Chemokine Regulation of Oligodendrocyte Development in the Spinal Cord. Bob Avino Saint Louis University Senior Honors Thesis April 19, 2011

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1 Chemokine Regulation of Oligodendrocyte Development in the Spinal Cord Bob Avino Saint Louis University Senior Honors Thesis April 19, 2011

2 Richard J. Miller, PhD Northwestern University Feinberg School of Medicine Chicago, IL Department of Molecular Pharmacology and Biological Chemistry Grant Funding: National Institute on Drug Abuse(NIDA) Fletcher A. White, MS, PhD Loyola University Medical Center, Maywood, IL Department of Cell Biology, Neurobiology Grant Funding: & Anatomy and Anesthesiology National Institute of Neurological Disorders and Stroke(NINDS)

3 Central Nervous System Control Center of the Body Integrates signals Regulates motor outputs Brain and Spinal Cord Brain: Site of Cognition Somatic V. Autonomic Control

4 Spinal Cord Anatomy

5 Spinal Cord Development COMPLEX REGULATION Genes turned on at specific time points Homeobox transcription factors become active Notochord induces folding of the neural plate Neural progenitor cells active Regulation cell typespecific differentiation

6 Overview of Developing Spinal Cord in Mouse By E14.5 ~E14

7 Chemokines & the Spinal Cord Chemokines play a critical role during brain development (CNS) CXCR4 (Chemokine Receptor) signaling has also been shown to regulate the migration and development of neural stem cells Cells migrate toward SDF-1 SDF-1 and CXCR4 are both expressed in the spinal cord Could SDF-1 play a role in the development of cells in the spinal cord?

8 Oligodendrocytes Myelinate the Central Nervous System Myelinate multiple neurons and provide axon support Originate from ventral ventricular zone of the spinal cord at E12 By E16.5 oligodendrocytes are spread throughout the spinal cord, concentrated in white matter

9 Oligodendrogenesis: Localized? Olig2 transcription factor involved with Oligodendrogenesis from the pmn zone

10 Chemokines Family of small cytokines, or proteins secreted by cells (approx kd in size) Chemokines are largely considered to be pro-inflammatory and exhibit chemotactic effects on immune system cells Chemokines are also involved in controlling the migration of neural stem cells during the normal processes of development. (e.g. SDF-1 is essential for normal development of the hippocampus and cerebellum)

11 MIP-1α,δ RANTES MCP MCP-1 MCP-2 MCP-3 MCP-4 RANTES MIP-3 MIP-1δ MCP RANTES MDC RANTES MCP-2 MIP-1α,β CCR1 CCR3 CCR4 CCR5 CCR11 CCR10 CCR9 MCP-1 MCP-2 MCP-4 CXCR2 CXCR3 CXCR5 IL-8 GRO α,β,γ IP10 MIG CX3R1 XCR1 Fractalkine Lymphotactin α,β β chemokines -weakly expressed in normal brain α chemokines unknown TECK BLR1 RANTES MCP-2 MIP-1α,β IP10 MIG CXCR4 SDF1-α,β,γ SDF1-α

12 Chemokines Receptors Divided into different families according to which family of chemokines they bind (CC, CXC, C, or CX3C) G protein-coupled receptors which trigger a flux in intracellular Ca 2+ upon interaction with chemokines 7 transmembrane proteins

13 Chemokine Signaling Pathways

14 SDF-1 and CXCR4 Distribution in the Spinal Cord SDF-1 concentrated in the meninges and around central canal CXCR4 positive cells populate the ventral ventricular zone (pmn) CXCR4 E12

15 SDF-1 Interaction with Oligodendrocytes SDF-1 interacts with CXCR4 positive cells CXCR4 cells located in pmn region Do chemokines facilitate the production of oligodendrocytes? Specifically does SDF-1 play a role in Oligodendrocyte progenitor regulation?

16 Olig2 Basic helix-loop-helix transcription factors that are expressed in the pmn region Olig2 positive cell have been shown to be progenitors for oligodendrocytes Olig2 progenitor cells have also been shown to be localized in the pmn zone

17 Methods Transgenic Mice Using recombinant DNA technology GFP-Olig2 Transcription factor expressed by oligodendrocytes and oligodendrocyte precursors RFP-SDF-1 Chemokine Embryological time points E11, E13, E14, E15.5, E16.5

18 Methods Genotyping Harvesting Embryos Embedding and Sectioning Sectioned on a cryostat at 15 m Microscopy Flouview 10i Olympus confocal microscope 10x oil emersion and 60x oil emersion Digital zoom to 120x magnification

19 E11 Spinal Cord at 10x GFP-Olig2 labeling in ventral ventricular zone of oligodendrocyte progenitor cells SDF-1 localized in central canal SDF-1 not in Olig2 cells

20 E11 at 120x Central Canal Meningeal Edge

21 E13 Spinal Cord at 10x pmn zone still present Radial glia processes GFP positive Individual oligodendrocytes cells now present High SDF-1 expression along central canal SDF-1 expression located in ventral meninges SDF-1 present along Radial Glia

22 E13 at 120x Central Canal Meningeal Edge

23 E14 Spinal Cord at 10x Similar to E13 Increased SDF-1 expression around ventral meninges and central canal SDF-1 present in dorsal meninges Interaction between SDF-1 and Olig2 progenitor cells along mendigeal edge

24 E14 at 120x Central Canal Meningeal Edge

25 E15.5 Spinal Cord at 10x SDF-1 concentrated along entire perimeter of spinal cord Oligodendrocytes lineage present throughout spinal cord pmn zone no longer mass of green Cells are well defined Height of SDF-1 expression in Olig2 positive cells

26 E15.5 at 120x Central Canal Meningeal Edge

27 E16.5 Spinal Cord at 10x Oligodendrocytes lineage cells spread throughout spinal cord SDF-1 along meningeal edge pmn zone not apparent at all Less interaction between chemokine and oligodendrocyte progenators

28 E16.5 at 120x Central Canal Meningeal Edge

29 Discussion From E11 to E15.5 steady increase in SDF-1 interaction with Olig2 positive cells Olig2 cells seem to be regulated by SDF-1 when they give rise to oligodendrocyte lineage cells Dual color mice very useful Suggests further experiments with CXCR4 or SDF-1 knock out mice for oligodendrocyte development

30 Why Study this Mechanism? Learn more about what factors regulate CNS development Create interventions to improve spinal cord healing after injury Combat demyelinating diseases such as Multiple Sclerosis Reduce chronic pain (White Lab Studies)

31 Thanks to Thesis Sponsor William S. Stark, PhD Department of Biology, Saint Louis University Student Grant Funding Children s Memorial Hospital Chicago, IL Northwestern University Richard J. Miller, PhD Department of Molecular Pharmacology and Biological Chemistry, Feinberg School of Medicine