Dynamics of β-Amyloid Reductions in Brain, Cerebrospinal Fluid, and Plasma of β-Amyloid Precursor Protein Transgenic Mice Treated with a γ-Secretase Inhibitor
Donna M. BartenValerie GussJason A. CorsaAlice LooSteven HanselMing ZhengB. MuñozK. SrinivasanBinghe WangBarbara RobertsonCraig PolsonJue WangSusan B. RobertsJoseph P. HendrickJeffrey J. AndersonJames LoyRoss M. DentonTodd A. VerdoornDavid W. SmithKevin M. Felsenstein
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γ-Secretase inhibitors are one promising approach to the development of a therapeutic for Alzheimer9s disease (AD). γ-Secretase inhibitors reduce brain β-amyloid peptide (Aβ), which is believed to be a major contributor in the etiology of AD. Transgenic mice overexpressing the human β-amyloid precursor protein (APP) are valuable models to examine the dynamics of Aβ changes with γ-secretase inhibitors in plaque-free and plaque-bearing animals. BMS-299897 2-[(1R)-1-[[(4-chlorophenyl)sulfony](2,5-difluorophenyl)amino]ethyl]-5-fluorobenzenepropanoic acid, a γ-secretase inhibitor, showed dose- and time dependent reductions of Aβ in brain, cerebrospinal fluid (CSF), and plasma in young transgenic mice, with a significant correlation between brain and CSF Aβ levels. Because CSF and brain interstitial fluid are distinct compartments in composition and location, this correlation could not be assumed. In contrast, aged transgenic mice with large accumulations of Aβ in plaques showed reductions in CSF Aβ in the absence of measurable changes in plaque Aβ in the brain after up to 2 weeks of treatment. Hence, CSF Aβ levels were a valuable measure of γ-secretase activity in the central nervous system in either the presence or absence of plaques. Transgenic mice were also used to examine potential side effects due to Notch inhibition. BMS-299897 was 15-fold more effective at preventing the cleavage of APP than of Notch in vitro. No changes in the maturation of CD8+ thymocytes or of intestinal goblet cells were observed in mice treated with BMS-299897, showing that it is possible for γ-secretase inhibitors to reduce brain Aβ without causing Notch-mediated toxicity.Keywords:
Interstitial fluid
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Christian Haass1, Christoph Kaether2, Gopal Thinakaran3 and Sangram Sisodia3 DZNE—German Center for Neurodegenerative Diseases, 80336 Munich, Germany; and Adolf Butenandt-Institute, Biochemistry, Ludwig-Maximilians University, 80336 Munich, Germany Leibniz Institut für Altersforschung, D-07745 Jena, Germany Department of Neurobiology, University of Chicago, Chicago, Illinois 60637 Correspondence: christian.haass{at}dzne.lmu.de; ssisodia{at}bsd.uchicago.edu
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Amyloid-β peptide (AβP) that accumulates in the Alzheimer's diseased brain is derived from proteolytic processing of the amyloid precursor protein (APP) by means of β- and γ-secretases. The β-secretase APP cleaving enzyme (BACE), which generates the N terminus of AβP, has become a target of intense research aimed at blocking the enzyme activity, thus reducing AβP and, subsequently, plaque formation. The search for specific inhibitors of β-secretase activity as a possible treatment for Alzheimer's disease intensified with the discovery that BACE may be involved in processing other non-APP substrates. The presence of the APP–BACE complex in early endosomes highlights the cell surface as a potential therapeutic target, suggesting that interference in APP–BACE interaction at the cell surface may affect amyloid-β production. We present here a unique approach to inhibit AβP production by means of antibodies against the β-secretase cleavage site of APP. These antibodies were found to bind human APP overexpressed by CHO cells, and the formed immunocomplex was visualized in the early endosomes. Indeed, blocking of the β-secretase site by these antibodies interfered with BACE activity and inhibited both intracellular and extracellular AβP formation in these cells.
