RESUMEN
MMP13 (matrix metallopeptidase 13) plays a key role in bone metabolism and cancer development, but has no known functions in Alzheimer's disease. In this study, we used high-throughput small molecule screening in SH-SY5Y cells that stably expressed a luciferase reporter gene driven by the BACE1 (ß-site amyloid precursor protein cleaving enzyme 1) promoter, which included a portion of the 5' untranslated region (5'UTR). We identified that CL82198, a selective inhibitor of MMP13, decreased BACE1 protein levels in cultured neuronal cells. This effect was dependent on PI3K (phosphatidylinositide 3-kinase) signalling, and was unrelated to BACE1 gene transcription and protein degradation. Further, we found that eukaryotic translation initiation factor 4B (eIF4B) played a key role, as the mutation of eIF4B at serine 422 (S422R) or deletion of the BACE1 5'UTR attenuated MMP13-mediated BACE1 regulation. In APPswe/PS1E9 mice, an animal model of Alzheimer's disease, hippocampal Mmp13 knockdown or intraperitoneal CL82198 administration reduced BACE1 protein levels and the related amyloid-ß precursor protein processing, amyloid-ß load and eIF4B phosphorylation, whereas spatial and associative learning and memory performances were improved. Collectively, MMP13 inhibition/CL82198 treatment exhibited therapeutic potential for Alzheimer's disease, via the translational regulation of BACE1.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Benzofuranos/uso terapéutico , Disfunción Cognitiva/tratamiento farmacológico , Metaloproteinasa 13 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz/uso terapéutico , Morfolinas/uso terapéutico , Enfermedad de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Células Cultivadas , Factores Eucarióticos de Iniciación/genética , Técnicas de Silenciamiento del Gen , Hipocampo/metabolismo , Humanos , Ratones , Ratones Transgénicos , Mutación , Oligopéptidos/genética , Fosfatidilinositol 3-Quinasas/metabolismo , RatasRESUMEN
Neurexins are neuron-specific synaptic proteins, and abnormal structure of Neurexin1ß is closely associated with autism. To characterize the minimal promoter of autism-associated NRXN1ß gene and identify functional elements regulating its transcription, luciferase reporter plasmids containing different regulatory regions upstream of NRXN1ß gene were constructed. After transfecting HEK293 cells with these plasmids, the minimal promoter region of NRXN1ß gene was determined by detecting the transcriptional activity of luciferase reporter genes while the corresponding functional elements that significantly enhance or inhibit the activity of reporter genes were further screened out. To identify cis-acting elements, continuous nucleotide mutation within the functional regions and adjacent DNA sequences were generated using site-directed mutagenesis techniques and then transcriptional regulatory elements in corresponding regions were analyzed using transcription factor binding prediction tool. Our results showed for the first time that the minimal promoter region of human NRXN1ß gene is located between positions -88 and +156 (-88/+156); two regions -88/-73 and +156/+149 enhance while the region +229/+419 inhibits promoter activity. The region -84/-63 significantly enhances promoter activity as cis-acting elements, suggesting the presence of DBP and ABF1 transcription factor binding sites in this region.