identification of phenotypes suitable for drug … · for both cultures, addition of 1µm mg2+...

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IDENTIFICATION OF PHENOTYPES SUITABLE FOR DRUG TESTING IN A GENETIC OR TOXIN-INDUCED MODEL OF PARKINSON'S

DISEASE USING HUMAN IPSC-DERIVED DOPAMINERGIC NEURONS.

Erika Torchio, Teresa Ferraro, Elisa Bianchini, Alessandra Toti, Andrea Kuan Cie Wong, Cristiana Griffante and Rosaria Remelli

In Vitro Pharmacology department, Aptuit (Verona) S.r.L., An Evotec Company

Parkinson’s disease (PD) is a neurodegenerative disease caused by the progressive loss of midbrain

dopaminergic neurons (mDAn). Genetic mutations affecting mDA neurons account for up to 15% of PD

onsets, providing a valuable tool for studying basic pathophysiological mechanisms of the disease. One of

the most relevant mutations is the A53T mutation in the SNCA gene, which causes the production of a

misfolded form of alpha synuclein (αSyn), resulting in αSyn accumulation. It has been reported that PD

patient neurons display a ~20% reduction of mitochondrial complex I activity. Rotenone is known to

reduce mitochondrial complex I activity. Therefore testing Rotenone administration in vitro could mimic PD

effect on mDAn mitochondria functionality.

INTRODUCTION

AIM

The aim of this work is the identification of PD-relevant phenotypes for in vitro drug testing to be

applied in drug-discovery programs for Parkinson’s Disease

WT and A53T dopaminergic cultures differentiation in 384 well format

Dopaminergic neurons maturation in 384 well format: A. iPSC-derived DA-neurons maturation was confirmed by the

expression of the neuronal marker MAP2 and DA-specific markers tyrosine-hydroxylase (TH) and FOXA2. B. Bar graph

reports the % of TH-positive cells over live cells quantified by high content analysis (HCA). C. Viability of the cultures is

quantified by HCA at dd21 and 28 by measure of the number of live c/w. Bar graph represents mean ±SEM of at least 14

replicate wells.

A53TWT

TH Hoechst

FOXA2

MAP2 TH Hoechst

WTMAP2 TH Hoechst

A53T

50 µm 50 µm

A

dd21

dd28

WT A53T0

20

40

60

80

TH

+ c

ells [

%]

B DA maturation - dd21

Fold vs WT21 Nuclei # dd21-28

WT A53T WT A53T 0.0

0.5

1.0

1.5

Fo

ld v

s W

T d

d21 L

IVE

C Live cells count

dd21 dd28

TH Hoechst

FOXA2

Time dependent αSYN accumulation in A53T mDAn cultures: A. αSYN MSD quantifications performed at dd21 and

28 revealed time-dependent accumulation of αSYN in A53T versus WT mDAn cultures. The graph bars report the fold increase

of the αSYN MSD signal of A53T over the signal of WT at each time point. The maximum fold of 1.63 was detected at dd28. B.

αSYN MSD quantifications in four independent cultures lysates at dd28 confirmed higher αSYN MSD signal in A53T versus

WT (fold: 1.76 ±0.11, p≤0.001; t-test). Comparable GAPDH MSD signal in WT and A53T dd28 lysates (subset samples)

confirmed uniform sample loading in αSYN MSD studies. MSD assays were performed in technical duplicates.

Mesoscale assay for sensitive αSYN quantification from mDAn lysates

αSYN quantification assay set up: Mesoscale Discovery (MSD) 96-well

assay kit was employed for quantification of total αSYN from mDAns cell

lysates. Linearity test of A53T lysates (dd28) was performed with 1µg and

0.5µg of lysates from A53T mDAn. ECL signal revealed linear αSYN

detection within the linear range of the calibration curve. 0.25µg/well was

the condition selected for the following quantifications in mDAn cultures.

