This might be caused GW-572016 chemical structure by the conversion of phenylalanine to tyrosine by hydroxyl radicals generated during the decomposition of peroxynitrite (Ferger et al., 2001). In accordance with this, there was no reactivity toward Aβ1-42 bearing a Y10A mutation after incubation with peroxynitrite (Figure S1).

Using this antiserum, we were able to detect 3NTyr10-Aβ in the supernatant of NOS2 overexpressing HEK293 cells after exogenous addition of nonaggregated Aβ, demonstrating that NOS2 is able to induce this posttranslational modification before Aβ deposits form (Figure S1). Immunohistochemical analysis of AD and control brains by 3NTyr10-Aβ antiserum revealed a lack of immunoreactivity in control brains, whereas in AD brain, the core of amyloid plaques was intensively labeled, as confirmed by IC16 double staining (Figure 1C). Measuring the relative amounts of 3NTyr10-Aβ by sandwich ELISA in SDS-soluble fractions of human brain samples, we detected 3NTyr10-Aβ in the SDS fraction of AD patients and only to very low amount in Crizotinib nondemented controls (Figure 1D). Further, the relative signal ratio of 3NTyr10-Aβ between control and AD patients was comparable to that of Aβ1-42 (Figure 1D).

Of note, we failed to detect any 3NTyr10-Aβ in human cerebrospinal fluid (CSF) of control, mild cognitive impaired, and AD patients underlining the insoluble properties of this species (Figure S1). Analysis of brain sections from 5- and 12-month-old APP/PS1 mice revealed a colocalization of antibody IC16 against Aβ with the 3NTyr10-Aβ antiserum from beginning of plaque formation starting at 5 months of age in this AD mouse model (Figures 2A and 2B). This costaining was observed in all brain areas where amyloid

plaques are formed. Casein kinase 1 In addition, colocalization was observed independently of plaque size, since it was already detectable in tiny plaques of 10 μm diameter in 5-month-old animals (Figure 2C), suggesting that formation of 3NTyr10-Aβ is an early event in plaque development. Similar to human AD brain, in APP/PS1 the 3NTyr10-Aβ immunoreactivity was localized to the core of the plaque surrounded by IC16 immunoreactivity (Figure 2D). Evaluation of individual Aβ plaque sizes by immunohistochemistry with antibody IC16 and the area of the 3NTyr10-Aβ positive core of 5- and 9-month-old APP/PS1 mice revealed that there are no changes in the average 3NTyr10-Aβ core size (Figures 2E and 2F), suggesting that the core, once formed, does not substantially increase in size any further. Nevertheless, we observed plaque growth between 5 and 9 months that was solely caused by accumulation of nonnitrated Aβ, as detected by IC16 immunoreactivity (Figures 2E and 2F). As a consequence, there was a highly significant drop in the 3NTyr10-Aβ/Aβ ratio (Figure 2G). In addition, we were able to immunoprecipitate 3NTyr10-Aβ of brain homogenates sequentially extracted with PBS, RIPA, SDS, and HIFP using 3NTyr10-Aβ antiserum.