RESUMO
The (Z)-fluoro-olefin amide bioisosteric replacement is an effective tool for addressing various shortcomings of the parent amide. In an effort to fine tune ADME properties of BACE1 preclinical candidate AM-6494, a series of structurally distinct (Z)-fluoro-olefin containing analogs was developed that culminated in compound 15. Herein, we detail design considerations, synthetic challenges, structure activity relationship (SAR) studies, and in vivo properties of an advanced compound in this novel series of BACE1 inhibitors.
Assuntos
Alcenos/farmacologia , Amidas/farmacologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Desenvolvimento de Medicamentos , Inibidores Enzimáticos/farmacologia , Hidrocarbonetos Fluorados/farmacologia , Alcenos/síntese química , Alcenos/química , Amidas/síntese química , Amidas/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Células HEK293 , Humanos , Hidrocarbonetos Fluorados/síntese química , Hidrocarbonetos Fluorados/química , Conformação Molecular , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
ß-Site amyloid precursor protein cleaving enzyme 1 (BACE1) is an aspartyl protease that plays a key role in the production of amyloid ß (Aß) in the brain and has been extensively pursued as a target for the treatment of Alzheimer's disease (AD). BACE2, an aspartyl protease that is structurally related to BACE1, has been recently reported to be involved in melanosome maturation and pigmentation. Herein, we describe the development of a series of cyclopropylthiazines as potent and orally efficacious BACE1 inhibitors. Lead optimization led to the identification of 20, a molecule with biochemical IC50 BACE2/BACE1 ratio of 47. Administration of 20 resulted in no skin/fur color change in a 13-day mouse hypopigmentation study and demonstrated robust and sustained reduction of CSF and brain Aß40 levels in rat and monkey pharmacodynamic models. On the basis of a compelling data package, 20 (AM-6494) was advanced to preclinical development.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ciclopropanos/farmacologia , Inibidores Enzimáticos/farmacologia , Tiazinas/farmacologia , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Peptídeos beta-Amiloides/metabolismo , Animais , Ácido Aspártico Endopeptidases/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Ciclopropanos/química , Ciclopropanos/farmacocinética , Ciclopropanos/uso terapêutico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/uso terapêutico , Humanos , Masculino , Camundongos , Modelos Moleculares , Fragmentos de Peptídeos/líquido cefalorraquidiano , Fragmentos de Peptídeos/metabolismo , Ratos Sprague-Dawley , Tiazinas/química , Tiazinas/farmacocinética , Tiazinas/uso terapêuticoRESUMO
BACE1 inhibition to prevent Aß peptide formation is considered to be a potential route to a disease-modifying treatment for Alzheimer's disease. Previous efforts in our laboratory using a combined structure- and property-based approach have resulted in the identification of aminooxazoline xanthenes as potent BACE1 inhibitors. Herein, we report further optimization leading to the discovery of inhibitor 15 as an orally available and highly efficacious BACE1 inhibitor that robustly reduces CSF and brain Aß levels in both rats and nonhuman primates. In addition, compound 15 exhibited low activity on the hERG ion channel and was well tolerated in an integrated cardiovascular safety model.
