Supplementary figures

Overview:

Fig. S1 Overview of significantly altered metabolites determined

during untargeted analysis with the accurate-mass Q-TOF

LC-MS system – p 1

Fig. S2 Example-peaks of metabolites affected by MPP+ treatment

– p 2

Fig. S3 MPP+-induced changes of amount and location of

apoptosis-related proteins – p 3

Fig. S4 Time course of MPP+ induced ROS generation – p 4

Fig. S5 Principal component analysis (PCA) of regulated genes of

MPP+ treated LUHMES cells – p 5

Fig. S6 Regulation of proteins involved in cellular thiol supply by

MPP+ – p 6

Fig. S7 Transcriptional changes triggered by the complex I

inhibitor rotenone in LUHMES cells – p 7

Fig. S8 Integrated analysis of metabolomics and transcriptomics

data to identify affected pathways – p 8

Fig. S9 Overview of MPP+-induced adaptations in human

dopaminergic neurons – p 9

Fig. S10 Antibodies – p 10

Fig. S11 Primers – p 11

Transcriptional and metabolic adaptation of

neurons to the mitochondrial toxicant MPP+

Anne K. Krug1, Simon Gutbier1, Liang Zhao2, Dominik Pöltl1,3, Cornelius

Kullmann1, Violeta Ivanova3,4, Sunniva Förster1, Smita Jagtap5, Johannes

Meiser6, Gérman Leparc7, Stefan Schildknecht1, Martina Adam1, Karsten

Hiller6, Hesso Farhan8, Thomas Brunner9, Thomas Hartung2, Agapios

Sachinidis5, Marcel Leist1

Downregulated metabolites

ATP

Cyc

lic A

DP-rib

ose

Dec

anoic

aci

d

Deh

ydro

asco

rbat

e

Deo

xyurid

ine

D-E

ryth

rose

D-G

luco

se

Glu

tath

ione

Guan

ine

Inosi

ne

L-Ala

nine

L-Asp

arag

ine

L-Glu

tam

ate

L-Pro

line

L-Ser

ine

Mal

eam

ate

N-A

cety

l-L-a

spar

tate

N-A

cety

l-L-g

luta

mat

e

O-A

cety

l-L-h

omose

rine

O-A

cety

lneu

ram

inic

aci

d

Pan

toth

enat

e

Phosp

hocrea

tine

sn-g

lyce

ro-3

-Phosp

hoethan

olam

ine

Sorb

itol

Taurine

UDP-a

lpha-

D-g

alac

tose

UDP-g

luco

se

UDP-N

-ace

tyl-D

-gal

acto

sam

ine

UDP-N

-ace

tyl-D

-Glu

cosa

min

e

2,3-

Dim

ethyl

mal

eate

2,5-

Dio

xopen

tanoat

e

3-Oxo

propan

oate

4-Am

inobuta

noate

0

50

100

150

No

rmalized

In

ten

sit

y V

alu

es

[% o

f co

ntr

ol

SD

]

Upregulated metabolites

Aden

ine

ADP

AM

P

Chole

ster

ol sulfa

te

Cre

atin

e

Deo

xyribose

Dih

ydro

ptero

ate

Formyl

-N-a

cety

l-5-m

ethoxy

kynure

namin

e

L-Arg

inin

e

L-Cys

tath

ionin

e

L-Lac

tate

L-Lys

ine

L-Met

hionin

e S-o

xide

L-Phen

ylal

anin

e

L-Try

ptophan

L-Tyr

osine

Pyr

uvate

S-A

denosy

l-L-h

omocy

stei

ne

S-A

denosy

l-L-m

ethio

nine

S-M

ethyl

GSH

Thiam

ine

acet

ic a

cid

Ura

te-3

-rib

onucleo

side

2-Ace

tola

ctat

e

3-(4

-Hyd

roxy

phenyl

)lact

ate

3-M

ethyl

-2-o

xobuta

noic a

cid

4-Hyd

roxy

-4-m

ethyl

gluta

mat

e

0

100

200

300

400

2000

4000

control

24 h

36 h

No

rmalized

In

ten

sit

y V

alu

es

[% o

f c

on

tro

l

SD

]

B

A Significantly changed metabolites

Page 1

Figure S1

Overview of significantly altered metabolites determined during untargeted

analysis with the accurate-mass Q-TOF LC-MS system Cells were treated with 5 µM MPP+ (control, 24 h or 36 h) in four independent experiments.

