RESUMO
Alzheimer's disease (AD) is characterized by the accumulation of amyloid-ß (Aß). The genes that govern this process, however, have remained elusive. To this end, we combined distinct mouse strains with transcriptomics to directly identify disease-relevant genes. We show that AD model mice (APP-Tg) with DBA/2 genetic backgrounds have significantly lower levels of Aß accumulation compared with SJL and C57BL/6 mice. We then applied brain transcriptomics to reveal the genes in DBA/2 that suppress Aß accumulation. To avoid detecting secondarily affected genes by Aß, we used non-Tg mice in the absence of Aß pathology and selected candidate genes differently expressed in DBA/2 mice. Additional transcriptome analysis of APP-Tg mice with mixed genetic backgrounds revealed kinesin light chain-1 (Klc1) as an Aß modifier, indicating a role for intracellular trafficking in Aß accumulation. Aß levels correlated with the expression levels of Klc1 splice variant E and the genotype of Klc1 in these APP-Tg mice. In humans, the expression levels of KLC1 variant E in brain and lymphocyte were significantly higher in AD patients compared with unaffected individuals. Finally, functional analysis using neuroblastoma cells showed that overexpression or knockdown of KLC1 variant E increases or decreases the production of Aß, respectively. The identification of KLC1 variant E suggests that the dysfunction of intracellular trafficking is a causative factor of Aß pathology. This unique combination of distinct mouse strains and model mice with transcriptomics is expected to be useful for the study of genetic mechanisms of other complex diseases.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Isoformas de Proteínas/metabolismo , Doença de Alzheimer/genética , Animais , Encéfalo/metabolismo , Cruzamentos Genéticos , Perfilação da Expressão Gênica , Humanos , Cinesinas , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Isoformas de Proteínas/genética , Especificidade da EspécieRESUMO
The canonical pathway of Notch signaling is mediated by regulated intramembrane proteolysis (RIP). In the pathway, ligand binding results in sequential proteolysis of the Notch receptor, and presenilin (PS)-dependent intramembrane proteolysis at the interface between the membrane and cytosol liberates the Notch-1 intracellular domain (NICD), a transcription modifier. Because the degradation of the Notch-1 transmembrane domain is thought to require an additional cleavage near the middle of the transmembrane domain, extracellular small peptides (Notch-1 Abeta-like peptide (Nbeta)) should be produced. Here we showed that Nbeta species are indeed secreted during the process of Notch signaling. We identified mainly two distinct molecular species of novel Nbeta, Nbeta21 and C-terminally elongated Nbeta25, which were produced in an approximately 5:1 ratio. This process is reminiscent of the production of Alzheimer disease-associated Abeta. PS pathogenic mutants increased the production of the longer species of Abeta (Abeta42) from beta-amyloid protein precursor. We revealed that several Alzheimer disease mutants also cause a parallel increase in the secretion of the longer form of Nbeta. Strikingly, chemicals that modify the Abeta42 level caused parallel changes in the Nbeta25 level. These results demonstrated that the characteristics of C-terminal elongation of Nbeta and Abeta are almost identical. In addition, because many other type 1 membrane-bound receptors release intracellular domains by PS-dependent intramembrane proteolysis, we suspect that the release of Abeta- or Nbeta-like peptides is a common feature of the proteolysis during RIP signaling. We anticipate that this study will open the door to searches for markers of RIP signaling and surrogate markers for Abeta42 production.
Assuntos
Precursor de Proteína beta-Amiloide/química , Receptor Notch1/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Citosol/metabolismo , DNA/metabolismo , DNA Complementar/metabolismo , Genes Reporter , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Ligantes , Proteínas de Membrana/genética , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Peptídeos/química , Presenilina-1 , Ligação Proteica , Estrutura Terciária de Proteína , Receptor Notch1/química , Transdução de SinaisRESUMO
Gas6, a product of the growth-arrest-specific gene 6, protects cortical neurons from amyloid beta protein (Abeta)-induced apoptosis. Neuronal apoptosis is also caused by human group IIA secretory phospholipase A(2) (sPLA(2)-IIA), which is expressed in the cerebral cortex after brain ischemia. sPLA(2)-IIA induces Ca(2+) influx via L-type voltage-sensitive calcium channels (L-VSCCs), leading to its neurotoxicity. In the present study, we investigated effects of Gas6 on sPLA(2)-IIA-induced cell death in primary cultures of rat cortical neurons. sPLA(2)-IIA caused neuronal cell death in a concentration- and time-dependent manner. Gas6 significantly prevented neurons from sPLA(2)-IIA-induced cell death. Gas6 suppressed sPLA(2)-IIA-induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA. Prior to cell death, sPLA(2)-IIA increased the influx of Ca(2+) into neurons through L-VSCCs. Gas6 significantly inhibited the sPLA(2)-IIA-induced Ca(2+) influx. The blocker of L-VSCCs also suppressed sPLA(2)-IIA-induced neuronal cell death. The cortical cultures contained few non-neuronal cells, indicating that Gas6 affected the survival of neurons directly, but not indirectly via non-neuronal cells. In conclusion, we demonstrate that Gas6 rescues cortical neurons from sPLA(2)-IIA-induced apoptosis. Furthermore, the present study indicates that inhibition of L-VSCC contributes to the neuroprotective effect of Gas6.