RESUMEN
Imbalances in the amounts of amyloid-ß peptides (Aß) generated by the membrane proteases ß- and γ-secretase are considered as a trigger of Alzheimer's disease (AD). Cell-free studies of γ-secretase have shown that increasing membrane thickness modulates Aß generation but it has remained unclear if these effects are translatable to cells. Here we show that the very long-chain fatty acid erucic acid (EA) triggers acyl chain remodeling in AD cell models, resulting in substantial lipidome alterations which included increased esterification of EA in membrane lipids. Membrane remodeling enhanced γ-secretase processivity, resulting in the increased production of the potentially beneficial Aß37 and/or Aß38 species in multiple cell lines. Unexpectedly, we found that the membrane remodeling stimulated total Aß secretion by cells expressing WT γ-secretase but lowered it for cells expressing an aggressive familial AD mutant γ-secretase. We conclude that EA-mediated modulation of membrane composition is accompanied by complex lipid homeostatic changes that can impact amyloidogenic processing in different ways and elicit distinct γ-secretase responses, providing critical implications for lipid-based AD treatment strategies.
Asunto(s)
Enfermedad de Alzheimer , Secretasas de la Proteína Precursora del Amiloide , Humanos , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Lípidos de la Membrana/metabolismo , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Línea Celular , Precursor de Proteína beta-Amiloide/metabolismo , Presenilina-1/metabolismoRESUMEN
All over the world, societies are facing rapidly aging populations combined with a growing number of patients suffering from Alzheimer's disease (AD). One focus in pharmaceutical research to address this issue is on the reduction of the longer amyloid-ß (Aß) fragments in the brain by modulation of γ-secretase, a membrane-bound protease. R-Flurbiprofen (tarenflurbil) was studied in this regard but failed to show significant improvement in AD patients in a phase 3 clinical trial. This was mainly attributed to its low ability to cross the blood-brain barrier (BBB). Here, we present the synthesis and in vitro evaluation of a racemic meta-carborane analogue of flurbiprofen. By introducing the carborane moiety, the hydrophobicity could be shifted into a more favourable range for the penetration of the blood-brain barrier, evident by a logD7.4 value of 2.0. Furthermore, our analogue retained γ-secretase modulator activity in comparison to racemic flurbiprofen in a cell-based assay. These findings demonstrate the potential of carboranes as phenyl mimetics also in AD research.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Compuestos de Boro/farmacología , Flurbiprofeno/análogos & derivados , Compuestos de Boro/síntesis química , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Inhibidores de la Ciclooxigenasa/farmacología , Flurbiprofeno/química , Humanos , Concentración 50 InhibidoraRESUMEN
Neurotoxic amyloid-ß peptide (Aß) 42/43 species generated by ß-secretase and γ-secretase from the ß-amyloid precursor protein (APP) are believed to trigger Alzheimer's disease (AD). Relative increases of these species due to mutations in APP and presenilin/γ-secretase are associated with the vast majority of early onset familial AD cases. Important breakthroughs have recently been made in elucidating the mechanism(s) of these mutations, showing that altered substrate interactions and substrate-enzyme complex stabilities are underlying their pathogenic Aß generation. Moreover, first structures of γ-secretase in complex with APP and Notch1 substrates allow insight into how substrate cleavage could be initiated and further progress has been made in the mechanistic understanding of γ-secretase modulators, advanced Aß-lowering drugs. These insights could be exploited for future AD clinical trials.
Asunto(s)
Enfermedad de Alzheimer , Secretasas de la Proteína Precursora del Amiloide , Péptidos beta-Amiloides , Precursor de Proteína beta-Amiloide , Humanos , MutaciónRESUMEN
Abnormal generation of neurotoxic amyloid-ß peptide (Aß) 42/43 species due to mutations in the catalytic presenilin 1 (PS1) subunit of γ-secretase is the major cause of familial Alzheimer's disease (FAD). Deeper mechanistic insight on the generation of Aß43 is still lacking, and it is unclear whether γ-secretase modulators (GSMs) can reduce the levels of this Aß species. By comparing several types of Aß43-generating FAD mutants, we observe that very high levels of Aß43 are often produced when presenilin function is severely impaired. Altered interactions of C99, the precursor of Aß, are found for all mutants and are independent of their particular effect on Aß production. Furthermore, unlike previously described GSMs, the novel compound RO7019009 can effectively lower Aß43 production of all mutants. Finally, substrate-binding competition experiments suggest that RO7019009 acts mechanistically after initial C99 binding. We conclude that altered C99 interactions are a common feature of diverse types of PS1 FAD mutants and that also patients with Aß43-generating FAD mutations could in principle be treated by GSMs.
