Using Genetically Diverse Collaborative Cross Mouse Strains

to Model Alzheimer’s Disease

Asli Uyar for the MODEL-AD Consortium

AD/PD 2021– 03.13.2021

• I have no disclosures to declare.

Mouse Models of AD on the C57BL/6J (B6J) Genetic Background

• Mouse models of Alzheimer’s disease (AD), carrying well-known early onset AD mutations, such as APP and PS1 (APP/PS1), or LOAD risk factors, such as APOE4, have usually been created on the C57BL/6J (B6J) genetic background.

• While these strains often exhibit amyloid accumulation and neuroinflammation, many additional molecular alterations present in human AD are absent in the mouse models.

• Previous work by our labs, and others, reveal the benefits of incorporating diverse mouse strains to model human AD.

• Mouse models of AD on genetically distinct wild-derived mouse strains demonstrated robust differences in immune response to amyloid and neurodegeneration, better recapitulating the heterogeneity in human AD than previous models.

Background and Motivation

Collaborative Cross (CC) lines

Saul et al 2019

• To broaden the phenotypes of mouse models further, we introduced genetic diversity by incorporating Collaborative Cross (CC) lines.

Objective

• CC lines is a recombinant inbred mouse panel created from eight highly diverse founder strains.

• Together, these founder strains recapitulate about 90% of the genetic variation observed in Mus musculus and represent genotypic variation comparable to human populations.

• CC strains are considered genetically homogenous once it reached a minimum level of inbreeding, typically once at least 90% of the genome is fixed after ~20 generations of inbreeding.

CC lines

Expression levels of AD risk genes varies across CC strains

AJB6129NODNZOCASTPWKWSB

2.5

3.0

3.5

4.0

4.5

log2(TP

M)

Trem2

F M F M F M F M F MCC002 CC006 CC013 CC037 CC041

Trem2 p = 1.91e-08

4.0

4.5

5.0

5.5

log2(TP

M)

Tyrobp

F M F M F M F M F MCC002 CC006 CC013 CC037 CC041

Tyrobp p = 0.0007

1.5

2.0

2.5

3.0

log2(TP

M)

Cd33

F M F M F M F M F MCC002 CC006 CC013 CC037 CC041

Cd33 p = 0.008

Five CC mouse strains were selected for maximal genetic and gene expression

variation at twelve late-onset GWAS loci, including TREM2, TYROBP, and CD33.

Genomic heterogeneity across CC strainsCC

002

CC01

3

CC04

1

CC00

6

CC03

7

Transcriptomic similarity

AJB6129NODNZOCASTPWKWSB

Model generation• Transgenic APP and PS1 alleles with a humanized APOE4 allele on a B6J background were

crossed with each CC line.

CC013CC006CC002B6.APOE4<HOM>.APPPS1<HEMI>

APOE4-KIAPP/PS1-Tg

CC037 CC041

B6 AD Model WT CC strains

F1

lethal

B6.CCn(F1).APOE4<HET>.APPPS1<HEMI> B6.CCn(F1).APOE4<HET>.APPPS1<WT>

• PC1 separates CC002 and CC013 from other strains; PC2 is Sex

Brain transcriptomes are separated by strain and sex

−5

0

5

0 10PC1: 39% variance

PC2:

12%

var

ianc

efactor(pcaData$Sex)

F

M

factor(pcaData$Strain)B6

CC002

CC006

CC013

CC037

CC041

• RNA-seq transcriptomes of brain hemisphere were assessed at 8-months for male and female mice.

• Six biological replicates per group.

Sex

Strain

Differential expression - Genotype effectB6.CCn(F1).APOE4<HET>.APPPS1<HEMI> vs. B6.CCn(F1).APOE4<HET>.APPPS1<WT>

269

73 6544

33 25 208 5 5 4 4 3 3 1

0

100

200

300

Inte

rsec

tion

Size

CC002 CC037 CC006 CC041

0100200300400Set Size

App Trem2 Gfap Tyrobp Itgb2 C1qa Ctsd Ctsz Ctss

Cd52 Ccl3 Prnp Mpeg1 Lpl Fcrls Cd68 Ccl6 Vsir

Ly86 Hexb Csf1r Gusb Slc111a1 Ptprc Olfml3 Laptm5 Cd9

Itgax St14 Arpp21 Grn Dhcr24 C1qc C1qb C3ar1 Rims1

Tent5c Fcgr3 Cts7 Lyz2 Cd300c2 C4b Clec7a Lamr1-ps1

Common DE genes across CC strains

• Number of DE genes in response to APP/PS1 transgenes on APOE4 background varies:• highest in CC002 (383) and lowest in

CC041 (104)

• Genes that are differentially expressed in all four strains are associated with microglia.

padj < 0.1

Sex specific differential expression

padj < 0.1

Itgb2

Cd52

Man2b1

Ctsd

Cybb

Cd48

Ccl6

Vsir

Plek

GfapLy86

Cd180

Hexb

Plau

Srebf2

Itgb5

App

Trem2

Myo1f

Csf1r Slc15a3

Gusb

Slc11a1

Usp50

Olfml3

Tnfsf8

Csf3r

Fgr

Gpnmb

Tbxas1

St14

Arpp21

Tlr13

Grn

C1qa

C1qc

C1qb

CtssC3ar1

Irf8

Rims1

Tent5c

Lncpint

Selplg

Ch25h

Cx3cr1

Capg

Fcer1g

Fcgr3

B2m

Hvcn1

Lyz1

Lyz2

C4b

Il4i1

Prnp

Bcl2a1b

Gm170870

5

10

15

−2 −1 0 1 2log2FoldChange

−log10(padj) diffexpressed

aaa

DOWN

NO

UP

Itgb2

Ccl3

Ctsd

Lnx2

Cd68

Ccl6

Gfap

Ly86

App

Tmem176a

Trem2

Tcf7l2

Hexa Laptm5

Tyrobp

Arpp21

Grn

Lysmd3

C1qc

Islr2

Fcgr3

Lyz2

C4b

Prnp

Gm5936Gm4767

0.0

2.5

5.0

7.5

10.0

−2 −1 0 1 2log2FoldChange

−log10(padj_scaled)

