CW

T Mo n o m

e r

GB A M

o n o me r

WT P

F F

GB A P

F F0

5

1 0

1 5

G C a s e 1 8 0 D P I B ra in

GC

as

e A

cti

vit

y(n

mo

l 4M

U/h

ou

r/m

g±

SE

M) * *** ****

WT M

o n o me r

GB A M

o n o me r

WT P

F F

GB A P

F F0

2 0

4 0

6 0

G lc C e r1 8 0 D P I B ra in

Glc

Ce

r L

ev

els

(ug

Glc

Ce

r/g

tis

su

e±

SE

M)

W T M o n o m e r

G B A M o n o m e r

W T P F F

G B A P F F

WT M

o n o me r (

M)

WT M

o n o me r (

F )

GB A M

o n o me r (

M)

GB A M

o n o me r (

F )

WT P

F F (M)

WT P

F F (F)

GB A P

F F (M)

GB A P

F F (F)

0

5 0 0

1 0 0 0

1 5 0 0

G lc S p h 1 8 0 D P I B ra in

Glc

Sp

h L

ev

els

(ug

Glc

Sp

h/g

tis

sue

± S

EM

)

* *******

********

**** *

The Michael J. Fox Foundation’s efforts to understand the relationship between GBA1 and alpha-synuclein through the development and characterization of preclinical models

Bradford H. Casey1, Nicole K. Polinski1, Terina N. Martinez1, Sean W.Clark2, Sean M. Smith3, Robert C. Switzer III4, S.O. Ahmad5, Sylvie Ramboz6, Michael Sasner7, Mark T. Herberth8, Liliana Menalled1, Marco Baptista1, Kuldip D. Dave1

The Michael J. Fox Foundation for Parkinson’s Research1, Amicus Therapeutics2, Merck & Co.3, NeuroScience Associates4, Saint Louis University5, PsychoGenics, Inc.6, The Jackson Laboratory7, Charles River Laboratories8.

742.15

IntroductionHeterozygous mutations in the GBA1 gene, which encodes lysosomal glucocerebrosidase (GCase), are the most common genetic risk factor for Parkinson’s disease (PD). Inaddition, decreased GCase activity has been reported in both genetic and sporadic cases of PD. Experimental evidence suggests a correlation between decreased GCaseactivity and accumulation of alpha-synuclein (aSyn). Thus, understanding the potential synergistic effect of increased aSyn and decreased GCase activity is important forunderstanding how alterations in GCase activity may contribute to or exacerbate PD-related pathology. To enable a better understanding of the relationship between aSynand GCase activity, The Michael J. Fox Foundation (MJFF) has developed and characterized two mouse models allowing investigation of aSyn pathology in the context ofreduced GCase activity. The first model analyzes the neurodegeneration/pathology induced through constitutive overexpression of wild type human alpha-synuclein directedby the murine Thy-1 promoter (hemizygous transgenic) in the context of the GCase activity-reducing D409V mutant form of GBA (homozygous knock-inGBA1). The secondmodel analyzes the level of nigrostriatal degeneration and synuclein pathology in the GBA D409V knock-in model versus wildtype mice following stereotaxic injection of aSynpreformed fibrils into the striatum. Here, we outline and discuss the preliminary results of these model characterization efforts. Together, these models provide importantplatforms for understanding the mechanisms underlying GCase and aSyn dynamics, and for evaluating therapeutics targeting this pathway/relationship.

Characterization of a GBA D409V KI x Thy-1 aSyn Overexpression Mouse Model

Characterization of the aSyn PFF Model in GBA D409V KI Mice

Figure 1. Assessments of motor phenotype demonstrate a potential decrease in grip strength of GBA D409V KI x Thy-1 aSyn mice at 12 months of age but otherwise no significant, sustained differences from C57Bl/6WT mice. Twelve cohorts of mice were used (4 genotypes at 3 ages) for this characterization study. A) Body weight measures were taken at the designated ages, with animals weighed using a properly calibrated electronic scale. Forelimb (B)and hindlimb (C) grip strength were measured by allowing the mice to grip a bar and pulling back gently to measure the force to release by continued pulling (average of three valid measurements are reported). D) Hindlimb foots play wasassessed by painting the heel pads of the hindfeet with nontoxic paint and dropping the mouse over a test sheet on a table while holding to measure the distance between the inner edges of the ink blots (average of three valid measurements arereported). E) Rotarod was performed on a 3cm rotating rod (rotation rate 12 rpm) for a maximum of 120 s intervals with the test session performed after a training session. F-G) Open field locomotor activity was obtained at 5-min intervals for a totalof 60 minute test sessions in an open-field using Kinder Scientific Monitor System in a sound-attenuated room with white noise set to operate at 70 ± 10 db. Bars represent mean ± SEM (n= 22 males/group for C57Bl/6 WT, GBA D409V KI, andGBA D409V KI x Thy-1 aSyn; n=15 males/group for Thy-1 aSyn). Two-way ANOVAs were performed for graphs A-E with Bonferroni post hoc tests for significance within ages between genotypes. Two-way repeated measures ANOVAs wereperformed for graphs F-H with Bonferroni post hoc tests for significance between genotypes. *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001.