After metabolite extraction, samples were run with the accurate-mass Q-TOF LC-MS system

(Agilent, Santa Clara, CA). Metabolites were determined by the MassHunter Acquisition

software (Agilent Technologies). ‘Areas under the Curve’ (AUC) for every peak/metabolite of

the ion chromatogram were calculated and used as basis for a relative quantification. AUCs

were normalized to the mean of the AUCs of control samples (untreated cells) of

4 independent experiments. Only metabolites that were significantly regulated (FDR adjusted

p-value of ≤ 0.05) and that could be unambiguously identified, are displayed.

Creatine

Counts vs. Aquisition Time

Phosphocreatine

Counts vs. Aquisition Time

L-methionine-S-oxide

Counts vs. Aquisition Time

S-Adenosyl-L-methionine

Counts vs. Aquisition Time

Example-peaks of significantly changed metabolites

Control

MPP+

Page 2

Figure S2

Example-peaks of metabolites affected by MPP+ treatment Cells were treated with MPP+ or solvent for 24 h. Samples were run with the accurate-mass

Q-TOF LC-MS system. Peak overlays are displayed by the Agilent MassHunter Acquisition

software. The areas under the curve (integral of the peak curve) were used for quantification.

Data are examples of typical primary data in a representative experiment.

Marker expression

0 12 24 36 48

0

50

100

Time [h]

Bcl-xL

[%

of

contr

ol S

D]

[10 h,

200 nM]

STS w/o 0 12 24 36 48

Cytosolic

cytochrome c

5 µM MPP+

GAPDH

Bcl-xL

GAPDH

0 6 12 24 36 48

5 µM MPP+

PSPC1

Tuji

0 6 12 24 36 48

5 µM MPP+

0 12 24 36 48

0

10

20

30

40

Time [h]

Cyto

solic

cyto

chro

me

c

[% o

f positiv

e c

ontr

ol S

D]

0 12 24 36 48

0

50

100

Time [h]

PS

PC

1

[%

of

contr

ol S

D]

B

A

C

Representative blots: Densitometric quantifications:

Time [h]:

Time [h]:

Time [h]:

Page 3

*

*

*

* *

Figure S3

MPP+-induced changes of amount and location of apoptosis and cell stress-

related proteins LUHMES cell lysates were prepared at different times after exposure to MPP+ as illustrated in

Fig. 1A. Proteins were quantified by western blot. To the left, representative blots are shown.

To the right, densitometric quantifications of the respective proteins are displayed as means

± SD of 3 independent differentiations. Calibration was relative to loading control and

untreated cell samples. A) Bcl-xl levels. B) The cytosolic cytochrome c levels were determined

by extraction of soluble cytosolic proteins after permeabilisation of the outer cell membrane

with 50 µg/ml digitonin. This procedure did not permeabilize the outer mitochondrial

membrane (Single et al. 1998). Controls were healthy cells without digitonin (w/o), healthy

cells with digitonin (0) and a positive control of cells exposed to 200 nM staurosporine for 10 h

(STS). C) PSPC1 (paraspeckle component 1). *: p < 0.05.

