RESUMO
A recent genome-wide association meta-analysis for Alzheimer's disease (AD) identified 19 risk loci (in addition to APOE) in which the functional genes are unknown. Using Drosophila, we screened 296 constructs targeting orthologs of 54 candidate risk genes within these loci for their ability to modify Tau neurotoxicity by quantifying the size of >6000 eyes. Besides Drosophila Amph (ortholog of BIN1), which we previously implicated in Tau pathology, we identified p130CAS (CASS4), Eph (EPHA1), Fak (PTK2B) and Rab3-GEF (MADD) as Tau toxicity modulators. Of these, the focal adhesion kinase Fak behaved as a strong Tau toxicity suppressor in both the eye and an independent focal adhesion-related wing blister assay. Accordingly, the human Tau and PTK2B proteins biochemically interacted in vitro and PTK2B co-localized with hyperphosphorylated and oligomeric Tau in progressive pathological stages in the brains of AD patients and transgenic Tau mice. These data indicate that PTK2B acts as an early marker and in vivo modulator of Tau toxicity.
Assuntos
Quinase 2 de Adesão Focal/genética , Proteínas tau/metabolismo , Doença de Alzheimer/genética , Animais , Biomarcadores , Modelos Animais de Doenças , Drosophila/genética , Quinase 2 de Adesão Focal/metabolismo , Loci Gênicos/genética , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Humanos , Fatores de Risco , Proteínas tau/genéticaRESUMO
Genome-wide association studies (GWAS) have identified a region upstream the BIN1 gene as the most important genetic susceptibility locus in Alzheimer's disease (AD) after APOE. We report that BIN1 transcript levels were increased in AD brains and identified a novel 3 bp insertion allele â¼28 kb upstream of BIN1, which increased (i) transcriptional activity in vitro, (ii) BIN1 expression levels in human brain and (iii) AD risk in three independent case-control cohorts (Meta-analysed Odds ratio of 1.20 (1.14-1.26) (P=3.8 × 10(-11))). Interestingly, decreased expression of the Drosophila BIN1 ortholog Amph suppressed Tau-mediated neurotoxicity in three different assays. Accordingly, Tau and BIN1 colocalized and interacted in human neuroblastoma cells and in mouse brain. Finally, the 3 bp insertion was associated with Tau but not Amyloid loads in AD brains. We propose that BIN1 mediates AD risk by modulating Tau pathology.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Predisposição Genética para Doença/genética , Proteínas Nucleares/genética , Proteínas Supressoras de Tumor/genética , Proteínas tau/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Doença de Alzheimer/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Endofenótipos , Expressão Gênica/genética , Humanos , Camundongos , Degeneração Neural/genética , Degeneração Neural/patologia , Proteínas Nucleares/biossíntese , Placa Amiloide/patologia , Polimorfismo de Nucleotídeo Único/genética , Sinaptossomos/patologia , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/biossíntese , Proteínas tau/antagonistas & inibidoresRESUMO
The peptidyl prolyl cis-trans isomerase Pin1 and the Inhibitor of Apoptosis Protein (IAP) Survivin are two major proteins involved in cancer. They both modulate apoptosis, mitosis, centrosome duplication and neuronal development but until now no functional relationship has been reported between these two proteins. We tested Pin1-induced regulation of Survivin in neuroblastoma cells. Pin1 overexpression in SY5Y neuroblastoma cells decreased Survivin levels. Immunocytochemical studies indicated that they partially co-localized in interphase and mitotic cells. Co-immunoprecipitation further demonstrates the existence of a Pin1/Survivin complex. Pin1-induced effect on Survivin was confirmed in COS cells. RT-PCR and mutagenesis experiments suggested that this Pin1-induced decrease of Survivin occurred at the protein level. Survivin downregulation depended on the binding ability of Pin1 but was not related to the single Thr-Pro site, suggesting an indirect relationship into a protein complex. Finally, this functional regulation of Survivin by Pin1 is reciprocal since Pin1 silencing led to an increase in Survivin levels. The characterization of this functional relationship between Pin1 and Survivin might help to better understand mitosis control and cancer mechanisms.
Assuntos
Regulação para Baixo , Proteínas Inibidoras de Apoptose/metabolismo , Peptidilprolil Isomerase/metabolismo , Animais , Sítios de Ligação , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , DNA Complementar , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Mutação , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Peptidilprolil Isomerase/genética , Testes de Precipitina , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Irradiated or injured cells enter apoptosis, and in turn, promote proliferation of surrounding unaffected cells. In Drosophila, apoptotic cells have an active role in proliferation, where the caspase Dronc and p53 induce mitogen expression and growth in the surrounding tissues. The Drosophila p53 gene structure is conserved and encodes at least two protein isoforms: a full-length isoform (Dp53) and an N-terminally truncated isoform (DΔNp53). Historically, DΔNp53 was the first p53 isoform identified and was thought to be responsible for all p53 biological activities. It was shown that DΔNp53 induces apoptosis by inducing the expression of IAP antagonists, such as Reaper. Here we investigated the roles of Dp53 and DΔNp53 in apoptosis and apoptosis-induced proliferation. We found that both isoforms were capable of activating apoptosis, but that they each induced distinct IAP antagonists. Expression of DΔNp53 induced Wingless (Wg) expression and enhanced proliferation in both 'undead cells' and in 'genuine' apoptotic cells. In contrast to DΔNp53, Dp53 did not induce Wg expression in the absence of the endogenous p53 gene. Thus, we propose that DΔNp53 is the main isoform that regulates apoptosis-induced proliferation. Understanding the roles of Drosophila p53 isoforms in apoptosis and in apoptosis-induced proliferation may shed new light on the roles of p53 isoforms in humans, with important implications in cancer biology.