RESUMEN
While amyloid-ß (Aß) plaques are considered a hallmark of Alzheimer's disease, clinical trials focused on targeting gamma secretase, an enzyme involved in aberrant Aß peptide production, have not led to amelioration of AD symptoms or synaptic dysregulation. Screening strategies based on mechanistic, multi-omics approaches that go beyond pathological readouts can aid in the evaluation of therapeutics. Using early-onset Alzheimer's (EOFAD) disease patient lineage PSEN1A246E iPSC-derived neurons, we performed RNA-seq to characterize AD-associated endotypes, which are in turn used as a screening evaluation metric for two gamma secretase drugs, the inhibitor Semagacestat and the modulator BPN-15606. We demonstrate that drug treatment partially restores the neuronal state while concomitantly inhibiting cell cycle re-entry and dedifferentiation endotypes to different degrees depending on the mechanism of gamma secretase engagement. Our endotype-centric screening approach offers a new paradigm by which candidate AD therapeutics can be evaluated for their overall ability to reverse disease endotypes.
Asunto(s)
Enfermedad de Alzheimer , Células Madre Pluripotentes Inducidas , Humanos , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Placa Amiloide/patología , Células Madre Pluripotentes Inducidas/metabolismoRESUMEN
Alzheimer's disease (AD) is pathologically characterized by the deposition of the ß-amyloid (Aß) peptide in senile plaques in the brain, leading to neuronal dysfunction and eventual decline in cognitive function. Genome-wide association studies have identified the bridging integrator 1 (BIN1) gene within the second most significant susceptibility locus for late-onset AD. BIN1 is a member of the amphiphysin family of proteins and has reported roles in the generation of membrane curvature and endocytosis. Endocytic dysfunction is a pathological feature of AD, and endocytosis of the amyloid precursor protein is an important step in its subsequent cleavage by ß-secretase (BACE1). In vitro evidence implicates BIN1 in endosomal sorting of BACE1 and Aß generation in neurons, but a role for BIN1 in this process in vivo is yet to be described. Here, using biochemical and immunohistochemistry analyses we report that a 50% global reduction of BIN1 protein levels resulting from a single Bin1 allele deletion in mice does not change BACE1 levels or localization in vivo, nor does this reduction alter the production of endogenous murine Aß in nontransgenic mice. Furthermore, we found that reduction of BIN1 levels in the 5XFAD mouse model of amyloidosis does not alter Aß deposition nor behavioral deficits associated with cerebral amyloid burden. Finally, a conditional BIN1 knockout in excitatory neurons did not alter BACE1, APP, C-terminal fragments derived from BACE1 cleavage of APP, or endogenous Aß levels. These results indicate that BIN1 function does not regulate Aß generation in vivo.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Predisposición Genética a la Enfermedad , Proteínas del Tejido Nervioso/genética , Proteínas Supresoras de Tumor/genética , Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Endocitosis , Endosomas/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones NoqueadosRESUMEN
γ-Secretase is a multisubunit complex that catalyzes intramembranous cleavage of transmembrane proteins. The lipid environment forms membrane microdomains that serve as spatio-temporal platforms for proteins to function properly. Despite substantial advances in the regulation of γ-secretase, the effect of the local membrane lipid microenvironment on the regulation of γ-secretase is poorly understood. Here, we characterized and quantified the partitioning of γ-secretase and its substrates, the amyloid precursor protein (APP) and Notch, into lipid bilayers using solid-supported model membranes. Notch substrate is preferentially localized in the liquid-disordered (Ld) lipid domains, whereas APP and γ-secretase partition as single or higher complex in both phases but highly favor the ordered phase, especially after recruiting lipids from the ordered phase, indicating that the activity and specificity of γ-secretase against these two substrates are modulated by membrane lateral organization. Moreover, time-elapse measurements reveal that γ-secretase can recruit specific membrane components from the cholesterol-rich Lo phase and thus creates a favorable lipid environment for substrate recognition and therefore activity. This work offers insight into how γ-secretase and lipid modulate each other and control its activity and specificity.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide , Membrana Dobles de Lípidos , Precursor de Proteína beta-Amiloide , Lípidos de la Membrana , Microdominios de MembranaRESUMEN