RESUMO
The ß-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is one of the most hotly pursued targets for the treatment of Alzheimer's disease. We used a structure- and property-based drug design approach to identify 2-aminooxazoline 3-azaxanthenes as potent BACE1 inhibitors which significantly reduced CSF and brain Aß levels in a rat pharmacodynamic model. Compared to the initial lead 2, compound 28 exhibited reduced potential for QTc prolongation in a non-human primate cardiovascular safety model.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Xantenos/química , Xantenos/farmacologia , Doença de Alzheimer/tratamento farmacológico , Animais , Linhagem Celular , Células HEK293 , Humanos , Inibidores de Proteases/síntese química , Ratos , Xantenos/síntese químicaRESUMO
We have previously shown that the aminooxazoline xanthene scaffold can generate potent and orally efficacious BACE1 inhibitors although certain of these compounds exhibited potential hERG liabilities. In this article, we describe 4-aza substitution on the xanthene core as a means to increase BACE1 potency while reducing hERG binding affinity. Further optimization of the P3 and P2' side chains resulted in the identification of 42 (AMG-8718), a compound with a balanced profile of BACE1 potency, hERG binding affinity, and Pgp recognition. This compound produced robust and sustained reductions of CSF and brain Aß levels in a rat pharmacodynamic model and exhibited significantly reduced potential for QTc elongation in a cardiovascular safety model.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Benzopiranos/síntese química , Inibidores de Proteases/síntese química , Piridinas/síntese química , Compostos de Espiro/síntese química , Peptídeos beta-Amiloides/metabolismo , Animais , Benzopiranos/farmacologia , Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Células HEK293 , Humanos , Concentração Inibidora 50 , Microssomos Hepáticos/metabolismo , Piridinas/farmacologia , Ratos Sprague-Dawley , Compostos de Espiro/farmacologia , Relação Estrutura-AtividadeRESUMO
The optimization of a series of aminooxazoline xanthene inhibitors of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) is described. An early lead compound showed robust Aß lowering activity in a rat pharmacodynamic model, but advancement was precluded by a low therapeutic window to QTc prolongation in cardiovascular models consistent with in vitro activity on the hERG ion channel. While the introduction of polar groups was effective in reducing hERG binding affinity, this came at the expense of higher than desired Pgp-mediated efflux. A balance of low Pgp efflux and hERG activity was achieved by lowering the polar surface area of the P3 substituent while retaining polarity in the P2' side chain. The introduction of a fluorine in position 4 of the xanthene ring improved BACE1 potency (5-10-fold). The combination of these optimized fragments resulted in identification of compound 40, which showed robust Aß reduction in a rat pharmacodynamic model (78% Aß reduction in CSF at 10 mg/kg po) and also showed acceptable cardiovascular safety in vivo.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/metabolismo , Oxazolona/análogos & derivados , Inibidores de Proteases/síntese química , Xantenos/síntese química , Animais , Cristalografia por Raios X , Células HEK293 , Humanos , Concentração Inibidora 50 , Masculino , Microssomos Hepáticos/metabolismo , Oxazolona/síntese química , Oxazolona/farmacologia , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Xantenos/farmacologiaRESUMO
γ-Secretase modulators (GSMs) are potentially disease-modifying treatments for Alzheimer's disease. They selectively lower pathogenic Aß42 levels by shifting the enzyme cleavage sites without inhibiting γ-secretase activity, possibly avoiding known adverse effects observed with complete inhibition of the enzyme complex. A cell-based HTS effort identified the sulfonamide 1 as a GSM lead. Lead optimization studies identified compound 25 with improved cell potency, PKDM properties, and it lowered Aß42 levels in the cerebrospinal fluid (CSF) of Sprague-Dawley rats following oral administration. Further optimization of 25 to improve cellular potency is described.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Amidas/farmacologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Picolinas/farmacologia , Doença de Alzheimer/enzimologia , Amidas/química , Animais , Células HEK293 , Humanos , Picolinas/química , Ratos , Ratos Sprague-DawleyRESUMO
A structure- and property-based drug design approach was employed to identify aminooxazoline xanthenes as potent and selective human ß-secretase inhibitors. These compounds exhibited good isolated enzyme, cell potency, and selectivity against the structurally related aspartyl protease cathepsin D. Our efforts resulted in the identification of a potent, orally bioavailable CNS penetrant compound that exhibited in vivo efficacy. A single oral dose of compound 11a resulted in a significant reduction of CNS Aß40 in naive rats.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Encéfalo/metabolismo , Oxazóis/síntese química , Xantenos/síntese química , Administração Oral , Peptídeos beta-Amiloides/metabolismo , Animais , Linhagem Celular , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Modelos Moleculares , Estrutura Molecular , Oxazóis/farmacocinética , Oxazóis/farmacologia , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Xantenos/farmacocinética , Xantenos/farmacologiaRESUMO
