P3-288: Gamma-secretase inhibitors have intrinsically different potencies against Aβ production and Notch signaling
Catherine R. BurtonJere E. MeredithJeremy H. ToynJodi D. BradleyQian WangDiane M. SharpLisa M. SiskValerie GussCraig PolsonCarol KrauseJudith Wardwell-SwansonQi GuoKevin FitzgeraldDietmar SeiffertAndrew M. SternRichard E. OlsonJohn E. MacorRobert ZaczekJohn G. HoustonCharles F. Albright
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Abstract:
Alzheimer's disease (AD) is the most prevalent form of dementia in the elderly and currently only symptomatic treatments are available. The underlying biochemistry of the disease appears to involve accumulation of the toxic amyloid-β peptide (Aβ) in the brain. Aβ is formed through proteolytic cleavage of the amyloid precursor protein (APP) by the proteases BACE and γ-secretase making development of inhibitors to both enzymes attractive therapeutic approaches for Alzheimer's disease. In addition to APP, γ-secretase cleaves other proteins thereby complicating the development of γ-secretase inhibitors. Of particular concern are the Notch-family of proteins which require γ-secretase cleavage to control cell fate decisions in several tissues, including the gastrointestinal tract and spleen. Here we describe γ-secretase inhibitors possessing intrinsically different inhibitory potencies for Aβ production and Notch signaling, observing a 500-fold range in the Notch/Aβ IC50 ratios among the set of inhibitors studied. To explore the biological relevance of these findings, a cellular model for differentiation of intestinal goblet cells was developed using the HT29 colon cancer cell line. Using this assay, we found that induction of goblet cell-like differentiation could be induced by γ-secretase inhibitors at potencies similar to those measured for inhibition of Notch signaling and that Notch signaling was required for this induction. Taken together, these findings demonstrate that inhibition of Aβ formation and Notch signaling can be partially separated in cultured cells and raise the possibility that Aβ can be inhibited without Notch toxicity in vivo leading to an increased therapeutic index with select γ-secretase inhibitors.Keywords:
Gamma secretase
Notch proteins
In neuronal cells, presenilin-dependent gamma-secretase activity cleaves amyloid precursor proteins to release Abeta peptides, and also catalyzes the release of the intracellular domain of the transmembrane receptor Notch. Accumulation of aberrant Abeta peptides appears to be causally related to Alzheimer's disease. Inhibition of Abeta peptide production is therefore a potential target for therapeutic intervention. Notch proteins play an important role in cell fate determination in many different organisms and at different stages of development, for example in mammalian T cell development. We therefore addressed whether structurally diverse gamma-secretase inhibitors impair Notch function by studying thymocyte development in murine fetal thymic organ cultures. Here we show that high concentrations of the most potent inhibitors blocked thymocyte development at the most immature stage. In contrast, lower concentrations or less potent inhibitors impaired differentiation at a later stage, most notably suppressing the development of CD8 single-positive T cells. These phenotypes are consistent with an impairment of Notch signaling by gamma-secretase inhibitors and define a strict Notch dose dependence of consecutive stages during thymocyte development.
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The generation of gamma-secretase inhibitors which block the release of beta-amyloid peptide (Abeta) has long been an attractive therapeutic avenue for treatment or prevention of Alzheimer's disease (AD). Such inhibitors would reduce levels of Abeta available for aggregation into toxic assemblies that lead to the plaque pathology found in affected brain tissue. Cumulative evidence suggests that the S3 cleavage of Notch is also dependent on presenilins (PS) and is carried out by the multimeric PS-containing gamma-secretase complex. It is therefore possible that Notch function could be affected by gamma-secretase inhibitors. To assess the relationship between the cleavage of these substrates in the same system, Western blot cleavage assays have been established using a human cell line stably expressing both the beta-amyloid precursor protein (beta-APP) and the truncated Notch1 receptor fragment NotchDeltaE. Thus, a direct correlation may be made, following inhibitor treatment, of the decrease in the levels of the cleavage products, Abeta peptide and the Notch intracellular domain (NICD), as well as the increase in stabilized levels of both substrates. This analysis has been performed with a range of selected gamma-secretase inhibitors from six distinct structural classes. Changes in all four species usually occur in concert and with remarkably good agreement. A significant cleavage window is not clearly apparent in any case. Thus, these Notch and beta-APP cleavages cannot be dissected apart easily since they show the same pharmacological profile of inhibition. Whether this translates into proportionally reduced Notch signaling in vivo, however, remains to be seen.
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Alzheimer's disease (AD) is the most prevalent form of dementia in the elderly and currently only symptomatic treatments are available. The underlying biochemistry of the disease appears to involve accumulation of the toxic amyloid-β peptide (Aβ) in the brain. Aβ is formed through proteolytic cleavage of the amyloid precursor protein (APP) by the proteases BACE and γ-secretase making development of inhibitors to both enzymes attractive therapeutic approaches for Alzheimer's disease. In addition to APP, γ-secretase cleaves other proteins thereby complicating the development of γ-secretase inhibitors. Of particular concern are the Notch-family of proteins which require γ-secretase cleavage to control cell fate decisions in several tissues, including the gastrointestinal tract and spleen. Here we describe γ-secretase inhibitors possessing intrinsically different inhibitory potencies for Aβ production and Notch signaling, observing a 500-fold range in the Notch/Aβ IC50 ratios among the set of inhibitors studied. To explore the biological relevance of these findings, a cellular model for differentiation of intestinal goblet cells was developed using the HT29 colon cancer cell line. Using this assay, we found that induction of goblet cell-like differentiation could be induced by γ-secretase inhibitors at potencies similar to those measured for inhibition of Notch signaling and that Notch signaling was required for this induction. Taken together, these findings demonstrate that inhibition of Aβ formation and Notch signaling can be partially separated in cultured cells and raise the possibility that Aβ can be inhibited without Notch toxicity in vivo leading to an increased therapeutic index with select γ-secretase inhibitors.
