Eta-secretase-like processing of the amyloid precursor protein (APP) by RHBDL4

Abstract

The amyloid precursor protein (APP) has been extensively studied with regards to its contribution to the pathology of Alzheimer's disease. APP is an ubiquitously expressed type I transmembrane protein synthesized in the endoplasmic reticulum (ER) and translocated to the plasma membrane where it undergoes proteolytic cleavages by several identified proteases. Conversely to other known proteases, we previously elucidated human rhomboid protease RHBDL4 as a novel APP processing enzyme where several cleavages likely occur already in the ER. Interestingly, the pattern of RHBDL4-derived large APP C-terminal fragments resemble those generated by the η-secretase or MT5-MMP, which was described to generate so called Aη fragments. The similarity in large APP C-terminal fragments between both proteases raised the question whether RHBDL4 may contribute to η-secretase activity and Aη-like fragments. Here, we identified two cleavage sites of RHBDL4 in APP by mass spectrometry, which, intriguingly, lie in close proximity to the cleavage sites of MT5-MMP. Indeed, we observed that RHBDL4 generates Aη-like fragments in vitro without contributions of α-, β-, or γ-secretases. Such Aη-like fragments are likely generated in the ER since RHBDL4-derived APP-C-terminal fragments do not reach the cell surface. Inherited, familial APP mutations appear to not affect this processing pathway. In RHBDL4 knockout mice, we observed increased cerebral full length APP levels in comparison to WT brains in support of RHBDL4 being a physiologically relevant protease for APP. Furthermore, we found secreted Aη fragments in dissociated mixed cortical cultures from wild type mice, however significantly less Aη fragments in cultures from RHBDL4 knockout mice. Our data underscores that RHBDL4 contributes to η-secretease-like processing of APP and that RHBDL4 is a physiologically relevant protease for APP.

Loss of the APP regulator RHBDL4 preserves memory in an Alzheimer’s disease mouse model

“…First, we confirmed our findings that RHBDL4 KO mice show increased endogenous APP levels as compared to WT (Fig. 2C, Penalva et al, 2023). In addition, in the APPtg model, RHBDL4 loss resulted in higher total APP protein expression (Fig.…”

“…We showed in HEK293T cells that RHBDL4 cleavage of APP led to the production of RHBDL4-specific APP fragments and, decreased cell surface levels of APP and Aβ production . We further confirmed that RHBDL4 cleavage of APP occurs in primary mixed cortical cultures (Penalva et al, 2023). Specifically, we found that RHBDL4 knockout (R4 KO) mixed cortical cultures did not produce an RHBDL4-specific APP fragment termed "Aη-like" for its resemblance to the APP fragment originally described to be produced by MT5-MPP by Willem et al (Penalva et al, 2023;Willem et al, 2015).…”

“…We have previously described RHBDL4 as a regulator of APP biology in cell culture. We showed that RHBDL4 was a major producer of physiologically relevant Aη-like peptides and competed with other significant APP processing pathways such as the amyloidogenic pathway Penalva et al, 2023). Herein, we demonstrate the relevance of RHBDL4 in APP homeostasis in vivo and more importantly describe a new role of RHBDL4 in AD through its β-catenin regulating function.…”

“…Specifically, we found that RHBDL4 knockout (R4 KO) mixed cortical cultures did not produce an RHBDL4-specific APP fragment termed "Aη-like" for its resemblance to the APP fragment originally described to be produced by MT5-MPP by Willem et al (Penalva et al, 2023;Willem et al, 2015). We also observed that APP expression was elevated in brain lysates from R4 KO mice in comparison to controls, suggesting a physiological relevance of RHBDL4 for APP in vivo (Penalva et al, 2023).…”