RESUMO
Efforts to develop drugs for Alzheimer's disease (AD) have shown promise in animal studies, only to fail in human trials, suggesting a pressing need to study AD in human model systems. Using human neurons derived from induced pluripotent stem cells that expressed apolipoprotein E4 (ApoE4), a variant of the APOE gene product and the major genetic risk factor for AD, we demonstrated that ApoE4-expressing neurons had higher levels of tau phosphorylation, unrelated to their increased production of amyloid-ß (Aß) peptides, and that they displayed GABAergic neuron degeneration. ApoE4 increased Aß production in human, but not in mouse, neurons. Converting ApoE4 to ApoE3 by gene editing rescued these phenotypes, indicating the specific effects of ApoE4. Neurons that lacked APOE behaved similarly to those expressing ApoE3, and the introduction of ApoE4 expression recapitulated the pathological phenotypes, suggesting a gain of toxic effects from ApoE4. Treatment of ApoE4-expressing neurons with a small-molecule structure corrector ameliorated the detrimental effects, thus showing that correcting the pathogenic conformation of ApoE4 is a viable therapeutic approach for ApoE4-related AD.
Assuntos
Apolipoproteína E4/toxicidade , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Apolipoproteína E3/metabolismo , Linhagem Celular , Células Cultivadas , Neurônios GABAérgicos/efeitos dos fármacos , Neurônios GABAérgicos/metabolismo , Edição de Genes , Homozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Degeneração Neural/patologia , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Proteínas tau/metabolismoRESUMO
Apolipoprotein E4 (apoE4), the major genetic risk factor for late onset Alzheimer disease, assumes a pathological conformation, intramolecular domain interaction. ApoE4 domain interaction mediates the detrimental effects of apoE4, including decreased mitochondrial cytochrome c oxidase subunit 1 levels, reduced mitochondrial motility, and reduced neurite outgrowth in vitro. Mutant apoE4 (apoE4-R61T) lacks domain interaction, behaves like apoE3, and does not cause detrimental effects. To identify small molecules that inhibit domain interaction (i.e. structure correctors) and reverse the apoE4 detrimental effects, we established a high throughput cell-based FRET primary assay that determines apoE4 domain interaction and secondary cell- and function-based assays. Screening a ChemBridge library with the FRET assay identified CB9032258 (a phthalazinone derivative), which inhibits domain interaction in neuronal cells. In secondary functional assays, CB9032258 restored mitochondrial cytochrome c oxidase subunit 1 levels and rescued impairments of mitochondrial motility and neurite outgrowth in apoE4-expressing neuronal cells. These benefits were apoE4-specific and dose-dependent. Modifying CB9032258 yielded well defined structure-activity relationships and more active compounds with enhanced potencies in the FRET assay (IC(50) of 23 and 116 nm, respectively). These compounds efficiently restored functional activities of apoE4-expressing cells in secondary assays. An EPR binding assay showed that the apoE4 structure correction resulted from direct interaction of a phthalazinone. With these data, a six-feature pharmacophore model was constructed for future drug design. Our results serve as a proof of concept that pharmacological intervention with apoE4 structure correctors negates apoE4 detrimental effects in neuronal cells and could be further developed as an Alzheimer disease therapeutic.
Assuntos
Apolipoproteína E4/antagonistas & inibidores , Apolipoproteína E4/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Apolipoproteína E4/química , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , Modelos Moleculares , Neurônios/metabolismo , Ftalazinas/química , Ftalazinas/farmacologia , Estrutura Terciária de Proteína , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer disease (AD) and likely contributes to neuropathology through various pathways. Here we report that the intracellular trafficking of apoE4 is impaired in Neuro-2a cells and primary neurons, as shown by measuring fluorescence recovery after photobleaching. In Neuro-2a cells, more apoE4 than apoE3 molecules remained immobilized in the endoplasmic reticulum (ER) and the Golgi apparatus, and the lateral motility of apoE4 was significantly lower in the Golgi apparatus (but not in the ER) than that of apoE3. Likewise, the immobile fraction was larger, and the lateral motility was lower for apoE4 than apoE3 in mouse primary hippocampal neurons. ApoE4 with the R61T mutation, which abolishes apoE4 domain interaction, was less immobilized, and its lateral motility was comparable with that of apoE3. The trafficking impairment of apoE4 was also rescued by disrupting domain interaction with the small-molecule structure correctors GIND25 and PH002. PH002 also rescued apoE4-induced impairments of neurite outgrowth in Neuro-2a cells and dendritic spine development in primary neurons. ApoE4 did not affect trafficking of amyloid precursor protein, another AD-related protein, through the secretory pathway. Thus, domain interaction renders more newly synthesized apoE4 molecules immobile and slows their trafficking along the secretory pathway. Correcting the pathological structure of apoE4 by disrupting domain interaction is a potential therapeutic approach to treat or prevent AD related to apoE4.
