Novel presenilin 1 and 2 double knock-out cell line for in vitro validation of PSEN1 and PSEN2 mutations

Abstract

Mutations in APP (amyloid precursor protein), PSEN1 (presenilin 1) or PSEN2 (presenilin 2) are the main cause of early-onset familial forms of Alzheimer's disease (autosomal dominant AD or ADAD). These genes affect γ-secretase-dependent generation of Amyloid β (Aβ) peptides, the main constituent of amyloid plaques and one of the pathological hallmarks of AD. Evaluation of patients with ADAD includes assessment of family history, clinical presentation, biomarkers, neuropathology when available and DNA sequencing data. These analyses frequently uncover novel variants of unknown significance in ADAD genes. This presents a barrier to recruitment of such individuals into clinical trials, unless a biochemical test can demonstrate that a novel mutation results in altered APP processing in a manner consistent with pathogenicity. Here we describe generation and characterization of a novel presenilin 1 and 2 double knock-out in N2A mouse neuroblastoma cells using CRISPR/Cas9, which results in complete ablation of Aβ production, decreased Pen-2 expression and Nicastrin glycosylation. Because of the absence of background Aβ secretion from endogenous γ-secretases, these cells can be used for validation of PSEN1 and PSEN2 variant effects on production of Aβ or other γ-secretase substrates and for biochemical studies of γ-secretase function using novel variants. We examined several PSEN1 and PSEN2 mutations of known and unknown pathogenicity. Known mutants increased Aβ42/Aβ40 ratio with varying effect on Aβ40, Aβ42, total Aβ levels and Pen-2 expression, which aligns with previous work on these mutants. Our data on novel PSEN1 V142F, G206V and G206D mutations suggest that these mutations underlie the reported clinical observations in ADAD patients. We believe our novel cell line will be valuable for the scientific community for reliable validation of presenilin mutations and helpful in defining their pathogenicity to improve and facilitate evaluation of ADAD patients, particularly in the context of enrollment in clinical trials.

Keywords: Autosomal dominant Alzheimer's disease; Aβ pathogenicity; Presenilin 1 and 2 double knock-out; Presenilin 1 knock-out; Variants of unknown significance; γ-Secretase.

Figure 1.. Deletion of Psen1 in N2A cells

(A) Design for CRISPR/Cas9-mediated knock-out of Psen1 and PCR screening assay using F1 and R1 primers. (B) Functional validation of single and dual gRNA activity for Psen1 targeting. (C) PCR screening of genomic DNA from Psen1 knock-out clones. Black arrows point to the PCR band from wild type or unedited clone. Red arrows point to the bands produced by CRISPR/Cas9-mediated editing. (D) Genomic DNA sequencing results of the N2A-PS1KO-8 clone for two alleles as assessed by PCR in (C). Nucleotides in bold belong to the coding exon. Start codon nucleotides are labeled in red.

Figure 2.. Deletion of Psen2 in N2A-PS1KO-8 cells

(A) Design for CRISPR/Cas9-mediated knock-out of Psen2 and PCR screening assay with F2 and R2 primers. (B) Functional validation of single and dual gRNA activity for Psen2 targeting. (C) PCR screening of genomic DNA from Psen1 and Psen2 knock-out clones. Black arrows point to the PCR band that is from wild type or unedited clone. Red arrows point to the bands produced by CRISPR/Cas9-mediated editing. (D) Genomic DNA sequencing results of the N2A-PS1/2KO-8/71 clone for both alleles as assessed by PCR in (C). Nucleotides in bold belong to the coding exon. Start codon nucleotides are labeled in red.

Figure 3.. Validation of double Psen1 and Psen2 knock-out in N2A cells and rescue with exogenous PSEN1 expression

(A) Schematic representation of editing in the genomic loci of Psen1 and Psen2 that created a double knock-out of mouse presenilin 1 and 2 in N2A cells by CRISPR/Cas9-mediated genome engineering. Nucleotides in bold belong to the coding exon. Start codon nucleotides are labeled in red. (B) qPCR for genes of γ-secretase subunits, Adam10, Bace1, and App in wild type (2 and 4) and knock-out (8, 35 and 8/71) clones. (C) Analysis of γ-secretase subunits and APP processing by western blotting in wild type (2 and 4) and knock-out (8, 35 and 8/71) clones rescued with wild type human presenilin 1 (PS1-WT). Quantification of western blotting data is in Supplementary Figure S1A. (D) Quantification of endogenous presenilin 2 protein levels in N2A-PS1KO clones (8 and 35) versus wild type N2A clones (2 and 4) based on (C). (E - H) Quantification of Aβ40, Aβ42, Aβ42/Aβ40 ratio by ELISA (E, F), and total Aβ by western blotting (G, H) in PS1KO clones (8 and 35) versus wild type N2A clones (2 and 4) (E, G) and in PS1/2KO-8/71 cells versus the wild type (2) and parental (8) lines (F, H). Values shown are mean ± SD of n = 3 independent experiments with one (D, G, H) or two (E, F) technical replicates each. * P < 0.05, ** P < 0.01, *** P < 0.001, one-way ANOVA with Dunnett’s post-hoc test against the control condition (WT in D, E, G and 2 in F, H).

Figure 4.. Rescue of double Psen1/Psen2 knock-out cells with exogenous PSEN2 expression

(A) Analysis of γ-secretase subunits and APP processing by western blotting in wild type (2) and double knock-out (8/71) cells rescued with wild type human presenilin 2 (PS2-WT). Quantification of western blotting data is in Supplementary Figure S1B. (B) Quantification of Aβ40, Aβ42, Aβ42/Aβ40 ratio by ELISA, and (C) total Aβ by western blotting based on (A) versus N2A-WT-2 cells. Values shown are mean ± SD of n = 3 independent experiments with one (C) or two (B) technical replicates each. ** P < 0.01, *** P < 0.001, one-way ANOVA with Dunnett’s post-hoc test against the control condition (2).

Figure 5.. The effect of PSEN1 coding mutations associated with AD clinical diagnosis on γ-secretase complex and Aβ production

(A) Analysis of γ-secretase subunits, APP and Aβ protein levels in cell lysates and conditioned medium from N2A-PS1/2KO-8/71 line transfected with wild type and mutant PSEN1. Quantification of western blotting data is in Supplementary Figure S2A. (B) Changes in Aβ40, Aβ42, and Aβ42/Aβ40 ratio caused by mutations in PSEN1 compared to wild type PSEN1 measured by ELISA. (C) Quantification of total Aβ (C) based on (A). Values shown are mean ± SD of n = 3 independent experiments with one (C) or two (B) technical replicates each. * P < 0.05, ** P < 0.01, *** P < 0.001, one-way ANOVA with Dunnett’s post-hoc test against control condition (8/71 + PS1-WT).

Figure 6.. The effect of PSEN2 coding mutations associated with AD clinical diagnosis on γ-secretase complex and Aβ production

(A) Analysis of γ-secretase subunits, APP and Aβ protein levels in cell lysates and conditioned medium from N2A-PS1/2KO-8/71 line transfected with wild type and mutant PSEN2. Quantification of western blotting data is in Supplementary Figure S2B. (B, C) Changes in Aβ40, Aβ42, Aβ42/Aβ40 ratio, and total Aβ introduced by mutations in PSEN2 compared to wild type PSEN2 measured by ELISA (B) and western blotting (C) based on (A). Values shown are mean ± SD of n = 3 independent experiments with one (C) or two (B) technical replicates each. * P < 0.05, ** P < 0.001, *** P < 0.001, one-way ANOVA with Dunnett’s post-hoc test against control condition (8/71 + PS2-WT).