Single, B., et al. (1998). "Simultaneous release of adenylate kinase and cytochrome c in cell death." Cell

Death Differ 5(12): 1001-1003.

control 2 h MPP+

4 h MPP+ 6 h MPP+

C 6 12 24 36 48

Par

GAPDH

250 kDA

C 6 12 24 36 48

NQO 1

GAPDH

A B

C

Figure S4: Time course of MPP+ induced ROS generation LUHMES were seeded at a density of 1.5*105 cells/cm2 at day 2. Medium was exchanged at day 5 and cells

were treated with MPP+ at various times.

a) Images of live cell staining of LUHMES (day 6) cultured with 5 µM MPP+ for 2-6 h. Untreated control cells

and treated cells were loaded for 45 minutes with a fluorescin-based ROS detection dye (ENZO, Total ROS

detection kit, ENZ-51011), medium was replaced and cells were imaged immediately. Cells that showed

fluorescence at 530 nm, when excited with 488 nm light were counted as positive for ROS production. Data are

means ± SD of triplicates.

b) Whole cell lysates of LUHMES treated with 5 µM MPP+ for 0, 6, 12, 24, 36 and 48 h were separated by 8%

SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were incubated with anti PAR (H10

monoclonal AB from 10H hybridoma cells (1:300) (Kawamitsu, Hoshino et al. 1984)., mouse) primary antibody

overnight and with anti-mouse (Jackson, 1:2500) secondary antibody for one hour. (n=3), before the Western

blot was developed.

c) Whole cell lysates of LUHMES treated with 5 µM MPP+ for 0, 12, 24, 36, and 48 h, were separated by 12%

SDS-PAGE and transferred to a nitrocellulose membrane. For Western blot detection, membranes were

incubated with anti NQO1 (Cell Signaling, 1:1000) primary antibody overnight and with anti-rabbit (GE

Healthcare, IgG,1:5000) secondary antibodies for one hour. Protein levels (scans) were normalized to loading

controls (GAPDH), and data are averages from 5 experiments.

Page 4

PCA analysis of gene array data

Page 5

Figure S5

Principal component analysis (PCA) of regulated genes of MPP+-treated

LUHMES cells Cells were treated with MPP+ (5 µM) for different times and samples were prepared for

microarray analysis as described in Fig. 1A. The signal of all probesets significantly regulated

(FDR adjusted p-value of ≤ 0.05; fold change values ≥ 2) was used for principal component

analysis (PCA). The first two dimensions of the respective data are displayed.

48 h

Control

36 h

24 h

Prin

cip

al c

om

po

ne

nt 2

(17

%)

Principal component 1 (23 %)

Figure S6:

Regulation of proteins involved in cellular thiol supply by MPP+ LUHMES were seeded at a density of 1.5*105 cells/cm2 at day 2. Medium was exchanged at day 5 and cells

were treated at various times with 5 µM MPP+.

A) Whole cell lysates of LUHMES treated with 5 µM MPP+ for 0, 12, 24, 36, and 48 h, were separated by 8%

SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were incubated with anti-ASS1 (Sigma

Aldrich, mouse, 1:250) overnight and with anti-mouse (Jackson,1:5000) ) secondary antibody for one hour.

Protein amounts were quantified by a luminescence imager relative to the loading control (GAPDH). These

relative protein amounts were then normalized to the amount of protein in control cells. Data are means ± SD of

5 determinations.

B) Whole cell lysates of LUHMES treated with 5 µM MPP+ for 0, 12, 24, 36, and 48 h, were separated by 12%

SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were incubated with anti-CTH (Abcam,

mouse, 1:250) overnight and with anti-mouse (Jackson,1:5000) ) secondary antibody for one hour (n=3).

Protein was quantified as in a.

C) Cells (350.000/well), treated with or without 5 µM MPP+ for 0, 12 or 24 h, were washed 1× with Hank's

balanced salt solution (HBSS), containing Ca2+, pH 7.4. Then 200 µl containing 0.2 µCi/ml [14C]-cystine (110

mCi/mmol stock solution; Perkin Elmer, Waltham, MA, USA) was added. The supernatants were removed after

20 min, cells were washed 3× gently with warm HBSS and then lysed with PBS/0.1% Triton X-100.

Radioactivity in cell lysates was measured using a Beckman LS-6500 scintillation counter. Experiments were

performed in parallel in the presence of a 100-fold excess (500 µM) of unlabelled (cold) cystine.

