Functional role of transmembrane domain interactions and intramembrane cleavage of microglial innate immunity receptors

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This project addresses a central question of this research unit, namely what defines a substrate to be cleaved by intramembrane cleaving proteases. Although many substrates of gamma-secretase have been identified until today, we still do not know the exact determinants, which make a transmembrane domain of a type 1 protein accessible for gamma-secretase processing or not. This question is not only of academic interest, but is also of greatest importance for therapeutic gamma-secretase inhibition in Alzheimer's disease patients. TREM2 processing is highly relevant for the physiological activity of microglia cells, and mutations, which have been linked to Alzheimer's disease and many other neurodegenerative disorders, were shown in our previous work to affect proteolytic processing of TREM2 and the phagocytic activity of microglia cells. TREM2 binds in vivo to TYROBP (DAP12). Whereas unbound TREM2 appears to be a good substrate for intramembrane proteolysis after shedding, DAP12, which lacks a N-terminal domain, whose removal appears to be an absolute requirement for all gamma-secretase substrates, may not be cleaved by gamma-secretase. DAP12 is known to form a highly stable homodimer. By this, we have potential substrates available, the interaction and processing of which play an essential role in microglial phagoytosis. Thus, by investigating the physiologically and pathologically highly relevant TREM2/DAP12 and DAP12/DAP12 interactions as an in vivo model we can address the questions if interactions of transmembrane domains influence intramembrane proteolysis and if transmembrane domains, which occur under physiological conditions as dimers, can be cleaved by gamma-secretase. We hypothesize that the tight interaction of the transmembrane domains of TREM2 and DAP12 may prevent or even regulate subsequent intramembrane proteolysis. This may also further affect signaling processes, which are required to initiate microglial phagocytosis. Moreover, we may be able to identify DAP12 as a gamma-secretase non-substrate although it apparently fulfills all requirements of a bona fide gamma-secretase substrate. DAP12 will then be used as a scaffold to confirm requirements of gamma-secretase substrates identified by the members of the research unit. Thus our project provides a naturally occurring biologically highly relevant test system to address central questions of this research unit.Taken together, we will elucidate determinants for gamma-secretase-cleavage (goal 1), as well as the role of transmembrane domain dimerization for intramembrane cleavage and signaling (goal 2) in vivo.