Análisis de las vías de inducción de autofagia en neuronas, y su papel neuroprotector en un modelo transgénico de la enfermedad de Alzheimer

Resumen

Alzheimer’s disease (AD), the most common neurodegenerative disorder, is considered a proteinopathy characterized by deposits of aggregated amyloid-beta (A) and hyperphosphorylated tau. The accumulation of autophagic vesicles within dystrophic neurites in the brain suggests a progressive dysfunction of the protein turnover mediated by macroautophagy. In this process, a double-membrane vesicle or autophagosome sequesters a cytoplasmic portion for its delivery to lysosomes and degradation. This project aimed to study the effect of different previously described autophagy inductors on the A secretion, trying to correlate autophagy and amyloid secretion/generation. For this purpose, we used the AD mouse model B6.Cg-Tg (APPSwe, PSEN1dE9)/J for experiments in vivo or primary neuronal cultures obtained from these mice or their wild-type littermates. According to our results, we only appreciated a quite modest autophagic induction effect by the inhibition of mTORC1 with rapamycin in neurons, contrary to the drastic effects described in tumour cells. The administration of rapamycin showed a decrease on A levels in vivo as well as in cultured neurons. The pharmacological prevention of autophagy in neurons increased the A secretion and reversed the lowering effect of rapamycin. Consequently, we concluded that autophagy induction decreases the A secretion through its degradation. Therefore, we proceed to analyse the modulation of autophagy in neuronal cells by other main regulators such as the protein kinase AMPK. Indeed, its blockage with Compound C generated the expected decrease in autophagy induction, though surprisingly this inhibition did not increase the A secretion. Moreover, various chemical compounds which activate AMPK through different mechanisms (Metformin, AICAR and 2DG tested) not only did not induce autophagy, but also seemed to damper it. Some of these compounds appeared to affect differently the A secreted levels, not reversed by the prevention of autophagosome formation. Whereas Metformin could diminish the A secreted levels, AICAR enhanced them. These results suggest different cellular responses through AMPK dependent and independent mechanisms, and do not permit to conclude a key role of AMPK in neuronal autophagy per se. Aside from the Metformin’s activation of PP2A, Metformin and to a lesser extent AICAR induced tau dephosphorylation in PP2B sensitive epitopes and generated a tau fragmentation, at least partially due to a non-apoptotic caspase-3 activation. These events could be related to the modulation of A levels, even though it should be further analysed. However, due to the delicate regulation of the AMPK pathway in neurons, as well as to the presumed independence of AMPK mediated autophagy in the modulation of beta-amyloid levels, we decided to analyse other strategies unrelated to this pathway, such as the disaccharide Trehalose. We observed a relevant A lowering effect with Trehalose, which has been described as an efficient mTORC1 independent autophagy inductor. Nevertheless, our results support an anti-amyloidogenic effect independent of autophagy induction, through diminishing the APP cleavage by beta-secretase. Preliminary data suggest a segregation of APP and BACE1 at the hand of the trans-Golgi network, presumably because of modifying the endomembrane system and gen-related expression levels. More experiments should be performed to confirm it. In summary, our data confirmed the anti-amyloidogenic effects of autophagy in neurons, though the highly regulated Akt-mTORC1 pathway, as mTORC1 inhibition just led to a slight induction of autophagy. Our data indicated that a certain level of AMPK activity appears to be necessary, whereas its enhancement disrupts the normal progression. In addition, we have identified a relevant anti-amyloidogenic effect of Trehalose through segregating APP and BACE1 in the cell, which could help the development of new strategies to fight Alzheimer’s Disease.