The evolution of gamma-secretase modulators (GSMs) through the introduction of novel heterocycles with the goal of aligning activity for reducing the levels of Aß42 and properties consistent with a drug-like molecule are described. The insertion of a methoxypyridine motif within the tetracyclic scaffold provided compounds with improved activity for arresting Aß42 production as well as improved properties, including solubility. In vivo pharmacokinetic analysis demonstrated that several compounds within the novel series were capable of crossing the BBB and accessing the therapeutic target. Treatment with methoxypyridine-derived compound 64 reduced Aß42 levels in the plasma of J20 mice, in addition to reducing Aß42 levels in the plasma and brain of Tg2576 mice.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Piridinas/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/análisis , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/biosíntesis , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Estructura Molecular , Piridinas/síntesis química , Piridinas/química , Relación Estructura-ActividadRESUMEN
Alzheimer's disease is the most common form of dementia, characterized by two pathological hallmarks: amyloid-ß plaques and neurofibrillary tangles. The amyloid hypothesis of Alzheimer's disease posits that the excessive accumulation of amyloid-ß peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau. However, to date, no single disease model has serially linked these two pathological events using human neuronal cells. Mouse models with familial Alzheimer's disease (FAD) mutations exhibit amyloid-ß-induced synaptic and memory deficits but they do not fully recapitulate other key pathological events of Alzheimer's disease, including distinct neurofibrillary tangle pathology. Human neurons derived from Alzheimer's disease patients have shown elevated levels of toxic amyloid-ß species and phosphorylated tau but did not demonstrate amyloid-ß plaques or neurofibrillary tangles. Here we report that FAD mutations in ß-amyloid precursor protein and presenilin 1 are able to induce robust extracellular deposition of amyloid-ß, including amyloid-ß plaques, in a human neural stem-cell-derived three-dimensional (3D) culture system. More importantly, the 3D-differentiated neuronal cells expressing FAD mutations exhibited high levels of detergent-resistant, silver-positive aggregates of phosphorylated tau in the soma and neurites, as well as filamentous tau, as detected by immunoelectron microscopy. Inhibition of amyloid-ß generation with ß- or γ-secretase inhibitors not only decreased amyloid-ß pathology, but also attenuated tauopathy. We also found that glycogen synthase kinase 3 (GSK3) regulated amyloid-ß-mediated tau phosphorylation. We have successfully recapitulated amyloid-ß and tau pathology in a single 3D human neural cell culture system. Our unique strategy for recapitulating Alzheimer's disease pathology in a 3D neural cell culture model should also serve to facilitate the development of more precise human neural cell models of other neurodegenerative disorders.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Técnicas de Cultivo de Célula/métodos , Modelos Biológicos , Células-Madre Neurales/metabolismo , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Diferenciación Celular , Evaluación Preclínica de Medicamentos/métodos , Espacio Extracelular/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Células-Madre Neurales/patología , Neuritas/metabolismo , Fosforilación , Presenilina-1/metabolismo , Agregación Patológica de Proteínas , Reproducibilidad de los Resultados , Proteínas tau/química , Proteínas tau/metabolismoRESUMEN
Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterized by pathological brain lesions and a decline in cognitive function. ß-Amyloid peptides (Aß), derived from proteolytic processing of amyloid precursor protein (APP), play a central role in AD pathogenesis. ß-Site APP cleaving enzyme 1 (BACE1), the transmembrane aspartyl protease which initiates Aß production, is axonally transported in neurons and accumulates in dystrophic neurites near cerebral amyloid deposits in AD. BACE1 is modified by S-palmitoylation at four juxtamembrane cysteine residues. S-palmitoylation is a dynamic posttranslational modification that is important for trafficking and function of several synaptic proteins. Here, we investigated the in vivo significance of BACE1 S-palmitoylation through the analysis of knock-in mice with cysteine-to-alanine substitution at the palmitoylated residues (4CA mice). BACE1 expression, as well as processing of APP and other neuronal substrates, was unaltered in 4CA mice despite the lack of BACE1 S-palmitoylation and reduced lipid raft association. Whereas steady-state Aß levels were similar, synaptic activity-induced endogenous Aß production was not observed in 4CA mice. Furthermore, we report a significant reduction of cerebral amyloid burden and BACE1 accumulation in dystrophic neurites in the absence of BACE1 S-palmitoylation in mouse models of AD amyloidosis. Studies in cultured neurons suggest that S-palmitoylation is required for dendritic spine localization and axonal targeting of BACE1. Finally, the lack of BACE1 S-palmitoylation mitigates cognitive deficits in 5XFAD mice. Using transgenic mouse models, these results demonstrate that intrinsic posttranslational S-palmitoylation of BACE1 has a significant impact on amyloid pathogenesis and the consequent cognitive decline.