Identification of calcium and integrin‐binding protein 1 as a novel regulator of production of amyloid β peptide using CRISPR/Cas9‐based screening system

Abstract

The aberrant metabolism of amyloid β peptide (Aβ) has been implicated in the etiology of Alzheimer disease (AD). Aβ is produced via the sequential cleavage of amyloid precursor protein (APP) by β‐ and γ‐secretases. However, the precise regulatory mechanism of Aβ generation still remains unclear. To gain a better understanding of the molecular mechanism of Aβ production, we established a genetic screening method based on the CRISPR/Cas9 system to identify novel regulators of Aβ production. We successfully identified calcium and integrin‐binding protein 1 (CIB1) as a potential negative regulator of Aβ production. The disruption of Cib1 significantly upregulated Aβ levels. In addition, immunoprecipitation experiments demonstrated that CIB1 interacts with the γ‐secretase complex. Moreover, the disruption of Cib1 specifically reduced the cell‐surface localization of mature Nicastrin (Nct), which is a component of the γ‐secretase complex, without changing the intrinsic activity of γ‐secretase. Finally, we confirmed using the single‐cell RNA‐seq data in human that CIB1 mRNA level in neuron was decreased in the early stage of AD. Taken together, our results indicate that CIB1 regulates Aβ production via controlling the subcellular localization of γ‐secretase, suggesting CIB1 is involved in the development of AD.

EphA4 regulates Aβ production via BACE1 expression in neurons

“…We found loss of CALM decreases the endocytosed γ‐secretase, resulting in the decreased production ratio of the pathogenic Aβ species, Aβ42 11 . Recently, we also identified calcium and integrin‐binding protein 1 (CIB1) as a novel negative regulator of Aβ production by CRISPR/Cas9 screening 12 …”

“…For secreted Aβ from primary cells, Aβ levels were analyzed by two-site enzymelinked immunosorbent assay (ELISA) using Human/Rat β Amyloid 30 ELISA Kit (294-64701, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and Human/Rat β -Amyloid 31 ELISA Kit (High Sensitivity [292-64501, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan]), as described. 12,32 The secreted Aβ from N2a cells was analyzed by ELISA using a homemade Aβ detecting plate based on the same principle of manufacturer's ELISA Kit. 9 Aβ levels measured by ELISA were then standardized by protein concentrations of the cell lysates and further normalized to the control in each experiment as indicated.…”

“…11 Recently, we also identified calcium and integrin-binding protein 1 (CIB1) as a novel negative regulator of Aβ production by CRISPR/Cas9 screening. 12 Eph receptors are synaptic adhesion molecules and are large receptors with tyrosine kinase activity. Eph receptors are classified into two subclasses, EphA and EphB, depending on their structural similarities and binding affinities to their ligands, the ephrins.…”

EphA4 regulates Aβ production via BACE1 expression in neurons

“…For the measurement of the secreted Aβ, conditioned media were collected, and cell debris was removed by the centrifugation at 240 x g for 3 min. For secreted Aβ from primary cells, Aβ levels were analyzed by two-site enzyme-linked immunosorbent assay (ELISA) using Human/Rat β Amyloid (40) ELISA Kit (294-64701, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and Human/Rat β -Amyloid (42) ELISA Kit, High Sensitivity (292-64501, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), as described (23,24). The secreted Aβ from N2a cells was analyzed by ELISA using a homemade Aβ detecting plate based on the same principle of manufacturer's ELISA Kit (9).…”

“…All procedures of immunoblotting were performed as previously described (24). Brie y, for sample preparation, cells were lysed by Laemlli 1X sample buffer (2% sodium dodecyl sulfate (SDS; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 80 mM Tris-HCl with pH 6.8, 15% glycerol (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 0.0025% Brilliant green (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 0.00625% Coomassie Brilliant Blue G-250 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan)) and sonicated (BRANSON, Danbury, CT, USA).…”