bog2022

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1. none Ab+ next_Ab+ pT+ next_pT+ both next_both V10A27106_B1_Br3854 none Ab+ next_Ab+ pT+ next_pT+ both next_both V10A27106_C1_Br3873 none Ab+ next_Ab+ pT+ next_pT+ both next_both V10A27106_D1_Br3880 none Ab+ next_Ab+ pT+ next_pT+ both next_both V10T31036_B1_Br3854 none Ab+ next_Ab+ pT+ next_pT+ both next_both V10T31036_C1_Br3873 none Ab+ next_Ab+ pT+ next_pT+ both next_both V10T31036_D1_Br3880 none Ab+ next_Ab+ pT+ next_pT+ both next_both V10A27004_D1_Br3880 0.0 2.5 5.0 7.5 10.0 V10A27106_A1_Br387402%3 0 1 2 3 4 5 V10T31036_A1_Br387402%3 0 1 2 3 V10A27004_A1_Br387402%3 0 50 100 150 0 10 20 30 V10A27106_A1_Br3874 SNAP25 0 10 20 30 10T31036_A1_Br3874 SNAP25 10A27004_A1_Br3874 SNAP25 Spatial Transcriptomics Analysis of Aβ-tau Synergy in the Inferior Temporal Cortex of the Human Brain in Alzheimer’s Disease SH Kwon1, M Tippani1, S Parthiban2, AB Spangler1, HR Divecha1, KD Montgomery1, C Bruce3, S Williams3, M Mak3, G Yu3, J Avalos-Gracia3, TM Hyde1, SC Page1, SC Hicks2, K Martinowich1, KR Maynard1,*, L Collado-Torres1,* *: Kristen.Maynard@libd.org lcolladotor@gmail.com 1: Lieber Institute for Brain Development, Baltimore, MD. 2: Department of Biostatistics, JHBPSH. 3: 10x Genomics, Inc. Pleasanton, CA. INTRODUCTION VISIUM-IF AD STUDY DESIGN PATHOLOGY DATA FROM VISIUM-IF CONCLUSIONS REFERENCES IDENTIFYING WHITE/GRAY MATTER Background: Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by senile plaques of β-amyloid (Aβ) and neurofibrillary tangles of phosphorylated tau (pTau). Accumulating evidence suggests that Aβ and tau act in concert to orchestrate complex pathobiological events underlying AD, but their additive effect remains poorly understood. We investigated Aβ-tau synergy at the level of gene expression in the inferior temporal cortex (ITC). 10x Genomics Visium ImmunoFluorescence (Visium-IF) is a new platform in which IF staining is paired with on-slide cDNA synthesis using spatially-barcoded arrays. Methods: We employed Visium-IF to generate a proteomic-based, spatially-resolved, transcriptomic-scale map of the human ITC in AD. ITC brain blocks were dissected from 3 brain donors with late-onset AD (Amyloid C/Braak III) and 1 age-matched neurotypical donor. We validated laminar patterning in each block using RNAscope in situ hybridization with Layer I and VI marker genes (RELN and NR4A2). Cryosections from each block were collected in triplicate on Visium arrays and immunostained for Aβ, pTau, MAP2 and GFAP to visualize plaques, tangles, neurites, and astroglial processes. Each section was scanned using multispectral imaging and sequenced: whole-genome and targeted gene expression using the 10X Genomics human neuroscience panel. Results: After quality-control, we constructed SpatialExperiment R objects with 38,115 spots across 27,853 genes, one for each expression panel. We segmented the IF images using VistoSeg and used these metrics to categorize spots into seven groups: 1) no pathology, 2) Aβ only, 3) pTau only, 4) Aβ/pTau, 5) Aβ adjacent, 6) pTau adjacent, 7) Aβ/pTtau adjacent. We identified spatially-aware gene expression k clusters using BayesSpace across all samples, with a k range from 2 to 28. Using limma we identified differentially expressed genes between the pathology groups in the gray matter of the cortex only. We further spatially-registered the BayesSpace clusters to the cortex layers and created an interactive website using spatialLIBD. PATHOLOGY SPOTS LABELING GENES ASSOCIATED WITH AD PATHOLOGY • Our experiments and data analysis strategies can enable us to identify molecular, cellular, and morphological associations with spatially- localized