PSEN1 Gly417Ala by CRISPR/Cas9 in functional pathogenicity

Abstract

The many of genetic variants in Amyloid precursor protein (APP), presenilin1 (PSEN1), and presenilin2 (PSEN2) from patients with early-onset Alzheimer's disease (EOAD) are considered to be pathological mutations. Largest number of pathological mutations was found in PSEN1, which is a subunit of gamma secretase in processing APP to amyloid-beta (Aβ). Previously, PSEN1 Gly417Ala was found from a 37-year-old Korean male patient. Clinical manifestations presented memory deteriorations for over three years and was diagnosed with EOAD with mild symptoms of Parkinson's disease. To further establish the gene's implications in the pathology, a stable cell-line of PSEN1 Gly417Ala was produced by using CRISPR/Cas9 technology protocol to investigate its functional pathogenicity. HEK293 cells were transfected with gRNA complex of PSEN1 Gly417Ala by CRISPR/Cas9 technology. Functional study was performed using ELISAs of Aβ40, Aβ42 and Aβ oligomers by Multiple Detection System (MDS). The activities gamma and beta secretase were measured by employing their specific substrates. Successful transfection of gRNA complex of PSEN1 Gly417Ala was confirmed by the restriction enzyme and Sanger sequencing. Ratio of Aβ and elevated levels oligomers were overserved in PSEN1 Gly417Ala in comparison with control cells (P<0.05). Functional studies also indicated the elevated activities of both gamma secretase (P<0.05) and beta secretase statistically in PSEN1 G417A in comparison with the control cells. These results may elaborate the pathogenic nature of PSEN1 G417A in Alzheimer's disease. CRISPR/Cas9 technology is a powerful tool in studying the pathogenicity of PSEN1 G417A variant. Our results in the functional studies supported variant's potential pathogenicity in AD as indicated. The amyloid cytotoxicity and causes of presenting Parkinsonism in the patient are currently being studied in our laboratory.