Research reportElevated transglutaminase-induced bonds in PHF tau in Alzheimer's disease
Introduction
Alzheimer's disease (AD) is characterized neuropathologically by an accumulation of insoluble protein aggregates known as amyloid plaques and neurofibrillary tangles. These neuropathological lesions develop in selectively vulnerable cortical regions including the entorhinal and orbital cortices as well as in the hippocampus, with relative sparing of the motor cortex and the cerebellum 1, 2. While amyloid plaques are rich in β-amyloid (Aβ), neurofibrillary tangles are composed of straight and paired helical filaments (PHF) which are primarily made up of hyperphosphorylated tau proteins [3]. The mechanism(s) responsible for the aggregation and stabilization of proteins into neurofibrillary tangles in AD brain are not known. Other post-translational modifications of tau are likely to be involved in the assembly of tau into neurofibrillary tangles. For example, tau filaments in AD are glycated, however glycation does not appear to inhibit the ability of tau to promote microtubule assembly 4, 5. Although disulfide bridges between tau proteins can cause dimer formation, higher molecular weight complexes do not form 6, 7. In vitro, tau proteins will form structures resembling straight and PHFs in the presence of free fatty acids, RNA, or sulfated glycosaminoglycans 8, 9, 10, 11. It is not known if any of these molecules interact in vivo with tau to form PHF. Interestingly, the physical and biochemical properties of plaques and tangles resemble those of protein polymers containing transglutaminase-induced ε-(γ-glutamyl)lysine cross-links [12].
Transglutaminase enzymes are a family of calcium-dependent enzymes that specifically introduce intra-molecular and inter-molecular covalent ε-(γ-glutamyl)lysine bonds in peptides, forming very stable high molecular weight polymers [13]. ε-(γ-glutamyl)lysine cross-linked polymers form protein aggregates that are extremely resistant to proteolysis and degradation and are insoluble in detergents and reducing agents, all of which are properties that characterize neurofibrillary tangles in AD 14, 15, 12. Previous in vitro studies have demonstrated that the microtubule-associated tau proteins are excellent substrates for transglutaminase enzymes forming insoluble aggregates composed of high molecular weight protein polymers 14, 16. Recent studies have demonstrated that transglutaminase enzymes are present within degenerating neurons in AD [17]. Furthermore, transglutaminase activity is elevated in the neocortex in AD as compared with age-matched non-AD controls [18].
We hypothesized that a neuropathological increase in transglutaminase-induced ε-(γ-glutamyl)lysine covalent cross-links induces an aberrant polymerization of tau leading to the formation of stable high molecular weight protein polymers in the brain. Therefore in the following study, we measured transglutaminase-induced ε-(γ-glutamyl)lysine covalent cross-links in human AD and in control postmortem cortical tissue.
Section snippets
Human brain tissue
Fresh-frozen (−80°C) postmortem human AD (70–87 years of age) and control (45–85 years of age) brain tissue was obtained from the Loyola University/Hines VA Brain Bank. Brain tissue consisted of gray matter from the frontal and parietal cortices. All AD cases contained moderate to severe neuropathology as defined by the CERAD criteria using sections stained by a modification of the Beilshowsky method [19]. All control cases lacked any known history of neurological deficits or neurodegenerative
Levels of ε-(γ-glutamyl)lysine cross-links in PHF tau and normal CNS tau
To determine if the levels of the ε-(γ-glutamyl)lysine cross-linking is higher in PHF tau compared to normal tau, the levels of ε-(γ-glutamyl)lysine cross-links were measured using immunoblots. Protein fractions rich in normal soluble tau were isolated from both AD and control frontal and parietal cortices. Immunoblots prepared with normal soluble tau from both AD and controls failed to demonstrate any 81D1c2 MAb immunolabelling. Next, the levels of ε-(γ-glutamyl)lysine cross-links in PHF tau
Discussion
Cytoskeletal proteins are natural substrates for transglutaminase-induced protein cross-linking 37, 38, and our results identified ε-(γ-glutamyl)lysine cross-links in neurofilament proteins and very low levels of cross-links in normal CNS tau proteins within cortical regions of non-AD controls. However, in AD, degenerating cortical regions with numerous plaques and tangles contained significantly elevated levels of ε-(γ-glutamyl)lysine cross-linked proteins as compared to cortical tissue from
Acknowledgements
The authors would like to thank Dr. Peter Davies (Albert Einstein School of Medicine) for his generous donation of several antibodies including PHF-1 and Dr. Lester Binder (Northwestern University Medical School, Chicago, IL) for donating the Tau-5 MAb. We would also like to extend our gratitude to Dr. Benjamin Wolozin, Dr. Wilfred Pinto, Dr. Shih-Yen Tsai, and Dr. Robert Handa (Loyola University Chicago Medical Center, IL), for their valuable advice and technical support. This work was
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2020, Handbook of Behavioral NeuroscienceCitation Excerpt :There is abundant evidence that proteins that aggregate and form inclusion bodies in neurodegenerative diseases are transamidated by TGase (Muma, 2007). The proteins that have been reported to be substrates for TGase include microtubule associated tau protein (Citron et al., 2002; Norlund, Lee, Zainelli, & Muma, 1999; Singer, Zainelli, Norlund, Lee, & Muma, 2002; Zemaitaitis, Lee, Troncoso, & Muma, 2000) which forms neurofibrillary tangles in tauopathies such as Alzheimer's disease and progressive supranuclear palsy, beta amyloid which forms amyloid plaques in Alzheimer's disease (Schmid et al., 2011), alpha-synuclein which forms Lewy bodies in Parkinson's disease (Andringa et al., 2004; Junn, Ronchetti, Quezado, Kim, & Mouradian, 2003), and mutant huntingtin protein which forms inclusion bodies in the nucleus and perinuclear regions of neurons in Huntington's disease (Karpuj, Becher, & Steinman, 2002; Zainelli, Ross, Troncoso, & Muma, 2003). These transamidated proteins form high-molecular aggregates of cross-linked proteins.
Identification of brain substrates of transglutaminase by functional proteomics supports its role in neurodegenerative diseases
2017, Neurobiology of DiseaseCitation Excerpt :Intracellular aggregates containing the protein Tau crosslinked by transglutaminase accumulate in the brain of transgenic mice that simulate tauopathies (Halverson et al., 2005). ε-(γ-glutamyl)lysine crosslinks between Tau and neurofilament proteins are detected in neurons of patients with Alzheimer's disease (Norlund et al., 1999). Tau and neurofilament proteins have been shown to be TG-2 substrates in transfected COS cells (Grierson et al., 2001).
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2013, Protein Expression and PurificationNovel role of transglutaminase 1 in corpora amylacea formation?
2011, Neurobiology of AgingCitation Excerpt :Interestingly, proteins such as Aβ (Dudek and Johnson, 1994), (hyperphosphorylated) tau (Dudek and Johnson, 1993; Dudek and Johnson, 1994), as well as α-synuclein (Andringa et al., 2004; Segers-Nolten et al., 2008) are substrates for TG-catalyzed cross-linking. TG-catalyzed cross-linking of these proteins generally results in the acceleration of their aggregation rate and increases their resistance towards proteolytic breakdown (Hartley et al., 2007; Konno et al., 2005a,b; Norlund et al., 1999; Segers-Nolten et al., 2008; Singer et al., 2002). Furthermore, cytoskeletal proteins, which are suggested to be abundantly present in CA, are well-known substrates for TGs (Miller and Anderton, 1986).
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