Novel Role of RanBP9 in BACE1 Processing of Amyloid Precursor Protein and Amyloid β Peptide Generation

Abstract

Accumulation of the amyloid ␤ (A␤) peptide derived from the proteolytic processing of amyloid precursor protein (APP) is the defining pathological hallmark of Alzheimer disease. We previously demonstrated that the C-terminal 37 amino acids of lipoprotein receptor-related protein (LRP) robustly promoted A␤ generation independent of FE65 and specifically interacted with Ran-binding protein 9 (RanBP9). In this study we found that RanBP9 strongly increased BACE1 cleavage of APP and A␤ generation. This pro-amyloidogenic activity of RanBP9 did not depend on the KPI domain or the Swedish APP mutation. In cells expressing wild type APP, RanBP9 reduced cell surface APP and accelerated APP internalization, consistent with enhanced ␤-secretase processing in the endocytic pathway. The N-terminal half of RanBP9 containing SPRY-LisH domains not only interacted with LRP but also with APP and BACE1. Overexpression of RanBP9 resulted in the enhancement of APP interactions with LRP and BACE1 and increased lipid raft association of APP. Importantly, knockdown of endogenous RanBP9 significantly reduced A␤ generation in Chinese hamster ovary cells and in primary neurons, demonstrating its physiological role in BACE1 cleavage of APP. These findings not only implicate RanBP9 as a novel and potent regulator of APP processing but also as a potential therapeutic target for Alzheimer disease. The major defining pathological hallmark of Alzheimer disease (AD)2 is the accumulation of amyloid ␤ protein (A␤), a neurotoxic peptide derived from ␤-and ␥-secretase cleavages of the amyloid precursor protein (APP). The vast majority of APP is constitutively cleaved in the middle of the A␤ sequence by ␣-secretase (ADAM10/TACE/ADAM17) in the non-amyloidogenic pathway, thereby abrogating the generation of an intact A␤ peptide. Alternatively, a small proportion of APP is cleaved in the amyloidogenic pathway, leading to the secretion of A␤ peptides (37-42 amino acids) via two proteolytic enzymes, ␤-and ␥-secretase, known as BACE1 and presenilin, respectively (1).The proteolytic processing of APP to generate A␤ requires the trafficking of APP such that APP and BACE1 are brought together in close proximity for ␤-secretase cleavage to occur. We and others have shown that the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytosis receptor (2), binds to APP and alters its trafficking to promote A␤ generation. The loss of LRP substantially reduces A␤ release, a phenotype that is reversed when full-length (LRP-FL) or truncated LRP is transfected in LRP-deficient cells (3, 4). Specifically, LRP-CT lacking the extracellular ligand binding regions but containing the transmembrane domain and the cytoplasmic tail is capable of rescuing amyloidogenic processing of APP and A␤ release in LRP deficient cells (3). Moreover, the LRP soluble tail (LRP-ST) lacking the transmembrane domain and only containing the cytoplasmic tail of LRP is sufficient to enhance A␤ secretion (5). This activity of LRP-ST is achieved by promoting APP/BACE1 intera...

Pivotal role of the RanBP9-cofilin pathway in Aβ-induced apoptosis and neurodegeneration

“…Although RanBP9 has previously been implicated in the promotion of cell death, the mechanisms of such activity are unknown. 25 We have previously shown that RanBP9 accelerates APP, LRP, and b1-integrin endocytosis, simultaneously leading to increased Ab generation in vitro and in vivo 6,7 and disruption of focal adhesions. 9 As such, our observation that RanBP9 activates/dephosphorylates cofilin is consistent with the known role of the integrin-LIMK pathway in the inactivation/phosphorylation of cofilin, although the Slingshot pathway cannot be ruled out.…”

“…We previously showed that increased RanBP9 expression promotes BACE1 processing of APP and Ab generation by scaffolding APP/BACE1/LRP complexes together and accelerating APP endocytosis. 6 To determine whether RanBP9-induced cell death is dependent on its ability to promote Ab generation, we treated HT22 cells with or without 5 mM g-secretase inhibitor, DAPT, for 24 h under normal cell culture conditions (10% FBS). In the absence of Ab oligomer treatment, no detectable Annexin V or PI staining could be observed in control empty vector-transfected cells in the presence or absence of g-secretase inhibition ( Figure 6).…”

“…Such interactions of RanBP9 promote the endocytosis of APP and strongly increase BACE1 cleavage of APP to generate Ab in vitro and in vivo. 6,7 Conversely, siRNA knockdown of RanBP9 reduces BACE1 cleavage of APP and Ab generation, indicating that endogenous RanBP9 normally functions in this capacity. 6 In addition, a 60-kD proteolytic fragment of RanBP9 is increased by more than sixfold in brains of AD patients, and this fragment potentiates Ab generation via BACE1 processing of APP.…”

“…6,7 Conversely, siRNA knockdown of RanBP9 reduces BACE1 cleavage of APP and Ab generation, indicating that endogenous RanBP9 normally functions in this capacity. 6 In addition, a 60-kD proteolytic fragment of RanBP9 is increased by more than sixfold in brains of AD patients, and this fragment potentiates Ab generation via BACE1 processing of APP. 8 In addition to promoting Ab generation by accelerating APP endocytosis, RanBP9 also potently disrupts integrin-dependent focal adhesion signaling and assembly by accelerating b1-integrin and LRP endocytosis.…”

Trafficking and Proteolytic Processing of APP

“…Lack of LRP expression causes reduced APP internalization and Ab secretion (Ulery et al 2000;Pietrzik et al 2002;Cam et al 2005), leading to the conclusion that endocytosis of LRP is coupled to APP internalization and processing, and Fe65 acts as a functional linker between APP and LRP in modulating endocytic APP trafficking (Pietrzik et al 2004). In addition, Ran-binding protein 9 promotes APP interaction with APP and facilitates APP internalization in a Fe65-independent manner (Lakshmana et al 2009). Finally, the type I transmembrane protein sorLA/LR11 (a member of the VPS10p-domain receptor family), which functionally interacts with cytosolic adaptors GGA and PACS-1, regulates Ab production by acting as a Golgi/TGN retention factor for APP (Andersen et al 2005;Offe et al 2006;Schmidt et al 2007).…”