Bethea, John


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Bethea, John

  1. 1. Astrocytes are central regulators of demyelination following SCIJohn R. BetheaPh.D<br />
  2. 2. Valery<br />Josh<br />
  3. 3.
  4. 4. Possible Explanations<br />Reduced Oligodendrocyte Death<br />Enhanced Oligogenesis and Remyelination<br />Does astroglial NF-κB activation contribute to oligodendrocytedeath after injury?<br />
  5. 5. Oligodendrocyte Death 1 Week Post-SCI<br />Astroglial NF-κB inactivation reduces oligodendrocyte death 1 week following SCI . (a) Quantification of the number of TUNEL and GSTπ co-labeled cells at 1 week following SCI (n = 3-4). (b) Total GSTπ counts in naïve animals revealed no significant change in total number of oligodendrocytes between WT (n=5) and IκBα-dn (n=4) animals. *, p < 0.05. <br />
  6. 6. Fate of oligodendrocytes after spinal cord injury<br />Early mechanism of OL loss<br /> - Proteolytic enzymes released from necrotic cells<br /> - Free radicals OL particularly vulnerable to oxidative stress<br />- Excitotoxicity(Glu / ATP)<br />- Infiltrating neutrophils and activated microglia (free radicals, cytokines, Glu, and proteases)<br />Chronic OL loss<br /> -Wallerian degeneration<br />Almad A. et al. (2011) Neurotherapeutics 8:262-73<br />
  7. 7. NADPH Oxidase<br /><br />
  8. 8. Astroglial NF-κB inactivation reduces gp91 phox protein expression 1 week following SCI. <br />
  9. 9. Oxidative Injury Following SCI<br />
  10. 10. In Vitro Injury Model<br />Shepard et al. (1991) Journal of Surgical Research. 51: 417-424.<br />
  11. 11. Injured Astrocyte Media Induce Oligodendrocyte Death<br />
  12. 12. Astrocyte-induced oligodendrocyte death is mediated by AMPA receptor signaling in vitro. <br />
  13. 13. AstroglialNF-κB inactivation does not alter extracellular glutamate levels following trauma in vitro<br />
  14. 14. Zinc can inhibit glutamate-mediated inward currents<br />Zhang et al. (2002) J Neurophysiol88: 1245-1251.<br />
  15. 15. Astrocyte injury reduces extracellular zinc concentrations<br />
  16. 16. Solute Carrier 39 (SLC39) helps regulate extracellular zinc concentrations<br />Eide, D.J. (2006). Biochimica et BiophysicaActa. <br />Microarray analysis revealed over a 2-fold reduction in SLC39 gene expression in TG animals following SCI.<br />
  17. 17. Astroglial NF-κB activation induces expression of SLC39a10 after trauma.<br />
  18. 18. Oligodendrocyte death occurs as a result of a zinc deficiency <br />in vitro. <br />
  19. 19. Injured WT astrocytes induce NADPH oxidase activity in oligodendrocytes in vitro. <br />
  20. 20. Genetic Deletion of gp91 phoxreduces astrocyte-mediated oligodendrocyte death. <br />
  21. 21. gp91Phox -/-oligodendrocytesare immune to glutamate-mediated toxicity. <br />
  22. 22. Apocynin reduces oligodendrocyte death following treatment with injured astrocyte media in vitro. <br />
  23. 23. Combined treatment significantly increases hind limb motor score following SCI. Animals received treatment [vehicle (water), NBQX (30 mg/kg), apocynin (10 mg/kg), or NBQX + apocynin)] 1 hr after injury and daily for 1 week after trauma. <br />
  24. 24. Combined treatment significantly increases stepping coordination. Assessment of hind limb coordination by grid walk analysis. <br />
  25. 25. Combined inhibition of AMPA receptors and NADPH oxidase reduces descending serotonergic fiber loss after spinal cord injury: 3 mm caudal to lesion epicenter. <br />
  26. 26.