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Abstract Accumulation of amyloid beta peptide (Abeta) in brain is a hallmark of Alzheimer's disease (AD). Inhibition of beta‐site amyloid precursor protein (APP)‐cleaving enzyme‐1 (BACE1), the enzyme that initiates Abeta production, and other Abeta‐lowering strategies are commonly tested in transgenic mice overexpressing mutant APP. However, sporadic AD cases, which represent the majority of AD patients, are free from the mutation and do not necessarily have overproduction of APP. In addition, the commonly used Swedish mutant APP alters APP cleavage. Therefore, testing Abeta‐lowering strategies in transgenic mice may not be optimal. In this study, we investigated the impact of BACE1 inhibition in non‐transgenic mice with physiologically relevant APP expression. Existing Abeta ELISAs are either relatively insensitive to mouse Abeta or not specific to full‐length Abeta. A newly developed ELISA detected a significant reduction of full‐length soluble Abeta 1–40 in mice with the BACE1 homozygous gene deletion or BACE1 inhibitor treatment, while the level of x‐40 Abeta was moderately reduced due to detection of non‐full‐length Abeta and compensatory activation of alpha‐secretase. These results confirmed the feasibility of Abeta reduction through BACE1 inhibition under physiological conditions. Studies using our new ELISA in non‐transgenic mice provide more accurate evaluation of Abeta‐reducing strategies than was previously feasible.
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Abstract FE65 is an adaptor protein that interacts with the cytoplasmic tail of the amyloid precursor protein (APP). In cultured non‐neuronal cells, the formation of the FE65‐APP complex is a key element for the modulation of APP processing, signalling and β‐amyloid (Aβ) production. The functions of FE65 in vivo , including its role in the metabolism of neuronal APP, remain to be investigated. In this study, transgenic mice expressing human FE65 were generated and crossbred with APP transgenic mice, known to develop Aβ deposits at 6 months of age. Compared with APP mice, APP/FE65 double transgenic mice exhibited a lower Aβ accumulation in the cerebral cortex as demonstrated by immunohistochemistry and immunoassay, and a lower level of APP‐CTFs. The reduced accumulation of Aβ in APP/FE65 double transgenics, compared with APP mice, could be linked to the low Aβ42 level observed at 4 months of age and to the lower APP‐CTFs levels. The present work provides evidence that FE65 plays a role in the regulation of APP processing in an in vivo model.
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The deficits characteristic of Alzheimer's disease (AD) are believed to result, at least in part, from the neurotoxic effects of β-amyloid peptides, a set of 39–43 amino acid fragments derived proteolytically from β-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 β-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.
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The deposition of amyloid-beta is a pathological hallmark of Alzheimer's disease. Amyloid-beta is derived from amyloid precursor protein through sequential proteolytic cleavages by β-secretase (beta-site amyloid precursor protein-cleaving enzyme 1) and γ-secretase. To further elucidate the roles of beta-site amyloid precursor protein-cleaving enzyme 1 in the development of Alzheimer's disease, a yeast two-hybrid system was used to screen a human embryonic brain cDNA library for proteins directly interacting with the intracellular domain of beta-site amyloid precursor protein-cleaving enzyme 1. A potential beta-site amyloid precursor protein-cleaving enzyme 1-interacting protein identified from the positive clones was divalent cation tolerance protein. Immunoprecipitation studies in the neuroblastoma cell line N2a showed that exogenous divalent cation tolerance protein interacts with endogenous beta-site amyloid precursor protein-cleaving enzyme 1. The overexpression of divalent cation tolerance protein did not affect beta-site amyloid precursor protein-cleaving enzyme 1 protein levels, but led to increased amyloid precursor protein levels in N2a/APP695 cells, with a concomitant reduction in the processing product amyloid precursor protein C-terminal fragment, indicating that divalent cation tolerance protein inhibits the processing of amyloid precursor protein. Our experimental findings suggest that divalent cation tolerance protein negatively regulates the function of beta-site amyloid precursor protein-cleaving enzyme 1. Thus, divalent cation tolerance protein could play a protective role in Alzheimer's disease.
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Neuritic plaques in the brain are a major neuropathological hallmark of Alzheimer’s disease. They are formed by the deposition and aggregation of extracellular amyloid-β protein (Aβ). Aβ is derived from the sequential cleavage of amyloid-β precursor protein (APP) by β-secretase and γ-secretase. β-Site APP cleaving enzyme 1 (BACE1) functions as the primary, if not sole, β-secretase in vivo and is essential for Aβ production. Regulation of APP processing is a major focus of research into AD pathogenesis. The trafficking systems of APP and its cleavage enzymes are complex. Transporting APP and secretases into the same subcellular organelles facilitates their interaction and favors APP processing. The role of APP and BACE1 trafficking in the amyloidgenic pathway and the underlying mechanisms for Aβ production are discussed in this review. In addition, the distinct mechanisms of amino- and carboxy-terminal Aβ generation are reviewed.
