Esquer, H. | Search bioRxiv

bioRxiv2026

High-content imaging of live microglia-containing Alzheimer's disease brain organoids

Alzheimer's disease (AD) is the most common progressive neurodegenerative disease for which there is currently no safe and effective treatment. AD is characterized by the pathological aggregation of amyloid-{beta} (A{beta}) in the brain resulting in neuronal dysfunction/death and cognitive decline. Modeling these processes in vitro remains challenging due to the lack of an immune-competent 3D in vitro human model of AD amenable to high-throughput analyses. Conventional cerebral organoids (COs) lack microglia and are optically dense which precludes real-time longitudinal analyses. Here, we employ the fluorescent amyloid probe PyrPeg to selectively monitor A{beta} aggregation, and calcein AM and propidium iodide to assess cellular viability within microglia-containing COs (MCOs). We identify age-dependent DS-AD-associated phenotypes within T21 MCOs including A{beta} neuropathology, neurodegeneration, and glial cell dysregulation. Our platform supports real-time, multiplexed tracking of disease progression and provides a scalable platform for longitudinal analyses studying disease mechanisms and targeted approaches to treatment.

neuroscience↗

bioRxiv2026

Trisomy 21 cerebral organoids exhibit Alzheimer's disease amyloid and apolipoprotein E co-pathologies

Adults with Down syndrome (DS) develop Alzheimers disease (AD) brain pathology by age 40 due to triplication of the Amyloid Precursor Protein (APP) gene on chromosome 21. Inheritance of the apolipoprotein E-e4 (APOE4) allele of the APOE gene on chromosome 19 remains the greatest genetic risk factor for AD in the typical population, yet its role in DS-associated AD (DS-AD) neuropathogenesis in people with DS is unclear. We generated human induced pluripotent stem cell (hiPSC)-derived neurons, astrocytes, and cerebral organoids (COs) using cells from people with DS and from euploid individuals. Aged DS COs were smaller than aged euploid COs and showed robust amyloid-{beta} neuropathology that was positively correlated with the levels of apoE expression. We then captured extracellular vesicles (EVs) from the conditioned media of COs and observed a decrease in the levels of secreted AD-related proteins, including amyloid, contained within the EVs and in the media from which the EVs were isolated. We also identified distinct neuronal and astrocytic gene expression signatures in DS COs relative to euploid COs, including a set of genes known to interact with both APOE and APP at the gene and/or protein levels. Lastly, we determined that, despite differences in the expression levels of the specific genes involved, several common pathways were upregulated in T21 hiPSC-derived neurons, astrocytes, and COs, including apoptosis, the endolysosome, and structural stabilization pathways. Taken together, our findings provide novel insights into molecular mechanisms that may contribute to DS-AD and indicate that apoE plays an important role in the disease process.

neuroscience↗