Cholesterol-mediated Lysosomal Dysfunction in APOE4 Astrocytes Promotes α-Synuclein Pathology in miBrains, a Human Brain Microphysiological System

Technical Summary: Cholesterol-mediated Lysosomal Dysfunction in APOE4 Astrocytes Promotes α-Synuclein Pathology in miBrains

Problem Statement
The pathological hallmark of neurodegenerative diseases involves the aberrant post-translational modification and aggregation of proteins, resulting in insoluble inclusion bodies. While genetic factors such as the APOE4 allele are established to increase the prevalence and severity of tau, amyloid, and α-synuclein inclusions, mechanistic understanding remains limited. This gap exists primarily because the human brain is largely inaccessible during the disease process, hindering the direct observation of these pathological mechanisms in a human context.

Methodology
To address the inaccessibility of the human brain, the authors developed an induced pluripotent stem cell (iPSC)-based three-dimensional model termed "miBrain." This system integrates multiple cell types—neurons, glia, myelin, and cerebrovascular cells—into a functional, human brain-like tissue. The study utilized this model to investigate the interplay between APOE4 genotypes and α-synuclein pathology.

Key experimental approaches included:

• Single-nucleus RNA sequencing (snRNA-seq): Performed on miBrains to confirm the presence of diverse cell populations and to identify cell-type-specific transcriptional responses to α-synuclein pathology.
• Comparative Genotyping: Analysis of miBrains with different APOE genotypes (specifically comparing APOE4/4 against other variants) to assess differences in protein aggregation.
• Combinatorial Experiments: Interventions designed to isolate the specific mechanisms by which astrocyte dysfunction influences neuronal pathology, focusing on cholesterol accumulation and endolysosomal pathways.

Key Results

1. Model Validation and Transcriptional Profiling: snRNA-seq confirmed that miBrains contain diverse, functional cell populations. The analysis revealed distinct cell-type-specific responses to α-synuclein pathology:
   • Neurons: Exhibited activation of apoptotic and synaptic gene programs.
   • Astrocytes: Displayed signatures of lipid dysregulation and reactivity.
2. APOE4-Dependent Aggregation: Consistent with human disease observations, the APOE4/4 miBrains showed increased levels of pathogenic phosphorylation and aggregation of α-synuclein compared to other genotypes.
3. Mechanistic Pathway: The study identified a specific cell-cell interaction mechanism:
   • APOE4/4 astrocytes accumulate cholesterol, leading to endolysosomal dysfunction.
   • This dysfunction impairs the astrocytes' ability to degrade α-synuclein.
   • The accumulated α-synuclein undergoes a pathogenic transformation within the astrocytes.
   • These transformed species subsequently seed the formation of α-synuclein inclusions in neighboring neurons.

Significance and Claims
The paper claims to establish a robust, human iPSC-derived brain tissue model ("miBrain") capable of investigating protein inclusions in a physiologically relevant context. By utilizing this system, the study elucidates a specific mechanism wherein astrocytes, driven by APOE4-mediated cholesterol accumulation and subsequent lysosomal failure, act as a source of pathogenic α-synuclein seeds that drive neuronal pathology. The authors posit that these findings highlight the critical role of astrocytes and cholesterol metabolism in APOE4-mediated neurodegeneration, thereby identifying specific biological targets that may open new therapeutic opportunities.