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Trastornos de la Memoria/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas Amiloidogénicas/metabolismo , Amiloidosis/metabolismo , Animales , Axones/metabolismo , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Femenino , Lipoilación/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/metabolismo , Procesamiento Proteico-Postraduccional/fisiologíaRESUMEN
Alzheimer's disease (AD) is characterized neuropathologically by an abundance of 1) neuritic plaques, which are primarily composed of a fibrillar 42-amino-acid amyloid-ß peptide (Aß), as well as 2) neurofibrillary tangles composed of aggregates of hyperphosporylated tau. Elevations in the concentrations of the Aß42 peptide in the brain, as a result of either increased production or decreased clearance, are postulated to initiate and drive the AD pathologic process. We initially introduced a novel class of bridged aromatics referred tγ-secretase modulatoro as γ-secretase modulators that inhibited the production of the Aß42 peptide and to a lesser degree the Aß40 peptide while concomitantly increasing the production of the carboxyl-truncated Aß38 and Aß37 peptides. These modulators potently lower Aß42 levels without inhibiting the γ-secretase-mediated proteolysis of Notch or causing accumulation of carboxyl-terminal fragments of APP. In this study, we report a large number of pharmacological studies and early assessment of toxicology characterizing a highly potent γ-secretase modulator (GSM), (S)-N-(1-(4-fluorophenyl)ethyl)-6-(6-methoxy-5-(4-methyl-1H-imidazol-1-yl)pyridin-2-yl)-4-methylpyridazin-3-amine (BPN-15606). BPN-15606 displayed the ability to significantly lower Aß42 levels in the central nervous system of rats and mice at doses as low as 5-10 mg/kg, significantly reduce Aß neuritic plaque load in an AD transgenic mouse model, and significantly reduce levels of insoluble Aß42 and pThr181 tau in a three-dimensional human neural cell culture model. Results from repeat-dose toxicity studies in rats and dose escalation/repeat-dose toxicity studies in nonhuman primates have designated this GSM for 28-day Investigational New Drug-enabling good laboratory practice studies and positioned it as a candidate for human clinical trials.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/toxicidad , Fragmentos de Péptidos/antagonistas & inhibidores , Fenetilaminas/farmacología , Fenetilaminas/toxicidad , Piridazinas/farmacología , Piridazinas/toxicidad , Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Células Cultivadas , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacocinética , Humanos , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Placa Amiloide/tratamiento farmacológico , Ratas , Ratas Sprague-Dawley , Proteínas tau/metabolismoRESUMEN
Soluble γ-secretase modulators (SGSMs) selectively decrease toxic amyloid ß (Aß) peptides (Aß42). However, their effect on the physiologic functions of γ-secretase has not been tested in human model systems. γ-Secretase regulates fate determination of neural progenitor cells. Thus, we studied the impact of SGSMs on the neuronal differentiation of ReNcell VM (ReN) human neural progenitor cells (hNPCs). Quantitative PCR analysis showed that treatment of neurosphere-like ReN cell aggregate cultures with γ-secretase inhibitors (GSIs), but not SGSMs, induced a 2- to 4-fold increase in the expression of the neuronal markers Tuj1 and doublecortin. GSI treatment also induced neuronal marker protein expression, as shown by Western blot analysis. In the same conditions, SGSM treatment selectively reduced endogenous Aß42 levels by â¼80%. Mechanistically, we found that Notch target gene expressions were selectively inhibited by a GSI, not by SGSM treatment. We can assert, for the first time, that SGSMs do not affect the neuronal differentiation of hNPCs while selectively decreasing endogenous Aß42 levels in the same conditions. Our results suggest that our hNPC differentiation system can serve as a useful model to test the impact of GSIs and SGSMs on both endogenous Aß levels and γ-secretase physiologic functions including endogenous Notch signaling.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Diferenciación Celular/fisiología , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Células Cultivadas , Proteínas de Dominio Doblecortina , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Neuropéptidos/metabolismo , Transducción de Señal/fisiología , Tubulina (Proteína)/metabolismoRESUMEN