Figure 4. Numbers of tyrosine hydroxylase-positive neurons are significantly decreased in theipsilateral hemisphere at 180 days post-aSyn PFF injection, regardless of genotype. Stereologywas performed in the SNpc of mice using tyrosine hydroxylase (TH) immunostained and Nissl immunostainedtissue. A) At 180 DPI, the number of TH-immunoreactive (TH+) neurons in the SNpc were significantlydecreased in the ipsilateral hemisphere following aSyn PFF, but not aSyn monomer, injection. No differenceswere observed between genotypes. B) At 180 DPI, the number of Nissl-immunoreactive (Nissl+) cells in theSNpc that were not TH-immunoreactive were not significantly different between genotype, hemisphere, orinjectate. Two-way ANOVA with Tukey post hoc tests for differences between injected vs uninjectedhemisphere and differences between genotypes were performed. N=11-12 mice/group. Bars representmean ± standard error of the mean. *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001.

Figure 3. Striatal neurochemistry at 90 and 180 days post-injection of alpha-synucleinpreformed fibrils (PFF) reveals decreases in dopamine and DOPAC levels agnostic of genotype,with less consistent changes in HVA between genotypes following PFF administration.Homozygous GBA D409V KI mice (GBA) and wildtype (WT) littermate controls were injected intrastriatallywith alpha-synuclein (aSyn) monomers or aSyn PFFs and analyzed 90 (A, C, E) and 180 (B, D, F) days post-injection (DPI) for levels of dopamine (A-B) and the dopamine metabolites DOPAC (C-D) and HVA (E-F) viastriatal HPLC. A-B) Dopamine levels were significantly decreased at 90 and 180 DPI following aSyn PFF, butnot aSyn monomer, injection in WT and GBA D409V KI mice. No differences were observed between WTand GBA D409V KI mice at either time point. C-D) The dopamine metabolite DOPAC was significantlydecreased at 90 and 180 DPI following aSyn PFF, but not aSyn monomer, injection in GBA D409V KI mice. InWT mice, aSyn PFF administration resulted in a significant reduction in DOPAC at 90, but not 180, DPI. Nostatistically significant differences were observed between WT and GBA D409V KI mice at either time point.E) At 90 DPI (5 months of age), HVA levels were statistically elevated in GBA D409V KI vs WT mice in theipsilateral and contralateral hemisphere post-PFF injection and in the ipsilateral hemisphere post-monomerinjection. HVA was only significantly decreased in WT mice post-PFF injection. F) At 180 DPI (8 months ofage), all differences in HVA levels between genotypes and between hemispheres were absent. Two-wayANOVA with Tukey post hoc tests for differences between injected vs uninjected hemisphere anddifferences between genotypes were performed. N=11-12 mice/group. Bars represent mean ± standarderror of the mean. *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001.

Model Description JAX IDC57Bl/6 WT Wild type C57Bl/6NJ mice obtained from JAX. 5304

GBA D409V KI Targeted KI of D409V mutation in mouse GBA gene on B6N. Homozygous. 19106Thy-1 aSyn Human wildtype aSyn driven by the murine Thy-1 promoter (line 15) on B6N. Hemizygous. 17682

GBA D409V KI x Thy-1 aSyn Cross of above GBA D409V KI and Thy-1 aSyn lines. Hom GBA x Hemi Thy-1 aSyn. 29124

Table 1. Summary of the models included in the characterization summary with the abbreviated name, short description of the line, and line identification number at Jackson Laboratories.

