The E693Δ Mutation in Amyloid Precursor Protein Increases Intracellular Accumulation of Amyloid β Oligomers and Causes Endoplasmic Reticulum Stress-Induced Apoptosis in Cultured Cells

Abstract

The E693Delta mutation within the amyloid precursor protein (APP) has been suggested to cause dementia via the enhanced formation of synaptotoxic amyloid beta (Abeta) oligomers. However, this mutation markedly decreases Abeta secretion, implying the existence of an additional mechanism of neuronal dysfunction that is independent of extracellular Abeta. We therefore examined the effects of this mutation on both APP processing to produce Abeta as well as subcellular localization and accumulation of Abeta in transfected HEK293 and COS-7 cells. Both beta- and gamma-cleavage of mutant APP increased, indicating a lack of inhibition in Abeta production. Instead, this mutation promoted Abeta accumulation within cells, including the endoplasmic reticulum (ER), Golgi apparatus, early and late endosomes, lysosomes, and autophagosomes, all of which have been proposed as intracellular sites of Abeta generation and/or degradation, suggesting impairment of APP/Abeta trafficking. Notably, the intracellular mutant Abeta was found to predominantly form oligomers. Concomitant with this accumulation, the ER stress markers Grp78 and phosphorylated eIF2alpha were both strongly induced. Furthermore, the activation of caspase-4 and -3 as well as DNA fragmentation were detected in these cells. These results suggest that mutant Abeta induces alteration of Abeta trafficking and subsequent ER stress-induced apoptosis via enhancement of its intracellular oligomerization. Our findings suggest that Abeta oligomers exhibit toxicity in the extracellular space and within the cells themselves.

A Mouse Model of Amyloid β Oligomers: Their Contribution to Synaptic Alteration, Abnormal Tau Phosphorylation, Glial Activation, and Neuronal LossIn Vivo

“…Biochemical analysis confirmed the accumulation of A␤ dimers and possibly trimers in their brains. This is consistent with our previous findings that mutant A␤ E22⌬ peptides neither formed amyloid fibrils in vitro nor were deposited in amyloid plaques in AD patient brains, and instead formed abundant oligomers in vitro and accumulated in oligomeric forms within transfected cells (Nishitsuji et al, 2009). The enhanced formation of A␤ oligomers and the lack of amyloid plaques in our APP E693⌬ -Tg mice indicate that this mouse model is suitable for study of the contribution of A␤ oligomers to the pathogenesis of AD.…”

“…The APP WT -Tg mice did not exhibit such intracellular staining despite having higher expression of APP than the APP E693⌬ -Tg mice. This finding is consistent with our previous observation that, in transfected cells, mutant A␤ E22⌬ accumulated within cells more abundantly than WT A␤ (Nishitsuji et al, 2009).…”

“…Alternatively, intraneuronal thioflavin S staining may reflect the presence of fibrillar aggregates of hyperphosphorylated tau. In our previous study, A␤ was found to localize largely at endosomes, and to a lesser extent in the ER, Golgi, lysosomes, and autophagosomes in cultured cells (Nishitsuji et al, 2009). In AD and Tg2576 mouse brains, A␤ was found to accumulate in multivesicular bodies, which are a type of endosome (Takahashi et al, 2002).…”

“…Since the mutant A␤ E22⌬ that accumulated in transfected cells tended to form oligomers (Nishitsuji et al, 2009), it is likely that intraneuronal A␤ in the APP E693⌬ -Tg mice also forms oligomers. To test whether this is the case, brain sections from mice at various ages were stained with a monoclonal antibody NU-1 selective to A␤ oligomers .…”

“…The mutant A␤ peptide does not form amyloid fibrils in vitro and patients with the mutation lack deposits of amyloid plaques . The mutant peptide, however, readily forms abundant oligomers in vitro and accumulates in oligomeric forms within transfected cells (Nishitsuji et al, 2009). When injected into rat cerebral ventricle, synthetic mutant A␤ E22⌬ peptide inhibits hippocampal long-term potentiation (LTP) more potently than wild-type (WT) peptide in vivo .…”