N-terminally truncated amyloid-b (Ab) peptides are present in early and diffuse plaques of individuals with Alzheimer's disease (AD), are overproduced in early onset familial AD and their amount seems to be directly correlated to the severity and the progression of the disease in AD and Down's syndrome (DS). The pyroglutamate-containing isoforms at position 3 [AbN3(pE))40/42] represent the prominent form among the N-truncated species, and may account for more than 50% of Ab accumulated in plaques. In this study, we compared the toxic properties, fibrillogenic capabilities, and in vitro degradation profile of Ab1-40, Ab1-42, AbN3(pE))40 and AbN3(pE))42. Our data show that fibre morphology of Ab peptides is greatly influenced by the C-terminus while toxicity, interaction with cell membranes and degradation are influenced by the N-terminus. AbN3(pE))40 induced significantly more cell loss than the other species both in neuronal and glial cell cultures. Aggregated AbN3(pE) peptides were heavily distributed on plasma membrane and within the cytoplasm of treated cells. AbN3(pE))40/42 peptides showed a significant resistance to degradation by cultured astrocytes, while fulllength peptides resulted partially degraded. These findings suggest that formation of N-terminally modified peptides may enhance b-amyloid aggregation and toxicity, likely worsening the onset and progression of the disease.

The proteolytic processing of amyloid precursor protein (APP) through the formation of membrane-bound C-terminal fragments (CTFs) and of soluble ␤-amyloid peptides likely influences the development of Alzheimer's disease (AD). We show that in human brain a subset of CTFs are tyrosine-phosphorylated and form stable complexes with the adaptor protein ShcA. Grb2 is also part of these complexes, which are present in higher amounts in AD than in control brains. ShcA immunoreactivity is also greatly enhanced in patients with AD and occurs at reactive astrocytes surrounding cerebral vessels and amyloid plaques. A higher amount of phospho-ERK1,2, likely as result of the ShcA activation, is present in AD brains. In vitro experiments show that the ShcACTFs interaction is strictly confined to glial cells when treated with thrombin, which is a well known ShcA and ERK1,2 activator and a regulator of APP cleavage. In untreated cells ShcA does not interact with either APP or CTFs, although they are normally generated. Altogether these data suggest that CTFs are implicated in cell signaling via Shc transduction machinery, likely influencing MAPK activity and glial reaction in AD patients.The cytoplasmic region of the amyloid precursor protein contains an NPXY motif, which is present in the cytodomains of several tyrosine kinase receptors and in non-receptor tyrosine kinase (1, 2). In tyrosine kinase receptors the tyrosine residue of this motif is phosphorylated upon tyrosine kinase activation, and the NPXpY motif (where pY is phosphotyrosine) functions as a docking site for the phosphotyrosine-binding domain present in several adaptor proteins interacting with tyrosine kinase receptors and non-receptor tyrosine kinase, such as the proteins belonging to the Shc family (3, 4). In APP 1 and in APPrelated proteins APLP1 and APLP2 the NPTY motif interacts with several adaptor proteins, such as Fe65 (5), X11 (6), mDab1 (7), and JIP-1 (8), but this interaction has been demonstrated to be independent of tyrosine phosphorylation (9, 10). Recent data (11) show that in human brain CTFs can be tyrosine-phosphorylated and that in vitro the APP cytodomain is tyrosine-phosphorylated by the non-receptor tyrosine kinase Abl, which phosphorylates a tyrosine residue upstream (Tyr-682), the NPTY motif (11,12). This phosphorylation generates a motif pYXXP that is recognized by the SH2 domain of Abl itself and that might be a docking site for SH2-containing adaptors such as Shc and Grb2 proteins (11). Here we describe that in human brain tyrosine-phosphorylated CTFs represent docking sites for Shc and Grb2 proteins and generate stable complexes with these adaptors that are up-regulated in AD cases. ShcA formation is strictly confined to activated astroglial cells only, and its levels are highly enhanced in AD brains in comparison to control subjects. In AD brains it is also up-regulated in the expression of Erk1,2 kinase, likely as a consequence of ShcA activation. In vitro experiments show that thrombin triggers the ShcA-CTFs interaction and Erk ph...

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