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Abstract The amyloid cascade hypothesis proposes that excessive accumulation of amyloid beta-peptides is the initiating event in Alzheimer’s disease. These neurotoxic peptides are generated from the amyloid precursor protein via sequential cleavage by β- and γ-secretases in the ‘amyloidogenic’ proteolytic pathway. Alternatively, the amyloid precursor protein can be processed via the ‘non-amyloidogenic’ pathway which, through the action of the α-secretase a d isintegrin a nd m etalloproteinase (ADAM) 10, both precludes amyloid beta-peptide formation and has the additional benefit of generating a neuroprotective soluble amyloid precursor protein fragment, sAPPα. In the current study, we investigated whether the orphan drug, dichloroacetate, could alter amyloid precursor protein proteolysis. In SH-SY5Y neuroblastoma cells, dichloroacetate enhanced sAPPα generation whilst inhibiting β-secretase processing of endogenous amyloid precursor protein and the subsequent generation of amyloid beta-peptides. Over-expression of the amyloid precursor protein partly ablated the effect of dichloroacetate on amyloidogenic and non-amyloidogenic processing whilst over-expression of the β-secretase only ablated the effect on amyloidogenic processing. Similar enhancement of ADAM-mediated amyloid precursor protein processing by dichloroacetate was observed in unrelated cell lines and the effect was not exclusive to the amyloid precursor protein as an ADAM substrate, as indicated by dichloroacetate-enhanced proteolysis of the Notch ligand, Jagged1. Despite altering proteolysis of the amyloid precursor protein, dichloroacetate did not significantly affect the expression of α-, β- or γ-secretases. In conclusion, dichloroacetate can inhibit amyloidogenic and promote non-amyloidogenic proteolysis of the amyloid precursor protein. As the drug is already used for the treatment of lactic acidosis and is known to cross the blood-brain-barrier, it might represent a cheap and effective therapy for slowing the progression of Alzheimer’s disease.
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The amyloid cascade hypothesis proposes that excessive accumulation of amyloid beta-peptides is the initiating event in Alzheimer's disease. These neurotoxic peptides are generated from the amyloid precursor protein via sequential cleavage by β- and γ-secretases in the 'amyloidogenic' proteolytic pathway. Alternatively, the amyloid precursor protein can be processed via the 'non-amyloidogenic' pathway which, through the action of the α-secretase a disintegrin and metalloproteinase (ADAM) 10, both precludes amyloid beta-peptide formation and has the additional benefit of generating a neuroprotective soluble amyloid precursor protein fragment, sAPPα. In the current study, we investigated whether the orphan drug, dichloroacetate, could alter amyloid precursor protein proteolysis. In SH-SY5Y neuroblastoma cells, dichloroacetate enhanced sAPPα generation whilst inhibiting β-secretase processing of endogenous amyloid precursor protein and the subsequent generation of amyloid beta-peptides. Over-expression of the amyloid precursor protein partly ablated the effect of dichloroacetate on amyloidogenic and non-amyloidogenic processing whilst over-expression of the β-secretase only ablated the effect on amyloidogenic processing. Similar enhancement of ADAM-mediated amyloid precursor protein processing by dichloroacetate was observed in unrelated cell lines and the effect was not exclusive to the amyloid precursor protein as an ADAM substrate, as indicated by dichloroacetate-enhanced proteolysis of the Notch ligand, Jagged1. Despite altering proteolysis of the amyloid precursor protein, dichloroacetate did not significantly affect the expression/activity of α-, β- or γ-secretases. In conclusion, dichloroacetate can inhibit amyloidogenic and promote non-amyloidogenic proteolysis of the amyloid precursor protein. Given the small size and blood-brain-barrier permeability of the drug, further research into its mechanism of action with respect to APP proteolysis may lead to the development of therapies for slowing the progression of Alzheimer's disease.
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Amyloid beta-peptide (Abeta) is implicated as the major causative agent in Alzheimer's disease (AD). Abeta is produced by the processing of the amyloid precursor protein (APP) by BACE1 (beta-secretase) and gamma-secretase. Many inhibitors have been developed for the secretases. However, the inhibitors will interfere with the processing of not only APP but also of other secretase substrates. In this study, we describe the development of inhibitors that prevent production of Abeta by specific binding to the beta-cleavage site of APP. We used the hydropathic complementarity (HC) approach for the design of short peptide inhibitors. Some of the HC peptides were bound to the substrate peptide (Sub W) corresponding to the beta-cleavage site of APP and blocked its cleavage by recombinant human BACE1 (rhBACE1) in vitro. In addition, HC peptides specifically inhibited the cleavage of Sub W, and not affecting other BACE1 substrates. Chemical modification allowed an HC peptide (CIQIHF) to inhibit the processing of APP as well as the production of Abeta in the treated cells. Such novel APP-specific inhibitors will provide opportunity for the development of drugs that can be used for the prevention and treatment of AD with minimal side effects.
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