Transglutaminase-Induced Cross-Linking of Tau Proteins in Progressive Supranuclear Palsy

Abstract

The mechanisms leading to the abnormal self-polymerization of tau into straight and paired helical filaments (PHFs) and neurofibrillary tangles (NFT) in Alzheimer disease (AD) and progressive supranuclear palsy (PSP) are not known. However, transglutaminase-induced cross-linking of PHF-tau was observed in AD and thus may also contribute to the formation of NFT in other neurodegenerative disorders including PSP. Tissue homogenates from PSP and normal age-matched controls were used to immunoaffinity-purify proteins containing transglutaminase-induced epsilon-(gamma-glutamyl) lysine cross-links. The immunoaffinity-purified proteins were then examined on immunoblots with a PHF-tau antibody, PHF-1. There were significantly higher levels of epsilon-(gamma-glutamyl) lysine cross-linking of PHF-tau in globus pallidus and pons regions of PSP cases compared to barely detectable cross-links in controls. The occipital cortex, an area spared from neurofibrillary pathology in PSP, showed no detectable cross-linking of PHF-tau protein in either PSP cases or control cases. Double-label immunofluorescence demonstrated the colocalization of the cross-link and PHF-tau in NFT in pons of PSP Previous studies and present data are consistent with the hypothesis that transglutaminase-induced cross-linking may be a factor contributing to the abnormal polymerization and stabilization of tau in straight and PHFs leading to neurofibrillary tangle formation in neurodegenerative diseases, including PSP and AD.

Tau Protein Is Cross-Linked by Transglutaminase in P301L Tau Transgenic Mice

“…1 A). This protein smear is characteristic of aggregated tau protein in human tauopathies (Norlund et al, 1999;Sergeant et al, 1999;Zemaitaitis et al, 2000;Zhukareva et al, 2002) and is demonstrated in Figure 1 B, in which we loaded an increased amount of cross-linked proteins from P30lL animals on gels in conjunction with cross-linked PHF from the Sarkosyl-insoluble fraction of AD temporal cortex. Both P301L tau transgenic mice and AD tissue exhibit a smear of high-molecular mass crosslinked tau protein, with some cross-linked tau remaining in the stacking gel (Fig.…”

“…Additionally, the 81D4 antibody has been used to quantitate ⑀-(␥-glutamyl) lysine bonds in AD tissue using a competitive ELISA (Nemes et al, 2004). This antibody has also been used extensively in immunoprecipitation experiments that demonstrate the presence of ⑀-(␥-glutamyl) lysine cross-links in AD, PSP, and Parkinson's disease tissue but not in control tissue (Norlund et al, 1999;Zemaitaitis et al, 2000;Andringa et al, 2004). The most convincing support for the specificity of the 81D4 antibody for ⑀-(␥-glutamyl) lysine cross-links comes from in vitro and cell culture experiments.…”

“…Protein concentration was determined using the BCA Protein Assay kit (Pierce). Immunopurification of proteins containing ⑀-(␥-glutamyl) lysine bonds was performed using 81D4 mAb prebound to Sepharose beads (CovalAb) according to the manufacturer protocol and published methods of our laboratory (Norlund et al, 1999;Zemaitaitis et al, 2000). Immunopurified cross-linked proteins were eluted in 25 l of sample buffer containing 2% SDS at 90°C and stored at Ϫ80°C until Western blot analysis, in which 15 or 25 l was loaded in each lane.…”

“…Specifically, two glutamine and 10 lysine residues in tau are modified by transglutaminase in vitro (Murthy et al, 1998). Transglutaminase activity (Johnson et al, 1997;Zemaitaitis et al, 2003) and transglutaminase-catalyzed bonds in PHFs and NFTs (Balin et al, 1999;Norlund et al, 1999;Zemaitaitis et al, 2000;Singer et al, 2002) are significantly elevated in brain regions with abundant neurofibrillary pathology in both AD and PSP. Furthermore, transglutaminase-catalyzed bonds are present in PHF tau before NFTs are microscopically detectable, implicating transglutaminase cross-linking of tau protein as an early event in NFT formation (Singer et al, 2002).…”

Transglutaminase Activity as a Possible Molecular Mechanism in the Etiopathogenesis of Neurodegenerative Diseases

“…By these molecular mechanisms, TGs could contribute to AD symptoms and progression [38] . Moreover, there is evidence that TGs also contribute to the formation of proteinaceous deposits in Parkinson's disease (PD) [39,40] , in supranuclear palsy [41,42] and in HD, a neurodegenerative disease caused by a CAG expansion in the affected gene [43] . For example, expanded polyglutamine domains have been reported to be substrates of TG2 [44][45][46] and therefore aberrant TG activity could contribute to CAG-expansion diseases, including HD (Figure 3).…”