**4. Discussion**

In this study we compared the staining characteristics of three small D-amino acid peptides (i.e., D1, D2, and D3) that were designed to specifically bind Aβ42 (D1, Wiesehan and Willbold, 2003; Wiesehan et al., 2003; D3, ) with the two traditional histochemical methods for amyloid (thioflavine-S and Congo red) and two newer techniques. We examined the labeling of Aβ deposits in Tg AD model mouse brain, in the hippocampus, cortex and in blood vessel walls. The data demonstrate that all dense Aβ deposits (plaques) are labeled with the D-peptides, but not diffuse deposits. This corresponds to the distribution of the Aβ staining in the brain when it is labeled with Aβ42 specific antibodies. Finally, the binding of the D-peptides corresponds closely to the localization of Aβ42 in the brain, more closely than to the localization of Aβ40.

Similarly, in brain tissue sections derived from AD patients, amyloid β plaques and leptomeningeal vessels containing Aβ are stained positively with the fluorescence-labeled derivative of D1 (Wiesehan et al, 2003). In contrast, fibrillar deposits derived from other amyloidosis are not labeled by D1 (Wiesehan et al, 2003). It should be noted that none of the D-

Fig. 4. Three photomicrographs of coronal sections of the parietal cortex of an 18-months-old APP/PS1 mouse stained with the D1 peptide conjugated with different fluorophores, showing staining with D1\*FITC, D1\*Bodipy, and D1\*Oregon green, respectively. Please note the increased background staining with the D1\*Bodipy, also note similarity of staining

In this study we compared the staining characteristics of three small D-amino acid peptides (i.e., D1, D2, and D3) that were designed to specifically bind Aβ42 (D1, Wiesehan and Willbold, 2003; Wiesehan et al., 2003; D3, ) with the two traditional histochemical methods for amyloid (thioflavine-S and Congo red) and two newer techniques. We examined the labeling of Aβ deposits in Tg AD model mouse brain, in the hippocampus, cortex and in blood vessel walls. The data demonstrate that all dense Aβ deposits (plaques) are labeled with the D-peptides, but not diffuse deposits. This corresponds to the distribution of the Aβ staining in the brain when it is labeled with Aβ42 specific antibodies. Finally, the binding of the D-peptides corresponds closely to the localization of Aβ42 in the brain, more closely

Similarly, in brain tissue sections derived from AD patients, amyloid β plaques and leptomeningeal vessels containing Aβ are stained positively with the fluorescence-labeled derivative of D1 (Wiesehan et al, 2003). In contrast, fibrillar deposits derived from other amyloidosis are not labeled by D1 (Wiesehan et al, 2003). It should be noted that none of the D-

between D1\*FITC and D1\*Oregon green.

than to the localization of Aβ40.

**4. Discussion** 

peptides showed any binding to Aβ deposits in the brains of mice which express the APPswe/dutch/iowa mutation van Groen et al, 2009). This is to be expected since the structure of the Aβ peptide with these mutations (i.e., the Dutch and Iowa mutations) is predicted to be different from the "normal" Aβ peptide (Demeester et al., 2001; Kumar-Singh et al., 2002; Tsubuki et al., 2003; Watson et al., 1999). It should be noted that these mutations are in the Aβ peptide sequence of APP, in contrast to the Swedish mutation (Selkoe, 2001).

The data demonstrate that none of the three D-peptides binds to diffuse Aβ deposits, whereas they do bind to dense Aβ deposits, i.e., plaques. Earlier we have shown that the diffuse Aβ deposits do not stain with thioflavine S, Congo red or thiazine red, whereas the core of plaques does. Furthermore, the diffuse deposits consist primarily of N-terminal fragments of Aβ, they contain some Aβ40 but do not contain stainable amounts of Aβ42 (Van Groen et al., 2003), in contrast to plaques that consist of significant amounts of both Aβ40 and Aβ42. We have suggested earlier that the diffuse deposits consist of Aβ that has a different length (and structure) from the Aβ42 and Aβ40 that is present in plaques, even if Aβ fibrils are present in the diffuse deposits (Van Groen et al., 2003). Together these data indicate that the D-peptides bind very specifically to only Aβ42.

Furthermore, it has been shown that Aβ42 is actively taken up by astrocytes and microglia (Nagele et al., 2003; Rogers and Lue, 2001. In contrast, surprisingly, no D-peptides are visible in astrocytes and microglia, the phagocytosing cells in the brain (Rogers et al, 2002). Activated microglial cells are present in the brains of AD model mice but these cells never show any presence of intracellular Aβ (e.g., Stalder et al., 2003; but see Paresce et al., 1996).

We have used these peptides to treat AD model mice and we have shown that a brain infusion with D3 significantly reduces pathology and cognitive deficits in AD model mice (van Groen et al, 2009, Funke et al, 2011). In contrast D1 infusion does not improve cognition (van Groen et al, 2009). Similarly it has been demonstrated that Congo red (Inouye and Kirschner, 2005, Lee, 2002) and thioflavine-S improve pathology (Alavez et al, 2011).

Together, we have demonstrated that 1) D-peptides that specifically bind to Aβ42 and, 2) that the D-peptides staining is similar, but more specific, to most traditional histochemical amyloid staining methods. Thus, our data strongly suggest that these novel and highly specific Abeta42 ligands have potential application(s) in the diagnosis and therapy of Alzheimer's disease (Masters and Beyreuther, 2006; Monaco et al., 2006), especially since these D-peptides are much more resistant to proteolysis than natural L-peptides.

### **5. Acknowledgements**

We thank Dr. Egon von Schnier for his excellent comments on an earlier version of this manuscript, and Pasi Miettinen for his assistance with the histology. This study was partially supported by TEKES project 40043/01 and partially by NIH AG10836.