The design and construction of a series of novel aminothiazole-derived γ-secretase modulators is described. The incorporation of heterocyclic replacements of the terminal phenyl D-ring of lead compound 1 was conducted in order to align potency with favorable drug-like properties. γ-Secretase modulator 28 displayed good activity for in vitro inhibition of Aß42, as well as substantial improvement in ADME and physicochemical properties, including aqueous solubility. Pharmacokinetic evaluation of compound 28 in mice revealed good brain penetration, as well as good clearance, half-life, and volume of distribution which collectively support the continued development of this class of compounds.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Diseño de Fármacos , Tiazoles/química , Secretasas de la Proteína Precursora del Amiloide/química , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Animales , Semivida , Humanos , Cinética , Masculino , Ratones , Microsomas Hepáticos/metabolismo , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/metabolismo , Ratas , Solubilidad , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/farmacocinéticaRESUMEN
The γ-secretase complex, composed of presenilin, nicastrin (NCT), anterior pharynx-defective 1 (APH-1), and presenilin enhancer 2 (PEN-2), is assembled in a highly regulated manner and catalyzes the intramembranous proteolysis of many type I membrane proteins, including Notch and amyloid precursor protein. The Notch family of receptors plays important roles in cell fate specification during development and in adult tissues, and aberrant hyperactive Notch signaling causes some forms of cancer. γ-Secretase-mediated processing of Notch at the cell surface results in the generation of the Notch intracellular domain, which associates with several transcriptional coactivators involved in nuclear signaling events. On the other hand, γ-secretase-mediated processing of amyloid precursor protein leads to the production of amyloid ß (Aß) peptides that play an important role in the pathogenesis of Alzheimer disease. We used a phage display approach to identify synthetic antibodies that specifically target NCT and expressed them in the single-chain variable fragment (scFv) format in mammalian cells. We show that expression of a NCT-specific scFv clone, G9, in HEK293 cells decreased the production of the Notch intracellular domain but not the production of amyloid ß peptides that occurs in endosomal and recycling compartments. Biochemical studies revealed that scFvG9 impairs the maturation of NCT by associating with immature forms of NCT and, consequently, prevents its association with the other components of the γ-secretase complex, leading to degradation of these molecules. The reduced cell surface levels of mature γ-secretase complexes, in turn, compromise the intramembranous processing of Notch.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Anticuerpos de Cadena Única/inmunología , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/inmunología , Especificidad de Anticuerpos , Células HEK293 , Humanos , Espacio Intracelular/metabolismo , Glicoproteínas de Membrana/química , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteolisis , Receptores Notch/metabolismo , Anticuerpos de Cadena Única/genéticaRESUMEN
Alzheimer's disease (AD) is characterized pathologically by an abundance of extracellular neuritic plaques composed primarily of the 42-amino acid amyloid ß peptide variant (Aß42). In the majority of familial AD (FAD) cases, e.g., those harboring mutations in presenilin 1 (PS1), there is a relative increase in the levels of Aß42 compared to the levels of Aß40. We previously reported the characterization of a series of aminothiazole-bridged aromates termed aryl aminothiazole γ-secretase modulators or AGSMs [Kounnas, M. Z., et al. (2010) Neuron 67, 769-780] and showed their potential for use in the treatment of FAD [Wagner, S. L., et al. (2012) Arch. Neurol. 69, 1255-1258]. Here we describe a series of GSMs with physicochemical properties improved compared to those of AGSMs. Specific heterocycle replacements of the phenyl rings in AGSMs provided potent molecules with improved aqueous solubilities. A number of these soluble γ-secretase modulators (SGSMs) potently lowered Aß42 levels without inhibiting proteolysis of Notch or causing accumulation of amyloid precursor protein carboxy-terminal fragments, even at concentrations approximately 1000-fold greater than their IC50 values for reducing Aß42 levels. The effects of one potent SGSM on Aß peptide production were verified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, showing enhanced production of a number of carboxy-truncated Aß species. This SGSM also inhibited Aß42 peptide production in a highly purified reconstituted γ-secretase in vitro assay system and retained the ability to modulate γ-secretase-mediated proteolysis in a stably transfected cell culture model overexpressing a human PS1 mutation validating the potential for use in FAD.