Modeling neuropathology of Alzheimer’s disease in aged patient‐derived neurons through 3D direct neuronal reprogramming

Abstract Background Alzheimer’s disease (AD) is the most common form of adult‐onset neurodegenerative diseases characterized by three hallmarks of neuropathology including the extracellular deposition of amyloid β (Aβ), intracellular tau tangles, and neurodegeneration. The mechanisms that underlying the late‐onset AD (LOAD), the most common form of AD, remain largely unresolved due to the complexity of risk factors underlying the disease. Currently, endogenously recapitulating these events in patients‐derived neurons remains a major challenge, limiting our ability to understand the biology of the disease. Method By optimizing the potency of microRNA‐mediated direct neuronal conversion of patient fibroblasts, we developed a matrigel‐based three‐dimensional (3D) culture system for generating cortical neurons, a subtype known to be susceptible to develop AD pathologies. For characterizing AD‐associated phenotypes such as amyloid β(Aβ) plaques, tau pathology, and neurodegeneration in the reprogrammed cortical neurons, we carried out immunofluorescence staining, RNA‐sequencing and other biochemistry‐based assays. Results The 3D reprogrammed neurons derived from familial AD and LOAD patients endogenously displayed all three AD hallmark pathologies, including increased extracellular Aβ deposition, phosphorylated tau, dystrophic (beaded) neurites characterized by the colocalization between Ubquitin‐K63 and pTAU, as well as spontaneous neuronal cell death in culture without additional cellular insult. By using this 3D reprograming model, we found that treating AD neurons with β or γ secretase inhibitors at pre‐Aβ aggregation phase, significantly ameliorated Aβ formation and subsequent tauopathy and neuronal cell death, whereas treating during the post‐Aβ aggregation phase only had a mild effect. Additionally, comparative transcriptomic analysis between AD neurons and age‐matched healthy control neurons showed enrichment of genes involved in inflammatory pathways. Conclusion We established a novel patient derived neuronal 3D direct reprogramming model which robustly recapitulates key AD hallmarks including the extracellular deposition of Aβ plaques, formation of intracellular Tau tangles, and neurodegeneration. By using this 3D platform, we further demonstrated that inhibition of Amyloid β formation reduces tauopathy and neuronal cell death. Our unique patient specific 3D model system provides opportunities to investigate the interplay between AD pathological events and could serve as a platform for screening compounds and developing personalized therapeutics for AD.