Sequential proteolytic cleavage of the amyloid precursor protein (APP) by ß-site APP-cleaving enzyme 1 (BACE1) and the γ-secretase complex produces the amyloid-ß peptide (Aß), which is believed to play a critical role in the pathology of Alzheimer's disease (AD). The aspartyl protease BACE1 catalyzes the rate-limiting step in the production of Aß, and as such it is considered to be an important target for drug development in AD. The development of a BACE1 inhibitor therapeutic has proven to be difficult. The active site of BACE1 is relatively large. Consequently, to achieve sufficient potency, many BACE1 inhibitors have required unfavorable physicochemical properties such as high molecular weight and polar surface area that are detrimental to efficient passage across the blood-brain barrier. Using a rational drug design approach we have designed and developed a new series of hydroxyethylamine-based inhibitors of BACE1 capable of lowering Aß levels in the brains of rats after oral administration. Herein we describe the in vitro and in vivo characterization of two of these molecules and the overall relationship of compound properties [e.g., in vitro permeability, P-glycoprotein (P-gp) efflux, metabolic stability, and pharmacological potency] to the in vivo pharmacodynamic effect with more than 100 compounds across the chemical series. We demonstrate that high in vitro potency for BACE1 was not sufficient to provide central efficacy. A combination of potency, high permeability, low P-gp-mediated efflux, and low clearance was required for compounds to produce robust central Aß reduction after oral dosing.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Etilaminas/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Administração Oral , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Proteínas Sanguíneas/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacocinética , Etilaminas/farmacocinética , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Técnicas In Vitro , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes , Relação Estrutura-AtividadeRESUMO
A series of potent hydroxyethyl amine (HEA) derived inhibitors of ß-site APP cleaving enzyme (BACE1) was optimized to address suboptimal pharmacokinetics and poor CNS partitioning. This work identified a series of benzodioxolane analogues that possessed improved metabolic stability and increased oral bioavailability. Subsequent efforts focused on improving CNS exposure by limiting susceptibility to Pgp-mediated efflux and identified an inhibitor which demonstrated robust and sustained reduction of CNS ß-amyloid (Aß) in Sprague-Dawley rats following oral administration.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Encéfalo/efeitos dos fármacos , Dioxolanos/síntese química , Etilaminas/síntese química , Fragmentos de Peptídeos/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Administração Oral , Animais , Disponibilidade Biológica , Encéfalo/metabolismo , Cristalografia por Raios X , Dioxolanos/farmacocinética , Dioxolanos/farmacologia , Cães , Desenho de Fármacos , Etilaminas/farmacocinética , Etilaminas/farmacologia , Humanos , Macaca mulatta , Masculino , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Conformação Proteica , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
We have previously shown that hydroxyethylamines can be potent inhibitors of the BACE1 enzyme and that the generation of BACE1 inhibitors with CYP 3A4 inhibitory activities in this scaffold affords compounds (e.g., 1) with sufficient bioavailability and pharmacokinetic profiles to reduce central amyloid-ß peptide (Aß) levels in wild-type rats following oral dosing. In this article, we describe further modifications of the P1-phenyl ring of the hydroxyethylamine series to afford potent, dual BACE1/CYP 3A4 inhibitors which demonstrate improved penetration into the CNS. Several of these compounds caused robust reduction of Aß levels in rat CSF and brain following oral dosing, and compound 37 exhibited an improved cardiovascular safety profile relative to 1.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Fragmentos de Peptídeos/metabolismo , Compostos de Espiro/síntese química , Tiazóis/síntese química , Administração Oral , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Animais , Proteínas Sanguíneas/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular , Cristalografia por Raios X , Citocromo P-450 CYP3A , Inibidores das Enzimas do Citocromo P-450 , Cães , Desenho de Fármacos , Humanos , Técnicas In Vitro , Masculino , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Fragmentos de Peptídeos/líquido cefalorraquidiano , Ligação Proteica , Conformação Proteica , Ratos , Ratos Sprague-Dawley , Compostos de Espiro/farmacocinética , Compostos de Espiro/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Suínos , Tiazóis/farmacocinética , Tiazóis/farmacologiaRESUMO
ß-Secretase inhibitors are potentially disease-modifying treatments for Alzheimer's disease. Previous efforts in our laboratory have resulted in hydroxyethylamine-derived inhibitors such as 1 with low nanomolar potency against ß-site amyloid precursor protein cleaving enzyme (BACE). When dosed intravenously, compound 1 was also shown to significantly reduce Aß40 levels in plasma, brain, and cerebral spinal fluid. Herein, we report further optimizations that led to the discovery of inhibitor 16 as a novel, potent, and orally efficacious BACE inhibitor.