Page 6

ASS1

GAPDH

C 6 12 24 36 48

C 6 12 24 36 48

CTH

GAPDH

control 12 h MPP+

24 h MPP+

0

100

200

300

400

500 total cystine uptake

uptake in presence of competitor

% u

pta

ke o

f co

ntr

ol

A

B

C

0 20 40 60

0

1

2

3

4

6

8

10

Time [h]

mR

NA

leve

l [fo

ld c

hang

e r

ela

tive

to

co

ntr

ol

SE

M]

Complex I inhibitor rotenone

0 24 48 72

0

50

100

0.02 µM0.1 µM0.5 µM

Time [h]

AT

P c

on

ce

ntr

atio

n

[% o

f co

ntr

ol

SE

M]

0 24 48 72

0

50

100

0.02 µM0.1 µM0.5 µM

Time [h]

GS

H c

on

ce

ntr

atio

n

[% o

f co

ntr

ol

SE

M]

0 24 48 72

0

20

40

600.02 µM

0.1 µM

0.5 µM

Time [h]

Ext

race

llula

r L

DH

[% o

f co

ntr

ol

SE

M]

Sampling

d-1 d0 d2 d4 d6 d7 d8 d9

48 h 24 h 72 h Replate

Medium

change Start diff.

0.1 µM Rotenone

ATF4 – Activating transcription factor 4

DDIT3 – DNA damage inducible

transcript 3 (CHOP, GADD153)

ASS1 – Argininosuccinate synthase 1

ASNS – Asparagine synthase

NOXA – Phorbol-12-myristate-13-

acetate-induced protein 1 (PMAIP1)

CTH – Cystathionase

(cystathionine gamma-lyase)

CBS – Cystathionine-β-synthase

TXNIP – Thioredoxin-interacting protein

HNRNPM – Heterogeneous nuclear

ribonucleoprotein M

TYMS – Thymidylate synthetase

Page 7

biosynthetic processes oxidative stress

chromosomal changes/paraspeckles

ER stressmitochondrial function

Color coding of biological process:

B

A

* * * *

* *

* *

* *

*

Figure S7

Transcriptional changes triggered by the complex I inhibitor rotenone in

LUHMES cells Cells were treated with different concentrations of rotenone (20, 100, 500 nM) for different

times (24, 48, 72 h) and all samples were taken at day 9 (d9), as indicated in the treatment

scheme. A) General cytotoxicity was evaluated by measurement of LDH release into the

medium. GSH and ATP concentrations were determined in cell lysates. B) Several genes,

which had been found to be regulated in the MPP+ toxicity model, were examined. The mRNA

from samples treated with 100 nM rotenone was quantified by RT-qPCR. All Data are means

± SEM of three independent experiments. They were normalized to untreated control. For

easier comparison, the 48 h data for cytotoxicity and gene regulation are highlighted by a

dashed box.

Combined Omics pathway-analysis

Regulated metabolites

Regulated genes

Page 8

Figure S8

Integrated analysis of metabolomics and transcriptomics data to identify affected

pathways The pathway-analysis is based on transcriptomics and metabolomics data of the cells treated for

24 h with 5 µM MPP+. The integration of both data sets for multi-omics analysis was performed

using Mass Profiler Professional (MPP) software (version 12.6, Agilent Technologies). The MPP

multi-omics capability allows two different omic experiments to be mapped and seen on the same

pathway. Metabolites, identified by Q-TOF LC-MS and transcripts of the DNA microarray analysis

were combined and reanalyzed together for pathway enrichment. A fold change cut-off for

transcripts and metabolites of 1.5 and a significance threshold of 0.05 (FDR corrected) were used.

