Proteolytic Processing of APP and its family members APLP1 and APLP2
APP (amyloid precursor protein) is the precursor of the Aβ peptide, which accumulates in plaques in brains of Alzheimer's disease (AD) patients. The canonical processing of APP is described by the amyloidogenic and the non-amyloidogenic pathway (Fig. 1). The non-amyloidogenic pathway starts with cleavage of full-length APP by α-secretase activity. Cleavage by α-secretase (ADAM 10) results in the release of the ectodomain of APP -soluble APPα (sAPPα)- and the concomitant generation of a membrane retained C-terminal fragment (CTF) consisting of 83 amino acids (aa) (C83 or α-CTF). C83 is further processed by γ-secretase, a transmembrane multiprotein complex consisting of four subunits: presenilin 1 or 2, nicastrin, APH-1, and PEN-2. y-Secretase cleaves within the transmembrane domain, a mechanism termed regulated intramembrane proteolysis (RIP). APP CTFs are proteolytically processed at three positions: first at the ε-cleavage site, then the ζ-cleavage site, and finally, at the γ-cleavage site. This leads to the release of a short peptide termed p3 and of the APP intracellular domain (AICD).
Instead of α-/γ-secretase processing, APP can also be cleaved in the amyloidogenic pathway. Here, it is first cleaved N-terminally of the Aβ sequence by the β-secretase β-site APP cleaving enzyme 1 (BACE1). This results in shedding of the APP ectodomain -soluble APPβ (sAPPβ)-and the production of a 99 aa CTF (C99 or β-CTF). C99 is subsequently cleaved by γ-secretase, releasing the Aβ peptide and AICD.
We were able to identify in this pathway a new cleavage site in APP, the so called ε-cleavage site, which is carried out by the γ-secretase complex and the starting point to the generation of the APP intracellular domain (AICD) and the Aβ/p3 peptides (Weidemann, Eggert et al., 2003). APP has two mammalian homologues, the APP gene family members APLP1 and APLP2. We demonstrated that APLP1 and APLP2 are processed by the same secretases as APP, at a time when the only other known γ-secretase substrate was the Notch receptor (Eggert et al., 2004). Furthermore, we investigated APP processing at the synapse and could demonstrate that shedding of APP limits its synaptogenic activity and cell-cell adhesion properties (Stahl et al., 2014).
Interestingly, APP familial Alzheimer’s disease (FAD) mutations are located around all three secretase cleavage sites in APP and lead to an early onset of the disease process before the age of 65. We are interested to find out the underlying mechanism of APP FAD mutations in more detail.
Induced dimerization of APP, APLP1, and APLP2 via the FKBP-rapamycine system
APP can dimerize in cis- and trans- orientation in a homotypic or heterotypic manner with its family members APLP1 and APLP2. We are working with a system, which allows induction of APP/APLP dimerization via an APP-FKBP fusion protein after the addition of an analogue of rapamycine, which binds two FKBP molecules (Eggert et al., 2009). This study includes Blue-Native gel analyses to identify APP complexes. Using this system we could demonstrate, that induced dimerization of APP leads to decreased Ab production (Eggert et al., 2009). We extended our studies on APP cis- dimerization and described via different immunocytochemical assays with different markers and via live cell imaging analyses in primary neurons the localization of APP cis- dimers, which are accumulating in endosomes while velocities are unchanged (Eggert et al., 2018). We are interested to intensify our research with this system using proteomic approaches to identify APP signaling partners and to elucidate the physiological function of APP dimerization at the synapse.
The APP family members as synaptic adhesion proteins
APP and its family members can dimerize in trans-orientation to mediate cell-cell adhesion (Soba et al., 2005). We were able to show that APP, APLP1, and APLP2 belong to the family of synaptic adhesion molecules (SAMs) (Schilling et al., 2017). APP/APLPs fulfill the features of SAMs, like upregulated expression during synaptogenesis, pre- and postsynaptic localization, synapse inducing properties, and loss of function influences synapse formation (Schilling et al. 2017, Tyan et al., 2012, Midthune et al., 2012). Further studies showed that the trans- interaction of APP is regulated by its proteolytic processing (Stahl et al., 2014). We are interested to analyze the signaling pathways of the APP family members, which modulate the differentiation of pre- and postsynapses. Furthermore, we aim to investigate the interplay of the APP family members with other synaptic adhesion molecules.