RESUMO
Using fragment-based screening of a focused fragment library, 2-aminoquinoline 1 was identified as an initial hit for BACE1. Further SAR development was supported by X-ray structures of BACE1 cocrystallized with various ligands and molecular modeling studies to expedite the discovery of potent compounds. These strategies enabled us to integrate the C-3 side chain on 2-aminoquinoline 1 extending deep into the P2' binding pocket of BACE1 and enhancing the ligand's potency. We were able to improve the BACE1 potency to subnanomolar range, over 10(6)-fold more potent than the initial hit (900 µM). Further elaboration of the physical properties of the lead compounds to those more consistent with good blood-brain barrier permeability led to inhibitors with greatly improved cellular activity and permeability. Compound 59 showed an IC(50) value of 11 nM on BACE1 and cellular activity of 80 nM. This compound was advanced into rat pharmacokinetic and pharmacodynamic studies and demonstrated significant reduction of Aß levels in cerebrospinal fluid (CSF).
Assuntos
Aminoquinolinas/síntese química , Aminoquinolinas/farmacologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Aminoquinolinas/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Animais , Ácido Aspártico Endopeptidases/metabolismo , Biocatálise/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Domínio Catalítico , Linhagem Celular , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Masculino , Modelos Químicos , Modelos Moleculares , Estrutura Molecular , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Cortical development is disrupted in presenilin-1 null mutant (Psen1-/-) mice. Prior studies have commented on similarities between Psen1-/- and reeler mice. Reelin induces phosphorylation of Dab1 and activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Psen1 is known to modulate PI3K/Akt signaling and both known reelin receptors (apoER2 and VLDLR) are substrates for Psen1 associated gamma-secretase activity. The purpose of this study was to determine whether reelin signaling is disrupted in Psen1-/- mice. We show that, while Dab1 is hypophosphorylated late in cortical development in Psen1-/- mice, it is normally phosphorylated at earlier ages and reelin signaling is intact in Psen1-/- primary neuronal cultures. gamma-secretase activity was also not required for reelin-induced phosphorylation of Dab1. Unlike reeler mice the preplate splits in Psen1-/- brain. Thus cortical development in Psen1-/- mice fails only after splitting of the preplate and is not due to an intrinsic failure of reelin signaling.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Córtex Cerebral/embriologia , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Presenilina-1/fisiologia , Serina Endopeptidases/metabolismo , Transdução de Sinais/fisiologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Western Blotting , Linhagem Celular , Células Cultivadas , Córtex Cerebral/metabolismo , Humanos , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Presenilina-1/genética , Proteína Reelina , Transdução de Sinais/genéticaRESUMO
Mice with a null mutation of the presenilin 1 gene (Psen1(-/-)) die during late intrauterine life or shortly after birth and exhibit multiple CNS and non-CNS abnormalities, including cerebral hemorrhages and altered cortical development. The cellular and molecular basis for the developmental effects of Psen1 remain incompletely understood. Psen1 is expressed in neural progenitors in developing brain, as well as in postmitotic neurons. We crossed transgenic mice with either neuron-specific or neural progenitor-specific expression of Psen1 onto the Psen1(-/-) background. We show that neither neuron-specific nor neural progenitor-specific expression of Psen1 can rescue the embryonic lethality of the Psen1(-/-) embryo. Indeed neuron-specific expression rescued none of the abnormalities in Psen1(-/-) mice. However, Psen1 expression in neural progenitors rescued the cortical lamination defects, as well as the cerebral hemorrhages, and restored a normal vascular pattern in Psen1(-/-) embryos. Collectively, these studies demonstrate that Psen1 expression in neural progenitor cells is crucial for cortical development and reveal a novel role for neuroectodermal expression of Psen1 in development of the brain vasculature.
