1) Oxidative stress and mitochondrial dysfunction play a key role in neuronal apoptosis following acute brain injuries like spinal cord injury and traumatic brain injury. 2) The STAT3 transcription factor regulates the expression of the mitochondrial antioxidant enzyme MnSOD (SOD2) which reduces oxidative stress. 3) Activating the IL-6/STAT3/MnSOD pathway may be neuroprotective by reducing mitochondrial oxidative stress and neuronal death following brain injuries.

1. Pak H. Chan, PhD James R. Doty Professor of Neurosurgery and Neurosciences Stanford University Medical School National Neurotrauma Symposium July 12, 2011 Fort Lauderdale, Florida Oxidative Stress, Mitochondrial Dysfunction and Neuronal Apoptosis in Acute Brain Injuries

3. Procaspase-9 Caspase - Cytochrome C Smac IAPs Smac mitochondria AIF Activation of CAD Intrinsic Mitochondria-Dependent Pathway of Apoptosis DNA Damage Activation of Downstream (2,6,8,10) PARP Cleavage cytosol AIF Cell Death Trigger (ischemia & trauma) Caspase-9 Caspase-3 (-) Smac ROS mitochondria DNA Damage Cell Death Activation of Downstream Caspases (2,6,8,10) PARP Cleavage nucleus Cytochrome c (-)

4. Oxidants as triggers for mitochondrial-dependent apoptosis in brain trauma and ischemia

5. Clip compression (5sec, 15g) Lumbar enlargement Spinal Cord Injury in Rats (SOD1 Tg rats) Motor neuron counting at 0, ±1, 2, 3 mm from the center Sugawara et al, FASEBJ , 2002

6. Double Fluorescence Staining of Ethidium and NeuN, GFAP, MitoTracker in Wt rats 1 day after SCI NeuN GFAP MT Sugawara et al, FASEBJ , 2002

7. Superoxide Radicals in Spinal Cord Sugawara et al, FASEBJ , 2002

8. Neuronal Histology (a,c,d,e) and NeuN (b,g) TUNEL (f) staining, and DNA laddering (h) in SCI 3d 1d 3d 3d Sugawara et al, FASEBJ , 2002

9. Western Blots of Cytochrome C and Cleaved Caspase 9 in SCI Sugawara et al, FASEBJ , 2002

10. Apoptotic Stimulus Procaspase-9 Caspase-9 Caspase-3 DNA Damage Cell Death Cytochrome C Cytochrome c Smac IAPs (-) (-) Smac Superoxide Overproduction Activation of Downstream Caspases (2,6,8,10) PARP Cleavage C:DNA Damage, Cell Death (3d) B:Cytochrome c Release, Caspase-9 Cleavage (6h-1d) A: Superoxide Production (6h) A B B mitochondria cytosol ? C Spinal Cord Injury Necrotic Cell Death (6h-1d) Within 0.3mm 0.3mm-2.5mm from the lesion In the present study

11. Mitochondrion, a life and death executor Wallace, Science 1999

12. Cytochrome C Release in Wt and SOD2 -/+ Mice After TBI (Controlled Cortical Impact, Dixon 1991) Lew é n et al, JCBFM, 2001

13. Superoxide radical and infarction volume in SOD2 -/+ mice after cerebral ischemia and reperfusion Kim et al, Stroke , 2002

14. SOD2-KO mice are more susceptible to hemorrhage SOD2-KO WT Hemoglobin  -Actin 72h Rate of HT SOD2-KO: 8/9 WT: 3/8 Maier et al., Ann Neurol 2006; 59:929-938