SYN CRC

1 10 100 1000 10000 10000010

100

1000

10000

100000

1000000

pg/mL

EC

L s

ign

al

0.5µg A53T

1µg A53T

αSYN GAPDH

A BαSYN time course MSD analysis dd28

αSYN accumulation in A53T DaN cultures

WT A53T0.0

0.5

1.0

1.5

2.0WT

A53T

-SYN MSD

***

-S

YN

fo

ld v

s W

T

WT A53T0.0

0.5

1.0

1.5WT

A53T

GAPDH MSD

GA

PD

H f

old

vs W

T

cultura #9

dd21 dd280

20000

40000

60000

80000WT

A53T

EC

L s

ign

al

A B

Cal 520

Different pattern of spontaneous Calcium oscillations in WT and A53T mDAn

Comparable Ionomycin responseIono addition

WT A53T0

5100 5

1100 6

2100 6

WT

IONO: initial phase representedmobilization of intracellular stores andthe sustained component representedCa2+ influx

A53T

AU

C

0 200 400 600 8000

1000

2000

3000

4000

5000

E7

E18

Time (s)

RF

U

+Mg2+

RL

UR

LU

WT

A53T +Mg2+

1200 sec

A53T

WTWT

A53T

Increased frequency of Calcium oscillations in A53T DaNs compared to WT detected with FLIPR

measurements in 384 well-format: Calcium oscillations with increased frequency were detected with FLIPR

measurements in A53T cultures compared to WT at dd21 and dd28. A. Representative Calcium traces detected in WT and

A53T mDAn. For both cultures, addition of 1µM Mg2+ inhibited spontaneous oscillations. Comparable cultures viability was

confirmed by Cal520 staining and evaluation of the response to addition of 1µM Ionomycin (B).

C. Peak analysis revealed peak frequency (BMP) 1.61 fold higher in A53T versus WT (p≤0.001; t-test), 0.57 fold reduced peak

spacing (p≤0.001; t-test) and comparable peak amplitude. FLIPR tests performed in 4 different cultures under comparable cell

viability revealed average increase of peak frequency of A53T versus WT of 1.52 ±0.1 and variable peak amplitude (fold A53T

versus WT: 1.26 ±0.26).

C Peak frequency Peak spacing Peak amplitudePeak Amplitude

WT A53T0

500

1000

1500

2000

2500WT

A53T

Avera

ge P

eak a

mp

litu

de

Peak Frequency

WT A53T0.0

0.2

0.4

0.6

0.8WT

A53T

BM

P

CDI d28_TMRE peripheral mitochondria

WT A53T0.6

0.7

0.8

0.9

1.0

1.1

1.2 ***

TM

RE

peri

f. m

ito

ch

on

dri

a

Inte

nsit

y (

fold

vs W

T)

Peak Spacing

WT A53T0

100

200

300WT

A53T

Avera

ge P

eak S

PA

CIN

G


WT A53T0.6

0.7

0.8

0.9

1.0

1.1

1.2 ***

TM

RE

peri

f. m

ito

ch

on

dri

a

Inte

nsit

y (

fold

vs W

T)

BM

P

Avg

. p

eak s

pacin

g (

sec)

Avg

. p

eak a

mp

litu

de

In this study, an extensive phenotypic characterization of iPSC-derived mDAn cultures carrying a PD-

relevant mutation (A53T) was performed in parallel to isogenic healthy control cultures (WT). Moreover,

acute and chronic Rotenone administration on WT mDAn was used to induce mitochondrial stress.

Key cellular features related to PD pathogenesis were explored:

mDAn differentiated in 384 well plates express neuronal marker MAP2 and DA-specific markers

tyrosine-hydroxylase (TH) and FOXA2

Compared to WT mDAn, an accumulation of αSYN was detected in A53T cells after 28 days in culture

A53T mDAn display a different pattern of spontaneous Calcium oscillations compared to WT cells,

potentially revealing a dysregulation of intracellular Calcium homeostasis (FLIPR 384-well assay)

A reduced mitochondrial membrane potential was detected in A53T mDAn compared to WT cells

A53T mDAn show higher basal respiration compared to WT in Seahorse XF metabolic assay

Rotenone administration results in reduction of cell viability and MMP (acute treatment), as well as in

reduction of mitochondrial respiration (chronic treatment). Inhibition of Complex I was compensated by

induction of other mitochondrial complexes and of glycolysis.