diffexpressedaaa

DOWN

NO

UP

B6.CC2(F1).APOE4<HET>.APPPS1<HEMI> vs. B6.CC2(F1).APOE4<HET>.APPPS1<WT>

#down=160

Female

#up=253 #down=7

Male

#up=63

KEGG pathways - CC lines and wild strains

Th17 cell differentiationViral myocarditis

AsthmaCircadian rhythm

Steroid biosynthesisSystemic lupus erythematosus

Glycosaminoglycan degradationRheumatoid arthritis

Viral protein interaction with cytokine and cytokine receptorB cell receptor signaling pathway

Cell adhesion moleculesAntigen processing and presentation

Chemokine signaling pathwayFc gamma R−mediated phagocytosis

Chagas diseaseStaphylococcus aureus infection

PertussisHematopoietic cell lineage

TuberculosisComplement and coagulation cascades

Proteoglycans in cancerLeishmaniasis

PhagosomeCoronavirus disease − COVID−19

Osteoclast differentiationLysosome

Acute myeloid leukemia

CC002_F(191)

CC006_F(37)

CC037_F(99)

CC041_F(63)

B6_F(40)

CAST_F(90)

PWK_F(278)

WSB_F(55)

GeneRatio0.05

0.10

0.15

0.20

0.04

0.03

0.02

0.01

p.adjust

B6 and wild strain data: Onos et al, 2020

Female

KEGG pathways - CC lines and wild strains

Thyroid hormone synthesisGlutamatergic synapse

Phospholipase D signaling pathwayCholinergic synapse

Gastric acid secretionSynaptic vesicle cycle

Neuroactive ligand−receptor interactionCalcium signaling pathway

AsthmaAntigen processing and presentation

Rheumatoid arthritisVarious types of N−glycan biosynthesis

Terpenoid backbone biosynthesisViral protein interaction with cytokine and cytokine receptor

Hematopoietic cell lineageProteoglycans in cancer

LeishmaniasisGlycosphingolipid biosynthesis − globo and isoglobo series

Glycosphingolipid biosynthesis − ganglio seriesPhagosome

Chagas diseaseOsteoclast differentiation

TuberculosisGlycosaminoglycan degradation

Steroid biosynthesisOther glycan degradation

PertussisComplement and coagulation cascades

Staphylococcus aureus infectionLysosome

CC002_M(40)

CC006_M(58)

CC037_M(55)

CC041_M(22)

CAST_M(72)

PWK_M(118)

WSB_M(244)

GeneRatio0.1

0.2

0.04

0.03

0.02

0.01

p.adjust

B6 and wild strain data: Onos et al, 2020

Male

AMP-AD gene co-expression modules

Wan et al, 2020

B6_FCAST_FPWK_FWSB_F

CC002_FCC006_FCC037_FCC041_FCC002_MCC006_MCC037_MCC041_M

AMP-AD module mapping

B6_MCAST_MPWK_MWSB_M

CC lines

B6 and wild strains

• Mouse models of AD on diverse CC line background demonstrate • significant strain and sex-specific differences in transcriptomic signatures in presence of APP/PS1

mutant transgenes and APOE4 allele• higher variation than common B6 background

• Using diverse genetic backgrounds introduce further variation to AD related pathologies at molecular level.

• Each mouse model strain recapitulates a different transcriptomic pattern in human data.

• These novel strains has the potential to enhance the translatability of AD mouse models.

Summary

National Institute on AgingU54 AG054345, U54 AG054349

Contactwww.model-ad.orgmodelad@iupui.edu@Model_ad_alz

Indiana University Bruce Lamb, Program Director

Paul Territo, PTC HeadAndrew Saykin, BDMC Co-HeadAdrian Oblak, Project Manager

Kwangsik NhoTatiana Foroud

Dino GhettiAndi Masters

Carla Biesdorf De AlmeidaSara Quinney

Deborah DeBusk, Administrator

The Jackson Laboratory Gareth Howell, DMP HeadGreg Carter, BDMC Head

Mike Sasner, DMP Co-HeadHarriet WilliamsDylan GarceauKevin Kotredes

Christoph PreussAsli Uyar

Yi LiRavi PandeyNikhil MilindKristen Onos

Martha Abbott, AdministratorSage Bionetworks

Lara Mangravite, BDMC Co-HeadLarsson Omberg

Ben LogsdonMette Peters

Solveig SiebertsYooree Chae

Anna GreenwoodKara Woo

Zoe Leanza

The MODEL-AD ConsortiumUC Irvine

Frank LaFerla, Program DirectorAndrea Tenner, Program Director

Grant MacGregor, DMP HeadAli Mortazavi, BDMC HeadKim Green, DMP Co-Head

Marcelo Wood, DMP Co-HeadStefania Forner, Project Manager

David Baglietto-Vargas Shan Jiang

Shimako KawauchiSherrilyn Collins

Jonathan NeumannEniko KramarEdna HingcoDina MatheosMaria Fonseca

Andrea Wasserman, Administrator

University of PittsburghStacey Rizzo, PTC Co-Head

Zackary CopeGabi Little

Sean Williams