Assuntos
Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Hemorragia Cerebral/metabolismo , Hemorragia Cerebral/patologia , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Células-Tronco/metabolismo , Animais , Movimento Celular , Córtex Cerebral/embriologia , Hemorragia Cerebral/genética , Perda do Embrião , Embrião de Mamíferos/irrigação sanguínea , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas de Filamentos Intermediários/genética , Íntrons/genética , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Nestina , Neurônios/patologia , Presenilina-1 , Ratos , Células-Tronco/patologia , Telencéfalo/embriologia , Telencéfalo/metabolismo , Telencéfalo/patologiaRESUMO
Mice with a null mutation of the presenilin-1 (PS1-/-) gene die during late intrauterine life or shortly after birth and exhibit defects in cortical development. A previous report suggested that neurons differentiate prematurely in PS1-/- brain [M. Handler, X. Yang, J. Shen, Presenilin-1 regulates neuronal differentiation during neurogenesis, Development 127 (2000) 2593-2606]. Here we reexamined the issue of whether premature neuronal differentiation occurs in PS1-/- brain using fresh cell suspensions from embryonic E11.5 and E13.5 telencephalon where individual cell phenotypes can be easily determined with cell type specific markers. Immunostaining with seven neuronal specific markers (MAP2, beta-III tubulin, GABA, reelin, GluR2/3, calbindin, and calretinin) failed to reveal any evidence of premature neuronal differentiation in PS1-/- telencephalon. We also determined the fraction of cells expressing the neural progenitor marker nestin and found no evidence for premature depletion of neural progenitor cells in PS1-/- telencephalon. Moreover, based on MAP2 staining of tissue sections from E12.5 embryos the topography of newly generated neurons also appeared to be undisturbed in the telencephalon of PS1-/- embryos. These studies thus argue that premature neuronal differentiation is unlikely to be a core pathophysiological feature underlying the aberrant cortical development that occurs in PS1-/- brain.
Assuntos
Diferenciação Celular/fisiologia , Proteínas de Membrana/deficiência , Neurônios/citologia , Células-Tronco/citologia , Animais , Células Cultivadas , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Presenilina-1 , Proteína Reelina , Células-Tronco/metabolismo , Telencéfalo/citologia , Telencéfalo/embriologia , Telencéfalo/metabolismoRESUMO
The functions of presenilin 1 (PS1) and how PS1 mutations cause familial Alzheimer's disease (FAD) are incompletely understood. PS1 expression is essential for neurogenesis during embryonic development and may also influence neurogenesis in adult brain. We examined how increasing PS1 expression or expressing an FAD mutant would affect neurogenesis in the adult hippocampus. A neuron-specific enolase (NSE) promoter was used to drive neuronal overexpression of either wild-type human PS1 or the FAD mutant P117L in transgenic mice, and the animals were studied under standard-housing conditions or after environmental enrichment. As judged by bromodeoxyuridine (BrdU) labeling, neural progenitor proliferation rate was mostly unaffected by increasing expression of either wild-type or FAD mutant PS1. However, in both housing conditions, the FAD mutant impaired the survival of BrdU-labeled neural progenitor cells leading to fewer new beta-III-tubulin-immunoreactive neurons being generated in FAD mutant animals during the 4-week postlabeling period. The effect was FAD mutant specific in that neural progenitor survival and differentiation in mice overexpressing wild-type human PS1 were similar to nontransgenic controls. Two additional lines of PS1 wild-type and FAD mutant transgenic mice showed similar changes indicating that the effects were not integration site-dependent. These studies demonstrate that a PS1 FAD mutant impairs new neuron production in adult hippocampus by decreasing neural progenitor survival. They also identify a new mechanism whereby PS1 FAD mutants may impair normal neuronal function and may have implications for the physiological functioning of the hippocampus in FAD.
Assuntos
Doença de Alzheimer/genética , Hipocampo/patologia , Proteínas de Membrana/genética , Neurônios/patologia , Substituição de Aminoácidos/genética , Peptídeos beta-Amiloides/biossíntese , Animais , Antígenos de Diferenciação/biossíntese , Bromodesoxiuridina , Contagem de Células , Sobrevivência Celular/genética , Modelos Animais de Doenças , Meio Ambiente , Expressão Gênica , Hipocampo/metabolismo , Humanos , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Transgênicos , Mutação , Neurônios/metabolismo , Fragmentos de Peptídeos/biossíntese , Presenilina-1 , Células-Tronco/metabolismo , Células-Tronco/patologiaRESUMO
Aberrant accumulation of neurofilaments is a feature of human motor neuron diseases. Experimentally motor neuron disease can be induced in transgenic mice by overexpressing the mouse neurofilament light subunit (NF-L), the human heavy subunit (NF-H), or mouse peripherin. Here we describe that mice harboring a bacterial artificial chromosome (BAC) transgene containing the human midsized neurofilament subunit (NF-M) gene develop a progressive hind limb paralysis associated with neurofilamentous accumulations in ventral horn motor neurons and axonal loss in ventral motor roots. Biochemical studies revealed that all three mouse neurofilament subunits along with the human NF-M contributed to filament formation, although filaments contained less peripherin. In addition the endogenous mouse NF-M became less phosphorylated in the presence of the human protein and accumulated in the cell bodies of affected neurons even though phosphorylated human NF-M did not. Remaining motor axons contained an increased density of neurofilaments and morphometric studies showed that principally small myelinated axons were lost in the transgenic animals. Removing half of the mouse NF-M by breeding the transgene onto the mouse NF-M heterozygous null background offered no protection against the development of disease, arguing that the effect is not simply due to elevation of total NF-M. Collectively these studies argue that the human and mouse NF-M proteins exhibit distinct biochemical properties and within mouse neurons are not interchangeable and that indeed the human protein may be toxic to some mouse neurons. These studies have implications for the use of human neurofilament transgenic mice as models of amyotrophic lateral sclerosis.