The above mentioned assays provide a panel of readouts suitable for testing potential therapeutics acting

on different PD pathophysiological mechanisms.

METHODS

mDAn cultures: iCell® DopaNeurons (WT) and MyCell® DopaNeurons (A53T) were seeded according to vendor’s instruction in

iCell Neural medium (Fujfilm Cellular Dynamics) on PDL/PLO/Laminin coated plates. Cells were seeded at the density of 65,000

(WT) and 50,000 (A53T) c/w for imaging studies (384w plates); at 130,000 (WT) or 100,000 (A53T) c/w (96w plates) for FLIPR

and Seahorse studies. Culture medium was replaced with BrainPhys™ Neuronal Medium (StemCell Technology) after 3 days in

culture and maintained for up to dd21 or dd28 with half medium volume exchange twice/week.

Immunofluorescence and Imaging: Cells were fixed in paraformaldehyde, permeabilized and incubated with primary

antibodies, followed by fluorescent secondary antibodies. Primary antibodies: Ms-anti-αSYN BD-610786; Ch-anti-TH (Abcam

ab76442), Rb-anti-FOXA2 (Cell Signalling 8186), Ms-anti-MAP2 (Abcam ab32454). All images were captured with InCell Analyzer

2200 and analyzed with Columbus Image Analysis System (PerkinElmer).

Mesoscale Immunoassay: Mesoscale Discovery (MSD) 96-well assay kit U-PLEX human αSYN K151WKK and GAPDH-

K151PWD were used according to manufacturer instructions. For testing αSYN, 0.25µg/well was used, for GAPDH 0.125µg/well.

FLIPR Ca2+ assay: Cells were stained with 10µM Cal520 calcium indicator (Abcam) in Tyrode’s buffer without Mg2+.

Measurements were carried out for 20 minutes. FLIPR Screenworks® Peak Pro™ Software was used for Peak analysis.

TMRE assay: cells were incubated with 30nM TMRE (Invitrogen) for 30 minutes in culture media without phenol-red, followed by

5 minutes of incubation with HOECHST for nuclei staining.

Seahorse XF assay: Cells were pre-incubated for 1h with assay buffer before the experiment (Agilent Seahorse XF DMEM, 25mM

Glucose, 2mM Glutamine, 1mM Na-Pyruvate). All stimuli were prepared as 10x solutions and added automatically by Seahorse XF

instrument. 3 measurements were taken for baseline and 4 measurement were taken after every stimulus.

REFERENCES: (1) Kweon et al. 2004, DOI 10.1074/jbc.M407336200. (2) Zheng et al. 2016, DOI: 10.7554/eLife.13374

Reduced mitochondrial membrane potential in A53T vs WT mDAn cultures

A TMRE staining Image analysis

20 µm

A53TWTTMRE Hoechst

SOMATIC TMRE PERIPHERAL TMRE

TMRE intensity quantifications performed in the cell soma and in the peripheral mitochondria showed lower

Mitochondrial Membrane Potential (MMP) in A53T cultures versus WT: A. TMRE assay was performed on WT and

A53T cultures at dd21 and dd28 and HCA was employed for analysis. Nuclear masks were identified based on Hoechst staining

and cell masks were segmented on the TMRE signal. For peripheral mitochondria identification, TMRE spot masks were

identified in the area surrounding the cells. B. Average data for each time-point from two independent cultures of WT and

A53T mDAn are reported. Comparable cell area was quantified in live WT and A53T cells. TMRE intensity quantified in the cell

soma of A53T displayed significantly lower signal at both time points (fold A53T vs WT: dd21, 0.87±0.01, p≤0.001; dd28

0.88±0.02, p≤0.01; t-test). Additionally, lower TMRE average intensity was detected in peripheral mitochondria (fold A53T vs

WT: dd21, 0.84±0.01, p≤0.001; dd28, 0.87±0.02, p≤0.001; t-test ).