  27. 27. Summary<br /><ul><li> Inhibition of astroglial NF-kB promotes oligo survival following SCI
  28. 28. Astrocyte mediated oligo death is dependent in part upon glutamate-zinc induced NADPH oxidase activity in oligodendrocytes
  29. 29. Combination strategies targeting NADPH oxidase and AMPA receptor inhibition may be therapeutic following SCI.</li></li></ul><li>Possible Explanations<br />Reduced Oligodendrocyte Death<br />Enhanced Oligogenesis and Remyelination<br />Does Oligogenesis Play a Role in Increased White Matter Preservation in GFAP-IkBadn Mice Following SCI? <br />
  30. 30. Oligogenesis: what is it, what are the important players, how do we study it?<br />(OPC)<br />CC1+<br />CC1+/-<br />BrdU: Bromodeoxyuridineis an analog of Thymidine<br />
  31. 31. Robust proliferative response 7 weeks post-SCI<br />Serial sections (coronal)<br />Sections are 250mm apart<br />Stereoinvestigator<br />1 cm<br />Moderate SCI<br />T9<br />BrdU<br />perfuse and analyze<br />BrdU<br />6<br />3<br />4<br />5<br />7 weeks post-SCI<br />0<br />1<br />2<br />500 mm<br />E<br />1000 mm<br />-500 mm<br />-1000 mm<br />IkBa-dn<br />Caudal<br />Rostral<br />Sham<br />
  32. 32. No difference in the number of BrdU+/NG2+ cells<br /> WT<br />NG2/ BrdU<br />
  33. 33. Increased oligogenesis in GFAP-IkBa-dn mice<br /> WT<br />IkBa-dn<br />CC1/ BrdU<br />
  34. 34. Microarray Analysis of spinal cords from WT and TG mice following SCI<br />T9 moderate contusion<br />1.5 cm<br />
  35. 35. Lingo-1: Leucine rich repeat and Ig domain containing 1<br />Lingo-1 is an NgR co-receptor expressed on the surface of oligodendrocytes<br />O/E inhibits oligodendrocyte differentiation and myelination.<br />Mi S. et al. (2005) Nat. Neurosci. 8: 745-751.<br />Lingo-1 antagonists promote CNS remyelination in EAE,<br />Mi S. et al. (2007) Nat. Med. 13: 1228-33.<br />and functional recovery and axonal sprouting in after SCI.<br />Ji B. et al. (2006) Mol. Cell Neurosci. 33: 311-320.<br />
  36. 36. Increased cell proliferation and transcriptional activity<br />WT<br />IkBa-dn<br />Ki67<br />Ki67 marker for cell in cycle<br />SOX-17 : Transcription factor that<br />regulates oligodendrocyte development<br />Sohn J et al. (2006) J. Neuroscience 26: 9722-35<br />Tcf4 : Oligodendrocyte-related transcription factor<br /> Fu H et al. (2009) J. Neurosci29: 11399-408<br />Sox17<br />Tcf4<br />
  37. 37. Innate immunity-related gene expression<br />WT<br />IkBa-dn<br />CD11b<br />CD11b: Integrin alpha M is a marker expressed by microglia and macrophages.<br />Microglia and mΦ have both destructive and reparative properties after SCI.<br />Jones TB et al. (2005) Curr. Pharm. Des. 11:1223-1236.<br />Macrophage depletion impairs oligodendrocyteremyelination.<br />Kotter MR et al. (2001) Glia 35: 204-212.<br />TLR2 and TLR4 regulate myelin sparing after SCI.<br />Kigerl KA et al. (2007) J Neurochem. 37-50<br />TLR2<br />TLR4<br />
  38. 38. No difference in microglia/macrophages cell number<br /><ul><li> Significant increase in CD11b+ cells 6 wks post-SCI vs naïve
  39. 39. Significant reduction in the number of microglial cells in naïve TG vs WT
  40. 40. But no difference in microglia number at 6 weeks post-injury between WT and TG mice</li></ul>WT<br />IkBa-dn<br />Same number but different state of activation?<br />