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-Secretase is a transmembrane aspartyl protease involved in processing of a multitude of functionally diverse substrates within their transmembrane domains. -Secretase cleavage of the amyloid precursor protein (APP) generates the APP intracellular domain (AICD) and the amyloid -peptide (A). A is a key player in the pathogenesis of Alzheimer disease (AD) and the main constituent of senile plaques, one of the hallmarks found in the brain of AD patients. Various lengths of A peptides are produced but the most common forms are 40 or 42 residues long (A40 or A42). Secretase cleavage of Notch results in the release of the Notch intracellular domain (NICD) which is known to activate transcription. Notch signaling is important for cell differentiation during embryonic development as well as in adulthood. In analogy with APP and Notch, -secretase cleavage of other substrates also generates secreted peptides and intracellular domains (ICDs). -Secretase is a protein complex consisting of presenilin (PS), nicastrin, anterior pharynx defective-1 and presenilin enhancer-2, where PS is suggested to harbor the active site. The work of this thesis has focused on studies of -secretase activity and its products in AD brain and model systems such as cell lines and rat brain. In paper I, we examined the effect of subcellular localization, pH and detergents, on -secretase activity, i.e. AICD production, in rat brain membranes. A fraction containing Golgi, endoplasmic reticulum, endosomes and synaptic vesicles demonstrated the highest AICD production and the optimal pH was found to be around 7.0. In addition, the -secretase activity was highly affected by detergents and CHAPSO at a concentration of 0.4% was found to enhance the activity. In paper II, the -secretase dependent production of A40 as well as of ICDs from APP, Notch1, N-cadherin, ephrinB and p75-neurotrophin receptor was found to be down-regulated in adult compared to embryonic rat brain membranes. Processing of all of the substrates was observed in embryonic rat brain membranes while only APP and Notch1 processing was detected in the adult rat brain membranes. In paper III, the large hydrophilic loop of PS1 was examined with respect to its effect on APP and Notch processing. Deletion of the PS1 loop resulted in a striking decrease in production of A38, A39, A40, whereas production of A42 was affected to a lesser extent, and the production of AICD and NICD was not impaired. In particular, the most C-terminal amino acids of the loop were important for the differential effect on APP processing. In paper IV, C-terminal A variants deposited in sporadic and familial AD brains were identified and quantified. Both A deposited in plaque cores and in total amyloid preparations was examined. Apart from A40 and A42, a longer A species, A43, was detected. A43 was detected more frequently than A40, especially in plaque cores. Taken together, we have optimized conditions for studies of -secretase processing of APP in rat brain, demonstrated an age-dependent production of ICDs from several substrates in rat brain and showed that the most C-terminal part of the PS1 loop influences the A profile. Finally, a longer A variant, A43 was frequently detected in amyloid depositions in AD brains. LIST OF PUBLICATIONS This thesis is based on the following publications, which will be referred to by their roman numerals. I. Jenny Franberg, Hedvig Welander, Mikio Aoki, Bengt Winblad, Lars O. Tjernberg and Susanne Frykman Rat brain -secretase activity is highly influenced by detergents Biochemistry (2007) 46, 7647-7654 II. Jenny Franberg, Helena Karlstrom, Bengt Winblad, Lars O. Tjernberg and Susanne Frykman -Secretase dependent production of intracellular domains is reduced in adult compared to embryonic rat brain membranes Manuscript III. Johanna Wanngren, Jenny Franberg, Annelie I. Svensson, Hanna Laudon, Bengt Winblad, Frank Liu, Jan Naslund, Johan Lundkvist and Helena Karlstrom The large hydrophilic loop of presenilin 1 is important for regulating secretase complex assembly and for dictating the A profile without affecting Notch processing Journal of Biological Chemistry, Epub 2010 jan 27, doi:10.1074 IV. Hedvig Welander, Jenny Franberg, Caroline Graff, Erik Sundstrom, Bengt Winblad and Lars O. Tjernberg A43 is more frequent than A40 in amyloid plaque cores from Alzheimer disease brains Journal of Neurochemistry (2009) 110, 697-706
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The β-amyloid precursor protein (APP) and the Notch receptor undergo intramembranous proteolysis by the Presenilin-dependent γ-secretase. The cleavage of APP by γ-secretase releases amyloid-β peptides, which have been implicated in the pathogenesis of Alzheimer's disease, and the APP intracellular domain (AID), for which the function is not yet well understood. A similar γ-secretase-mediated cleavage of the Notch receptor liberates the Notch intracellular domain (NICD). NICD translocates to the nucleus and activates the transcription of genes that regulate the generation, differentiation, and survival of neuronal cells. Hence, some of the effects of APP signaling and Alzheimer's disease pathology may be mediated by the interaction of APP and Notch. Here, we show that membrane-tethered APP binds to the cytosolic Notch inhibitors Numb and Numb-like in mouse brain lysates. AID also binds Numb and Numb-like, and represses Notch activity when released by APP. Thus, γ-secretase may have opposing effects on Notch signaling; positive by cleaving Notch and generating NICD, and negative by processing APP and generating AID, which inhibits the function of NICD.
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