CDI d28_Cytoplasm area

WT A53T0.6

0.7

0.8

0.9

1.0

1.1

1.2

Cell A

rea

(fo

ld v

s W

T)


WT A53T0.6

0.7

0.8

0.9

1.0

1.1

1.2 ***

TM

RE

peri

f. m

ito

ch

on

dri

a

Inte

nsit

y (

fold

vs W

T)

CDI d28_TMRE soma cytoplasm

WT A53T0.6

0.7

0.8

0.9

1.0

1.1

1.2 **

TM

RE

Cell In

ten

sit

y

(fo

ld v

s W

T)

CDI d21_Cytoplasm area

WT A53T0.6

0.7

0.8

0.9

1.0

1.1

1.2

Cell A

rea

(fo

ld v

s W

T)


WT A53T0.6

0.7

0.8

0.9

1.0

1.1

1.2

***T

MR

E p

eri

f. m

ito

ch

on

dri

a

Inte

nsit

y (

fold

vs W

T)

CDI d21_TMRE soma cytoplasm

WT A53T0.6

0.7

0.8

0.9

1.0

1.1

1.2

***

TM

RE

Cell In

ten

sit

y

(fo

ld v

s W

T)

BCell area

dd

21

dd

28

Somatic

mitochondria intensity

Peripheral

mitochondria intensity

Higher basal mitochondrial respiration in A53T mDAn compared to WT Chronic Rotenone administration reduces basal mitochondrial respiration

NT Rot. 1nM Rot. 5nM Rot. 10nM

0

200

400

600

800

Cell count

NT

Rot. 1nM

Rot. 5nM

Cell n

um

ber


0

20

40

60

80

Total OCR Capacity

OC

R %

Acute administration of Rotenone reduces cell viability and MMP in WT mDAn

WT mDAn viability and MMP is reduced upon acute administration of Rotenone: Rotenone was administered on

WT mDAn for 24, 48 or 72h. A. Rotenone reduced the cell viability in a time-dependent manner with EC50 2.3nM at both 24

and 48h, but with a maximal effect at 48h. To assess cell viability, RT-Glo luminescence were measured using CLARIOstar

(BMG LABTECH) B. TMRE assay was performed on cells treated with Rotenone dilution curve after 48h of treatment.

Rotenone reduced the MMP with an EC50 of 9.5nM.

Metabolic analysis of Mitochondrial functionality of A53T and WT mDAn at dd21 and dd28 by Seahorse XF

Analyzer (Agilent). A. OCR traces at dd21 and dd28 and Seahorse protocol. B. Quantification of variation of OCR (OCR %)

induced by applied stimuli. Live cell number quantified by HCA (HOECHST) was used to normalize basal levels of respiration

and glycolysis. Dot plot graph represents mean ±SEM.

• A53T cells have higher basal respiration compared to WT mDAn (i).

• Both lines have similar response to OligomycinA (ii) and similar maximum respiratory capacity (iii), suggesting similar

functionality of mitochondria in WT and A53T cells. Further studies will be performed to investigate whether the difference

in basal respiration is due to differences in mitochondrial number.

• Complex I (iv) is more used than Complex III (v), which is inactive in both WT and A53T mDAn.

• Comparing dd21 with dd28, In both lines there is an increase in basal respiration (i), an increased utilization of respiratory

capacity (~50% at dd21, ~100% at dd28) (iii), and a loss of glycolytic spare capacity (iii ECAR), suggesting metabolic

maturation at dd28.

WT d

d21

WT d

d28

A53

T dd21

A53

T dd28

0

200

400

600

800

1000

Cell number

Bonferroni's Multiple Comparison Test

WT dd21 vs WT dd28 8

WT dd21 vs A53T dd21 1




A53T dd21 vs A53T dd28 8

Mean Diff.

209.0

-102.0

129.8

-311.0

-79.19

231.8

t

6.213

3.034

4.026

8.649

2.286

6.690

Significant? P < 0.05?

