Supplementary Information
Global lipidomics identifies cardiolipin oxidation as a mitochondrial target for redox therapy of acute brain injury Jing Ji1,2,7,8, Anthony E Kline3,8,9, Andrew Amoscato2,7, Alejandro S Arias2,7, Louis J Sparvero2,7, Vladimir A Tyurin2,7, Yulia Y Tyurina2,7, Bruno Fink10, Mioara D Manole5, 8, Ava M Puccio6, David O Okonkwo6, Jeffrey P Cheng3,8, Henry Alexander1,8, Robert SB Clark1,8, Patrick M Kochanek1,8, Peter Wipf4, Valerian E Kagan2,7 & Hülya Bayır1,2,7,8 Correspondence should be addressed to H.B. (
[email protected]).
Major CL species undergoing oxidation m/z Molecular species 1476 18:1/18:1/18:2/20:4 1476 18:0/18:2/18:2/20:4 1500 18:0/18:2/18:2/22:6 1502 18:0/18:1/18:2/22:6 1502 18:0/18:1/18:2/22:6 1504 18:0/18:0/18:2/22:6
m/z 1524 1572 1580 1550 1582 1552
Oxygenated CL molecular species Plus three oxygens Plus five oxygens Plus six oxygens + + + + + +
Supplementary Table 1. Major CL species that underwent oxidation and oxidation products generated after TBI
a
PE 760
Control
760
CCI
722 746 788 750 762
722 746 788
750 778
718
0
0
10
20
30 Time (min)
700
700
760 m/z
PE ox 820
792
b
20
30 Time (min)
700
850 m/z
1000 700
760 m/z
CCI
764
820
820 10
30 Time (min)
20
0
10
20
30 Time (min)
792
792
PC PC ox
PC
950 700
PC ox
818
764
850 m/z
820
792
764
750
790 774
722 718 738
PC
Control
0
760
750
722 718 738 1000
850 m/z
10
PE
790 774
750
PE ox
0
760
PE
778
718
764 800 m/z
900
750
850 m/z
700
950
818 800 m/z
900
c PG
PI 747
885
747
Control
CCI
Control
857 883 775
857 883
775 6
12 18 Time (min)
CCI
769
769
0
885
24
0
6
12 18 Time (min)
24
Supplementary Figure 1. 2D-LCMS characterization of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol (PI) and their oxidation products after TBI. (a, b) Representative reverse phase chromatograms and MS characterization of non-oxidized (shown in blue) and oxidized (shown in red) species of PC and PE in control and TBI brains. There was no accumulation of oxidation products in these two most abundant classes of phospholipids in rat brain after TBI. (c) Representative reverse phase chromatograms for PG and PI in control and TBI brain. No changes in chromatographic profiles for PG and PI between control and TBI were detectable.
a
1480
1478
1450
1500 10 12 14 16 18 20 Time (min)
CLox
CL
1428 1524
1496 1464
1508
1562
1578
1404
1460 1480 1500 1520 1540 1560 1580 m/z
1400
1548 1440
1480
1520 m/z
1560
750 766
b
PE
PC
790
744
778
818
718
792 772
760
700
820
700
800
m/z
800 m/z
900
m/z
PE ox 700
846
PC ox 900
1000
700
800
900
1000
m/z
Supplementary Figure 2. 2D-LCMS analysis of phospholipids in the penumbra of contused brain tissue taken from a TBI patient with intractable hypertension. (a) Analysis of CL and CLox by 2D-LCMS in human brain tissue after TBI. The tissue was obtained by right temporal lobe resection from a patient with intractable intracranial hypertension in the acute stage following severe TBI. Upper insert: 2nd dimension chromatographic separation of non-oxidized and oxidized CL. The latter eluted during the 10-12 min retention time window. Lower panel demonstrates nonoxidized (blue) and the appearance of numerous oxidized (red) CL (CLox) species after TBI. (b) Spectra obtained from oxidized and non-oxidized fractions after reverse phase chromatography for PC and PE in the same brain tissue. In contrast to CL, no oxidation products were detected in PC or PE.