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/biosíntesis , Fragmentos de Péptidos/biosíntesis , Derivados del Benceno/síntesis química , Derivados del Benceno/química , Derivados del Benceno/farmacología , Línea Celular Tumoral , Pruebas de Enzimas , Humanos , Imidazoles/síntesis química , Imidazoles/química , Imidazoles/farmacología , Mutación , Presenilina-1/genética , Presenilina-1/metabolismo , Proteolisis , Receptor Notch1/metabolismo , Solubilidad , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/química , Tiazoles/farmacologíaRESUMEN
Alzheimer disease (AD), the leading cause of dementia, is characterized by the accumulation of ß-amyloid peptides (Aß) in senile plaques in the brains of affected patients. Many cellular mechanisms are thought to play important roles in the development and progression of AD. Several lines of evidence point to the dysregulation of Ca(2+) homeostasis as underlying aspects of AD pathogenesis. Moreover, direct roles in the regulation of Ca(2+) homeostasis have been demonstrated for proteins encoded by familial AD-linked genes such as PSEN1, PSEN2, and APP, as well as Aß peptides. Whereas these studies support the hypothesis that disruption of Ca(2+) homeostasis contributes to AD, it is difficult to disentangle the effects of familial AD-linked genes on Aß production from their effects on Ca(2+) homeostasis. Here, we developed a system in which cellular Ca(2+) homeostasis could be directly manipulated to study the effects on amyloid precursor protein metabolism and Aß production. We overexpressed stromal interaction molecule 1 (STIM1) and Orai1, the components of the store-operated Ca(2+) entry pathway, to generate cells with constitutive and store depletion-induced Ca(2+) entry. We found striking effects of Ca(2+) entry induced by overexpression of the constitutively active STIM1(D76A) mutant on amyloid precursor protein metabolism. Specifically, constitutive activation of Ca(2+) entry by expression of STIM1(D76A) significantly reduced Aß secretion. Our results suggest that disruptions in Ca(2+) homeostasis may influence AD pathogenesis directly through the modulation of Aß production.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Canales de Calcio/metabolismo , Calcio/metabolismo , Regulación de la Expresión Génica , Señalización del Calcio , Ensayo de Inmunoadsorción Enzimática , Células HEK293 , Homeostasis , Humanos , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Proteínas de Neoplasias/metabolismo , Proteína ORAI1 , Molécula de Interacción Estromal 1RESUMEN
The recently discovered interaction between presenilin 1 (PS1), a catalytic subunit of γ-secretase responsible for the generation of amyloid-ß(Aß) peptides, and GLT-1, the major glutamate transporter in the brain (EAAT2 in the human) may provide a mechanistic link between two important pathological aspects of Alzheimer's disease (AD): abnormal Aßoccurrence and neuronal network hyperactivity. In the current study, we employed a FRET-based approach, fluorescence lifetime imaging microscopy (FLIM), to characterize the PS1/GLT-1 interaction in its native environment in the brain tissue of sporadic AD (sAD) patients. There was significantly less interaction between PS1 and GLT-1 in sAD brains, compared to tissue from patients with frontotemporal lobar degeneration (FTLD), or non-demented age-matched controls. Since PS1 has been shown to adopt pathogenic "closed" conformation in sAD but not in FTLD, we assessed the impact of changes in PS1 conformation on the interaction. Familial AD (fAD) PS1 mutations which induce a "closed" PS1 conformation similar to that in sAD brain and gamma-secretase modulators (GSMs) which induce a "relaxed" conformation, reduced and increased the interaction, respectively. This indicates that PS1 conformation seems to have a direct effect on the interaction with GLT-1. Furthermore, using biotinylation/streptavidin pull-down, western blotting, and cycloheximide chase assays, we determined that the presence of PS1 increased GLT-1 cell surface expression and GLT-1 homomultimer formation, but did not impact GLT-1 protein stability. Together, the current findings suggest that the newly described PS1/GLT-1 interaction endows PS1 with chaperone activity, modulating GLT-1 transport to the cell surface and stabilizing the dimeric-trimeric states of the protein. The diminished PS1/GLT-1 interaction suggests that these functions of the interaction may not work properly in AD.