WikiPathways served as pathway source. Differentially detected metabolites and genes are

highlighted in color and have an adjacent heat strip for the relative abundances across the different

conditions (red = control samples, yellow = 24 h samples, blue = 36 h samples, grey = 48 h

samples). Regulated metabolites are indicated in yellow (cysteine was detected only in the targeted

analysis); blue highlights regulated genes. (AHCY = adenosylhomocysteinase, AHCYL1 =

adenosylhomocysteinase-like 1, AHCYL2 = adenosylhomocysteinase-like 2, AMT =

aminomethyltransferase, BHMT = betaine—homocysteine

S-methyltransferase, CBS = cystathionine-β-synthase, CTH = cystathionase, DHFR =

dihydrofolatereductase, DHFRL1 = dihydrofolatereductase-like 1, DNMT1 = DNA (cytosine-5-)-

methyltransferase 1, DNMT3A = DNA (cytosine-5-)-methyltransferase 3 alpha, DNMT3B = DNA

(cytosine-5-)-methyltransferase 3 beta, DNMT3L = DNA (cytosine-5-)-methyltransferase 3-like, dTMP =

deoxythymidine monophosphate, dUMP = deoxyuridine monophosphate, GCLC = glutamate-cysteine

ligase, GCLM = glutamate-cysteine ligase, modifier subunit, GSS = glutathione synthetase, MAT2B =

methionine adenosyltransferase II beta, MAT1A = methionine adenosyltransferase I alpha, MAT2A =

methionine adenosyltransferase II alpha, MTHFD1 = methylenetetrahydrofolate dehydrogenase 1,

MTHFD1L = methylenetetrahydrofolate dehydrogenase 1-like, MTHFD2 = methylenetetrahydrofolate

dehydrogenase 2, MTHFD2L = methylenetetrahydrofolate dehydrogenase 2-like, MTHFR =

methylenetetrahydrofolatereductase, MTR = methionine synthase, PHGDH =

phosphoglyceratedehydrogenase, PSAT1 = phosphoserine aminotransferase 1, PSPH =

phosphoserinephosphatase, ROS = reactive oxygen species, SHMT1 = serine hydroxymethyl-transferase

1, SHMT2 = serine hydroxymethyl-transferase 2, THF = tetrahydrofolate, TYMS = thymidylatesynthetase)

Page 9

MPP+ induced cellular adaptations

Figure S9

Overview of MPP+-induced adaptations in human dopaminergic neurons The findings of this study (labeled here with *) have been incorporated into a network of adaptive

regulations. The molecular initiating event of MPP+ toxicity is inhibition of mitochondrial

respiratory chain complex I. Thus, NADH oxidation is hindered, and this leads to an arrest of the

TCA cycle. The subsequent slowdown of the pyruvate dehydrogenase* leads to an accumulation

of pyruvate* and lactate*. Cellular adaptations lead to increased glucose consumption* and

usage of alternative ATP synthesis sources*. Complex I inhibition also leads to a higher O2-•

production, as indicated by decreased levels of the antioxidant dehydroascorbate* and an

increase in methionine sulfoxide*. The two primary events cause a general increase in GSH

demand with adaptations on metabolite and transcriptome level*. Demand for alternative energy

sources and GSH results in changed amino acid metabolism* and higher expression of their

transporters*. Several of these changes contribute to a rise in ER stress, as indicated by

phosphorylation of eIF2a* and increases of ATF4*. Many of the observed changes in gene

regulation may be attributed to ATF-4 and related transcriptions factors. A DNA damage

response* and altered lipid metabolism* suggest that many further cellular changes take place

long before energy is depleted. At the point of no return the counter-regulation capacity of the

cell is exhausted, ATP and GSH drop steeply*, programmed cell death pathways (NOXA ↑,

PUMA ↑, Bcl-xL ↓) are activated* and loss of mitochondrial integrity ensues*.Pyr = pyruvate, Lac

= lactate, Gluc = glucose, TCA = tricarboxylic acid cycle, Dehydroasc = dehydroascorbate,

Met(SO) = methionine sulfoxide, GSH = reduced glutathione, GSSG = glutathione disulfide.

