Cholesterol-mediated Lysosomal Dysfunction in APOE4 Astrocytes Promotes α-Synuclein Pathology in miBrains, a Human iPSC-derived Brain Tissue

The pathological hallmark of neurodegenerative disease is the aberrant post-translational modification and aggregation of proteins, leading to the formation of insoluble protein inclusions. Genetic factors like APOE4 are known to increase the prevalence and severity of tau, amyloid, and α-synuclein inclusions. However, the human brain is largely inaccessible during this process, limiting our mechanistic understanding. Here, we developed an iPSC-based 3D model that integrates neurons, glia, myelin, and cerebrovascular cells into a functional human brain tissue (“miBrain”). Single-nucleus RNA sequencing of miBrains confirmed the presence of diverse cell populations and revealed cell-type-specific transcriptional responses to synuclein pathology. Like the human brain, we found that pathogenic phosphorylation and aggregation of α-synuclein are increased in the APOE4 miBrain. Combinatorial experiments revealed that endolysosomal dysfunction caused by cholesterol accumulation in APOE4 astrocytes impairs the degradation of α-synuclein and leads to a pathogenic transformation that seeds neuronal inclusions of α-synuclein. Collectively, this study establishes a robust model for investigating protein inclusions in human iPSC-derived brain tissue and highlights the role of astrocytes and cholesterol in APOE4-mediated pathologies, opening therapeutic opportunities.