Oligomerization of amyloid beta (Abeta) peptides is the decisive event in the development of Alzheimer's disease (AD), the most common neurogenerative disorder in developed countries. Recent evidence links this conformation-driven process to primary- and secondary-structure modifications of Abeta. The N and C terminus of deposited Abeta has been shown to possess conspicuous heterogeneity. While the C-terminally longer form of Abeta, i.e., Abeta (42), is considered more amyloidogenic, the role of the N-terminal modifications, e.g., truncation and glutamate cyclization accounting for the majority of the deposited peptides, is less understood. In the present study, we characterized the oligomerization and seeding capacity of pGlu-amyloid peptides using two unrelated techniques based on flow cytometry or flourescence dye binding. Under different conditions and irrespective of the C terminus of Abeta, i.e., Abeta40 or 42, pGlu-modified peptides displayed an up to 250-fold accelerated initial formation of aggregates compared to unmodified Abeta. The accelerated seed formation is accompanied by a change in the oligomerization kinetics because of N-terminal pGlu formation. Furthermore, the formation of mixed aggregates consisting of either pGlu-Abeta (3-42) or ADan or ABri and Abeta (1-42) was investigated by Abeta fluorescence labeling in flow cytometry. The results suggest that pGlu-modified peptides are potential seeding species of aggregate formation in vivo. The data presented here and the abundance of pGlu peptides in amyloidoses, such as FBD and AD, suggest pGlu-amyloid peptides as a species with biophysical characteristics that might be in particular crucial for the initiation of the disease.

Compelling evidence suggests that N-terminally truncated and pyroglutamyl-modified amyloid-beta (Abeta) peptides play a major role in the development of Alzheimer's disease. Posttranslational formation of pyroglutamic acid (pGlu) at position 3 or 11 of Abeta implies cyclization of an N-terminal glutamate residue rendering the modified peptide degradation resistant, more hydrophobic, and prone to aggregation. Previous studies using artificial peptide substrates suggested the potential involvement of the enzyme glutaminyl cyclase in generation of pGlu-Abeta. Here we show that glutaminyl cyclase (QC) catalyzes the formation of Abeta 3(pE)-40/42 after amyloidogenic processing of APP in two different cell lines, applying specific ELISAs and Western blotting based on urea-PAGE. Inhibition of QC by the imidazole derivative PBD150 led to a blockage of Abeta 3(pE)-42 formation. Apparently, the QC-catalyzed formation of N-terminal pGlu is favored in the acidic environment of secretory compartments, which is also supported by double-immunofluorescence labeling of QC and APP revealing partial colocalization. Finally, initial investigations focusing on the molecular pathway leading to the generation of truncated Abeta peptides imply an important role of the amino acid sequence near the beta-secretase cleavage site. Introduction of a single-point mutation, resulting in an amino acid substitution, APP(E599Q), i.e., at position 3 of Abeta, resulted in significant formation of Abeta 3(pE)-40/42. Introduction of the APP KM595/596NL "Swedish" mutation causing overproduction of Abeta, however, surprisingly diminished the concentration of Abeta 3(pE)-40/42. The study provides new cell-based assays for the profiling of small molecule inhibitors of QC and points to conspicuous differences in processing of APP depending on sequence at the beta-secretase cleavage site.

J. Neurochem. (2012) 121, 774–784. Abstract Pyroglutamate (pGlu)‐modified amyloid peptides have been identified in sporadic and familial forms of Alzheimer’s disease (AD) and the inherited disorders familial British and Danish Dementia (FBD and FDD). In this study, we characterized the aggregation of amyloid‐β protein Aβ37, Aβ38, Aβ40, Aβ42 and ADan species in vitro, which were modified by N‐terminal pGlu (pGlu‐Aβ3‐x, pGlu‐ADan) or possess the intact N‐terminus (Aβ1‐x, ADan). The pGlu‐modification confers rapid formation of oligomers and short fibrillar aggregates. In accordance with these observations, the pGlu‐modified Aβ38, Αβ40 and Αβ42 species inhibit hippocampal long term potentiation of synaptic response, but pGlu‐Aβ3‐42 showing the highest effect. Among the unmodified Aβ peptides, only Aβ1‐42 exhibites such propensity, which was similar to pGlu‐Aβ3‐38 and pGlu‐Aβ3‐40. Likewise, the amyloidogenic peptide pGlu‐ADan impaired synaptic potentiation more pronounced than N‐terminal unmodified ADan. The results were validated using conditioned media from cultivated HEK293 cells, which express APP variants favoring the formation of Aβ1‐x, Aβ3‐x or N‐truncated pGlu‐Aβ3‐x species. Hence, we show that the ability of different amyloid peptides to impair synaptic function apparently correlates to their potential to form oligomers as a common mechanism. The pGlu‐modification is apparently mediating a higher surface hydrophobicity, as shown by 1‐anilinonaphtalene‐8‐sulfonate fluorescence, which enforces potential to interfere with neuronal physiology.

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