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bog2022

 bog2022

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    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,* *: [email protected] [email protected]
    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 [email protected]
    • 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
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    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
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    −1
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    WM
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    1
    2
    k =2
    1: GM, 2: WM
    BayesSpace clusters
    1
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    Prop IF/Spot
    Prop pTau/spot Prop Aβ/spot
    −20
    −10
    0
    10
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    −20 0 20 40 60 80
    runPCA 01 (42%)
    runPCA 02 (10%)
    path_groups
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    runPCA 01 (42%)
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    sample_id
    V10A27004_D1_Br3880
    V10A27106_B1_Br3854
    V10A27106_C1_Br3873
    V10A27106_D1_Br3880
    V10T31036_B1_Br3854
    V10T31036_C1_Br3873
    V10T31036_D1_Br3880
    −20
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    runPCA 02 (17%)
    path_groups
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    V10A27106_B1_Br3854
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    p<0.05 in
    targeted
    sequencing
    panel

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