Processing of the amyloid precursor protein (APP) by b-and c-secretases leads to the generation of amyloid-b (Ab) peptides with varying lengths. Particularly Ab42 contributes to cytotoxicity and amyloid accumulation in Alzheimer's disease (AD). However, the precise molecular mechanism of Ab42 generation has remained unclear. Here, we show that an amino-acid motif GxxxG within the APP transmembrane sequence (TMS) has regulatory impact on the Ab species produced. In a neuronal cell system, mutations of glycine residues G29 and G33 of the GxxxG motif gradually attenuate the TMS dimerization strength, specifically reduce the formation of Ab42, leave the level of Ab40 unaffected, but increase Ab38 and shorter Ab species. We show that glycine residues G29 and G33 are part of a dimerization site within the TMS, but do not impair oligomerization of the APP ectodomain. We conclude that c-secretase cleavages of APP are intimately linked to the dimerization strength of the substrate TMS. The results demonstrate that dimerization of APP TMS is a risk factor for AD due to facilitating Ab42 production.

Formation of aberrant protein conformers is a common pathological denominator of different neurodegenerative disorders, such as Alzheimer's disease or prion diseases. Moreover, increasing evidence indicates that soluble oligomers are associated with early pathological alterations and that oligomeric assemblies of different disease-associated proteins may share common structural features. Previous studies revealed that toxic effects of the scrapie prion protein (PrP(Sc)), a β-sheet-rich isoform of the cellular PrP (PrP(C)), are dependent on neuronal expression of PrP(C). In this study, we demonstrate that PrP(C) has a more general effect in mediating neurotoxic signalling by sensitizing cells to toxic effects of various β-sheet-rich (β) conformers of completely different origins, formed by (i) heterologous PrP, (ii) amyloid β-peptide, (iii) yeast prion proteins or (iv) designed β-peptides. Toxic signalling via PrP(C) requires the intrinsically disordered N-terminal domain (N-PrP) and the GPI anchor of PrP. We found that the N-terminal domain is important for mediating the interaction of PrP(C) with β-conformers. Interestingly, a secreted version of N-PrP associated with β-conformers and antagonized their toxic signalling via PrP(C). Moreover, PrP(C)-mediated toxic signalling could be blocked by an NMDA receptor antagonist or an oligomer-specific antibody. Our study indicates that PrP(C) can mediate toxic signalling of various β-sheet-rich conformers independent of infectious prion propagation, suggesting a pathophysiological role of the prion protein beyond of prion diseases.

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N-terminally truncated A␤ peptides starting with pyroglutamate (A␤pE3) represent a major fraction of all A␤ peptides in the brain of Alzheimer disease (AD) patients. A␤pE3 has a higher aggregation propensity and stability and shows increased toxicity compared with full-length A␤. In the present work, we generated a novel monoclonal antibody (9D5) that selectively recognizes oligomeric assemblies of A␤pE3 and studied the potential involvement of oligomeric A␤pE3 in vivo using transgenic mouse models as well as human brains from sporadic and familial AD cases. 9D5 showed an unusual staining pattern with almost nondetectable plaques in sporadic AD patients and non-demented controls. Interestingly, in sporadic and familial AD cases prominent intraneuronal and blood vessel staining was observed. Using a novel sandwich ELISA significantly decreased levels of oligomers in plasma samples from patients with AD compared with healthy controls were identified. Moreover, passive immunization of 5XFAD mice with 9D5 significantly reduced overall A␤ plaque load and A␤pE3 levels, and normalized behavioral deficits. These data indicate that 9D5 is a therapeutically and diagnostically effective monoclonal antibody targeting low molecular weight A␤pE3 oligomers. Alzheimer disease (AD)3 represents the most frequent form of dementia and is characterized by the presence of extracellular amyloid plaques composed of amyloid-␤ (A␤) surrounded by dystrophic neurites and neurofibrillary tangles. The discovery that certain early-onset familial forms of AD may be caused by enhanced levels of A␤ peptides have led to the hypothesis that amyloidogenic A␤ is intimately involved in the AD pathogenic process (1). In the past extracellular A␤ has been regarded as the major culprit, whereas more recent evidence now points to toxic effects of A␤ in intracellular compartments (2-3). In addition, other concepts propose that the soluble oligomers and the ␤-sheet containing amyloid fibrils are the toxic forms of A␤ (4 -6). Supporting this notion, it has been demonstrated that soluble oligomeric A␤42, but not plaque-associated A␤, correlates best with cognitive dysfunction in AD (7-8). Oligomers are formed preferentially intracellulary within neuronal processes and synapses rather than extracellularly (9 -10). Besides full-length A␤ peptides starting with an aspartate at position 1, a variety of different N-truncated A␤ peptides have been identified in AD brains. Ragged peptides including phenylalanine at position 4 of A␤ have been reported as early as 1985 by Masters et al. (11). In contrast, no N-terminal sequence could be obtained from cores purified in a sodium dodecyl sulfate-containing buffer, which led to the assumption that the N terminus could be blocked (12-13). The presence of A␤pE3 (N-terminally truncated A␤ starting with pyroglutamate) in AD brain was subsequently shown using mass spectrometry of purified A␤ peptides, explaining at least partially initial difficulties in sequencing A␤ peptides purified from human brain tissue (14). The author...

The aggregation of the amyloid-␤ (A␤) peptide plays a pivotal role in the pathogenesis of Alzheimer's disease, as soluble oligomers are intimately linked to neuronal toxicity and inhibition of hippocampal long-term potentiation (LTP). In the C-terminal region of A␤ there are three consecutive GxxxG dimerization motifs, which we could previously demonstrate to play a critical role in the generation of A␤. Here, we show that glycine 33 (G33) of the central GxxxG interaction motif within the hydrophobic A␤ sequence is important for the aggregation dynamics of the peptide. A␤ peptides with alanine or isoleucine substitutions of G33 displayed an increased propensity to form higher oligomers, which we could attribute to conformational changes. Importantly, the oligomers of G33 variants were much less toxic than A␤ 42 wild type (WT), in vitro and in vivo. Also, whereas A␤ 42 WT is known to inhibit LTP, A␤ 42 G33 variants had lost the potential to inhibit LTP. Our findings reveal that conformational changes induced by G33 substitutions unlink toxicity and oligomerization of A␤ on the molecular level and suggest that G33 is the key amino acid in the toxic activity of A␤. Thus, a specific toxic conformation of A␤ exists, which represents a promising target for therapeutic interventions.