Ethan James1, Michael Ha1, Citlali Aguilera-Rico1, Anna Howell1, Karen Cusato1 and M. Natalia Vergara1,2,3 1CellSight Ocular Stem Cell and Regeneration Program, Sue Anschutz Rodgers Eye Center, University of Colorado School of Medicine, Aurora, CO 80045, 2Linda Crnic Institute for Down Syndrome, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, 3University of Colorado Alzheimer's and Cognition Center, University of Colorado School of Medicine. Aurora, CO 80045 Alzheimer’s Disease Neuropathology in the Human Trisomy 21 Retina Introduction Methods Acknowledgements Conclusions We would like to thank the following benefactors whose support made our research possible: Contact: ethan.james@colorado.edu Results 1) Increased AD histopathology in donor retinas from individuals with DS 2) DS hiPSC can generate ROs with all major retinal cell types 3) DS ROs exhibit phosphorylated Tau pathology 4) DS ROs exhibit amyloid-β pathology 5) Fluorescence-based assay for the quantitative assessment of amyloid plaques in ROs • Down syndrome (DS) results from an extra copy of chromosome 21, causing genetic imbalance and affecting brain development, leading to intellectual disabilities. • Individuals with DS face a heightened risk of Alzheimer's disease (AD), resulting in accelerated formation of amyloid plaques and neurofibrillary tangles in the brain and retina. • Human induced pluripotent stem cell (iPSC)-derived retinal organoids (ROs) offer a promising avenue for studying AD- related changes specific to DS, providing insights into retinal pathology and potential biomarkers for early diagnosis and interventions. CTR D21 T21 AT8/DAPI T231/DAPI A B C D E F CTR D21 T21 4G8/DAPI NIAD/DAPI 4G8/NIAD/DAPI A B D E G H C F I *** * CTR DS 4G8/DAPI 6E10/DAPI A C E B D F T231/DAPI NIAD/DAPI G H E I J Figure 3: (A-H) Comparison of AT8 (A-C) and T231 staining (D-F) targeting different phosphorylation sites of pTau, (G) quantification of average relative stained area for pTau (n=5, p<0.001***), (H) protein quantification by Western blot for pTau in human DS retinas (n=2) and a control retina (p<0.05*), error bars show SEM Figure 4: (A-K) Comparison of 4G8 staining for Aβ (A-C), NIAD staining for amyloid plaques (D-F), and colocalization (G-I). (J) Quantification of average relative stained area for Aβ (n=5, p<0.001***). (K) Percent of 4G8 colocalized with NIAD, indicative of dense core plaques. CTR D21 RCV/DAPI Otx2/DAPI A C E B D F AP2α /DAPI PROX1/DAPI G H E HuC/D/DAPI I J Figure 2: (A-J) (A-B) Recoverin (RCV) staining for photoreceptor cells, (C-D) Otx2 staining for photoreceptor cells, (E-F) PROX1 staining for horizontal and amacrine cells, (G- H) AP2α staining for amacrine cells, (I-J) HuC/D staining for ganglion cells Figure 1: (A-H) (A-B) 4G8 staining for Aβ, (C-D) 6E10 staining for Aβ , (E-F) NIAD staining for amyloid plaques, (G- H), T231 staining for phosphorylated Tau (pTau) J K G H *** Figure 5: 3D Automated Reporter Quantification (Vergara et al., 2017) of NIAD-4-stained whole-mount 3D retinal organoids shows a statistically significant increase in NIAD-4 fluorescence in DS organoids compared to controls. Hoescht staining was used for size normalization. * p<0.05. *** *** • Our study confirms the presence of AD histopathology in the human retina of individuals with DS. • Development and characterization of the first RO model of human T21 provide a valuable tool for studying the relationship between DS and AD within the context of the retina, offering new avenues for therapeutic interventions and biomarker discovery. • Utilizing human iPSC-derived ROs allow us to recapitulate AD- related changes, including Aβ deposition and pTau, providing a platform for investigating the underlying molecular mechanisms and potential therapeutic targets. CB DS