Transglutaminase 2 in calcium homeostasis and neurodegeneration

Transglutaminase 2 (TG2) is a calcium-dependent protein crosslinking enzyme activated in misfolding diseases and it is implicated in multiple disorders linked to calcium dysregulation, including neurodegeneration. In vitro, TG2 has been involved in the generation of toxic amyloid-β (Aβ) oligomers by post-translational modification (PTM), and literature data suggest that TG2 is activated in disease, e.g. the early stages of Alzheimer’s disease (AD). TG2 is also involved in cell-matrix dynamics and has been suggested to be a cargo of extracellular vesicles (EVs) in cancer and tissue fibrosis. EVs have been implicated in the spreading of pathogenic proteins in neurodegenerative diseases (e.g. Aβ and tau) and represent a new field of research in dementia. The aims of this study are to: i. investigate the role of extracellular TG2 in neuron-glia cross-talk in the context of neurodegeneration; ii. explore substrates of TG2 PTM in a cell model simulating AD; iii. evaluate TG2 as a potential marker of dementia. To this purpose, both primary cells (embryonic rat brain cells) and biological samples from dementia patients were analysed. We found that when raised at levels compatible with inflammatory states, extracellular TG2 consistently increased basal calcium concentration ([Ca2+]i) in hippocampal neurons, affecting calcium homeostasis, which is at the basis of neuronal functions. This effect was mediated by TG2- driven membrane depolarisation, which may be caused by the interaction of TG2 with plasma membrane ionic channels [i.e. Voltage Operated Calcium Channels (VOCCs) and Na+ /Ca2+ exchanger (NCX)]. We confirmed previous evidence showing that astrocytes are a rich source of extracellular TG2 in brain and showed for the first time that TG2 is released as a cargo of astrocytic EVs. Simulation of AD pathology in primary hippocampal neurons stimulated with Aβ1-42 led to the identification of 11 TG2 substrates (TG2 transamidome) using a global quantitative proteomic approach (SWATH™-MS/MS proteomics). These included proteins involved in ion transport [Plasma Membrane Ca2+ Transporting ATPase 2 (AT2B2) and Transmembrane Channel-like Protein 5 (TMC)], which could be involved in TG2-mediated alteration of calcium homeostasis in pathology. We also found that a number of neurotrophic proteins involved in neuronal growth and apoptosis were significantly decreased upon TG2 inhibition, suggesting that TG2 might play a dual role in neuronal survival during neurodegeneration. Finally, analysis of plasma from 45 dementia patients and healthy controls by an optimised ELISA assay revealed no significant changes in TG2 between the study groups. Our preliminary data suggest that quantitation of TG2 should be performed in plasma-derived EVs for a more accurate and sensitive evaluation