Three-dimensional direct neuronal reprogramming for modeling Alzheimer’s disease neuropathology

Late-onset Alzheimer’s disease (LOAD) accounts for over 95% of Alzheimer’s disease (AD) cases. However, modeling the age-dependent neuropathological hallmarks of LOAD has remained a major challenge. We recently developed a patient-based, three-dimensional (3D) neuronal culture system that endogenously recapitulates key neuropathological features of AD, including extracellular Aβ deposition, tau dysregulation and spontaneous neuronal death. This platform uses high-efficiency, microRNA- and chromatin-based direct somatic reprogramming of fibroblasts from patients with autosomal dominant AD or LOAD to cortical neurons in 3D thin gel (3D-CNs) or self-assembled neuronal spheroids. Here we provide stepwise instructions for generating 3D-CNs and cortical spheroids via neuronal reprogramming of patient fibroblasts, along with methods for analyzing downstream AD neuropathology. Within 3 weeks, fibroblasts are reprogrammed into neurons under either thin-gel or spheroid conditions. By 4 weeks, key AD neuropathology, such as Aβ deposition, tau dysregulation, and neuronal death, can be detected in AD neurons. This system provides an age-relevant, patient-specific neuronal model for investigating molecular events underlying AD-associated neurodegeneration and evaluating compounds or gene targets in the context of potential personalized therapeutic interventions. Successful implementation of this protocol requires prior experience in mammalian cell culture, plasmid preparation, lentiviral production and standard confocal imaging techniques.