RESUMEN
Several lines of evidence implicate lipid raft microdomains in Alzheimer disease-associated ß-amyloid peptide (Aß) production. Notably, targeting ß-secretase (ß-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1)) exclusively to lipid rafts by the addition of a glycosylphosphatidylinositol (GPI) anchor to its ectodomain has been reported to elevate Aß secretion. Paradoxically, Aß secretion is not reduced by the expression of non-raft resident S-palmitoylation-deficient BACE1 (BACE1-4C/A (C474A/C478A/C482A/C485A)). We addressed this apparent discrepancy in raft microdomain-associated BACE1 processing of APP in this study. As previously reported, we found that expression of BACE1-GPI elevated Aß secretion as compared with wild-type BACE1 (WTBACE1) or BACE1-4C/A. However, this increase occurred without any difference in the levels of APP ectodomain released following BACE1 cleavage (soluble APPß), arguing against an overall increase in BACE1 processing of APP per se. Further analysis revealed that WTBACE1 cleaves APP at ß- and ß'-sites, generating +1 and +11 ß-C-terminal fragments and secreting intact as well as N-terminally truncated Aß. In contrast, three different BACE1-GPI chimeras preferentially cleaved APP at the ß-site, mainly generating +1 ß-C-terminal fragment and secreting intact Aß. As a consequence, cells expressing BACE1-GPI secreted relatively higher levels of intact Aß without an increase in BACE1 processing of APP. Markedly reduced cleavage at ß'-site exhibited by BACE1-GPI was cell type-independent and insensitive to subcellular localization of APP or the pathogenic KM/NL mutant. We conclude that the apparent elevation in Aß secretion by BACE1-GPI is mainly attributed to preferential cleavage at the ß-site and failure to detect +11 Aß species secreted by cells expressing WTBACE1.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/genética , Péptidos beta-Amiloides/química , Animales , Ácido Aspártico Endopeptidasas/genética , Sitios de Unión , Membrana Celular/metabolismo , Regulación Enzimológica de la Expresión Génica , Células HEK293 , Humanos , Ratones , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutagénesis , Mutación , Fragmentos de Péptidos/metabolismo , Estructura Terciaria de Proteína , Solubilidad , Especificidad por SustratoRESUMEN
Alzheimer's disease (AD) manifested before age 65 is commonly referred to as early-onset AD (EOAD) (Reitz et al. Neurol Genet. 2020;6:e512). While the majority (> 90%) of EOAD cases are not caused by autosomal-dominant mutations in PSEN1, PSEN2, and APP, they do have a higher heritability (92-100%) than sporadic late-onset AD (LOAD, 70%) (Wingo et al. Arch Neurol. 2012;69:59-64, Fulton-Howard et al. Neurobiol Aging. 2021;99:101.e1-101.e9). Although the endpoint clinicopathological changes, i.e., Aß plaques, tau tangles, and cognitive decline, are common across EOAD and LOAD, the disease progression is highly heterogeneous (Neff et al. Sci Adv Am Assoc Adv Sci. 2021;7:eabb5398). This heterogeneity, leading to temporally distinct age at onset (AAO) and stages of cognitive decline, may be caused by myriad combinations of distinct disease-associated molecular mechanisms. We and others have used transcriptome profiling in AD patient-derived neuron models of autosomal-dominant EOAD and sporadic LOAD to identify disease endotypes (Caldwell et al. Sci Adv Am Assoc Adv Sci. 2020;6:eaba5933, Mertens et al. Cell Stem Cell. 2021;28:1533-1548.e6, Caldwell et al. Alzheimers Demen. 2022). Further, analyses of large postmortem brain cohorts demonstrate that only one-third of AD patients show hallmark disease endotypes like increased inflammation and decreased synaptic signaling (Neff et al. Sci Adv Am Assoc Adv Sci. 2021;7:eabb5398). Areas of the brain less affected by AD pathology at early disease stages-such as the primary visual cortex-exhibit similar transcriptomic dysregulation as those regions traditionally affected and, therefore, may offer a view into the molecular mechanisms of AD without the associated inflammatory changes and gliosis induced by pathology (Haroutunian et al. Neurobiol Aging. 