Screening for Small Molecule Inhibitors of Statin-Induced APP C-terminal Toxic Fragment Production.
ABSTRACT: Alzheimer's disease (AD) is characterized by neuronal and synaptic loss. One process that could contribute to this loss is the intracellular caspase cleavage of the amyloid precursor protein (APP) resulting in release of the toxic C-terminal 31-amino acid peptide APP-C31 along with the production of APP?C31, full-length APP minus the C-terminal 31 amino acids. We previously found that a mutation in APP that prevents this caspase cleavage ameliorated synaptic loss and cognitive impairment in a murine AD model. Thus, inhibition of this cleavage is a reasonable target for new therapeutic development. In order to identify small molecules that inhibit the generation of APP-C31, we first used an APP?C31 cleavage site-specific antibody to develop an AlphaLISA to screen several chemical compound libraries for the level of N-terminal fragment production. This antibody was also used to develop an ELISA for validation studies. In both high throughput screening (HTS) and validation testing, the ability of compounds to inhibit simvastatin- (HTS) or cerivastatin- (validation studies) induced caspase cleavage at the APP-D720 cleavage site was determined in Chinese hamster ovary (CHO) cells stably transfected with wildtype (wt) human APP (CHO-7W). Several compounds, as well as control pan-caspase inhibitor Q-VD-OPh, inhibited APP?C31 production (measured fragment) and rescued cell death in a dose-dependent manner. The effective compounds fell into several classes including SERCA inhibitors, inhibitors of Wnt signaling, and calcium channel antagonists. Further studies are underway to evaluate the efficacy of lead compounds - identified here using cells and tissues expressing wt human APP - in mouse models of AD expressing mutated human APP, as well as to identify additional compounds and determine the mechanisms by which they exert their effects.
Project description:The deficits characteristic of Alzheimer's disease (AD) are believed to result, at least in part, from the neurotoxic effects of beta-amyloid peptides, a set of 39-43 amino acid fragments derived proteolytically from beta-amyloid precursor protein (APP). APP also is cleaved intracytoplasmically at Asp-664 to generate a second cytotoxic peptide, APP-C31, but whether this C-terminal processing of APP plays a role in the pathogenesis of AD is unknown. Therefore, we compared elements of the Alzheimer's phenotype in transgenic mice modeling AD with vs. without a functional Asp-664 caspase cleavage site. Surprisingly, whereas beta-amyloid production and plaque formation were unaltered, synaptic loss, astrogliosis, dentate gyral atrophy, increased neuronal precursor proliferation, and behavioral abnormalities were completely prevented by a mutation at Asp-664. These results suggest that Asp-664 plays a critical role in the generation of Alzheimer-related pathophysiological and behavioral changes in human APP transgenic mice, possibly as a cleavage site or via protein-protein interactions.
Project description:Previous studies suggested that cleavage of the amyloid precursor protein (APP) at aspartate residue 664 by caspases may play a key role in the pathogenesis of Alzheimer's disease. Mutation of this site (D664A) prevents caspase cleavage and the generation of the C-terminal APP fragments C31 and Jcasp, which have been proposed to mediate amyloid-beta (Abeta) neurotoxicity. Here we compared human APP transgenic mice with (B254) and without (J20) the D664A mutation in a battery of tests. Before Abeta deposition, hAPP-B254 and hAPP-J20 mice had comparable hippocampal levels of Abeta(1-42). At 2-3 or 5-7 months of age, hAPP-B254 and hAPP-J20 mice had similar abnormalities relative to nontransgenic mice in spatial and nonspatial learning and memory, elevated plus maze performance, electrophysiological measures of synaptic transmission and plasticity, and levels of synaptic activity-related proteins. Thus, caspase cleavage of APP at position D664 and generation of C31 do not play a critical role in the development of these abnormalities.
Project description:Amyloid ?-protein (A?) toxicity is hypothesized to play a seminal role in Alzheimer's disease (AD) pathogenesis. However, it remains unclear how A? causes synaptic dysfunction and synapse loss. We hypothesize that one mechanism of A?-induced synaptic injury is related to the cleavage of amyloid ? precursor protein (APP) at position D664 by caspases that release the putatively cytotoxic C31 peptide. In organotypic slice cultures derived from mice with a knock-in mutation in the APP gene (APP D664A) to inhibit caspase cleavage, A?-induced synaptic injury is markedly reduced in two models of A? toxicity. Loss of dendritic spines is also attenuated in mice treated with caspase inhibitors. Importantly, the time-dependent dendritic spine loss is correlated with localized activation of caspase-3 but is absent in APP D664A cultures. We propose that the APP cytosolic domain plays an essential role in A?-induced synaptic damage in the injury pathway mediated by localized caspase activation.