Aβ and tau pathology. This project motivated the development of software for Visium-IF analysis. • Spatial clusters between whole genome and targeted sequencing panel are highly concordant. Join us! https://www.libd.org/careers/ • Research Associate: masters or undergrad with 3 years of experience • Staff Scientist: PhD or masters with 5 years of experience https://lcolladotor.github.io/bioc_team_ds/ Questions? @lcolladotor or lcolladotor@gmail.com • VisiumIF https://www.10xgenomics.com/products/spatial-proteogenomics • Targeted sequencing panel https://www.10xgenomics.com/products/targeted-gene- expression • SpatialExperiment https://doi.org/10.1093/bioinformatics/btac299 • VistoSeg https://doi.org/10.1101/2021.08.04.452489 • BayesSpace https://doi.org/10.1038/s41587-021-00935-2 • limma https://doi.org/10.1093/nar/gkv007 • DLPFC Visium data and layer registration https://doi.org/10.1038/s41593-020-00787-0 • spatialLIBD https://doi.org/10.1101/2021.04.29.440149 VISIUM IMMUNOFLUORESCENCE (IF) ACKNOWLEDGEMENTS Sang Ho Kwon @sanghokwon17 Madhavi Tippani @MadhaviTippani % pTau/spot % Abeta/spot BayesSpace k=28 none Ab+ next_Ab+ pT+ next_pT+ both next_both V10A27106_A1_Br3874 none Ab+ next_Ab+ pT+ next_pT+ both next_both V10T31036_A1_Br3874 none Ab+ next_Ab+ pT+ next_pT+ both next_both V10A27004_A1_Br3874 PATHOLOGY DIFFERENCES IN GRAY MATTER P1 P7 Pathology Pathology Groups Sowmya Parthiban @sowmyapartybun Case AgeDeath Race RIN Braak CERAD Neurotypical Br3874 73 EUR/CAUC 7.2 B2 C0 AD #1 Br3854 65 EUR/CAUC 7.0 B3 C3 AD #2 Br3873 88 EUR/CAUC 7.2 B3 C3 AD #3 Br3880 90 EUR/CAUC 7.1 B3 C3 −1 −0.9 −0.8 −0.7 −0.6 −0.5 −0.4 −0.3 −0.2 −0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 WM L6 L5 L4 L3 L2 L1 11 2 17 13 28 25 27 10 20 21 15 5 6 7 1 16 9 24 3 4 8 14 12 26 DLPFC Visium Data Maynard et al 2021 DLPFC LAYERS REGISTRATION Whole genome Targeted sequencing 640 155 49 6 55 17 709 15 438 43 156 309 7 709 104 581 11 1193 49 2 3 1 12 277 631 73 73 57 283 1776 21 666 68 148 3 301 20 376 46 30 10 44 14 1757 37 831 67 132 4 137 19 1448 102 279 21 883 73 4 11 6 219 524 15 36 17 141 2450 54 712 136 117 1 121 31 205 899 45 39 35 141 2112 22 556 101 173 1 216 13 S1_B1_3854 S1_C1_3873 S1_D1_3880 S2_B1_3854 S2_C1_3873 S2_D1_3880 S3_D1_3880 0.5 1.0 1.5 2.0 2.5 0.5 1.0 1.5 2.0 2.5 0.5 1.0 1.5 2.0 2.5 0.5 1.0 1.5 2.0 2.5 0.5 1.0 1.5 2.0 2.5 0.5 1.0 1.5 2.0 2.5 0.5 1.0 1.5 2.0 2.5 0% 25% 50% 75% 100% Percentage path_groups none Ab+ next_Ab+ pT+ next_pT+ both next_both BayesSpace k = 2 1: gray matter 2: white matter −1 −0.9 −0.8 −0.7 −0.6 −0.5 −0.4 −0.3 −0.2 −0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 WM L6 L5 L4 L3 L2 L1 1 2 k =2 1: GM, 2: WM BayesSpace clusters 1 2 V10A27106_A1_Br3874 1 2 V10T31036_A1_Br3874 1 2 V10A27004_A1_Br3874 1 2 V10A27106_B1_Br3854 1 2 V10A27106_C1_Br3873 1 2 V10A27106_D1_Br3880 1 2 V10T31036_B1_Br3854 1 2 V10T31036_C1_Br3873 1 2 V10T31036_D1_Br3880 1 2 V10A27004_D1_Br3880 Prop IF/Spot Prop pTau/spot Prop Aβ/spot −20 −10 0 10 20 −20 0 20 40 60 80 runPCA 01 (42%) runPCA 02 (10%) path_groups none Ab+ next_Ab+ pT+ next_pT+ both next_both −20 −10 0 10 20 −20 0 20 40 60 80 runPCA 01 (42%) runPCA 02 (10%) sample_id V10A27004_D1_Br3880 V10A27106_B1_Br3854 V10A27106_C1_Br3873 V10A27106_D1_Br3880 V10T31036_B1_Br3854 V10T31036_C1_Br3873 V10T31036_D1_Br3880 −20 0 20 40 −25 0 25 50 runPCA 01 (33%) runPCA 02 (17%) path_groups none Ab+ next_Ab+ pT+ next_pT+ both next_both −20 0 20 40 −25 0 25 50 runPCA 01 (33%) runPCA 02 (17%) sample_id V10A27004_D1_Br3880 V10A27106_B1_Br3854 V10A27106_C1_Br3873 V10A27106_D1_Br3880 V10T31036_B1_Br3854 V10T31036_C1_Br3873 V10T31036_D1_Br3880 p<0.05 in targeted sequencing panel