Making the final cut: pathogenic amyloid-β peptide generation by γ-secretase
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Making the final cut: pathogenic amyloid-β peptide generation by γ-secretase – Alzheimer´s disease (AD) is a devastating neurodegenerative disease of the elderly population. Genetic evidence strongly suggests that aberrant generation and/or clearance of the neurotoxic amyloid-β peptide (Aβ) is triggering the disease. Aβ is generated from the amyloid precursor protein (APP) by the sequential cleavages of β- and γ-secretase. The latter cleavage by γ-secretase, a unique and fascinating four-component protease complex, occurs in the APP transmembrane domain thereby releasing Aβ species of 37-43 amino acids in length including the longer, highly pathogenic peptides Aβ42 and Aβ43. The lack of a precise understanding of Aβ generation as well as of the functions of other γ-secretase substrates has been one factor underlying the disappointing failure of γ-secretase inhibitors in clinical trials, but on the other side also been a major driving force for structural and in depth mechanistic studies on this key AD drug target in the past few years. Here we (...)Keywords:
Amyloid (mycology)
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The amyloid precursor protein (APP) is subject to alternative pathways of proteolytic processing, leading either to production of the amyloid-beta (Abeta) peptides or to non-amyloidogenic fragments. Here, we report the first structural study of C99, the 99-residue transmembrane C-terminal domain of APP liberated by beta-secretase cleavage. We also show that cholesterol, an agent that promotes the amyloidogenic pathway, specifically binds to this protein. C99 was purified into model membranes where it was observed to homodimerize. NMR data show that the transmembrane domain of C99 is an alpha-helix that is flanked on both sides by mostly disordered extramembrane domains, with two exceptions. First, there is a short extracellular surface-associated helix located just after the site of alpha-secretase cleavage that helps to organize the connecting loop to the transmembrane domain, which is known to be essential for Abeta production. Second, there is a surface-associated helix located at the cytosolic C-terminus, adjacent to the YENPTY motif that plays critical roles in APP trafficking and protein-protein interactions. Cholesterol was seen to participate in saturable interactions with C99 that are centered at the critical loop connecting the extracellular helix to the transmembrane domain. Binding of cholesterol to C99 and, most likely, to APP may be critical for the trafficking of these proteins to cholesterol-rich membrane domains, which leads to cleavage by beta- and gamma-secretase and resulting amyloid-beta production. It is proposed that APP may serve as a cellular cholesterol sensor that is linked to mechanisms for suppressing cellular cholesterol uptake.
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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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Aβ production is influenced by intracellular trafficking of secretases and amyloid precursor protein (APP).
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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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Alzheimer disease (AD) is characterized by the accumulation of extracellular deposits (amyloid plaques) and intracellular protein aggregates (neurofibrillary tangles), which leads to synaptic loss and progressive neuronal death. Plaques mainly consist of the aggregated beta-amyloid (Aβ) peptide of, typically, 40 or 42 amino acids, which is a result of the consecutive cleavage of the amyloid precursor protein (APP) at β- and γ-secretase cleavage sites (Fig. 1). Such APP processing represents the amyloidogenic pathway. Frequently, AD is associated with an increase in activity of the β-site amyloid precursor protein cleaving enzyme 1, which can lead to Aβ accumulation in the brain (reviewed in De Strooper 2010). MMP-9-mediated cleavage of APP and other substrates may promote signaling through β1 integrins. APP can be consequently cleaved either by α- and γ-secretases (non-amyloidogenic pathway) or by β- and γ-secretases (amyloidogenic pathway). MMP-9 has α-secretase activity promoting the non-amyloidogenic cleavage of APP. Furthermore, MMP-9 can cut dystroglycan complex and intercellular adhesion molecule-5 (ICAM-5). The cleavage products of MMP-9 substrates can interact with β1 integrins and promote their activation. The signaling cascade activated by β1 integrins leads to reorganization of the cytoskeleton and formation and maintenance of new spines. However, under normal conditions the majority of APP is processed through the non-amyloidogenic pathway and cleaved consecutively by α- and γ-secretases, which does not generate Aβ peptide but results in the release of sAPPα extracellular fragment (Fig. 1). Importantly, this fragment was shown to be neurotrophic (Bell et al. 2008) and able to restore the deficit in hippocampal synaptic plasticity