visual space learning, using the Morris Water Maze Test. In addition, the study discussed the correlation between Aß accumulation and hypertensive WML cognitive impairment by adopting an enzyme-linked immunosorbent assay (ELISA) to detect the level of Aß 1-42, and by detecting the expression of amyloid precursor protein (APP) and Beta-secretase 1 (BACE1) using Western blot. Results of the study showed that at 4 weeks, 8 weeks, 12 weeks and 16 weeks after operation, the blood pressure and brain Aß expression in the rats of the model group notably increased (P less than 0.01), along with deformed and degenerated brain tissues, confirming that the unusual Aß accumulation may participate in the occurrence and development of hypertensive WML as well as the induction of cerebral cognitive decreases.

Electrochemical Activity Assay for Protease Analysis Using Carbon Nanofiber Nanoelectrode Arrays.

There is a strong demand for bioanalytical techniques to rapidly detect protease activities with high sensitivity and high specificity. This study reports an activity-based electrochemical method toward this goal. Nanoelectrode arrays (NEAs) fabricated with embedded vertically aligned carbon nanofibers (VACNFs) are functionalized with specific peptide substrates containing a ferrocene (Fc) tag. The kinetic proteolysis curves are measured with continuously repeated ac voltammetry, from which the catalytic activity is derived as the inverse of the exponential decay time constant based on a heterogeneous Michaelis-Menten model. Comparison of three peptide substrates with different lengths reveals that the hexapeptide H2N-(CH2)4-CO-Pro-Leu-Arg-Phe-Gly-Ala-NH-CH2-Fc is the optimal probe for cathepsin B. The activity strongly depends on temperature and is the highest around the body temperature. With the optimized peptide substrate and measuring conditions, the limit of detection of cathepsin B activity and concentration can reach 2.49 × 10-4 s-1 and 0.32 nM, respectively. The peptide substrates show high specificity to the cognate proteases, with negligible cross-reactions among three cancer-related proteases cathepsin B, ADAM10, and ADAM17. This electrochemical method can be developed into multiplex chips for rapid profiling of protease activities in cancer diagnosis and treatment monitoring.

Validation of a commercial antibody to detect endogenous human nicastrin by immunoblot.

Nicastrin (NCSTN) is a transmembrane glycoprotein that is part of the gamma-secretase complex. Gamma-secretase is a protease complex that cleaves type-I single-pass transmembrane proteins. There are many potential substrates for this complex, including NOTCH receptors and amyloid precursor proteins (APP). There are a number of commercial antibodies to nicastrin, but they do not agree on expected peptide size. We confirmed the specificity of a C-terminal binding rabbit anti-human antibody from Sigma-Aldrich (#N1660) using wildtype HEK293 cells and HEK293 cells deleted for nicastrin. The wildtype cells showed a prominent band at approximately 110 kDa. We confirmed this larger than expected sized was due to glycosylation by treating the lysate with peptide-N-glycosidase F (PNGase F), which reduced the band to less than 75 kDa. These data suggest that this polyclonal is specific for nicastrin and can detect endogenous levels of protein.

A peptide-WS2 nanosheet based biosensing platform for determination of ß-secretase and screening of its inhibitors.

ß-Secretase (BACE1) is an important drug target in the treatment of Alzheimer's disease (AD). Therefore, sensitive detection of BACE1 is essential for AD treatment and drug discovery. In this work, a facile and sensitive fluorescence biosensing platform was developed for BACE1 detection. This sensing platform was constituted based on the interaction between a WS2 nanosheet and a peptide sequence. In the absence of BACE1, a FAM-labeled peptide substrate could be adsorbed on the surface of the WS2 nanosheet, thereby quenching its fluorescence. However, in the presence of BACE1, the hydrolysis of the peptide substrate by BACE1 triggers could occur with the subsequent release of short FAM-linked peptide fragments which could not be adsorbed on the surface of the WS2 nanosheet. This resulted in weak fluorescence quenching, thus restoring the fluorescence signal. By measuring the change in the fluorescence of the FAM-labeled peptide substrate, the fluorescence sensing platform based on the WS2 nanosheet could monitor BACE1. The proposed WS2 nanosheet-based platform exhibited excellent specificity and high sensitivity with a detection limit of 66 pM for BACE1. Importantly, we also demonstrated that this platform was suitable for the screening of BACE1 inhibitors. The proposed sensing platform not only provides a novel strategy for the BACE1 assay but also offers a potential tool for screening drugs.

