Tissue Transglutaminase Regulatory Mechanism And Its Inhibitor Effects In Alzheimer’s Disease

Alzheimer’s disease(AD)is characterized by intracellular and extracellular protein aggregates,including microtubule associated protein Tau and cleavage product of amyloid precursor protein,β-amyloid(A(3).Tissue transglutaminase(tTG)is a calcium-dependent enzyme that cross-links proteins forming a γ-glutamyl-ε-lysine Isopeptide bond(representing for tTG enzyme activity).Highly resistant to proteolysis,this bond can induce protein aggregation and deposition.Previous studies have shown that tTG and Isopeptide are increased in advanced AD,but they have not addressed if this is an early or late feature of AD.So,we are intented to evaluate tTG alterations and effects in AD process.In the present study,we measured tTG expression levels and enzyme activity in brains of individuals with no cognitive impairment(NCI),mild cognitive impairment(MCI)and AD,as well as a transgenic mouse model of AD with Western Blot and Immunohistochemistry.We find that both enzyme expression and activity are increased in MCI as well as AD compared to NCI.In the transgenic model of AD,tTG expression and enzyme activity increased sharply with age and were relatively specific for the hippocampus.We also assessed overlap of Isopeptide immunoreactivity with neurodegeneration-related proteins with Multiple Immunoblot and find neurofilament,Tau and Aβ show co-localization with Isopeptide in both AD and transgenic mice.These results suggest that tTG might be a key factor in pathogenesis of abnormal protein aggregation in AD.It is reasonable to speculate tTG as a target in AD therapatic studies.In our further study,we examined the effect of an irreversible tTG inhibitor NTU283 on the condition of Aβ mimic AD pathogenesis in SH-SY5Y cells.Western blot and In-cell Western were applied to detect tTG and Isopeptide protein levels.Moreover,Hoechst and PI co-staining,Caspase-3 and Caspase-7 activities and Bax/Bcl-2 ratio were determined to evaluate the effect of NTU283 on apoptotic process.The results confirmed tTG activity was inhibited by NTU283 in a concentration dependent manner in SH-SY5Y cells.Contrary to our expectation,however,the Isopeptide bonds were increased when co-treated cells with Aβ and NTU283.In addition,NTU283 alone did not induce apoptosis of SH-SY5Y cell.But when co-treated cells with Aβ,NTU283 promoted rather than inhibited Aβ-induced apoptosis.Consistent with apoptotic rate,pretreating cells with different concentration of NTU283,Aβ significantly increased the activities of Caspase-3 and Caspase-7 as well as the ratio of Bax/Bcl-2.The results demonstrated for the first time that irreversible inhibition of tTG activity could not block,but promote Aβ-induced apoptosis,which might imply more complicated functions of tTG in AD pathogenesis.Thus,it is very important to choose inhibitor types of tTG in further AD therapeutic studies.