2009;30:561-73). To this end, we analyzed AD patient samples from the primary visual cortex (19 EOAD, 20 LOAD) using transcriptomic signatures to identify patient clusters and disease endotypes. Interestingly, although the clusters showed distinct combinations and severity of endotypes, each patient cluster contained both EOAD and LOAD cases, suggesting that AAO may not directly correlate with the identity and severity of AD endotypes.
Asunto(s)
Enfermedad de Alzheimer , Edad de Inicio , Anciano , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Encéfalo/patología , Perfilación de la Expresión Génica , Humanos , Transcriptoma/genéticaRESUMEN
The γ-secretase protein complex executes the intramembrane proteolysis of amyloid precursor protein (APP), which releases Alzheimer disease ß-amyloid peptide. In addition to APP, γ-secretase also cleaves several other type I membrane protein substrates including Notch1 and N-cadherin. γ-Secretase is made of four integral transmembrane protein subunits: presenilin (PS), nicastrin, APH1, and PEN2. Multiple lines of evidence indicate that a heteromer of PS-derived N- and C-terminal fragments functions as the catalytic subunit of γ-secretase. Only limited information is available on the domains within each subunit involved in the recognition and recruitment of diverse substrates and the transfer of substrates to the catalytic site. Here, we performed mutagenesis of two domains of PS1, namely the first luminal loop domain (LL1) and the second transmembrane domain (TM2), and analyzed PS1 endoproteolysis as well as the catalytic activities of PS1 toward APP, Notch, and N-cadherin. Our results show that distinct residues within LL1 and TM2 domains as well as the length of the LL1 domain are critical for PS1 endoproteolysis, but not for PS1 complex formation with nicastrin, APH1, and PEN2. Furthermore, our experimental PS1 mutants formed γ-secretase complexes with distinct catalytic properties toward the three substrates examined in this study; however, the mutations did not affect PS1 interaction with the substrates. We conclude that the N-terminal LL1 and TM2 domains are critical for PS1 endoproteolysis and the coordination between the putative substrate-docking site and the catalytic core of the γ-secretase.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Presenilina-1/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Precursor de Proteína beta-Amiloide , Animales , Cadherinas/genética , Cadherinas/metabolismo , Dominio Catalítico/fisiología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , Mutagénesis , Mutación , Presenilina-1/genética , Estructura Terciaria de Proteína , Receptor Notch1/genética , Receptor Notch1/metabolismo , Especificidad por Sustrato/fisiologíaRESUMEN
ADAM10, a member of a disintegrin and metalloprotease family, is an alpha-secretase capable of anti-amyloidogenic proteolysis of the amyloid precursor protein. Here, we present evidence for genetic association of ADAM10 with Alzheimer's disease (AD) as well as two rare potentially disease-associated non-synonymous mutations, Q170H and R181G, in the ADAM10 prodomain. These mutations were found in 11 of 16 affected individuals (average onset age 69.5 years) from seven late-onset AD families. Each mutation was also found in one unaffected subject implying incomplete penetrance. Functionally, both mutations significantly attenuated alpha-secretase activity of ADAM10 (>70% decrease), and elevated Abeta levels (1.5-3.5-fold) in cell-based studies. In summary, we provide the first evidence of ADAM10 as a candidate AD susceptibility gene, and report two potentially pathogenic mutations with incomplete penetrance for late-onset familial AD.