Overexpression of ADAM10 in oral squamous cell carcinoma with metastases.

ADAMs (a disintegrin and metalloproteinase) are important mediators of cell signalling events, which play a role in the pathogenesis and progression of cancers. Immunohistochemical method was used to examine the immunoexpression of ADAM10 and microvessel density in 80 cases of oral squamous cell carcinoma (OSCC): without metastases - OSCC M(-) (n = 38), and with metastases - OSCC M(+) (n = 42), in 24 cases of oral leukoplakia (OLK), (15 cases with low-grade dysplasia - OLK-LG, and 9 cases with high-grade dysplasia - OLK-HG), and 19 controls. The immunoexpression of ADAM10 and the mean number of vessels were significantly increased in both groups of OSCC in comparison to both groups of OLK and controls. Moreover, the immunoexpression of ADAM10 and microvessel density were significantly increased in the OSCC M(+) group in comparison to the OSCC M(-) group. No statistically significant differences were found between immunoexpression of ADAM10 and microvessels density in the OLK-LG, OLK-HG, and control cases. In conclusion, the present study revealed overexpression of ADAM10 in OSCCs, especially in OSCC with metastasis. These findings suggest that ADAM10 could potentially contribute to metastases of oral cancer. Although, our findings suggest that ADAM10 may be involved in angiogenesis of OSCC, further studies are required to determine the role of ADAM10 in this process.

Overexpression of Amyloid Precursor Protein Promotes the Onset of Seborrhoeic Keratosis and is Related to Skin Ageing.

Seborrhoeic keratosis (SK) is an age-related skin disease. Amyloid precursor protein (APP) plays an important role in the pathogenesis of age-related Alzheimer's disease. The aim of this study was to elucidate the expression characteristics of APP in SK tissues (n = 50), and explore whether the production of APP is related to the onset of SK and skin ageing, including ultraviolet (UV)-induced ageing, as observed in normal skin (n = 79). The results of immunohistochemistry, Western blotting and quantitative real-time PCR showed that APP and its downstream products (i.e. amyloid-ß42) were more highly expressed in SK than in paired adjacent normal skin tissues. In contrast, the expression of its key secretase (i.e. ß-secretase1) was generally low. Furthermore, APP expression was higher in UV-exposed than non-exposed skin sites, and expression in the older age group (61-85 years) was greater than that in the younger age group (41-60 years) in SK tissues (p<0.05). APP expression correlated positively with age in epidermis (p<0.05), but not in dermis. These findings suggest that overexpression of APP may promote the onset of SK and is a marker of skin ageing and UV damage. Further research will elucidate whether therapeutic mitigation of increased levels of APP in the skin might delay the onset of SK and skin ageing.

Quantitative Measurement of γ-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms.

We have developed a pair of cell-based reporter gene assays to quantitatively measure γ-secretase cleavage of distinct substrates. This manuscript describes procedures that may be used to monitor γ-secretase-mediated cleavage of either APP-C99 or Notch, using a Gal4 promoter-driven firefly luciferase reporter system. These assays were established by stably co-transfecting HEK293 cells with the Gal4-driven luciferase reporter gene and either the Gal4/VP16-tagged C-terminal fragment of APP (APP-C99; CG cells), or the Gal4/VP16-tagged Notch-ΔE (NΔE; NG cells). Using these reporter assays in parallel, we have demonstrated that an ErbB2 inhibitor, CL-387,785, can preferentially suppress γ-secretase cleavage of APP-C99 in CG cells, but not NΔE in NG cells. The differential responses exhibited by the CG and NG cells, when treated with CL-387,785, represent a preferred characteristic for γ-secretase modulators, and these responses are in stark contrast to the pan-inhibition of γ-secretase induced by DAPT. Our studies provide direct evidence that γ-secretase activities toward different substrates can be differentiated in a cellular context. These new assays may therefore be useful tools in drug discovery for improved AD therapies.