caused by APP gene deficiency (Ring et al. 2007). Thus, identification of proteases with α-secretase activity is of high importance to develop approaches for stimulation of the non-amyloidogenic pathway and to eventually reverse the imbalance between amyloidogenic and non-amyloidogenic APP processing in AD. In this issue of Journal of Neurochemistry, Fragkouli and colleagues published a study in which they have generated transgenic mice over-expressing matrix metalloproteinase MMP-9 (TgMMP9) under the platelet-derived-growth factor promoter (Fragkouli et al. 2012). TgMMP9 mice have enhanced cleavage of APP by the non-amyloidogenic pathway: The expression of sAPPα secreted fragment is enhanced in the brains of these mice, whereas the production of the membrane-spanning holoAPP is unaltered. This corroborates recent publications by the same group, which showed that MMP-9 possesses α-secretase activity and directly cleaves APP in vitro (Talamagas et al. 2007; Fragkouli et al. 2011). Another important question addressed by Fragkouli and colleagues is regarding the functional consequences of MMP-9 over-expression in vivo. TgMMP9 mice show improvement in spatial learning and reversal learning in the water maze task, and more stable late long-term potentiation of the CA3-CA1 synapses, as compared with wild-type mice (Fragkouli et al. 2011). This is in line with impaired hippocampal long-term potentiation and underperformance in hippocampus-dependent cognitive tasks upon pharmacological or genetic ablation of MMP-9 activity (reviewed by Wlodarczyk et al. 2011). Furthermore, TgMMP9 mice show enhanced performance in the non-spatial object recognition task. Analysis of dendritic spines after behavioral testing (but not in naive TgMMP9 mice) revealed an increase in dendritic spine density in the hippocampus and the somatosensory cortex, as compared with wild types. In the context of these observations demonstrating the elevated levels of physiological synaptic plasticity in TgMMP9 mice, it is noteworthy that transgenic rats with constitutive neuronal MMP-9 over-expression under the control of the synapsin I promoter show increased pathogenic neuroplasticity in the kindling model of epilepsy (Wilczynski et al. 2008). In summary, the study of Fragkouli and colleagues shows a correlative link between the increase in expression of sAPPα and beneficial effects of MMP-9 over-expression on synaptic plasticity. The obvious next question is which of these effects are due to the overproduced sAPPα rather than the excessive cleavage of other MMP-9 substrates. One approach to address this question could be to cross-breed TgMMP9 and APP-deficient mice and verify if ablation of APP would minimize beneficial effects of MMP-9 over-expression. This has not yet been performed. However, an indirect support for the role of sAPPα in TgMMP9 mice is coming from a study of mice over-expressing the disintegrin metalloprotease ADAM-10, which also has α-secretase activity in vivo and was found to promote structural synaptic plasticity (Bell et al. 2008). Strikingly, over-expression of ADAM-10 and exogenous infusion of sAPPα lead to significant elevations in the glutamatergic, cholinergic, and GABAergic cortical pre-synaptic boutons in adult mice (Bell et al. 2008). It would be very intriguing to see whether TgMMP9 mice would also show not only an increase in spine density, which is thought to reflect an increase in the number of excitatory synapses, but also an increased number of cholinergic and GABAergic synapses. Although the important role of overproduced sAPPα in TgMMP9 mice is quite plausible, there are other mechanisms to consider. Among these is the ability of MMP-9 to catabolize Aβ (Yin et al. 2006) and the cleavage of other numerous substrates of MMP-9, including β-dystroglycan and intercellular adhesion molecule-5. Interestingly, the cleavage products of these two proteins may interact with β1 integrins, and it is known that activation of β1 integrins may launch the cytoskeleton reorganization that leads to the generation of new spines (Michaluk et al. 2007; Tian et al. 2007). Excitingly, a study from the laboratory of Dennis Selkoe suggests that sAPPα may stimulate β1 integrin-mediated signaling by a relief of inhibitory activity of holoAPP (Young-Pearse et al. 2008). Thus, integrins might be the common receptor for several MMP-9 cleavage products, raising the question if over-expression of MMP-9 in TgMMP9 results in activation of β1 integrin and downstream signaling cascades (Fig. 1). In conclusion, the beneficial effects of MMP-9 overproduction on neuroplasticity stimulate further interest to study whether synaptic and cognitive deficits found in mouse models of AD can be abrogated by cross-breeding them with TgMMP9 mice or by administration of β1 integrin agonists. These studies may eventually open new avenues for the development of therapeutic strategies for the treatment of AD. The authors declare no conflicts of interest.
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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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