”Generation Of IPSC-derived neural organoids from an Alzheimer’s Disease patient carrying novel PSEN1 T119I mutation

ISAJA, LUCIANA; MARAZITA, MARIELA; RODRÍGUEZ VARELA, MARÍA SOLEDAD; MUCCI, SOFÍA; ITZCOVICH, TATIANA; CHEM-MENDEZ, PATRICIO; NIIKADO, MATIAS; FERRIOL LAFFOUILLERE, SOFÍA LUJÁN; ALLEGRI, RICARDO; MARTINETTO, HORACIO; SEVLEVER, GUSTAVO EMILIO; SCASSA, MARÍA ELIDA; SURACE, EZEQUIEL IGNACIO; ROMORINI, LEONARDO

Belem

Congreso; 3rd Congress of the Federation of Latin American and Caribbean Neuroscience Societies (FALAN) and the XLIII Congress of the Brazilian Society for Neuroscience and Behavior (SBNeC); 2022

FALAN IBRO

Introduction: Presenilin-1 (PSEN1) gene mutations are the most common cause of familial Alzheimer’s disease (AD). Previously, our group generated a human induced pluripotent stem cell (hiPSC) line (FLENIi001-A) from a carrier of the heterozygous PSEN1 p.T119I variant.Objectives: To functionally validate the PSEN1 (p.T119I) variant, we generate a 3D brain organoid (BO) model to study hallmarks of AD, such as Aβ deposition and tau hyperphosphorylation.Methods: BOs were generated from FLENIi001-A and control hiPSCs following a published protocol. Briefly, hiPSCs were first single-cell detached with Accutase and plated in 96-well ultra-low attachment plates at 21000 cells/well in human ESC medium and fiber solution. At day 6, medium was replaced by neural induction medium (DMEM/F12, N2 and heparin). On day 11, BOs were embedded in Matrigel and cultured in ultra-low attachment p60 plates (10-16 BOs/well) in differentiation medium (50% DMEM/F12-50% Neurobasal, N2, B27 without vitamin A and insulin). After 4-5 days, BOs were transferred to differentiation medium (with B27 with vitamin A) and plates swirled in an orbital shaker. Medium was replaced every 5 days and from day 40 Matrigel was added in every medium change. BOs were fixed at days 60 and 90 and immunostained for neural and cortical markers. At day 90 Aβ1-42, levels were quantified by ELISA in supernatants. Results: FLENIi001-A BOs showed expected morphology patterns such as neuroepithelial buds and smooth cortical lobes. Also, expression of neural (PAX-6, MAP-2, NeuN, GFAP and TUJ-1) and cortical (CTIP2 and TBR1) markers was detected in 60- and 90-days BOs. These BOs exhibited: increased Aβ and p-Ser-205 tau accumulation and increased supernatant Aβ1-42 levels compared to wild-type counterparts.ConclusionsOverall, we successfully obtained BOs from hiPSCs line FLENIi001-A carrying the AD PSEN1 p.T119I genotype that showed AD pathological features. These results add further functional evidence of pathogenicity for this variant.