Yes

Yes

Yes

Yes

No

Yes

Summary

***

*

**

***

ns

***

95% CI of diff

111.9 to 306.0

-199.1 to -4.965

36.74 to 222.8

-414.8 to -207.2

-179.2 to 20.81

131.8 to 331.8

***

***

Cell n

um

ber

WT d

d21

WT d

d28

A53

T dd21

A53

T dd28

0.0

0.1

0.2

0.3

Normalized Basal Respiration








Mean Diff.

-0.06485

-0.04984

-0.1784

0.01501

-0.1135

-0.1285

t

7.287

5.327

20.88

1.442

11.72

12.72


Yes

Yes

Yes

No

Yes

Yes

Summary

***

***

***

ns

***

***

95% CI of diff

-0.09005 to -0.03965

-0.07633 to -0.02335

-0.2025 to -0.1542

-0.01447 to 0.04449

-0.1409 to -0.08608

-0.1571 to -0.09990

***

***

# # #

OC

R/c

ell n

um

ber

WT d

d21

WT d

d28

A53

T dd21

A53

T dd28

0

20

40

60

80

100

ATPase Activity

OC

R %

WT d

d21

WT d

d28

A53

T dd21

A53

T dd28

-50

0

50

100

Maximum Respiratory Capacity








Mean Diff.

48.09

9.280

73.87

-38.81

25.78

64.59

t

10.19

2.030

15.65

8.489

5.461

14.13


Yes

No

Yes

Yes

Yes

Yes

Summary

***

ns

***

***

***

***

95% CI of diff

34.56 to 61.61

-3.816 to 22.38

60.34 to 87.40

-51.90 to -25.71

12.26 to 39.31

51.49 to 77.69

***

# # #

***

OC

R %

Live cell count

Normalized

Basal Respiration

ATP Synthase

Activity

Maximum

Respiratory Capacity

dd21 dd28

WT

dd21 dd28

A53T

dd21 dd28

WT

dd21 dd28

A53T

dd21 dd28

WT

dd21dd28

A53T

dd21 dd28

WT

dd21 dd28

A53T

B

i ii iii

WT d

d21

WT d

d28

A53

T dd21

A53

T dd28

0

20

40

60

80

100

Complex I Activity








Mean Diff.

-36.88

3.153

-45.24

40.03

-8.358

-48.39

t

12.99

1.156

16.58

13.18

2.751

16.49


Yes

No

Yes

Yes

No

Yes

Summary

***

ns

***

***

ns

***

95% CI of diff

-44.90 to -28.86

-4.552 to 10.86

-52.94 to -37.53

31.45 to 48.61

-16.94 to 0.2246

-56.68 to -40.10

******

OC

R %

WT d

d21

WT d

d28

A53

T dd21

A53

T dd28

0

2

4

6

8

10

Complex II Activity

OC

R %

WT d

d21

WT d

d28

A53

T dd21

A53

T dd28

0.00

0.02

0.04

0.06

Basal Glycolysis








Mean Diff.

-0.01280

-0.003609

-0.02415

0.009188

-0.01136

-0.02054

t

6.504

1.733

13.32

4.064

5.633

9.653


Yes

No

Yes

Yes

Yes

Yes

Summary

***

ns

***

**

***

***

95% CI of diff

-0.01845 to -0.007140

-0.009596 to 0.002378

-0.02937 to -0.01894

0.002689 to 0.01569

-0.01715 to -0.005560

-0.02666 to -0.01443

***

# # #

***

EC

AR

/cell n

um

ber

WT d

d21

WT d

d28

A53

T dd21

A53

T dd28

-50

0

50

100

150

Maximum Glycolytic Capacity








Mean Diff.

