| Alzheimer disease is a progressive neurodegenerative disease which involves a loss of cognition, memory, language, judgement, and behavior. The neuronal degeneration seen in Alzheimer disease is accompanied by the formation of intraneuronal inclusions known as neurofibrillary tangles, which consist of large aggregates of paired helical filaments (PHFs). The primary proteinaceous constituent of these PHFs is an abnormally phosphorylated form of the microtubule associated protein tau. Although tau is typically soluble, the PHFs contain tau in an insoluble, aggregated state that is resistant to proteolysis. The biochemical properties of PHFs suggest that a covalent cross-linking of tau may occur in Alzheimer disease. Tissue transglutaminase is a calcium-dependent cross-linking enzyme found in a variety of cell types, including neurons. As there are several reports of calcium dysregulation in Alzheimer disease, it is reasonable to hypothesize that increases in intracellular calcium concentrations may pathologically activate tissue transglutaminase, leading to a cross-linking of tau into PHFs. The current studies are designed to assess the tissue transglutaminase-mediated cross-linking of tau as a possible mechanism of neurofibrillary tangle formation in Alzheimer disease. The first study demonstrates that tau is an excellent substrate of tissue transglutaminase in vitro, displaying kinetic parameters that are potentially physiological. Bovine tau and recombinant human tau isoforms rapidly form high molecular weight, cross-linked polymers upon incubation with tissue transglutaminase, and cross-linked tau has an appearance similar to tau from Alzheimer disease brains when visualized on a western blot. The next study investigates protein levels and activity of tissue transglutaminase in Alzheimer disease and control postmortem brain tissue. In Alzheimer disease, both the levels and the activity of tissue transglutaminase are significantly elevated over controls in the prefrontal cortex, which is affected in Alzheimer disease, but not in the cerebellum, which is generally spared. The third study provides a protocol for isolating tissue transglutaminase-mediated epsilon-(gamma-glutamyl)lysine isopeptide bonds from cross-linked substrates, using exhaustive proteolytic digestion, derivatization, and separation with high performance liquid chromatography. Taken together, these studies support the hypothesis that tissue transglutaminase may be involved in the formation of insoluble tau aggregates in Alzheimer disease, and suggest experiments that may be used to investigate the potential involvement of tissue transglutaminase in other paradigms. |