Pulsatile stretch as a novel modulator of amyloid precursor protein processing and associated inflammatory markers in human cerebral endothelial cells.

Amyloid ß (Aß) deposition is a hallmark of Alzheimer's disease (AD). Vascular modifications, including altered brain endothelial cell function and structural viability of the blood-brain barrier due to vascular pulsatility, are implicated in AD pathology. Pulsatility of phenomena in the cerebral vasculature are often not considered in in vitro models of the blood-brain barrier. We demonstrate, for the first time, that pulsatile stretch of brain vascular endothelial cells modulates amyloid precursor protein (APP) expression and the APP processing enzyme, ß-secretase 1, eventuating increased-Aß generation and secretion. Concurrent modulation of intercellular adhesion molecule 1 and endothelial nitric oxide synthase (eNOS) signaling (expression and phosphorylation of eNOS) in response to pulsatile stretch indicates parallel activation of endothelial inflammatory pathways. These findings mechanistically support vascular pulsatility contributing towards cerebral Aß levels.

Development of a Reporter System for In Vivo Monitoring of γ-Secretase Activity in Drosophila.

The γ-secretase complex represents an evolutionarily conserved family of transmembrane aspartyl proteases that cleave numerous type-I membrane proteins, including the ß-amyloid precursor protein (APP) and the receptor Notch. All known rare mutations in APP and the γ-secretase catalytic component, presenilin, which lead to increased amyloid ßpeptide production, are responsible for early-onset familial Alzheimer's disease. ß-amyloid protein precursor-like (APPL) is the Drosophila ortholog of human APP. Here, we created Notch- and APPL-based Drosophila reporter systems for in vivo monitoring of γ-secretase activity. Ectopic expression of the Notch- and APPL-based chimeric reporters in wings results in vein truncation phenotypes. Reporter-mediated vein truncation phenotypes are enhanced by the Notch gain-of-function allele and suppressed by RNAi-mediated knockdown of presenilin. Furthermore, we find that apoptosis partly contributes to the vein truncation phenotypes of the APPL-based reporter, but not to the vein truncation phenotypes of the Notch-based reporter. Taken together, these results suggest that both in vivo reporter systems provide a powerful genetic tool to identify genes that modulate γ-secretase activity and/or APPL metabolism.

[Molecular markers of Alzheimer disease early diagnostic: investigation perspectives of peripheral tissues.]

Alzheimer's disease (AD) is a progressive neurodegenerative disorder of elderly and old age people. For intravital diagnosis of the expression of signaling molecules - AD markers, cerebrospinal fluid (CSF) and peripheral tissues are used: lymphocytes and blood platelets, buccal and olfactory epithelium, skin fibroblasts. There are several changes in the production of hyper phosphorylated form of τ-protein, BACE1 and peptide Аß42 in CSF in case of AD, but CSF taking may have a number of side effects. Less traumatic taking of sampling tissues for the diagnosis of AD is in use of epithelium biopsy and blood portion. An increase in the expression of the hyper phosphorylated form of τ-protein is shown in blood lymphocytes of AD patients. An increase in the content of high molecular weight forms of phosphorylated t-protein and amyloid precursor protein-APP was also revealed in blood platelets of AD patients. Changes in the amount of 2 miRNA families - miR-132 family and miR-134 family were revealed in blood cells 1-5 years before the manifestation of clinical signs of AD. An increase in the concentration of bound calcium, synthesis of peptides Aß40 and Aß42, τ protein was observed in AD skin fibroblasts. In the olfactory and buccal epithelium an increase in the expression of hyper phosphorylated form of τ-protein and Aß peptide was detected in patients with AD. Verification of AD markers in peripheral tissues for biopsy have the important significant for life diagnostics, prevention and and target AD treatment.

Hydrogen sulfide ameliorates learning memory impairment in APP/PS1 transgenic mice: A novel mechanism mediated by the activation of Nrf2.