89.46

-34.49

86.97

-123.9

-2.488

121.5

t

18.91

7.545

19.02

26.20

0.5259

26.57


Yes

Yes

Yes

Yes

No

Yes

Summary

***

***

***

***

ns

***

95% CI of diff

75.99 to 102.9

-47.51 to -21.48

73.95 to 99.98

-137.4 to -110.5

-15.96 to 10.98

108.4 to 134.5

***

# # #

***EC

AR

%

Complex I Activity Complex III ActivityNormalized

Basal Glycolysis

Maximum

Glycolytic Capacity

dd21 dd28

WT

dd21 dd28

A53T

dd21 dd28

WT

dd21 dd28

A53T

dd21 dd28

WT

dd21 dd28

A53T

dd21 dd28

WT

dd21 dd28

A53T

iv v i ECAR iii ECAR Metabolic analysis of Mitochondrial functionality of WT mDAn after chronic Rotenone treatment. WT mDAn

were treated with Rotenone from dd3 to dd21 to simulate chronic inhibition of Complex I.

5 and 10nM of chronic Rotenone treatment reduced cell viability and basal respiration (i) but had no effect on ATP Synthase

activity (ii). 10nM Rotenone stimulus was used to reduce Complex I activity by 20%, revealing increased cell vulnerability of

cells chronically pre-treated with Rotenone (iv). To compensate chronic reduction of Complex I, mDAn adopted two strategies:

induction of other mitochondrial complexes (v) and induction of glycolysis (i ECAR). Dot plot graph represents mean ±SEM,

**=p ≤0.01, ***=p≤0.001, ANOVA with Dunnett’s post-hoc test.

Maximum

Respiratory Capacity

Live cell count

iii

***

***


0.00

0.02

0.04

0.06

0.08

0.10

Normalized Basal Respiration

NT

Rot. 1nM

Rot. 5nM

OC

R/c

ell n

um

ber


0

20

40

60

80

ATPase Activity

OC

R %

i

Normalized

Basal Respiration

ATP Synthase

Activityii

***

***

***

***

***


0

20

40

60

Partial Inhibition of Complex I

OC

R %

NT Rot. 1 nM Rot. 5 nM Rot. 10 nM

-10

0

10

20

Spare Respiratory Capacity

NT

Rot. 1 nM

Rot. 5 nM

Rot. 10 nM

OC

R %

20% Inhibition

Complex I

Spare

Respiratory Capacityiv v**

**

CONCLUSIONS

Normalized

Basal Glycolysis

BASAL

ii

iv

v

i

iii

Oligomycin A

1µM

FCCP

1µM

Rotenone

10nM

Rotenone

0.5µM

● NT=Not Treated

● Rot 1nM

● Rot 5nM

● Rot 10nM

A

dd21 dd28

i

ii

iii

iv v

● WT

● A53T

● WT

● A53T

Oligomycin A

1µM

FCCP

1µM

Rotenone

0.5µM

Antimycin A

0.5µM

Oligomycin A

1µM

FCCP

1µM

Rotenone

0.5µM

Antimycin A

0.5µM

RESULTS

OCR (pmol/min) = oxygen consumption rate ECAR (mpH/min) = extracellular acidification rate

ANOVA with Bonferroni post-hoc test*** = p<0.001 (dd21 vs dd28)### = p<0.001 (WT vs A53T)

48h

-12 -11 -10 -9 -8 -7 -6 -5 -40

20

40

60

80

100

12024h

48h

72h

Rotenone log [M]%

of

CT

RL

-11 -10 -9 -8 -7 -6 -5 -40

20

40

60

80

100

120

Oligomycin log [M]

% o

f C

TR

L

-12 -11 -10 -9 -8 -7 -6 -5 -40

20

40

60

80

100

12024h

48h

72h

Rotenone log [M]%

of

CT

RL

Cell viability Mitochondrial Membrane Potential

-12 -11 -10 -9 -8 -7 -6 -50

2000

4000

6000

8000

10000

Rotenone log [M]

TM

RE

sig

nal in

ten

sit

y

A B


0.020

0.025

0.030

0.035

0.040

0.045

Normalized Basal Glycolysis

EC

AR

/cell n

um

ber


0.020

0.025

0.030

0.035

0.040

0.045

Normalized Basal Glycolysis

EC

AR

/cell n

um

ber

i ECAR

identification of phenotypes suitable for drug … · for both cultures, addition of 1µm mg2+...

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