Asunto(s)
Enfermedad de Alzheimer , Secretasas de la Proteína Precursora del Amiloide , Humanos , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide , Secretasas de la Proteína Precursora del Amiloide/genética , Mutación , Presenilina-1/genéticaRESUMEN
We report the novel presenilin 1 (PSEN1) single amino acid deletion mutation F175del. Comprehensive clinical work-up, including cerebral MRI, FDG-PET, and CSF analysis, was performed in a male who had developed forgetfulness at the age of 39. Alzheimer's disease dementia was diagnosed according to established criteria. The index patient manifested rapid progressive dementia, seizures, and myoclonus, and a Pisa syndrome as a side effect of donepezil treatment. The PSEN1 mutation F175del was found on genetic testing. It was rendered very likely pathogenic as amyloid-ß (Aß) peptide 42 was elevated in a cell culture model compared to presenilin 1 wild-type controls. An additional, unusual increase in Aß39 indicates a rarely observed product line deviation in the generation of the shorter Aß species. Our observations extend the range of PSEN1 mutations to be considered in familial dementia. We demonstrate that deletion of a single conserved amino acid, which is very rare compared to missense mutations as the common cause for PSEN1-associated Alzheimer's disease, can lead to an unusual profile of Aß species.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Mutación , Presenilina-1/genética , Enfermedad de Alzheimer/diagnóstico por imagen , HumanosRESUMEN
Epithelial cell-adhesion molecule (EpCAM) is a transmembrane protein that regulates cell cycle progression and differentiation and is overexpressed in many carcinomas. The EpCAM-induced mitogenic cascade is activated via regulated intramembrane proteolysis (RIP) of EpCAM by ADAM and γ-secretases, generating the signaling-active intracellular domain EpICD. Because of its expression pattern and molecular function, EpCAM is a valuable target in prognostic and therapeutic approaches for various carcinomas. So far, several immunotherapeutic strategies have targeted the extracellular domain of EpCAM. However, targeting the intracellular signaling cascade of EpCAM holds promise for specifically interfering with EpCAM's proliferation-stimulating signaling cascade. Here, using a yellow fluorescence protein-tagged version of the C-terminal fragment of EpCAM, we established a high-content screening (HCS) of a small-molecule compound library (n = 27,280) and characterized validated hits that target EpCAM signaling. In total, 128 potential inhibitors were initially identified, of which one compound with robust inhibitory effects on RIP of EpCAM was analyzed in greater detail. In summary, our study demonstrates that the development of an HCS for small-molecule inhibitors of the EpCAM signaling pathway is feasible. We propose that this approach may also be useful for identifying chemical compounds targeting other disorders involving membrane cleavage-dependent signaling pathways.
Asunto(s)
Molécula de Adhesión Celular Epitelial/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Proliferación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Molécula de Adhesión Celular Epitelial/metabolismo , Células HEK293 , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Bibliotecas de Moléculas Pequeñas/química , Transcripción Genética/efectos de los fármacosRESUMEN
As stated by the prevailing amyloid cascade hypothesis, Alzheimer's disease (AD) is caused by the aggregation and cerebral deposition of long amyloid-ß peptide (Aß) species, which are released from a C-terminal amyloid precursor protein fragment by γ-secretase. Mutations in its catalytic subunit presenilin-1 (PS1) increase the Aß42 to Aß40 ratio and are the major cause of familial AD (FAD). An opposing hypothesis states that loss of essential presenilin functions underlies the disease. A major argument for this hypothesis is the observation that the nearly inactive PS1 L435F mutant, paradoxically, causes FAD We now show that the very little Aß generated by PS1 L435F consists primarily of Aß43, a highly amyloidogenic species which was overlooked in previous studies of this mutant. We further demonstrate that the generation of Aß43 is not due to a trans-dominant effect of this mutant on WT presenilin. Furthermore, we found Aß43-containing plaques in brains of patients with this mutation. The aberrant generation of Aß43 by this particular mutant provides a direct objection against the presenilin hypothesis.
Asunto(s)
Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Proteínas Mutantes/metabolismo , Fragmentos de Péptidos/metabolismo , Presenilina-1/metabolismo , Lóbulo Frontal/patología , Humanos , Inmunohistoquímica , Microscopía , Proteínas Mutantes/genética , Presenilina-1/genéticaRESUMEN
Intramembrane proteolysis has emerged as a key mechanism required for membrane proteostasis and cellular signaling. One of the intramembrane-cleaving proteases (I-CLiPs), γ-secretase, is also intimately implicated in Alzheimer's disease, a major neurodegenerative disease and leading cause of dementia. High-resolution crystal structural analyses have revealed that I-CLiPs harbor their active sites buried deeply in the membrane bilayer. Surprisingly, however, the key kinetic constants of these proteases, turnover number kcat and catalytic efficiency kcat/KM, are largely unknown. By investigating the kinetics of intramembrane cleavage of the Alzheimer's disease-associated ß-amyloid precursor protein in vitro and in human embryonic kidney cells, we show that γ-secretase is a very slow protease with a kcat value similar to those determined recently for rhomboid-type I-CLiPs. Our results indicate that low turnover numbers may be a general feature of I-CLiPs.