Spatial Proteomic Analysis of Alzheimer’s Disease Human Brainusing Multiplexed Imaging
Arindam Bose1, Lisa Arvidson2, Richard W. Cho2, Supriya Singh2, Gabriella Spang2, Michael J. Smith1, Vasundhara Agrawal1
1Leica Microsystems, Waltham, MA, 2Cell Signaling Technology, Danvers, MA
1.A Control Brain

Results• The spatial profiling of AD brain reveals a significant
increase in phosphorylated tau containing neurofibrillarytangles and β-amyloid containing plaques, indicative ofneurodegeneration. Such structures are more abundantaround the outer periphery of the AD brain tissue. While celltypes like astrocytes marked by GFAP+ cells demonstrateclustered and heterogeneous expression in the periphery ofAD brain tissue, they are more enriched and homogenously
expressed in the inner core of the brain tissue (Figure 1A-C).
• Additionally, the β-amyloid+ plaques in the Alzheimer's brain
demonstrated a wide distribution of sizes in terms of crosssectional area compared to area occupied by the neuronalcell types (Figure 1D-E).
• There is a significant loss of various cell phenotypes in
Alzheimer’s brain, for instance, microglia marked by
TMEM119+ cells, and astroglia marked by GFAP+ cells andS100B+ cells. Interestingly, in addition to the Alzheimer’sassociated Tau markers, IBA1+ cells are increased inAlzheimer’s brain compared to the control. (Figure 1F).
• Although there is a global reduction in various astrocytes
and microglia cell types in Alzheimer’s brain, there is an
accumulation of astrocytes and microglia (GFAP+, S100B+,TMEM+ and IBA1+ cells) close to relatively larger plaques,potentially indicative of astrogliosis and microgliosis aroundthose regions of the brain (Figure 2A-D).

Clone

Conjugate

Concentration Dilution

Myelin Basic protein
AQP4 (D1F8E)
Iba1/AIF-1
p-Tau (T217)
TMEM119
Enolase2 (E2H9X)
GFAP
beta-Amyloid
PSD95
p-Tau (T181)
p-Tau (T217)
S100B
Tau (GT-38)
TMEM119
ApoE (pan)

D8X4Q
D1F8E
E404W
E9Y4S
E3E4T
E2H9X
GA5
D3D2N
D74D3
D9F4G
E9Y4S
E7C3A
GT-38
E3E4T
E8C2U

AF488
AF488
AF555
AF555
AF555
AF555
AF647
AF647
AF647
AF647
AF750
AF750
AF750
AF750
AF750

200 µg/mL
200 µg/mL
200 µg/mL
200 µg/mL
200 µg/mL
200 µg/mL
200 µg/mL
200 µg/mL
200 µg/mL
200 µg/mL
200 µg/mL
200 µg/mL
200 µg/mL
200 µg/mL
200 µg/mLSlideTissue

1:100
1:100
1:100
1:100
1:100
1:100
1:100
1:100
1:100
1:100
1:100
1:100
1:100
1:100
1:100

1 mm

D

1 mm

Microglia

Astrocytes

GFAP

TMEM119

Mature Neurons

AD-associated Markers

P-TAU217

200 µmF % ∆ Expression in Alzheimer's Brain Compared to Control Brain

Enolase2

Catalogue Number

AB-0054

Human Adult Normal: Brain

T2234035

AB-0055

Alzheimer's Disease: Brain

T2236035AlzAB-0057

Alzheimer's Disease: Brain: Amygdala

T2236036Alz

100 µm

S100B

IBA1

β -Amyloid

Methods & Materials
CST antibodies undergo a vigorous validation process toensure antibody performance on FFPE tissue. All antibodies
in this study were direct conjugates (Table 1). Following
preliminary validation, conjugated antibody solutions withthe optimum degree of labeling and concentration wererandomly assigned to a round, without optimization andused for subsequent staining of normal and Alzheimer
disease brain tissue. Tissue was obtained from acommercial source (BioChain; Table 1). Slides were imagedon the Cell DIVE imager using four channels plus DAPI, with
automatic AF removal, corrections, and stitching. Imagingrounds were conducted over a 2-week period. At round 5
slides were stored for long term at 4oC for future
experiments. Fully stitched images were imported, fused,and analyzed using AIVIA 13. Using AI-driven analysis, theexpression of markers (shown in Table 1) was quantified forthe segmented phenotypes and used for subsequent
analyses (Figure 1C).

2.A

Iterative staining and imaging of human adult brain tissues
with CST antibodies and Cell DIVE multiplexed imaging
solution enabled spatial characterization of AD human brain.
Cell DIVE multiplexing solution is tissue preserving, enabling
further probing to comprehensively characterize the ADassociated markers within the neuronal environment. Further
comprehensive studies focused on specific sections of thebrain including the amygdala (Figure 3) can reveal novel
spatial characteristics associated with the disease.

