Pivotal role of RanBP9 in integrin‐dependent focal adhesion signaling and assembly

Abstract

Accumulation of the amyloid β (Aβ) peptide derived from the amyloid precursor protein (APP) plays a central role in the pathogenesis of Alzheimer's disease (AD). We previously reported that the scaffolding protein RanBP9 is markedly increased in AD brains and promotes Aβ generation by scaffolding APP/BACE1/LRP complexes together and accelerating APP endocytosis. Because APP, LRP, and RanBP9 all physically interact with β-integrins, we investigated whether RanBP9 alters integrin-dependent cell adhesion and focal adhesion signaling. Here, we show that RanBP9 overexpression dramatically disrupts integrin-dependent cell attachment and spreading in NIH3T3 and hippocampus-derived HT22 cells, concomitant with strongly decreased Pyk2/paxillin signaling and talin/vinculin localization in focal adhesion complexes. Conversely, RanBP9 knockdown robustly promotes cell attachment, spreading, and focal adhesion signaling and assembly. Cell surface biotinylation and endocytosis assays reveal that RanBP9 overexpression and RanBP9 siRNA potently reduces and increases surface β1-integrin and LRP by accelerating and inhibiting their endocytosis, respectively. Primary hippocampal neurons derived from RanBP9-transgenic mice also demonstrate severely reduced levels of surface β1-integrin, LRP, and APP, as well as neurite arborization. Therefore, these data indicate that RanBP9 simultaneously inhibits cell-adhesive processes and enhances Aβ generation by accelerating APP, LRP, and β1-integrin endocytosis.

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

“…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. Therefore, we propose that disruption of cell adhesive processes via deregulation of integrin-dependent focal adhesions and activation of cofilin underlie both Ab and RanBP9-induced increase in the vulnerability of neurons to undergo neuritic dysfunction, synapse loss, and mitochondria-mediated apoptotic process (Figure 8).…”

“…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. 9 In this study, we found that RanBP9 levels are increased not only in brains of AD patients but also in APP transgenic mice. Increased RanBP9 expression significantly induces gliosis, neurodegeneration, synapse loss, and spatial memory deficits.…”

“…Given the observed activation of cell death pathways and neurodegeneration in RanBP9 transgenic mice, we next assessed whether RanBP9 promotes cell death in a cell culture model. Under serum withdrawal conditions (2% serum) for 12 h in HT22 cells (hippocampus-derived neuroblastoma cell line), 9,11 RanBP9 transfection effectively doubled the number of cells at the early stages of apoptosis (lower right quadrant) as measured by Annexin V/PI staining and FACS (Supplementary Figure S5A). Similar to that seen in HT22 cells, RanBP9 or GFP-RanBP9 transfections doubled more than the amount of LDH release compared with the vector control or EYFP transfections in H4 neuroglioma cells (Supplementary Figures S5B and C).…”

A C-Terminal Transmembrane Anchor Targets the Nuage-Localized Spermatogenic Protein Gasz to the Mitochondrial Surface

“…We thus cotransfected EGFP-Gasz with its protein partners and examined the effects on Gasz localization. RanBP9 is a scaffold protein that has been reported to function as a signaling platform and interact with a variety of partners including nuclear receptors, cytoplasmic kinases, receptor tyrosine kinases, and centrosome-localized proteins [20, 21]. When transfected into HeLa cells, we observed cytoplasmic and microtubular-appearing patterns of distribution, but no effect on the mitochondrial localization of Gasz was noted (Figure 4(a)).…”

COPS5 Protein Overexpression Increases Amyloid Plaque Burden, Decreases Spinophilin-immunoreactive Puncta, and Exacerbates Learning and Memory Deficits in the Mouse Brain

“…Importantly, COPS5 not only binds RanBP9, BACE1, LRP, and APP (27), which are all known to increase A␤ generation by targeting APP to lipid rafts, but we and others have shown previously that COPS5 binds LFA-1 (41,42), and LFA-1, in turn, binds LRP (43). Interestingly, we also showed that RanBP9 increases endocytosis of LFA-1, LRP, and APP, thereby regulating cell adhesion and A␤ generation (44). By increasing endocytosis of LFA-1 and other integrins that are known to transduce synaptic plasticity signals, the COPS5/RanBP9 pathway may indirectly regulate synaptic alterations and behavioral deficits.…”