1600
a
b
20
* 10
0
c
CCI
Naïve Sham 3 h
ΔPCox (3.8 ± 2.4)
24 h
3h
ΔPEox (3.8 ± 2.2)
90
* 60
30
0
24 h
CCI
120
PSH (nmol/mg protein)
GSH (nmol/mg protein)
30
Naïve Sham
3h
24 h
3h
24 h
d Ph BocHN O
.O ΔCL ox (127.9 ± 18.8)
O N
N H
N H
N H N O
O NHCbz
Supplementary Figure 3. Biochemical analysis after TBI and structure of XJB-5131.Quantification of reduced glutathione (GSH, a) and protein sulfhydryl (PSH, b) levels in the ipsilateral cortex after CCI . XJB-5-131 (red bars) attenuated CCIinduced GSH and PSH depletion in PND 17 rats (blue bars). *P < 0.01 vs. naïve and sham controls, CCI 3h, and CCI (3h and 24h) + XJB-5-131; error bars, standard deviation; n = 4 rats per group. (c) Increase in the contents of oxydized cardiolipin (CLox), phosphatidylcholine (PCox) and phosphatidylethanolamine (PEox) in rat cortical neurons 2 hours after stretch assessed by LC-MS. Data are presented as pmol of oxidized phospholipid per nmol of phospholipid; error bars, standard deviation; n = 4 experiments. (d) Structure of the hemigramicidin Speptidyl 4-amino TEMPO conjugate, XJB-5-131.
a
b 40
120
Stretch
*
90
* 20
* * 10
MTT (% control)
LDH (% cytoxicity)
30
Stretch
* *
60
30
0 0 1 2.5 5 10 25 0 1 2.5 5 10 25 XJB, µM
control
0
0
1 2.5 5 10 25 0
1 2.5 5 10 25
XJB, µM
Supplementary Figure 4. Effects of XJB-5-131 (1-25 μM) on primary rat cortical neurons in culture exposed to in vitro trauma by mechanical stretch. Vehicle or XJB-5-131 was added to the medium 10 min before stretch. (a) Percent cytotoxicity (lactate dehydrogenase (LDH) release relative to Triton exposure (corrected for background LDH). (b) 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) as a percentage of normal control conditions. Mechanical stretch induced approximately 35% neuronal death at 24 hours assessed by MTT and LDH. A marked reduction in neuronal death was produced by 5, 10, and 25 μM XJB-5-131 treatments. *P < 0.01 vs. stretch only neurons; error bars, standard deviation; data are from three independent experiments and include 8-11 wells for each condition.
Cell viability % by trypan blue
Stretch
a
120
siRNA
100 80
c
*
N
SC
40 20 0 C
b
*
60
CS CYC XJB
C
N
SC
CS
CYC
XJB
C
N
SC
CS
CYC
XJB
Supplementary Figure 5. Response of neurons treated with CL synthase-siRNA or cyt c-siRNA or XJB-5-131 to in vitro TBI. (a) Cell counts after mechanical stretch in cortical neurons transfected with cardiolipin synthase (CS) or Cytochrome c (CYC) or scrambled control (SC) siRNA as well as after treatment with XJB-5-131 were performed using Trypan Blue assay. (b, c) Immunostaining for cleaved caspase 3 (b) and neuronal cytoskeleton (microtubule-associated protein 2, MAP-2) (c) after mechanical stretch in cortical neurons transfected with cardiolipin synthase (CS) or Cytochrome c (CYC) or scrambled control (SC) siRNA as well as after treatment with XJB-5-131. C: control normal neurons; N: non-transfected neurons. * P < 0.01 vs. C, CS, CYC, and XJB; error bars, standard deviation; data are from three independent experiments and include 8-11 wells for each condition.