Assuntos
Glicoproteínas de Membrana , Doença dos Neurônios Motores/genética , Proteínas de Neurofilamentos/biossíntese , Proteínas de Neurofilamentos/genética , Animais , Axônios/ultraestrutura , Western Blotting , Tamanho Celular , Imunofluorescência , Membro Posterior/fisiopatologia , Humanos , Corpos de Inclusão/patologia , Proteínas de Filamentos Intermediários/biossíntese , Proteínas de Filamentos Intermediários/genética , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Doença dos Neurônios Motores/metabolismo , Neurônios Motores/patologia , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Proteínas de Neurofilamentos/metabolismo , Paralisia/induzido quimicamente , Paralisia/genética , Paralisia/fisiopatologia , Periferinas , FosforilaçãoRESUMO
Presenilin1 (PS1), a protein implicated in Alzheimer's disease (AD), forms complexes with N-cadherin, a transmembrane protein with important neuronal and synaptic functions. Here, we show that a PS1-dependent gamma-secretase protease activity promotes an epsilon-like cleavage of N-cadherin to produce its intracellular domain peptide, N-Cad/CTF2. NMDA receptor agonists stimulate N-Cad/CTF2 production suggesting that this receptor regulates the epsilon-cleavage of N-cadherin. N-Cad/CTF2 binds the transcription factor CBP and promotes its proteasomal degradation, inhibiting CRE-dependent transactivation. Thus, the PS1-dependent epsilon-cleavage product N-Cad/CTF2 functions as a potent repressor of CBP/CREB-mediated transcription. Importantly, PS1 mutations associated with familial AD (FAD) and a gamma-secretase dominant-negative mutation inhibit N-Cad/CTF2 production and upregulate CREB-mediated transcription indicating that FAD mutations cause a gain of transcriptional function by inhibiting production of transcriptional repressor N-Cad/CTF2. These data raise the possibility that FAD mutation-induced transcriptional abnormalities maybe causally related to the dementia associated with FAD.
Assuntos
Caderinas/metabolismo , Proteínas de Transporte/metabolismo , Transcrição Gênica , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Western Blotting , Caderinas/química , Linhagem Celular , Membrana Celular/metabolismo , Células Cultivadas , Cisteína Endopeptidases/metabolismo , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Endopeptidases/metabolismo , Genes Dominantes , Vetores Genéticos , Humanos , Immunoblotting , Proteínas de Membrana/metabolismo , Camundongos , Microscopia de Fluorescência , Complexos Multienzimáticos/metabolismo , Mutação , Neurônios/metabolismo , Peptídeos/química , Testes de Precipitina , Presenilina-1 , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares/metabolismo , Sinapses/metabolismo , Fatores de Tempo , Ativação Transcricional , Tubulina (Proteína)/metabolismoRESUMO
Mutations in the presenilin-1 (PS-1) gene are one cause of familial Alzheimer's disease (FAD). However, the functions of the PS-1 protein as well as how PS-1 mutations cause FAD are incompletely understood. Here we investigated if neuronal overexpression of wild-type or FAD mutant PS-1 in transgenic mice affects neurogenesis in the hippocampus of adult animals. We show that either a wild-type or an FAD mutant PS-1 transgene reduces the number of neural progenitors in the dentate gyrus. However, the wild-type, but not the FAD mutant PS-1 promoted the survival and differentiation of progenitors leading to more immature granule cell neurons being generated in PS-1 wild type expressing animals. These studies suggest that PS-1 plays a role in regulating neurogenesis in adult hippocampus and that FAD mutants may have deleterious properties independent of their effects on amyloid deposition.