Beta-amyloid (Aß) plaques and oxidative stress are associated with the pathogenesis of Alzheimer's disease (AD). Hydrogen sulfide (H2S) has been recognized as a cytoprotectant, which improves learning memory impairment and exerts antioxidant effects in neurodegenerative disorders, including AD. The experiment was projected to explore the effects of H2S on cognitive deficits, Aß levels and possible antioxidant mechanisms. Here, APP/PS1 transgenic mice were injected sodium hydrosulfide (NaHS, a H2S donor, 2.8mg/kg) once a day for three months. It was found that APP/PS1 transgenic mice exhibited cognitive deficits and a large number of senile plaques, along with neurons decrease and Aß increase. However, intraperitoneal (i.p.) injection of NaHS improved learning memory deficits, decreased the number of senile plaques, Aß1-40 and Aß1-42 levels, suppressed neurons loss, together with up-regulated the levels of cystathionine-ß-synthase (CBS) and 3-mercaptopyruvate-sulfurtransferase (3MST). Furthermore, the protein levels of beta-amyloid precursor (APP) and beta-secretase 1 (BACE1) were dramatically restrained after administration of H2S. In addition, H2S exerted antioxidant effects via up-regulation nuclear factor erythroid-2-related factor 2 (Nrf2), heme oxygenase-1(HO-1) and glutathione S-transferase (GST). Taken together, these findings suggest that H2S ameliorates learning memory impairment, decreases the number of senile plaques in APP/PS1 mice possibly through inhibition of Aß production and activation of Nrf2/antioxidant response element (ARE) pathway.

Gemcitabine inhibits immune escape of pancreatic cancer by down regulating the soluble ULBP2 protein.

Due to early onset of local invasion and distant metastasis, pancreatic cancer is the most lethal human malignant tumor, with a 5 year survival rate of less than 5%. As a effective chemotherapy drug for pancreatic cancer patients, gemcitabine is reported to inhibit cell proliferation as a nucleotide analog. In this study, we investigated the role of gemcitabine in immune regulation of pancreatic cancer. Our data showed that the level of soluble ULBP2 (sULBP2), a ligand of NKG2D receptor, decreased in the supernatants of pancreatic cancer cell lines when gemcitabine was added, and sULBP2 level correlated with NK92 cells cytotoxicity to pancreatic cancer cell lines. Importantly, our data showed that gemcitabine promoted PANC-1 cells and MIA PaCa-2 immune evasion by reducing ADAM10 expression, a metalloproteinase involved in sULBP2 shedding from cell membrane. Knockdown of ADAM10 clearly downregulated sULBP2 levels in the culture supernatants and cells became more susceptible to NK92 cytotoxicity. Serum samples and tumor samples were obtained from 45 patients with pancreatic ductal adenocarcinoma (PDAC). Statistical analysis showed a significant correlation between the serum level of sULBP2 with ADAM10 expression in PDAC tissues. In conclusion, our data demostrated that gemcitabine inhibits ULBP2 ectodomain shedding through the suppression of ADAM10 and enhance NK cells cytotoxicity by NKG2D-ULBP2 interaction. The results extends our understanding of gemcitabine in the treatment of pancreatic cancer from cell proliferation inhibition to immune regulation.

Restricted Location of PSEN2/γ-Secretase Determines Substrate Specificity and Generates an Intracellular Aß Pool.

γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aß that contains longer Aß; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aß further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aß42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis.

Age-Related Changes in the Synaptic Density of Amyloid-ß Protein Precursor and Secretases in the Human Cerebral Cortex.

Amyloid-ß protein precursor (AßPP) is involved in synaptic formation and function. In the human cingulate cortex, AßPP was preferentially located in the presynaptic active zone as in rodents, indicating a preserved subsynaptic AßPP distribution across species and brain regions. Synaptic AßPP immunoreactivity was decreased with aging in cortical samples collected from autopsies of males (20-80 years), whereas the synaptic levels of α-secretase (ADAM10) and ß-secretase (BACE1) did not significantly change. Decreased AßPP levels may be related to lower allostasis of synapses in the aged brain and their greater susceptibility to dysfunction characteristic of the onset of neurodegenerative disorders.

Identification of the novel activity-driven interaction between synaptotagmin 1 and presenilin 1 links calcium, synapse, and amyloid beta.

