RESUMO
Nicastrin is a type 1 transmembrane glycoprotein that interacts with presenilin, Aph-1, and Pen-2 proteins to form a high molecular complex with gamma secretase activity. Then, nicastrin has a central role in presenilin-mediated processing of beta-amyloid precursor protein and in some aspects of Notch/glp-1 signaling in vivo. Here, we isolated a rat nicastrin cDNA and investigated gene expression in embryonic and adult rat tissues. The predicted amino acid sequence is comprised of 708 residues and showed a high degree of identity with other vertebrate orthologs. Besides full-length nicastrin mRNA, we identified an alternative spliced variant lacking the whole exon 3 and predicted to encode a 62-residue-long truncated protein. Full-length nicastrin mRNA was observed to be ubiquitously expressed, while the spliced variant was preferentially transcribed in the nervous system, whether in embryonic or adult neural tissues. Studies performed on primary cell cultures demonstrated that the short isoform was expressed in neurons, but not in astrocyte and microglial cells. Further experiments performed to verify the presence of the variant in neuroblastoma culture failed to show any truncated protein. Treatments by cyclohexamide showed the involvement of a quality control-based surveillance mechanism, which selectively degrades the exon 3-skipped isoform. In summary, this is the first report describing a novel skipped isoform of nicastrin which may suggest a new possible control mechanism based on the alternative splicing and nonsense-mediated mRNA decay to regulate brain protein expression and provide newer insights into potential implication in Alzheimer's disease.
Assuntos
DNA Complementar/isolamento & purificação , Regulação Enzimológica da Expressão Gênica/fisiologia , Variação Genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Secretases da Proteína Precursora do Amiloide , Animais , Northern Blotting , Western Blotting/métodos , Células Cultivadas , Córtex Cerebral/citologia , Clonagem Molecular/métodos , Cicloeximida/farmacologia , Embrião de Mamíferos , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Humanos , Camundongos , Dados de Sequência Molecular , Neuroblastoma , Neuroglia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Homologia de Sequência de Aminoácidos , Fatores de TempoRESUMO
Nicastrin is a protein recently discovered associated to presenilins and involved in the production of amyloid beta peptide that accumulates in Alzheimer's disease (AD) brain. In this study the nicastrin gene was examined for unknown mutations and polymorphisms in 104 patients with familial AD (52 early-onset and 52 late-onset), 174 sporadic AD and 191 healthy neurological controls of Italian origin. The scanning of the nicastrin gene identified a missense mutation (N417Y) in two patients with sporadic AD, in an early-onset familial AD and in a young control subject. Furthermore, we found two silent mutations and four intronic polymorphisms, three of them co-segregating in a single haplotype. We found some differences in the distribution of genotype alterations in the AD population compared to the controls. However, our data together with other evidence did not support the pathological role of missense mutation N417Y.