********

************

******

4 Mo n th

s

8 Mo n th

s

1 2 Mo n th

s0

5 0

1 0 0

1 5 0

2 0 0

F o re lim b G r ip S tre n g th

Fo

reli

mb

Gri

p S

tre

ng

th(g

± S

EM

)

** ******

****

*

******

****

4 Mo n th

s

8 Mo n th

s

1 2 Mo n th

s0

2 0

4 0

6 0

8 0

1 0 0

H in d lim b G r ip S tre n g th

Hin

dli

mb

Gri

p S

tre

ng

th(g

± S

EM

)

**** ************

*******

*

4 Mo n th

s

8 Mo n th

s

1 2 Mo n th

s0

1 0

2 0

3 0

4 0

H in d lim b F o o ts p la y

Hin

dli

mb

Fo

ots

pla

y(m

m)

****

***

******

4 Mo n th

s

8 Mo n th

s

1 2 Mo n th

s0

2 0

4 0

6 0

8 0

R o ta ro d T im e to F a ll

Ro

taro

d T

ime

to

Fa

ll(s

eco

nd

s)

****

**

****

*

******** ****

********

C 5 7 B l/6 W T

G B A D 4 0 9 V K I

G B A D 4 0 9 V K Ix T h y -1 a S y n

T h y -1 a S y n

1 2 3 4 5 6 7 8 9 1 0 1 1 1 20

5 0 0

1 0 0 0

1 5 0 0

In te rv a l

Op

en

Fie

ld T

ota

l A

cti

vit

y

4 M o n th s

G B A D 4 0 9 V K I x T h y -1 a S y n > C 5 7 B l /6 ( * * * )

G B A D 4 0 9 V K I x T h y -1 a S y n > G B A D 4 0 9 V K I ( * * )

G B A D 4 0 9 V K I x T h y -1 a S y n > T h y - 1 a S y n ( * * * )

1 2 3 4 5 6 7 8 9 1 0 1 1 1 20

5 0 0

1 0 0 0

1 5 0 0

In te rv a l

Op

en

Fie

ld T

ota

l A

cti

vit

y

8 M o n th s

G B A D 4 0 9 V K I > C 5 7 B l /6 ( * * )

G B A D 4 0 9 V K I x T h y -1 a S y n > C 5 7 B l /6 ( * )

1 2 3 4 5 6 7 8 9 1 0 1 1 1 20

5 0 0

1 0 0 0

1 5 0 0

In te rv a l

Op

en

Fie

ld T

ota

l A

cti

vit

y

1 2 M o n th s

C 5 7 B l/6 W T

T h y -1 a S y n

G B A D 4 0 9 V K I

G B A D 4 0 9 V K Ix T h y -1 a S y n

C 5 7 B l /6 > T h y - 1 a S y n ( * * )

G B A D 4 0 9 V K I x T h y -1 a S y n > G B A D 4 0 9 V K I ( * )

G B A D 4 0 9 V K I x T h y -1 a S y n > T h y - 1 a S y n ( * * * )

**********

4 Mo n th

s

8 Mo n th

s

1 2 Mo n th

s0

1 0

2 0

3 0

4 0

B o d y W e ig h t

Bo

dy

We

igh

t(g

± S

EM

)

* **

****

******

A B C D

E F G H

4 Mo n th

s

8 Mo n th

s

1 2 Mo n th

s0

5

1 0

1 5

2 0

D o p a m in e

Do

pa

min

e(n

g/m

g t

issu

e)

* ***

***** *

4 Mo n th

s

8 Mo n th

s

1 2 Mo n th

s0 .0

0 .5

1 .0

1 .5

D o p a m in e T u r n o v e r

Do

pa

min

e T

urn

ov

er

(DO

PA

C+

HV

A)/

DA

± S

EM

* * ****

**** *

4 Mo n th

s

8 Mo n th

s

1 2 Mo n th

s0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

N ig ra l D o p a m in e N e u ro n N u m b e rs

TH

+ N

eu

ron

s i

n t

he

SN

pc

(Co

un

ts±

SE

M)

C 5 7 B l/6 W T

G B A D 4 0 9 V K I

G B A D 4 0 9 V K Ix T h y -1 a S y n

T h y -1 a S y n

A B C

calculated by dividing the summation of DOPAC and HVA levels fromDA. C) Stereology of tyrosine hydroxylase-immunoreactive (TH+) cellsin the substantia nigra pars compacta (SNpc) was performed toquantify the number of nigral dopamine neurons. Bars represent mean± SEM (n= 6-7 males/group). Two-way ANOVAs were performedwith Bonferroni post hoc tests for significance within ages betweengenotypes. *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001.