Project description:The cytoplasmic tail of the amyloid precursor protein (APP) contains two putatively cytotoxic peptides, Jcasp and C31, derived by caspase cleavage of APP. Jcasp is a fragment starting from the epsilon-secretase site to position 664, while C31 is a fragment from position 665 to the C-terminus. Our studies now showed that compared to C31, Jcasp appeared to play a minor role in cytotoxicity. In particular, inhibition of Jcasp generation by treatment of gamma-secretase inhibitor did not lead to any attenuation of C31-induced toxicity. Secondly, because C31 toxicity is largely absent in cells lacking endogenous APP, we determined, using a split beta-galactosidase complementary assay to monitor protein-protein interactions, the presence of APP associated complexes. Our results demonstrated that both APP homomeric and C31/APP heteromeric complexes were correlated with cell death, indicating that C31 complexes with APP to recruit the interacting partners that initiate the signals related to cellular toxicity.
Project description:Proteolytic cleavage of the amyloid precursor protein (APP) generates ?-amyloid (A?) peptides. Prolonged accumulation of A? in the brain underlies the pathogenesis of Alzheimer disease (AD) and is regarded as a principal target for development of disease-modifying therapeutics.Using Chinese hamster ovary (CHO) APP751SW cells, we identified and characterized effects of 2-([pyridine-2-ylmethyl]-amino)-phenol (2-PMAP) on APP steady-state level and A? production. Outcomes of 2-PMAP treatment on A? accumulation and associated memory deficit were studied in APPSW /PS1dE9 AD transgenic model mice.In CHO APP751SW cells, 2-PMAP lowered the steady-state APP level and inhibited A?x-40 and A?x-42 production in a dose-response manner with a minimum effective concentration???0.5?M. The inhibitory effect of 2-PMAP on translational efficiency of APP mRNA into protein was directly confirmed using a 35S-methionine/cysteine metabolic labeling technique, whereas APP mRNA level remained unaltered. Administration of 2-PMAP to APPSW /PS1dE9 mice reduced brain levels of full-length APP and its C-terminal fragments and lowered levels of soluble A?x-40 and A?x-42 . Four-month chronic treatment of APPSW /PS1dE9 mice revealed no observable toxicity and improved animals' memory performance. 2-PMAP treatment also caused significant reduction in brain A? deposition determined by both unbiased quantification of A? plaque load and biochemical analysis of formic acid-extracted A?x-40 and A?x-42 levels and the level of oligomeric A?.We demonstrate the potential of modulating APP steady-state expression level as a safe and effective approach for reducing A? deposition in AD transgenic model mice.
Project description:Alzheimer's disease (AD) is a progressive neurodegenerative disorder pathologically characterized by deposition of beta-amyloid (Abeta) peptides as senile plaques in the brain. Recent studies suggest that green tea flavonoids may be used for the prevention and treatment of a variety of neurodegenerative diseases. Here, we report that (-)-epigallocatechin-3-gallate (EGCG), the main polyphenolic constituent of green tea, reduces Abeta generation in both murine neuron-like cells (N2a) transfected with the human "Swedish" mutant amyloid precursor protein (APP) and in primary neurons derived from Swedish mutant APP-overexpressing mice (Tg APPsw line 2576). In concert with these observations, we find that EGCG markedly promotes cleavage of the alpha-C-terminal fragment of APP and elevates the N-terminal APP cleavage product, soluble APP-alpha. These cleavage events are associated with elevated alpha-secretase activity and enhanced hydrolysis of tumor necrosis factor alpha-converting enzyme, a primary candidate alpha-secretase. As a validation of these findings in vivo, we treated Tg APPsw transgenic mice overproducing Abeta with EGCG and found decreased Abeta levels and plaques associated with promotion of the nonamyloidogenic alpha-secretase proteolytic pathway. These data raise the possibility that EGCG dietary supplementation may provide effective prophylaxis for AD.
