Direct evidence of the presence of cross-linked Aβ dimers in the brains of Alzheimer's disease patients

Brain-derived amyloid-β (Aβ) dimers are associated with Alzheimer´s disease (AD). However, their covalent nature remains controversial. This feature is relevant, as a covalent cross-link would make brain-derived dimers (brain dimers) more synaptotoxic than Aβ monomers and would make them suitable candidates for biomarker development. To resolve this controversy, we here present a three-step approach. First, we validated a type of synthetic cross-linked Aβ (CL Aβ) dimers, obtained by means of the photo-induced cross-linking of unmodified proteins (PICUP) reaction, as well-defined mimics of putative brain CL Aβ dimers. Second, we used these PICUP CL Aβ dimers as standards to improve the isolation of brain Aβ dimers and to develop state-of-the-art mass spectrometry (MS) strategies to allow their characterization. Third, we applied these MS methods to the analysis of brain Aβ dimer samples allowing the detection of the CL [Aβ(6-16)]2 peptide comprising a dityrosine cross-link. This result demonstrates the presence of CL Aβ dimers in the brains of patients with AD and opens up avenues for establishing new therapeutic targets and developing novel biomarkers for this disease

Estudio de la apoptosis inducida por la inhibición de la vía de la PI3K/AKT

[spa] Una de las vías que se postula que tienen una mayor importancia en las enfermedades neurodegenerativas es la de los inositoles fosfato. Para el estudio de esta vía se ha utilizado un inhibidor farmacológico de la fosfoinositol 3 cinasa (PI3K), el LY294002, en un modelo in vitro de células granulares de cerebelo de rata (CGC). Al tratar las CGC con una dosis de 30μM de LY294002 se produce una muerte celular por apoptosis que es independiente de calpaínas y dependiente de caspasas, además no se observa la fragmentación de p35 ni de α espectrina que se da por activación de las calpaínas. Los ensayos de actividad caspasa nos muestran un incremento significativo de la actividad de las caspasas 6 y 9 pero no de la 3 como sucede en otros modelos de apoptosis como la deprivación de S/K+. Nuestros estudios muestran que aunque existen algunas similitudes entre los modelos de inhibición de la PI3K y la deprivación de S/K+ también existen importantes diferencias. En ambos se produce una desfosforilación de AKT en Ser476 y consecuentemente una desfosforilación de GSK3β en Ser9, lo que indica la activación de GSK3β. Respecto a la proteína Rb en ambos modelos se observa un incremento de su fosforilación, si bien su papel es distinto ya que en la deprivación de S/K+ conduce a la liberación del E2F y a la transcripción de proteínas relacionadas con el ciclo celular. Además, se observó un incremento de la síntesis de DNA. Por el contrario el tratamiento con LY294002, pese a provocar un incremento en la fosforilación del Rb, no lleva a la expresión de ciclinas, CDKs ni un aumento de la síntesis de DNA.. Sin embargo el uso de inhibidores de CDK como flavopiridol y roscovitina muestran una protección significativa frente a la apoptosis inducida por LY294002, nuestros estudios muestran por vez primera que, no solo flavopiridol sino también otros inhibidores de CDK como la roscovitina tienen capacidad para inhibir la actividad GSK3β. Rb puede ser fosforilado por p38, un miembro de la vía de las MAPK las cuales son inhibidas por AKT. Nuestros resultados indican que LY294002 produce un incremento de la actividad de p38, pero no de JNK. Además, los cultivos Knockout de JNK3 no muestran una protección frente al tratamiento con LY294002, lo que refuerza la idea de que JNK no juega un papel central en este modelo. El incremento de actividad de p38 fue revertido con SB203580, un inhibidor de p38, así como por SP600125, inhibidor de JNK. Ambos fármacos mostraron una protección significativa frente a la apoptosis inducida por LY294002 y una reducción de la fosforilación del factor de transcripción c‐Jun, implicado en la apoptosis. La activación de c‐Jun conduce a la expresión de genes proapoptóticos como dp5 relacionados con la vía intrínseca, la inhibición de p38 previno del aumento de expresión de dp5. Por el contrario otras proteínas implicadas en la vía como Bim no están reguladas por c‐Jun ya que la inhibición de esta vía no reduce su activación. En nuestro estudio podemos concluir que, LY294002 produce una apoptosis dependiente de caspasas 6 y 9, sin implicación ni de calpaínas ni de proteínas del ciclo celular. La inhibición de AKT lleva a la activación de GSK3β y de p38. Además, p38 es capaz de fosforilar c‐Jun que regula la expresión de genes relacionados con la apoptosis por la vía intrínseca.[eng] Study of the apoptosis induced by the inhibition of the PI3K/AKT in neurons The inositol pathway has been reported that plays a key role in neurodegenerative diseases We study the mechansims involved in the apoptosis induced by inhibiting the phosphoinositol 3 kinase (PI3K) using a pharmacological inhibitor named LY294002 in an in vitro model of rat cerebellar granule cells (CGC). LY294002 induced apoptotic cell death through calpain independent and caspase dependent. Furthermore, we could not observed neither fragmentation of of p35 or α espectrin which is caused by calpains. The caspase activity assays showed a significant increase in caspase 6 and 9 but not in caspasa 3, in contrast with other apoptotic models such as de S/K+ deprivation. Our studies show that although exist several common points between inhibition of PI3K and S/K+ deprivation, also exist important differences between them. In both cases it has been observed AKT dephosphorylation at Ser476 and consequently GSK3β dephosphorylation at Ser9, which indicates GSK3β activation. On the other side, it was observed an increase of Rb phosphorylation in both models. However, it seems that the role played by this protein is different since in the de S/K+ deprivation leads to E2F released which participates in the transcription of proteins related to cell cycle. Moreover, the BrdU assay showed an increase in DNA synthesis. On the contrary, the LY294002 treatment, in spite of the fact that induced an increase of Rb phosphorylation, it did not induce any change of the levels neither cell cycle proteins or However, CDK inhibitors such as flavopiridol and roscovitine protected from the apoptosis induced by LY294002, our studies showed for the first time, that not only flavopiridol, but also other CDK inhibitors such as roscovitine could inhibit the GSK3β activity. Furthermore Rb can be phosphorylated by p38, which is a protein of MAPK pathway that is down‐regulated by AKT. Our results showed that LY294002 produced an increase of p38 activity, but not of JNK. Moreover, JNK3 Knockout cultures were not significantly protected from LY294002 treatment, this reinforces the idea that JNK is not the main target involved in this model. The increase of p38 activity was prevented with SB203580, a specific p38 inhibitor, and either with SP600125, a JNK inhibitor. Both drugs shown a significant protection from the apoptosis induced by LY294002 and prevented from c‐Jun phosphorylation, a transcription factor implied in apoptosis. The activation of c‐Jun triggered the expression of proapoptotic genes such as dp5 which is related to the intrinsic pathway, p38 inhibition prevented from the increase in dp5 expression. On the contrary, other proapoptotic proteins related to this pathway such as Bim was not regulated by c‐Jun since the inhibition of p38 pathway did not reduce its expression. In our study we can conclude that LY294002 induced apoptosis mediated by caspasas 6 and 9. Neither calpains nor cell cycle proteins were involved in this apoptotic model. The inhibition of AKT leaded to GSK3β and p38 activation. Moreover, p38 was able to phosphorylate c‐Jun that triggers the expression of proapoptotic genes implied in the apoptotic intrinsic pathway