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