Antigen Antibody (supplier; clone) Dilution Blocking

(5%)

Species

ATF4

(CREB-2)

Anti-ATF4(D4B8, cell signaling) 1:1000 BSA rabbit

CTH Anti-CTH (abcam) 1:250 BSA mouse

GADD34 Anti-GADD34 (proteintech) 1:1000 BSA mouse

eIF2a-p Anti-eIF2a [pS52] (invitrogen) 1:1000 BSA rabbit

PSPC1 Anti-PSPC1 (sigma) 1:200 Milk rabbit

GAPDH Anti-GAPDH (Sigma; Clone GAPDH-

71.1)

1:5000 BSA mouse

Bcl-xl anti Bcl-xl (CellSignaling) 1:1000 Milk rabbit

Cytochrome c Anti-Cytochcrome c (BD Pharmingen;

Clone 7H8.2C12

1:1000 Milk mouse

anti-mouse anti-mouse HRP antibody (Jackson

Immuno Research)

1:2500 goat

anti-rabbit anti-rabbit HRP antibody (GE Healthcare ) 1:5000 goat

Figure 10– Antibodies used for western blot or immunostaining

Antibodies

Page 10

Primers

Name Forward sequence Reverse Sequence

ASNS GGGGCTTGGACTCCAGCTTG GAGCCTGAATGCCTTCCTCA

ASS1 TGCTCCCTGGAGGATGCCTG GTGTAGAGACCTGGAGGCGC

ATF2 AGAGCGAAATAGAGCAGCAG CATGGCGGTTACAGGGCAAT

ATF4 GGCTGGCTGTGGATGGGTTG CTCCTGGACTAGGGGGGCAA

CBS TCCTGGGAATGGTGACGCTT GTGCTGTGGTACTGGATCTG

CCNB TGGATGTGCCCCTGCAGAAG CAGTGACTTCCCGACCCAGT

CTH TGGATGATGTGTATGGAGGTACAAACAGG GCCTTCAATGTCAATCACCTTCTGGG

DDIT3 ATGGCAGCTGAGTCATTGCC TCCTCAGTCAGCCAAGCCAG

DDIT4 AGTCCCTGGACAGCAGCAAC AACTGGCTAGGCATCAGCAG

GADD34 GCATCACCCAGGCCCAGGAG AGACGAGCGGGAAGGTGTGG

GAPDH CACCATCTTCCAGGAGCGAGATC GCAGGAGGCATTGCTGATGATC

HNRNPM TGGTGTGGTGGTCCGAGCAG GGACGCTCAGGAGGGAAGAA

MLF1IP TTTGTAAGGCAGCCATCGCC CTGTGGCTCTAACCGAAGCA

NOXA CAGTGCCAACTCAGCACATTG CGCCCAACAGGAACACATTGA

NQO1 TGGAGTCGGACCTCTATGCCA CTTGTGATATTCCAGTTCCCCCTGC

PHGDH AAC TCC AGG TGG TGG GCA GG GCT CCC ATT TGC CGT CCT TC

PPA2 TGGAAAGCTACGCTATGTGG GCTTCAGGATCATTCGCATTG

PSAT1 AGC TGC CGC ACT CAG TGT TG AAC TGG CCG CAC CCA CCT CC

PSPC1 CAGCAGCGTGAGCAGGTTGA CGCCGATGCTCCTCTTCATG

PSPH CCC CGG CAT AAG GGA GCT GG GCT GTT GGC TGC GTC TCA TC

SFPQ TCAGGCAAATCTTTTGCGCC CTCTCTTTGGCGCCTCATTT

SHMT2 CAACCTGGCACTGACTGCTC GATGTCCGCGTGCTTGAAAG

SLC3A2 CAC CCT GCC AGG GAC CCC TG GCC GCC GGA ACA AGG AAA GG

SLC7A11 GCA GCG TGG GCA TGT CTC TG CAC AGC AGT AGC TGC AGG GC

TXNIP1 CATGGCGTGGCAAGAGCCTT CTCAGAGCTGGTTCGGCTGG

TYMS CAGCTTCAGCGAGAACCCAG ACCTCGGCATCCAGCCCAAC

Figure 11– Primers used for RT-qPCR

Page 11