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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
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