Transglutaminases and transglutaminase-catalyzed cross-links colocalize with the pathological lesions in Alzheimer's disease brain

Abstract

Alzheimer's disease (AD) is characterized by pathological lesions, in particular senile plaques (SPs), cerebral amyloid angiopathy (CAA) and neurofibrillary tangles (NFTs), predominantly consisting of self-aggregated proteins amyloid beta (Abeta) and tau, respectively. Transglutaminases (TGs) are inducible enzymes, capable of modifying conformational and/or structural properties of proteins by inducing molecular covalent cross-links. Both Abeta and tau are substrates for TG cross-linking activity, which links TGs to the aggregation process of both proteins in AD brain. The aim of this study was to investigate the association of transglutaminase 1 (TG1), transglutaminase 2 (TG2) and TG-catalyzed cross-links with the pathological lesions of AD using immunohistochemistry. We observed immunoreactivity for TG1, TG2 and TG-catalyzed cross-links in NFTs. In addition, both TG2 and TG-catalyzed cross-links colocalized with Abeta in SPs. Furthermore, both TG2 and TG-catalyzed cross-links were associated with CAA. We conclude that these TGs demonstrate cross-linking activity in AD lesions, which suggests that both TG1 and TG2 are likely involved in the protein aggregation processes underlying the formation of SPs, CAA and/or NFTs in AD brain.

Figure 1

Immunohistochemical staining of the neocortex of control brain for TG1, TG2 and TG‐catalyzed cross‐links. The anti‐TG1 antibody stained both neurons in the gray matter (A, arrow) and glial cells in the white matter (B, arrow). TG1 immunoreactivity was absent in vessels of the neocortex (C, arrow). TG2 was present in neurons in the gray matter (D, arrow) and in glial cells in the white matter (E, arrow). TG2 staining was observed in both capillaries and parenchymal vessels (F, arrow). The anti‐TG‐catalyzed cross‐link antibody (anti‐CL) was immunoreactive in astrocytes of both the white and the gray matter (G, arrow) and in the nucleus of glial cells and neurons of the neocortex (H, arrow). Weak immunoreactivity of the anti‐CL antibody was observed in parenchymal vessels (I, arrow). Original magnification: A, C, D, F, G, I×200; B, E, H×150. Abbreviations: TG = transglutaminase; TG1 = transglutaminase 1; TG2 = transglutaminase 2.

Figure 2

Immunohistochemical staining for TG1 of SPs, CAA and NFTs in the neocortex of Alzheimer's disease brain. Serial sections: A–B, C–D, E–F, J–K. The anti‐Aβ antibody stained both classic (A, arrow) and diffuse SPs (C, arrow), and CAA (C, large arrow). The anti‐TG1 antibody was absent in both classic (B, arrow) and diffuse (D, arrow) SPs, and in CAA (D, large arrow). The anti‐tau antibody‐stained NFTs (E, arrow). Anti‐TG1 immunoreactivity was demonstrated in NFTs (F, arrow). Double immunofluorescence staining demonstrated colocalization of TG1 with hyperphosphorylated tau in NFTs (G–I). Specificity of the anti‐TG1 antibody was demonstrated by preadsorption of the antibody with human recombinant TG1 (J,K). Staining of NFTs with anti‐TG1 (J) is absent after preadsorption (K). Original magnification: A–F, J, K×200; G–I×400. Abbreviations: TG1 = transglutaminase 1; SPs = senile plaques; CAA = cerebral amyloid angiopathy; NFTs = neurofibrillary tangles; Aβ = amyloid‐β.

Figure 3

Immunohistochemical staining of SPs, CAA and NFTs in the neocortex of Alzheimer's disease brain for TG2. Serial sections: A–B, C–D, E–F, J–K. The anti‐Aβ antibody stained both diffuse (A, arrow) and classic SPs (C), and CAA (E). TG2 staining was present in both diffuse (B) and classic (D) SPs. TG2 was present in endothelial cells in CAA vessels and as a halo surrounding the deposited Aβ in CAA (F). Double immunofluorescence staining demonstrated colocalization of TG2 with hyperphosphorylated tau in NFTs (arrows, G–I). Specificity of the TG2 staining was demonstrated by preadsorption of the antibody with recombinant human TG2 (J,K). Staining of both classic (arrow) and diffuse (arrow with asterisk) SPs with anti‐TG2 (J) was strongly reduced after preadsorption (K). Original magnification: A, B, J, K×200; C–F×250; G–I×400. Abbreviations: TG2 = transglutaminase 2; SPs = senile plaques; CAA = cerebral amyloid angiopathy; NFTs = neurofibrillary tangles; Aβ = amyloid‐β.

Figure 4

Immunohistochemical staining of SPs, CAA and NFTs in the neocortex of Alzheimer's disease brain for TG‐catalyzed cross‐links. The anti‐Aβ antibody stained both diffuse (A, arrow, and E, arrow with asterisk) and classic (C, arrow) SPs, and CAA (E, arrow). The anti‐TG‐catalyzed cross‐link antibody was demonstrated in both diffuse (B, arrow, and F, arrow with asterisk) and classic (D, large arrow) SPs. In addition, TG‐catalyzed cross‐links were also observed in reactive astrocytes associated with classic SPs (D, arrow). The anti‐TG‐catalyzed cross‐link antibody was demonstrated in CAA (F, arrow). Double immunofluorescence demonstrated TG‐catalyzed cross‐links in endothelial and possible perivascular cells in CAA and in deposited Aβ surrounding Aβ‐affected vessels (G–I). Double immunofluorescence also demonstrated TG‐catalyzed cross‐links in NFTs (J–L). Specificity of the anti‐TG‐catalyzed cross‐link antibody was demonstrated by preadsorption of the antibody with H‐Glu(H‐Lys‐OH)‐OH (M,N). Staining of SPs with anti‐TG‐catalyzed cross‐link (M) was strongly reduced after preadsorption (N). Original magnification: A–F, M–N×200; G–I×250; J–L×400. Abbreviations: TG = transglutaminase; SPs = senile plaques; CAA = cerebral amyloid angiopathy; NFTs = neurofibrillary tangles; Aβ = amyloid‐β.