Project description:Streptozotocin (STZ) is widely used to induce oxidative damage and to impair glucose metabolism, apoptosis, and tau/A? pathology, eventually leading to cognitive deficits in both in vitro and in vivo models of Alzheimer's disease (AD). In this study, we constructed a cell-based platform using STZ to induce stress conditions mimicking the complicated pathologies of AD in vitro, and evaluated the anti-amyloid effects of a small molecule, N-(1,3-benzodioxol-5-yl)-2-[5-chloro-2-methoxy(phenylsulfonyl)anilino]acetamide (LX2343) in the amelioration of cognitive deficits in AD model mice.Cell-based assays for screening anti-amyloid compounds were established by assessing A? accumulation in HEK293-APPsw and CHO-APP cells, and A? clearance in primary astrocytes and SH-SY5Y cells after the cells were treated with STZ in the presence of the test compounds. Autophagic flux was observed using confocal laser scanning microscopy. APP/PS1 transgenic mice were administered LX2343 (10 mg·kg-1·d-1, ip) for 100 d. After LX2343 administration, cognitive ability of the mice was evaluated using Morris water maze test, and senile plaques in the brains were detected using Thioflavine S staining. ELISA assay was used to evaluate A? and sAPP? levels, while Western blot analysis was used to measure the signaling proteins in both cell and animal brains.LX2343 (5-20 ?mol/L) dose-dependently decreased A? accumulation in HEK293-APPsw and CHO-APP cells, and promoted A? clearance in SH-SY5Y cells and primary astrocytes. The anti-amyloid effects of LX2343 were attributed to suppressing JNK-mediated APPThr668 phosphorylation, thus inhibiting APP cleavage on one hand, and inhibiting BACE1 enzymatic activity with an IC50 value of 11.43±0.36 ?mol/L, on the other hand. Furthermore, LX2343 acted as a non-ATP competitive PI3K inhibitor to negatively regulate AKT/mTOR signaling, thus promoting autophagy, and increasing A? clearance. Administration of LX2343 in APP/PS1 transgenic mice significantly ameliorated cognitive deficits and markedly ameliorated the A? pathology in their brains.LX2343 ameliorates cognitive dysfunction in APP/PS1 transgenic mice via both A? production inhibition and clearance promotion, which highlights the potential of LX2343 in the treatment of AD.
Project description:Evidence for a central role of amyloid ?-protein (A?) in the genesis of Alzheimer’s disease (AD) has led to advanced human trials of A?-lowering agents. The “amyloid hypothesis” of AD postulates deleterious effects of small, soluble forms of A? on synaptic form and function. Because selectively targeting synaptotoxic forms of soluble A? could be therapeutically advantageous, it is important to understand the full range of soluble A? derivatives. We previously described a Chinese hamster ovary (CHO) cell line (7PA2 cells) that stably expresses mutant human amyloid precursor protein (APP). Here, we extend this work by purifying an sodium dodecyl sulfate (SDS)-stable, ?8 kDa A? species from the 7PA2 medium. Mass spectrometry confirmed its identity as a noncovalently bonded A?40 homodimer that impaired hippocampal long-term potentiation (LTP) in vivo. We further report the detection of A?-containing fragments of APP in the 7PA2 medium that extend N-terminal from Asp1 of A?. These N-terminally extended A?-containing monomeric fragments are distinct from soluble A? oligomers formed from A?1-40/42 monomers and are bioactive synaptotoxins secreted by 7PA2 cells. Importantly, decreasing ?-secretase processing of APP elevated these alternative synaptotoxic APP fragments. We conclude that certain synaptotoxic A?-containing species can arise from APP processing events N-terminal to the classical ?-secretase cleavage site.
Project description:Mutations in the amyloid precursor protein (APP) gene and the aberrant cleavage of APP by γ-secretase are associated with Alzheimer's disease (AD). Here we have developed a simple and sensitive cell-based assay to detect APP cleavage by γ-secretase. Unexpectedly, most familial AD (FAD)-linked APP mutations make APP partially resistant to γ-secretase. Mutations that alter residues N terminal to the γ-secretase cleavage site Aβ42 have subtle effects on cleavage efficiency and cleavage-site selectivity. In contrast, mutations that alter residues C terminal to the Aβ42 site reduce cleavage efficiency and dramatically shift cleavage-site specificity toward the aggregation-prone Aβ42. Moreover, mutations that remove positive charge at residue 53 greatly reduce the APP cleavage by γ-secretase. These results suggest a model of γ-secretase substrate recognition, in which the APP region C terminal to the Aβ42 site and the positively charged residue at position 53 are the primary determinants for substrate binding and cleavage-site selectivity. We further demonstrate that this model can be extended to γ-secretase processing of notch receptors, a family of highly conserved cell-surface signaling proteins.
Project description:A mutation in the BRI2/ITM2b gene causes loss of BRI2 protein leading to familial Danish dementia (FDD). BRI2 deficiency of FDD provokes an increase in amyloid-? precursor protein (APP) processing since BRI2 is an inhibitor of APP proteolysis, and APP mediates the synaptic/memory deficits in FDD. APP processing is linked to Alzheimer disease (AD) pathogenesis, which is consistent with a common mechanism involving toxic APP metabolites in both dementias. We show that inhibition of APP cleavage by ?-secretase rescues synaptic/memory deficits in a mouse model of FDD. ?-cleavage of APP yields amino-terminal-soluble APP? (sAPP?) and ?-carboxyl-terminal fragments (?-CTF). Processing of ?-CTF by ?-secretase releases amyloid-? (A?), which is assumed to cause AD. However, inhibition of ?-secretase did not ameliorate synaptic/memory deficits of FDD mice. These results suggest that sAPP? and/or ?-CTF, rather than A?, are the toxic species causing dementia, and indicate that reducing ?-cleavage of APP is an appropriate therapeutic approach to treating human dementias. Our data and the failures of anti-A? therapies in humans advise against targeting ?-secretase cleavage of APP and/or A?.