Asunto(s)
Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Enfermedad de Alzheimer/enzimología , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Proteína ADAM10 , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/genética , Animales , Células CHO , Cricetinae , Cricetulus , Femenino , Humanos , Masculino , Persona de Mediana Edad , Linaje , Polimorfismo de Nucleótido SimpleRESUMEN
Alzheimer's disease (AD) is a genetically complex and heterogeneous disorder. To date four genes have been established to either cause early-onset autosomal-dominant AD (APP, PSEN1, and PSEN2(1-4)) or to increase susceptibility for late-onset AD (APOE5). However, the heritability of late-onset AD is as high as 80%, (6) and much of the phenotypic variance remains unexplained to date. We performed a genome-wide association (GWA) analysis using 484,522 single-nucleotide polymorphisms (SNPs) on a large (1,376 samples from 410 families) sample of AD families of self-reported European descent. We identified five SNPs showing either significant or marginally significant genome-wide association with a multivariate phenotype combining affection status and onset age. One of these signals (p = 5.7 x 10(-14)) was elicited by SNP rs4420638 and probably reflects APOE-epsilon4, which maps 11 kb proximal (r2 = 0.78). The other four signals were tested in three additional independent AD family samples composed of nearly 2700 individuals from almost 900 families. Two of these SNPs showed significant association in the replication samples (combined p values 0.007 and 0.00002). The SNP (rs11159647, on chromosome 14q31) with the strongest association signal also showed evidence of association with the same allele in GWA data generated in an independent sample of approximately 1,400 AD cases and controls (p = 0.04). Although the precise identity of the underlying locus(i) remains elusive, our study provides compelling evidence for the existence of at least one previously undescribed AD gene that, like APOE-epsilon4, primarily acts as a modifier of onset age.
Asunto(s)
Enfermedad de Alzheimer/genética , Apolipoproteínas E/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Edad de Inicio , Algoritmos , Alelos , Teorema de Bayes , Estudios de Casos y Controles , Cromosomas Humanos Par 14 , Marcadores Genéticos , Humanos , Modelos Lineales , Desequilibrio de Ligamiento , Linaje , Polimorfismo de Nucleótido Simple , Lectina 3 Similar a Ig de Unión al Ácido Siálico/genética , Población BlancaRESUMEN
A potent γ-secretase modulator (GSM) has been developed to circumvent problems associated with γ-secretase inhibitors (GSIs) and to potentially enable use in primary prevention of early-onset familial Alzheimer's disease (EOFAD). Unlike GSIs, GSMs do not inhibit γ-secretase activity but rather allosterically modulate γ-secretase, reducing the net production of Aß42 and to a lesser extent Aß40, while concomitantly augmenting production of Aß38 and Aß37. This GSM demonstrated robust time- and dose-dependent efficacy in acute, subchronic, and chronic studies across multiple species, including primary and secondary prevention studies in a transgenic mouse model. The GSM displayed a >40-fold safety margin in rats based on a comparison of the systemic exposure (AUC) at the no observed adverse effect level (NOAEL) to the 50% effective AUC or AUCeffective, the systemic exposure required for reducing levels of Aß42 in rat brain by 50%.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/prevención & control , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Fenetilaminas/administración & dosificación , Piridazinas/administración & dosificación , Transducción de Señal/efectos de los fármacos , Péptidos beta-Amiloides/metabolismo , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Femenino , Humanos , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuroblastoma/metabolismo , Neuroblastoma/patología , Fragmentos de Péptidos/metabolismo , Ratas , Ratas Sprague-Dawley , Resultado del TratamientoRESUMEN
Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.