MECHANISMS OF AUTOPHAGY-MEDIATED AB SECRETION

Abstract

We have recently shown that autophagy mediates the secretion of Aβ by studying autophagy-deficient APP transgenic (Tg) mice (Nilsson et al., Cell Reports). These findings have extended the previously established degradative role of autophagy in Aβ metabolism and add Aβ to the growing number of proteins secreted by autophagy. The genetic deletion of autophagy, obtained by conditional knockout (cKO) of autophagy-related protein 7 (Atg7), additionally led to accumulation of intracellular Aβ. Since the APP tg mice are based on APP overexpression, which may produce artifacts, our laboratory has developed a new generation of AD mouse models based on APP knock-in (KI) technology (Saito et al., unpublished). These mice exhibit endogenous levels of APP, but increased Aβ levels due to the introduced Swedish and Beyreuther mutations and develop Aβ plaque from 6 months of age. These mice will be used to confirm previous data obtained with APP Tg mice. In an attempt to elucidate the pathway of autophagy-mediated Aβ secretion, we used fluorescence and immunoelectron microscopy (IEM) to trace the intracellular Aβ accumulation that occurs in the autophagy deficient APP mice. By applying different tissue fixation methods we could establish protocols that allowed identification of Aβ in Golgi and in the multivesicular bodies (MVBs). To further confirm the role of autophagy in Aβ secretion, we generated autophagy-deficient APP-KI mice. Interestingly, we found that Aβ accumulated in Golgi upon deletion of autophagy. In contrast, the MVBs, which contain significant amounts of Aβ in the APP mice, exhibited drastically decreased Aβ levels in the autophagy-deficient APP mice. In agreement with the results obtained with the APP tg mice, the extracellular Aβ plaque load of autophagy-deficient APP-KI mice were drastically decreased. IEM data indicate that autophagy mediates transport of Aβ from Golgi and that secretion possibly occurs through MVBs. In addition, we could confirm that autophagy mediates the secretion of Aβ using a novel APP-KI mouse model without APP overexpression, which we use to further investigate the mechanisms of autophagy-mediated Aβ secretion.