Assuntos
Apolipoproteína E4/metabolismo , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Complexo de Golgi/metabolismo , Hipocampo/citologia , Humanos , Camundongos , Modelos Biológicos , Mutação , Neurônios/metabolismoRESUMO
The anaphase-promoting complex (APC) regulates cell division in eukaryotes by targeting specific proteins for destruction. APC substrates generally contain one or more short degron sequences that help mediate their recognition and poly-ubiquitination by the APC. The most common and well characterized degrons are the destruction box (D box) and the KEN box. The budding yeast Acm1 protein, an inhibitor of Cdh1-activated APC (APC(Cdh1)) also contains several conserved D and KEN boxes, and here we report that two of these located in the central region of Acm1 constitute a pseudosubstrate sequence required for APC(Cdh1) inhibition. Acm1 interacted with and inhibited substrate binding to the WD40 repeat domain of Cdh1. Combined mutation of the central D and KEN boxes strongly reduced both binding to the Cdh1 WD40 domain and APC(Cdh1) inhibition. Despite this, the double mutant, but not wild-type Acm1, was poly-ubiquitinated by APC(Cdh1) in vitro. Thus, unlike substrates in which D and KEN boxes promote ubiquitination, these same elements in the central region of Acm1 prevent ubiquitination. We propose that this unique property of the Acm1 degron sequences results from an unusually high affinity interaction with the substrate receptor site on the WD40 domain of Cdh1 that may serve both to promote APC inhibition and protect Acm1 from destruction.
Assuntos
Ciclo Celular/fisiologia , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitinação/fisiologia , Ciclossomo-Complexo Promotor de Anáfase , Proteínas Cdh1 , Proteínas de Ciclo Celular , Estrutura Terciária de Proteína/fisiologia , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Complexos Ubiquitina-Proteína Ligase/antagonistas & inibidores , Complexos Ubiquitina-Proteína Ligase/genéticaRESUMO
The anaphase-promoting complex (APC) regulates the eukaryotic cell cycle by targeting specific proteins for proteasomal degradation. Its activity must be strictly controlled to ensure proper cell cycle progression. The co-activator proteins Cdc20 and Cdh1 are required for APC activity and are important regulatory targets. Recently, budding yeast Acm1 was identified as a Cdh1 binding partner and APC(Cdh1) inhibitor. Acm1 disappears in late mitosis when APC(Cdh1) becomes active and contains conserved degron-like sequences common to APC substrates, suggesting it could be both an inhibitor and substrate. Surprisingly, we found that Acm1 proteolysis is independent of APC. A major determinant of Acm1 stability is phosphorylation at consensus cyclin-dependent kinase sites. Acm1 is a substrate of Cdc28 cyclin-dependent kinase and Cdc14 phosphatase both in vivo and in vitro. Mutation of Cdc28 phosphorylation sites or conditional inactivation of Cdc28 destabilizes Acm1. In contrast, inactivation of Cdc14 prevents Acm1 dephosphorylation and proteolysis. Cdc28 stabilizes Acm1 in part by promoting binding of the 14-3-3 proteins Bmh1 and Bmh2. We conclude that the opposing actions of Cdc28 and Cdc14 are primary factors limiting Acm1 to the interval from G(1)/S to late mitosis and are capable of establishing APC-independent expression patterns similar to APC substrates.
Assuntos
Proteína Quinase CDC28 de Saccharomyces cerevisiae/fisiologia , Proteínas de Ciclo Celular/fisiologia , Regulação Fúngica da Expressão Gênica , Proteínas Tirosina Fosfatases/fisiologia , Proteínas Repressoras/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Complexos Ubiquitina-Proteína Ligase , Proteínas 14-3-3/metabolismo , Sequência de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Mitose , Modelos Biológicos , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Cdh1 is a coactivator of the anaphase-promoting complex/cyclosome (APC/C) and contributes to mitotic exit and G1 maintenance by facilitating the polyubiquitination and subsequent proteolysis of specific substrates. Here, we report that budding yeast Cdh1 is a component of a cell cycle-regulated complex that includes the 14-3-3 homologs Bmh1 and Bmh2 and a previously uncharacterized protein, which we name Acm1 (APC/CCdh1 modulator 1). Association of Cdh1 with Bmh1 and Bmh2 requires Acm1, and the Acm1 protein is cell cycle regulated, appearing late in G1 and disappearing in late M. In acm1Delta strains, Cdh1 localization to the bud neck and association with two substrates, Clb2 and Hsl1, were strongly enhanced. Several lines of evidence suggest that Acm1 can suppress APC/CCdh1-mediated proteolysis of mitotic cyclins. First, overexpression of Acm1 fully restored viability to cells expressing toxic levels of Cdh1 or a constitutively active Cdh1 mutant lacking inhibitory phosphorylation sites. Second, overexpression of Acm1 was toxic in sic1Delta cells. Third, ACM1 deletion exacerbated a low-penetrance elongated-bud phenotype caused by modest overexpression of Cdh1. This bud elongation was independent of the morphogenesis checkpoint, and the combination of acm1Delta and hsl1Delta resulted in a dramatic enhancement of bud elongation and G2/M delay. Effects on bud elongation were attenuated when Cdh1 was replaced with a mutant lacking the C-terminal IR dipeptide, suggesting that APC/C-dependent proteolysis is required for this phenotype. We propose that Acm1 and Bmh1/Bmh2 constitute a specialized inhibitor of APC/CCdh1.