Transglutaminase 1 and its regulator tazarotene-induced gene 3 localize to neuronal tau inclusions in tauopathies

Abstract

Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), and Pick's disease (PiD) are commonly known as tauopathies. Neurodegeneration observed in these diseases is linked to neuronal fibrillary hyperphosphorylated tau protein inclusions. Transglutaminases (TGs) are inducible enzymes, capable of modifying conformational and/or structural properties of proteins by inducing molecular cross-links. Both transglutaminase 1 (TG1) and transglutaminase 2 (TG2) are abundantly expressed in the brain and are associated with fibrillary hyperphosphorylated tau protein inclusions in neurons of AD, so-called neurofibrillary tangles (NFTs). However, other data obtained by our group suggested that tau pathology in the brain may be primarily related to TG1 and not to TG2 activity. To obtain more information on this issue, we set out to investigate the association of TG1, TG2, and TG-catalysed cross-links with fibrillary hyperphosphorylated tau inclusions in tauopathies other than AD, using immunohistochemistry. We found strong TG1 and TG-catalysed cross-link staining in neuronal tau inclusions characteristic of PSP, FTDP-17 with mutations in the tau gene (FTDP-17T), and PiD brain, whereas, in contrast to AD, TG2 was only rarely observed in these inclusions. Furthermore, using a biochemical approach, we demonstrated that tau is a substrate for TG1-mediated cross-linking. Interestingly, we found co-localization of the TG1 activator, tazarotene-induced gene 3 (TIG3), in the neuronal tau inclusions of PSP, FTDP-17T, and PiD, but not in NFTs of AD cases, indicating that these tau-containing protein aggregates are not identical. We conclude that TG1-catalysed cross-linking, regulated by TIG3, might play an important role in the formation of neuronal tau inclusions in PSP, FTDP-17T, and PiD brain.

Figure 1

Immunoreactivity of TG1, TG2 and TG-catalyzed cross-link antibodies in thalamus of PSP cases. The anti-TG1 antibody demonstrated TG1 immunoreactivity in NFT-like neurons in the thalamus of PSP cases (A, arrow). The anti-tau (AT8) antibody stained NFTs (C, arrow). Double immunofluorescence staining of an anti-tau antibody with an anti-TG1, anti-TG2 or anti-TG-catalyzed cross-link antibody was performed. TG1 staining was present in NFTs (B-D, arrow). In contrast, only weak TG2 immunoreactivity was observed in NFTs (E-G, arrows). TG-catalyzed cross-link immunoreactivity was found in NFTs (H-J, arrow). Original magnification: A-J × 400. Abbreviations: TG1 = transglutaminase 1; TG2 = transglutaminase 2; AT8 = anti-hyperphosphorylated tau antibody; cross-links = TG-catalyzed cross-links.

Figure 2

Immunoreactivity of TG1, TG2 and TG-catalyzed cross-link antibodies in FTDP-17T cases. The anti-TG1 antibody demonstrated fibrillary tau containing neurons in the medial frontal gyrus of FTDP-17T cases (A, arrow). The AT8 antibody stained fibrillary tau containing neurons (C, arrow). Double immunofluorescence staining of an anti-tau antibody with an anti-TG1, anti-TG2 or anti-TG-catalyzed cross-link antibody was performed. TG1 staining was present in neurons showing fibrillary tau inclusions (B-D, arrow). TG-catalyzed cross-link immunoreactivity colocalized with fibrillary tau containing inclusions in neurons (E-G, arrow). Original magnification: A-G × 400. Abbreviations: TG1 = transglutaminase 1; AT8 = anti-hyperphosphorylated tau antibody; cross-links = TG-catalyzed cross-links

Figure 3

Immunoreactivity of TG1, TG2 and TG-catalyzed cross-link antibodies in PiD cases, and cross-linking of both 3R and 4R tau by TG1. The anti-TG1 antibody demonstrated TG1 immunoreactivity in Pick Bodies (PB)-containing neurons in the medial frontal gyrus of PiD cases (A, arrow). The AT8 antibody stained PBs (C, arrow). Double immunofluorescence staining of an anti-tau antibody with an anti-TG1, anti-TG2 or anti-TG-catalyzed cross-link antibody was performed. TG1 staining was present in PBs (B-D, arrow). Strong TG-catalyzed cross-link immunoreactivity was found in PBs (E-G, arrow). Both 3R tau (H) or 4R tau (I) demonstrate multimerization when co-incubated with TG1 at room temperature for 1 hour, according to the methods described in the materials and methods section. Co-incubation of 3R (H) and 4R (I) tau with TG1, in the presence of BAP (as described in the materials and methods section), demonstrated incorporation of BAP into both low and high molecular weight tau multimers. In contrast, no BAP incorporation was observed in both 3R tau (H) and 4R tau (I) in the absence of TG1. Experiment was repeated 3 times with similar results. Typical experiment is shown. Original magnification: A-G × 400. Abbreviations: TG1 = transglutaminase 1; AT8 = anti-hyperphosphorylated tau antibody; cross-links = TG-catalyzed cross-links; Av/str = avidine/streptavidine.

Figure 4

TIG3 staining in PSP, FTDP-17T and PiD cases. The anti-TIG3 immunoreactivity demonstrated NFT-like neurons in the substantia nigra (SN) of PSP cases (A, arrow). The AT8 antibody stained pretangles (B, arrow) and fibrillary tau containing neurons (E, H, arrow). Double immunofluorescence staining of the AT8 antibody with the anti-TIG3 antibody was performed in SN of PSP cases and the medial frontal gyrus of PiD and FTDP-17T cases. TIG3 was found present in pretangles of PSP cases (B-D, arrow), in fibrillary tau containing neurons in FTDP-17T cases (E-G, arrow), and in PBs in PiD cases (H-J, arrow). Double immunofluorescence staining of the anti-TG1 antibody with the anti-TIG3 antibody was performed in SN of PSP cases. Colocalisation of TG1 staining with TIG3 staining in NFTs in SN of PSP cases was observed (K-M, arrow). Original magnification: A-M × 400. Abbreviations: TIG3 = Tazarotene-induced gene 3; PSP = progressive supranuclear palsy; TG1 = transglutaminase 1; PiD = Pick’s Disease; FTDP-17T = frontotemporal dementia and parkinsonism linked to chromosome 17; AT8 = anti-hyperphosphorylated tau antibody.

Figure 5

Immunoreactivity of the TIG3 antibody in corpora amylacea (CA) in control brain, and its absence in NFTs in AD brain. The anti-TG-catalyzed cross-link antibody stained CA in neocortex of control cases (A, arrow). Double immunofluorescence staining of the anti-TG-catalyzed cross-linking antibody with anti-TIG3 antibody showed colocalization in CA (A-C, arrow). The AT8 antibody stained NFTs in the neocortex of AD cases (D, arrow). Double immunofluorescence staining of the AT8 antibody with the TIG3 antibody demonstrated no colocalization of TIG3 with hyperphosphorylated tau in NFTs of AD cases (D-F, arrow). Original magnification: A-F × 400. Abbreviations: TIG3 = Tazarotene-induced gene 3; AT8 = anti-hyperphosphorylated tau antibody; cross-links = TG-catalyzed cross-links, AD = Alzheimer’s disease.