Determining the relative amounts of 3NTyr10-Aβ in SDS fractions o

Determining the relative amounts of 3NTyr10-Aβ in SDS fractions of wild-type, APP/PS1, and APP/PS-1 NOS2 (−/−) animals by sandwich ELISA, we were unable to detect this species in wild-type mice, but in APP/PS1 mice. In turn,

APP/PS1 mice lacking NOS2 (−/−) showed a 74% reduction of 3NTyr10-Aβ (Figure 2I). Since N-terminal modifications of Aβ have been shown to induce its aggregation, we speculated whether nitration of Aβ exerts a similar effect. Indeed, incubation of synthetic Aβ1-42 with peroxynitrite or the NO donor Sin-1 resulted in increased generation of high molecular weight SDS-resistant oligomers (Figures 3A SCR7 price and S2). Using Aβ1-42 peptides with a tyrosine to alanine or phenylalanine mutation (Aβ42Y10A

or Aβ42Y10F) reduced aggregation to the level of untreated Aβ1-42 (Figures 3A and S2). In case of the nonmutated Aβ1-42, we observed the incorporation Dasatinib chemical structure of nitrated Aβ1-42 into oligomers (Figure 3C). There was a very low amount of nitrated Aβ1-42Y10F detectable using the 3NTyr10-Aβ antiserum. Finally, we confirmed our western blot results by detecting an increased formation rate of β sheet amyloid fibril structures of nitrated Aβ1-42 using thioflavin T (Figure 3D), which was prevented using the Aβ42Y10F peptide treated with peroxynitrite. Oxidative conditions can also result in the formation of dityrosine cross-linked proteins (Kato et al., 2000). We therefore investigated whether peroxynitrite is able to induce this modification as well. Using the dityrosine specific antibody IC3 we were able to detect dityrosine cross-linked Aβ in vitro after incubation with increasing concentrations of peroxynitrite (Figure 3E). High concentrations of peroxynitrite resulted in decreased formation of this species, whereas formation of 3NTyr10-Aβ

increased even further (Figure 3E). Dityrosine immunoreactivity nearly was also found to be present in the insoluble fractions of aggregated Aβ (Figure 3F). In addition, we performed immunohistochemical analysis of dityrosine with Aβ or 3NTyr10-Aβ in sections of APP/PS1 mice, revealing plaque localization and 3NTyr10-Aβ colocalization of dityrosine immunoreactivity (Figure 3G). These results suggest that dityrosine formation might also contribute to Aβ aggregation. Looking at effects on spatial memory formation by radial arm maze in 12-month-old APP/PS1 mice, we noticed a strong protection of the NOS2 gene knockout for memory deficits (Figure 4A). In addition, we conducted a therapeutic approach by treating plaque containing mice from 7–12 months with the selective NOS2 inhibitor L-NIL resulting in a reversion of APP/PS1 phenotype concerning reference memory errors (Figure 4A).

Leave a Reply

Your email address will not be published. Required fields are marked *

*

You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>