a CLS normalized to actin
120
CLS
*
60
COX IV TOM 40
30
* N
N
c
30
20
* 10
120
72 h
48 h
72 h
CS
8
4
N
96 h
SC
72 h
12
0 N
48 h
SC
16 nmoles ATP per mg protein
pmole CL per nmole total lipid Pi
actin
48 h 72 h 48 h 72 h
40
Cyt c normalized to actin
CS
90
0
d
CS SC
0
b
SC
72 h 96 h 72 h 96 h
CYC SC
CYC
90 cyt c 60
actin
*
30
0
N
N
*
48 h 72 h 48 h 72 h
48 h
72 h
48 h
72 h
Supplementary Figure 6. Generation of cardiolipin (CL) and cytochrome c (cyt c) deficient neurons. (a) Primary rat neurons were transfected with CL-synthase (CS) siRNA or a scrambled control (SC) siRNA. Insert: Representative western blot showing effective knockdown of CS at 48h and 72h after siRNA treatment. (b) Quantification of total CL content in control and CS knock down neurons by electrospray ionization MS (ESI-MS). Transfection with CS produced ~15% (72 h) and 56% (96 h) decrease in CL content, and did not change the mitochondrial member markers (as assessed by cytochrome c oxidase IV (COX IV) and transporter outer membrane 40 (TOM 40)). (c) Assessment of ATP levels in control and CS deficient neurons. CL deficiency did not change cellular ATP levels. (d) Primary rat neurons were transfected with Cytochrome c (CYC) or SC siRNA. Insert: Representative western blot showing effective knock down of cyt c at 48 h and 72 h after siRNA treatment. N, nontransfected neurons. *P < 0.01 vs. N and SC; error bars, standard deviation; n = 4 experiments.
a
b
100 80 60 40 20 0
CCI+Vehicle (N=10) CCI+XJB (N=9) * Sham (N=10) 30
31 32 33 Time after injury (day)
40 Cortical lesion volume (mm3)
Time to Platform (s)
120
30
*
20
10
0 34
CCI + XJB
CCI + Vehicle
Supplementary Figure 7. Assessments of sub-chronic neurobehavioral and histological outcome in PND 17 rats treated with XJB-5-131 after TBI. (a) Morris water maze test at 30-34 days after TBI in PND17 rats. XJB-5-131 enhanced the acquisition of spatial learning and memory vs vehicle-treated controls. A repeated measures ANOVA revealed significant group (F2,26 = 19.156, P < 0.001) and day (F4,104 = 42.509, P < 0.001) differences, as well as a significant group × day interaction (F8,104 = 2.723, P = 0.0091). Bonferroni post hoc analyses revealed that the TBI + XJB group performed significantly better than the TBI + vehicle group (P < 0.001; error bars, standard error; n = 9-10 rats per group) and did not differ from the sham controls (P = 0.69). (b) Analysis of cortical lesion volume at 36 days after TBI in PND 17 rats. Cortical lesion volume was significantly decreased at 5 weeks in the TBI group treated with XJB -5-131 vs. the TBI group that received vehicle. P = 0.0465, ANOVA, F1,13 = 4.840; error bars, standard error; n = 9-10 rats per group.
Caspase 3/7 activity (fold change)
CCI Vehicle
XJB
*
3
2
1
0
Naïve Sham
3h
24 h
3h
24 h
Supplementary Figure 8. Quantification of caspase 3/7 activity in the ipsilateral cortex. XJB-5-131 attenuated CCI-induced increase in caspase 3/7 activity in PND 17 rats. *P < 0.01 vs. naïve and sham controls, CCI 3h + Vehicle, and CCI + XJB-5-131; error bars, standard deviation; n = 4 rats per group.
Mean fluorescence of Mitosox (fold change)
a *
Control
2
Stretch + Vehicle Stretch + XJB
#
1 20x 0
b
Stretch
3
Control
Vehicle
XJB
Stretch
16
Cytosolic cyt c (normalized to actin)
* 12
Control
Stretch + Vehicle
Stretch + XJB
cyt c 8 actin 4
0
Control
Vehicle
XJB
Supplementary Figure 9. Biochemical effects of XJB-5-131 after in vitro TBI. (a) Assessment of mitochondrial superoxide generation in primary cortical neurons after mechanical stretch. Left panel shows quantitative analysis of mitochondrial superoxide generation by flow cytometry. Mechanical stretch injury resulted in increased superoxide formation in neuronal mitochondria at 2 hours after stretch injury compared with control. XJB-5-131 attenuated stretch induced mitochondrial superoxide generation. Right panel shows fluorescence microscope assessment of MitoSOX (red) staining in neurons. Co-staining of nuclei with Hoechst 33342 (blue). (b) Quantification of cytochrome c (cyt c) release from mitochondria into cytosol after mechanical stretch. Right insert: representative western blots showing control, stretch + vehicle, and stretch + XJB-5-131. XJB-5-131 attenuated stretch-induced cyt c release into the cytosol. *P < 0.01 vs. control and stretch + XJB-5-131; #P < 0.05 vs. control; error bars, standard deviation; n = 4 experiments.