  41. 41. Chemokine-chemokine receptor gene expression<br />CXCR4<br />CXCR4 promotes differentiation oligodendrocyte progenitors and remyelination<br />Patel JR et al. (2010) PNAS 107: 11062-7<br />SDF1/CXCL12 stimulates neurite growth on <br />inhibitory CNS myelin.<br />Opatz J et al. (2009) Mol. Cell Neurosci. 40: 293-300.<br />CXCL12<br />
  42. 42. CXCR4 is Upregulated following SCI in TG mice<br />IkBa-dn<br />WT<br />CXCR4 / GFAP<br />WT TG WT TG<br />CXCR4<br />b-tubulin<br />CC1 / CXCR4<br />
  43. 43. Foxc2 is Upregulated following SCI in TG mice<br />> Foxc2 : is a transcription factor regulating CXCR4 gene expression<br />Kume T. (2008) Trends Cardiovasc.Med. 18: 224-8.<br />Foxc2<br />6 wks post-SCI<br />*<br />WT<br />TG<br />Foxc2<br />b-actin<br />
  44. 44. Summary<br /><ul><li> Increase white matter sparing and improved locomotor function following SCI in GFAP-IkBa-dn mice
  45. 45. Strong proliferative response at 7 weeks post-injury
  46. 46. Inhibition of astroglial NF-kB promote oligogenesis following SCI
  47. 47. Up-regulation of the CXCL12-CXCR4 signaling pathway
  48. 48. Transgenic astrocytes may affect the state of activation of microglial cells </li></li></ul><li>Acknowledgements<br />The Miami Project to Cure Paralysis<br />Dr. Roberta Brambilla<br />Dr. Valery Bracchi-Ricard<br />Dr. Anna Dellarole<br />Jessica Ashbaugh<br />Winston Walters<br />ShaffiatKarmally<br />Holly Maslowski<br />Paul Morton<br />Joshua Johnstone<br />Surgery, Microscopy and Histology Core Facilities<br />Drs. BeataFrydel and Alex Marcillo<br />Funding<br />NIH: NINDS<br />Craig NeilsenFoundation<br />The Miami Project to Cure Paralysis<br />
  49. 49. NADPH oxidase inhibition reduces oligodendrocyte death <br />1 week after SCI. <br />
  50. 50. Thank you<br /><ul><li>Knockdown CXCL12 or CXCR4 pharmacologically or genetically
  51. 51. Evaluate the state of activation of microglia/macrophages between WT and TG mice after SCI by flow cytometry
  52. 52. To get more insights into how TG astrocytes promote oligogenesis we will perform in vitro studies using astrocytes from injured WT or TG spinal cord</li></li></ul><li>A work in progress<br />microglia<br />?<br />OPC<br />OL<br />CXCL12 (SDF1)<br />X?<br />(TNF, insulin)<br />CXCR4<br />Foxc2<br />CXCR4 gene<br />
  53. 53. Neurological disorders: Astrocyte dysfunction ?<br />Astrocytes from the contused spinal cord inhibit oligodendrocyte differentiation of adult oligodendrocyte precursor cells<br />Wang Y. et al. (2011) J. Neurosci. 31:6053-58.<br />Transplantation of Specific Human Astrocytes Promotes Functional Recovery after Spinal Cord Injury<br />Stephen J. A. Davies et al. (2011) PLoS One. 6:e17328<br />
  54. 54.
  55. 55. Injured Astrocyte Media Induce Oligodendrocyte Death<br />
  56. 56. CXCL12 expression in the injured spinal cord<br />WT<br />IkBa-dn<br />CD11b<br />CXCL12<br />GFAP<br />DAPI<br />
  57. 57.
  58. 58. 5-HT Immunofluorescence of Raphaespinal Projections<br />WT<br />TG<br />
  59. 59. BDA Labeling of the CST<br />WT<br />TG<br />
  60. 60. GAP-43 Co-Localizes with BDA Labeled CST Axons<br />
  61. 61. Production of reactive oxygen species<br />NADPH = Nicotinamide adenine dinucleotide phosphate<br /><br />