Figure 2. Nigrostriatal degenerationreadouts indicate minimal deficits at 12months of age in DA turnover of the GBAD409V KI x Thy-1 aSyn line. A) Levels ofdopamine and dopamine metabolites weremeasured in mouse striatum usingUHPLC/MS/MS. B) Dopamine turnover was

Mo n o m

e rP F F

0

5

1 0

1 5

D A 1 8 0 D P I

Do

pa

min

e(n

g/m

g t

issu

e±

SE

M)

* *** ****

Mo n o m

e rP F F

0

2

4

6

8

1 0

D A 9 0 D P I

Do

pa

min

e(n

g/m

g t

issu

e±

SE

M)

* *** ****

A B

Mo n o m

e rP F F

0 .0

0 .1

0 .2

0 .3

0 .4

0 .5

D O P A C 1 8 0 D P I

DO

PA

C(n

g/m

g t

issu

e±

SE

M)

***

Mo n o m

e rP F F

0 .0

0 .2

0 .4

0 .6

D O P A C 9 0 D P I

DO

PA

C(n

g/m

g t

issu

e±

SE

M)

* ****

C D

Mo n o m

e rP F F

0 .0

0 .5

1 .0

1 .5

H V A 1 8 0 D P I

HV

A(n

g/m

g t

issu

e±

SE

M)

Mo n o m

e rP F F

0 .0

0 .5

1 .0

1 .5

H V A 9 0 D P I

HV

A(n

g/m

g t

issu

e±

SE

M) *

** ****

E F

G B A : U n in je c te d

W T : U n in je c te d

G B A : In je c te d

W T : In je c te d

***

Mo n o m

e rP F F

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

T H S te re o lo g y 1 8 0 D P I

TH

+ N

eu

ron

s i

n t

he

SN

pc

(Co

un

ts±

SE

M)

****

Mo n o m

e rP F F

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

N is s l S te re o lo g y 1 8 0 D P I

Nis

sl+

Ce

lls

in

th

e S

Np

c(C

ou

nts

± S

EM

)

G B A : U n in je c te d

W T : U n in je c te d

G B A : In je c te d

W T : In je c te d

A B

Figure 5. GCase activity and GlcSph levels in the are significantly altered in the GBA D409V KImice, with no differences between aSyn monomer vs PFF injection. A) GCase activity wasmeasured via the CBE 4MU method in ipsilateral hemisphere brain homogenate lacking striatum (used forHPLC). GBA D409V KI mice exhibit significantly reduced GCase activity as compared to WT mice. aSynPFF injection had no effect on GCase activity at 180 DPI. B) Glucosylceramide (GlcCer) levels weremeasured by LC-MS/MS in ipsilateral hemisphere brain homogenate lacking striatum. No differenceswere observed between WT and GBA D409V KI mice or between aSyn monomer vs aSyn PFF injection.C) Glycosphingolipid (GlcSph) levels were measured by LC-MS/MS in ipsilateral hemisphere brainhomogenate lacking striatum. GBA D409V KI mice exhibit significantly greater GlcSph levels as comparedto WT mice, with female GBA D409V KI mice consistently displaying greater levels than males. aSyn PFFinjection had no effect on GlcSph at 180 DPI. One-way ANOVA with Tukey post hoc tests for differencesbetween injected vs uninjected hemisphere and differences between genotypes were performed. N=11-12 mice/group. Bars represent mean ± standard error of the mean. *P<0.05, ****P<0.0001.

A B

Figure 6. α-Syn striatal PFF injection inducesmidbrain synuclein aggregation in GBA D409Vmice Homozygous GBA D409V KI mice (GBA) andwildtype (WT) littermate controls were injectedintrastriatally with alpha-synuclein (aSyn) monomers oraSyn PFFs and analyzed 180 days post-injection. 40umsections of postfixed whole brain were mounted andstained for phospho-Serine129 (pS129). Asterisk (*)indicates treated hemisphere. Area shown magnified ininset is indicated against coronal section. Images shownare representative of overall results for the groupscompared. IHC utilized anti-pSer129 aSyn IHC, WakoCat#010-26481 at a primary dilution of 1:15,000.

WT Monomer WT PFF GBA Monomer GBA PFF

* * * *

MJFF is invested in providing the PD research community with high-quality tools and models to support rapid new discoveries and encourage reliable, reproducible data. Models and studies described in this poster are the result of recent collaborative efforts aimed at generating and characterizinganimal models for PD research. More information can be found in the Research Tools Catalog at www.michaeljfox.org/toolscatalog (questions can be sent to tools@michaeljfox.org). In addition, MJFF also offers patient biosamples (eg blood, CSF, plasma, serum, urine, iPSCs) and clinical data to theresearch community. Information on available biosamples can be found at www.michaeljfox.org/biosamples and data can be found at www.michaeljfox.org/datasets. Visit us at Booth #2030 to speak with us about these resources and our funding programs.