E

β-Amyloid Plaque Size
Distribution

Figure 1. Multiplexed Cell DIVE imaging of A. Adult Human Control and B. Adult Human
Alzheimer’s brain demonstrating a panel of 15 markers. C. Top: Spatial localization ofneuronal markers to understand heterogeneity in expression in periphery versus interior ofAD brain. Bottom: A pixel classifier was designed to segment all phenotypic markers in AD
Brain. D. Cell phenotypes characterized by expression of markers specific to astrocytes(GFAP, S100B), microglia (TMEM119, IBA1), mature neurons (Enolase2), and Alzheimer’sassociated markers (TauGT-38, p-Tau217,P-Tau181 and β-amyloid). DAPI show in blue. E.
Histogram of cross-sectional area (size) of β-amyloid plaques. F. Quantification of area ratio
to estimate % change in marker expression in Alzheimer's brain compared to control brain.

Spatial Landscape Around Plaques

BCPeriphery of AD Brain

20 µmFigure 2. Understanding the neuronal spatial landscape in AD brain. A. Correlation map of all neuronal components in AD brain
which shows the Pearson correlation coefficient between pairs of all measurements shown in a heatmap. B. Spatial landscaperelative to plaques in Alzheimer’s brain. Arrow indicates β -amyloid positive plaque segment, and 20 µm wide radial circleshighlight the neighboring cell types marked by GFAP+, S100B+, TMEM119+, and IBA1+ cells clustered around the plaques. C.
Clustering within the AD brain periphery based on β -amyloid plaque sizes reveals an enrichment of several cell populations aroundlarger plaques. D. Clustering analysis demonstrating all identified phenotypic clusters based on relative distance to β -amyloidplaques. Bottom Left: Representative image of GFAP+ cell segmentation color coded based on relative distance to plaques.

DConclusion

Questions?
Contact vasundhara.agrawal@leica-microsystems.com or arindam.bose@leica-microsystems.com
Contact support@cellsignal.com or lisa.arvidson@cellsignal.com

Interior of AD Brain

TargetPlaque Size-based clustering

Alzheimer’s disease (AD) is a genetic and sporadic
neurodegenerative disease and a common cause of cognitive
impairment acquired in midlife and late life. AD ispathologically defined by the presence of β-amyloidcontaining plaques and phosphorylated tau containing
neurofibrillary tangles. Effective therapies for the treatment
and/or prevention of AD are lacking. The examination of thehistopathological features of AD may reveal cellular
relationships that contribute to disease etiology leading topotential novel therapeutic strategies. The Cell DIVE
Multiplexed Imaging Solution, in combination with IF/IHCvalidated antibodies from Cell Signaling Technology (CST),
can be used to computationally examine synaptic processes
and spatially and molecularly define cells, such as glia andneurons, surrounding pathological hallmarks in AD.
Segmentation and clustering analysis can identify spatiallyco-localized populations of cells, including subpopulations of
microglia defined by specific disease-associated microglia
markers. CST’s broad portfolio of validated antibodies
enables the detection of altered synaptic processes and celltype populations in the context of human disease tissue. Here
we demonstrate multiplexed Cell DIVE imaging using a novel
CST® panel to probe AD brain. We examine the protein
landscape in diseased tissue in the context of Amyloid-β (Aβ)
and tau expression. The ability of cell-type specific markers,
combined with multiplexed tissue imaging will provide a new
approach for the neuroscience research community tounderstand spatial heterogeneity of the human brain and theircontribution to disease.

Table 1. Study Design: Antibodies and Tissues

C32 mmFigure 3. Alzheimer’s associated markers in Amygdala. The localizationof neurofibrillary tangles (magenta), Amyloid β peptide (Aβ) deposits inplaques (white), and GFAP+ (green) astrocytes in AD brain Amygdala.
Amygdala of Human AD brain reveals that medial nuclei are relatively
free of neurofibrillary tangles and Aβ plaques. The tangles and Aβplaques are significantly upregulated in other regions of the amygdala
like the accessory basal area in the Alzheimer’s brain.

Distance to Plaque-based clustering

Introduction & Aim

Brain
B Alzheimer's
B

Periphery of AD Brain

Poster # 10429

GFAP Mean
Nearest Distance toPlaque =11.6 ±9.7 µm

MC-0006363 - 11.12.2023. EN Copyright© by Leica Microsystems CMS GmbH, Wetzlar, Germany, 2021. Subject to modifications. LEICA and the Leica Logo are registered trademarks of Leica Microsystems IR GmbH.
Cell Signaling Technology, and CST, are registered Trademarks of Cell Signaling Technology, Inc