16. Putative binding motifs of STAT3 in promoter of mouse Mn-SOD gene A Mouse SOD2 promoter -1880 tgaagaccgc tttgacatca gccatggaga cgcagagttt ggagtttacc cagctggttt cctgtctttc tttggggatt acagttaagt gattggatgg atctcagaag agaccttgaa -1760 ctttggattt ttaatattgt tgagactgct atagactatg aggacttgaa aag tttgact aaa tgcattt ttttttgttt ttgtttttgt ttttttttga ggcagggttt ctctgtatag -1640 ccctggctgt c ttccggaaa tcactttgta gaccaggcta gccttgaact cagaaatccg cctgcctctg tctccggagt gctgggatca aaggcatgtg ccaccacacc accatagcat -1420 tttgcattat gctatgttta agtatggccc cccatagact catgtttttg aacaagtcta tggggggcca gggagtggag catgatggtt tgtatattct tgggccaggg agtggcacca -1300 tctggaggtg tggcc ttgtt ggaa taggtg tgacctggtt ggaatgggtg tgtcactgtg ggtgtgggca taagatcctc accctagttg cctggaagtc agtcttccac tagccgcctt -1180 tggatgagga catagaactc tcagctctgc ctgtgccatg cctgcctgga tactgccatg ctcccacctt aatgataatg gactgaacct ctgaacctgt aagccagcca caagtaactg -1060 ttgtttttta taagacttgc cttggtcatg gtgtctgttc acagcagtaa aaccctaact aagacacagg tcatccatac agctccggtc tgacgcctgt ggacaggttt ctccctaccg -940 gaaagcatcc tcttgacaat tcccc ttacc ctaa gccctg tcatccttag gaggcctgca aagggggtgg gaaaaaacca cc gggaacta ct cagccaac tgccttctgc t ttcttagtg -820 ttttctaatc agggaagaca taaaccca tt aagtaga aga ggaaagctgt ataattg tta t g gaaa catt tgatagccac tgc ttcttag ac taataagt ccacaaaggg aaacaatgtc -700 cttttacat t tggtcgag tg ggttggtttt tgaagtcact gaatg ttgca gagt ttaatc aag aaagatg aacctcgcct tctaatccgg actcttacta g ttaactggc aagctgcacc -580 cggagtccgc aaccccagtc tcaggggcaa caaagatgaa cacacgcaaa cctgcgacgt gatttaatgt caggttaggc cccagggaag gtcactccgg gcataaatta actgggtcag -460 aggggccctg attactccat aa ttctgacc a gcagcagag ccttggct tt ccggagg aaa gtctccaggg t ttcccagaa gcaggagtag aaatagagtg gaagctttgc agctcacagc -340 cagagctgga ca gggcggc c cg aggagggg cggacc gaca ccgcggggac c ggggttccc c gag gcgggc ggggc caagg ccgatggtg g gggcgtggc t gtagcaagca cgcggcctct -220 accaatttgg cacaggggag acgttgcc tt cccagga tgc cgctccgtta tgcgccgggc cgtccgtgtc gccgtcctcc cctccgctga tgggcgctgc gggcagggtc accgcttcgc -100 tgtgtccttg cggacgcc gg gcgg acgctg cctagcagac gcgcgcctgc gagcgaacgg ccgtg ttctg agga gagcag cggtcgtgta aacctcaata atg ttgtgtc gggcggcgtg NF-kB NF-kB SP-1 SP-1 SP-1 SP-1 SP-1 Start codon I II III IV V VI STAT3 STAT3 STAT3 STAT3 3.STAT3 7.STAT3 11.STAT3 STAT3 STAT3 1.STAT3 4.STAT3 5.STAT3 6.STAT3 8.STAT3 9.STAT3 10.STAT3 12.STAT3 2.STAT3 +1 STAT3: S ignal T ransducers and A ctivator of T ranscription 3 SPI: Promoter-selective transcription factor 1 NF  B: Nuclear Factor Kappa B Jung et al, J Neurosci , 2009

17. STAT3 is significantly down-regulated by ischemic reperfusion in mouse MCAO model Jung et al, J Neurosci , 2009

18. Cerebral ischemia/reperfusion STAT3 Hypothesis: Induction of Mn-SOD (SOD2) expression by STAT3 is neuroprotective after cerebral ischemia and reperfusion p-STAT3 (Y705) Nuclear translocation Mn-SOD promoter-DNA binding Mn-SOD expression Mitochondrial oxidative stress Neuronal survival/death

19. STAT3 regulates the transcription of the mouse Mn-SOD gene Jung et al, J Neurosci , 2009

20. Cytokine (IL-6) JAK2 activation Binding of cytokine receptor by IL-6 and the activation of STAT3 is neuroprotective STAT3 phosphorylation (Y705) Mn-SOD induction and expression Neuronal survival Jung et al, unpublished data

21. IL-6 increase p-STAT3 and MnSOD expression in the ischemic brain and in cortical neurons after OGD Jung et al, unpublished data

22. IL-6R knockdown induces cell death and ischemic infarction Jung et al, unpublished data

23. IL-6 preconditioning up-regulated Mn-SOD via STAT3 activation and induced cytoprotection for neural stem cells in vitro H Sakata et al, unpublished data

24. IL-6 preconditioning reduced grafted cell death in vivo H Sakata et al, unpublished data

25. Summary Can we block mitochondrial activation of apoptosis? 1) Enhanced mitochondrial anti-oxidative properties and functions, upregulation of the inducible mitochondrial anti-oxidant enzyme, MnSOD (SOD2) 2) Activation of the anti-apoptotic signaling pathway: IL-6  IL-6R/gp130  JAK2/STAT3  p-STAT3 (Y705)  MnSOD Promoter  MnSOD Expression  Anti-Mitochondrial Oxidative Stress  Neuronal Survival 3) Targeting IL-6/STAT3/MnSOD signaling in neural stem cells for neuroprotection in acute brain injuries?