BACKGROUND: Synaptic loss strongly correlates with memory deterioration. Local accumulation of amyloid ß (Aß) peptide, and neurotoxic Aß42 in particular, due to abnormal neuronal activity may underlie synaptic dysfunction, neurodegeneration, and memory impairments. To gain an insight into molecular events underlying neuronal activity-regulated Aß production at the synapse, we explored functional outcomes of the newly discovered calcium-dependent interaction between Alzheimer's disease-associated presenilin 1 (PS1)/γ-secretase and synaptic vesicle proteins. RESULTS: Mass spectrometry screen of mouse brain lysates identified synaptotagmin 1 (Syt1) as a novel synapse-specific PS1-binding partner that shows Ca(2+)-dependent PS1 binding profiles in vitro and in vivo. We found that Aß level, and more critically, conformation of the PS1 and the Aß42/40 ratio, are affected by Syt1 overexpression or knockdown, indicating that Syt1 and its interaction with PS1 might regulate Aß production at the synapse. Moreover, ß-secretase 1 (BACE1) stability, ß- and γ-secretase activity, as well as intracellular compartmentalization of PS1 and BACE1, but not of amyloid precursor protein (APP), nicastrin (Nct), presenilin enhancer 2 (Pen-2), or synaptophysin (Syp) were altered in the absence of Syt1, suggesting a selective effect of Syt1 on PS1 and BACE1 trafficking. CONCLUSIONS: Our findings identify Syt1 as a novel Ca(2+)-sensitive PS1 modulator that could regulate synaptic Aß, opening avenues for novel and selective synapse targeting therapeutic strategies.

Effect and mechanism of fuzhisan and donepezil on the sirtuin 1 pathway and amyloid precursor protein metabolism in PC12 cells.

The present study aimed to determine the effect and mechanism of fuzhisan (FZS) and donepezil on the SIRT1 signaling pathway and the metabolism of the amyloid precursor protein (APP) in PC12 cells. An experimental cell model of PC12 cells with Aß25­35­induced neurotoxicity was established and cell proliferation was determined by the MTT assay following treatment with donepezil and FZS. In addition, cell apoptosis was determined using DAPI staining and light microscopy. Furthermore, western blot analysis and ELISA were utilized to evaluate the expression levels of associated APP, Aß40, Aß42, sAPPα, sAPPß, ADAM10, sirtuin 1 (SIRT1) and forkhead box O (FoxO) protein. The results indicated that the cell model was successfully established and FZS protected the PC12 cells from the neurotoxic effects of Aß25­35, in a similar effect to donepezil, in a dose­dependent manner. The expression of APP remained at the same level during the experimental period. The levels of Aß40, Aß42 and sAPPß were downregulated, where as sAPPα, ADAM10, SIRT1 and FoxO expression levels were upregulated. In conclusion, FZS treatment attenuated the Aß25­35­induced neurotoxicity in vitro. The neuroprotective mechanism of FZS was determined, including the induction of ADAM10 and SIRT1­FoxO pathway, which participated in the process of neuroprotection. The present study identified the neuroprotective function of FZS, which may protect against Aß­induced toxicity. Therefore, FZS may be used clinically as a beneficial therapeutic drug for the development or progression of Alzheimer's disease.

Imaging and Functional Analysis of γ-Secretase and Substrate in a Proteolipobead System with an Activity-Based Probe.

Investigation of intramembranal protease catalysis demands the generation of intact biomembrane assemblies with structural integrity and lateral mobility. Here, we report the development of a microsphere supported-biomembrane platform enabling characterization of γ-secretase and substrate within proteolipobead assemblies via microscopy and flow cytometry. The active enzyme loading levels were tracked using an activity-based probe, with the biomembranes delineated by carbocyanine lipid reporters. Proteolipobeads formed from HeLa proteoliposomes gave rise to homogeneous distributions of active γ-secretase within supported biomembranes with native-like fluidity. The substrate loading into supported biomembranes was detergent-dependent, as evidenced by even colocalization of substrate and lipid tracers in confocal 3D imaging of individual proteolipobeads. Moreover, the loading level was tunable with bulk substrate concentration. γ-Secretase substrate cleavage and its inhibition within γ-secretase proteolipobeads were observed. This platform offers a means to visualize enzyme and substrate loading, activity, and inhibition in a controllable biomembrane microenvironment.