RESUMO
Alzheimer's disease (AD) is characterized pathologically by the abundance of senile plaques and neurofibrillary tangles in the brain. We synthesized over 1200 novel gamma-secretase modulator (GSM) compounds that reduced Abeta(42) levels without inhibiting epsilon-site cleavage of APP and Notch, the generation of the APP and Notch intracellular domains, respectively. These compounds also reduced Abeta(40) levels while concomitantly elevating levels of Abeta(38) and Abeta(37). Immobilization of a potent GSM onto an agarose matrix quantitatively recovered Pen-2 and to a lesser degree PS-1 NTFs from cellular extracts. Moreover, oral administration (once daily) of another potent GSM to Tg 2576 transgenic AD mice displayed dose-responsive lowering of plasma and brain Abeta(42); chronic daily administration led to significant reductions in both diffuse and neuritic plaques. These effects were observed in the absence of Notch-related changes (e.g., intestinal proliferation of goblet cells), which are commonly associated with repeated exposure to functional gamma-secretase inhibitors (GSIs).
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/imunologia , Precursor de Proteína beta-Amiloide/genética , Análise de Variância , Animais , Anticorpos/farmacologia , Butiratos/farmacologia , Caderinas/metabolismo , Células Cultivadas , Cricetinae , Cricetulus , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Transferência Ressonante de Energia de Fluorescência/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidrocarbonetos Halogenados/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fragmentos de Peptídeos/metabolismo , Presenilina-1/genética , Ratos , Receptores Notch/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Transfecção/métodosRESUMO
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.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Sequência Conservada/fisiologia , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Evolução Molecular , Humanos , Células Jurkat , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Neoplasias/genética , Técnicas de Patch-Clamp , Interferência de RNA , Molécula 1 de Interação Estromal , Tapsigargina/farmacologiaRESUMO
When mitosis is bypassed, as in some cancer cells or in natural endocycles, sister chromosomes remain paired and produce four-stranded diplochromosomes or polytene chromosomes. Cyclin/Cdk1 inactivation blocks entry into mitosis and can reset G2 cells to G1, allowing another round of replication. Reciprocally, persistent expression of Cyclin A/Cdk1 or Cyclin E/Cdk2 blocks Drosophila endocycles. Inactivation of Cyclin A/Cdk1 by mutation or overexpression of the Cyclin/Cdk1 inhibitor, Roughex (Rux), converts the 16(th) embryonic mitotic cycle to an endocycle; however, we show that Rux expression fails to convert earlier cell cycles unless Cyclin E is also downregulated. Following induction of a Rux transgene in Cyclin E mutant embryos during G2 of cell cycle 14 (G2(14)), Cyclins A, B, and B3 disappeared and cells reentered S phase. This rereplication produced diplochromosomes that segregated abnormally at a subsequent mitosis. Thus, like the yeast CKIs Rum1 and Sic1, Drosophila Rux can reset G2 cells to G1. The observed cyclin destruction suggests that cell cycle resetting by Rux was associated with activation of the anaphase-promoting complex (APC), while the presence of diplochromosomes implies that this activation of APC outside of mitosis was not sufficient to trigger sister disjunction.
