Possible Explanations Reduced Oligodendrocyte Death Enhanced Oligogenesis and Remyelination Does astroglial NF-κB activation contribute to oligodendrocytedeath after injury?
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.
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
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.
Astroglial NF-κB activation induces expression of SLC39a10 after trauma.
Oligodendrocyte death occurs as a result of a zinc deficiency in vitro.
Injured WT astrocytes induce NADPH oxidase activity in oligodendrocytes in vitro.
Genetic Deletion of gp91 phoxreduces astrocyte-mediated oligodendrocyte death.
gp91Phox -/-oligodendrocytesare immune to glutamate-mediated toxicity.
Apocynin reduces oligodendrocyte death following treatment with injured astrocyte media in vitro.
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.
Combined treatment significantly increases stepping coordination. Assessment of hind limb coordination by grid walk analysis.
Combined inhibition of AMPA receptors and NADPH oxidase reduces descending serotonergic fiber loss after spinal cord injury: 3 mm caudal to lesion epicenter.
Inhibition of astroglial NF-kB promotes oligo survival following SCI
Astrocyte mediated oligo death is dependent in part upon glutamate-zinc induced NADPH oxidase activity in oligodendrocytes
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?
Oligogenesis: what is it, what are the important players, how do we study it? (OPC) CC1+ CC1+/- BrdU: Bromodeoxyuridineis an analog of Thymidine
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
No difference in the number of BrdU+/NG2+ cells WT NG2/ BrdU
Increased oligogenesis in GFAP-IkBa-dn mice WT IkBa-dn CC1/ BrdU
Microarray Analysis of spinal cords from WT and TG mice following SCI T9 moderate contusion 1.5 cm
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.
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
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
No difference in microglia/macrophages cell number
Significant increase in CD11b+ cells 6 wks post-SCI vs naïve
Significant reduction in the number of microglial cells in naïve TG vs WT
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?
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
CXCR4 is Upregulated following SCI in TG mice IkBa-dn WT CXCR4 / GFAP WT TG WT TG CXCR4 b-tubulin CC1 / CXCR4
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
Increase white matter sparing and improved locomotor function following SCI in GFAP-IkBa-dn mice
Strong proliferative response at 7 weeks post-injury
Inhibition of astroglial NF-kB promote oligogenesis following SCI
Up-regulation of the CXCL12-CXCR4 signaling pathway
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
NADPH oxidase inhibition reduces oligodendrocyte death 1 week after SCI.
Knockdown CXCL12 or CXCR4 pharmacologically or genetically
Evaluate the state of activation of microglia/macrophages between WT and TG mice after SCI by flow cytometry
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
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