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David Mann Tammy Hibler Isaac Holmes Arun George Paul

Using Gene Knockout and Transgenic Approaches to Evaluate in vivo Functions of CNS Regeneration Inhibitors: How Important is Nogo ?. David Mann Tammy Hibler Isaac Holmes Arun George Paul. Road Map. Background Hypotheses Specific Aims Expected Results Conclusion Future Research.

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David Mann Tammy Hibler Isaac Holmes Arun George Paul

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  1. Using Gene Knockout and Transgenic Approaches to Evaluate in vivo Functions of CNS Regeneration Inhibitors: How Important is Nogo ? David Mann Tammy Hibler Isaac Holmes Arun George Paul

  2. Road Map • Background • Hypotheses • Specific Aims • Expected Results • Conclusion • Future Research

  3. Background The Big Picture • Alzheimer’s Disease • Parkinson’s Disease • Amyotrophic Lateral Sclerosis (Lou Gehrig’s Disease) • Huntington’s Disease http://content.karger.com/ProdukteDB/produkte.asp?Aktion=JournalHome&ProduktNr=229093

  4. Background Spinal Cord Injury SCI on CST Cervical (C1-8) Thoracic (T1-12) Lumbar (L1-5) Sacral (S1-5) Coccygeal (C1) Purves, Dale et.al. Neuroscience. Sinauer Associates, Inc.2000. p14.

  5. Background 66 Amino Acid Domain Nogo-A Nogo-C Nogo-B NgR Receptor OMgp MAG What are the myelin-derived inhibitors? CNS Neuron Fournier et. al. (2001) Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration. Nature 409: 341-346.

  6. Background Hypothesis 1 Hypothesis 2 Nogo Controversy

  7. Our Hypotheses Hypothesis 1: Modest axonal regeneration in Schwab’s Nogo-A- mice was due to the presence of Nogo-B Hypothesis 2: Absence of axonal regeneration in older mice was due to increased age-dependant inhibition by OMgp

  8. Specific Aims Schwab Nogo-A/B-/- Mice Aim 1 Hypothesis 1 Nogo-B Aim 2 WT OMgp (Young vs. Adult) Aim 3 Nogo-A/Nogo-B-/- -OMgp- Hypothesis 2 Aim 4 OMgp++

  9. Aim What we propose: Increased Axonal Regeneration Nogo-B SCI Schwab’s Nogo-A- mouse Aim 1:To studyinhibitory effects of Nogo-B on axonal regeneration in Nogo-A- Mice Rationale: The presence of Nogo-B should account for the modest axonal regeneration in Schwab’s Nogo-A- mouse

  10. Aim Nogo Gene 2 4-9 1 3 Schwab A A/B A/B/C A Exon 1 Nogo-B Gene Construct IRES-ß-geo PKG-NEO Creation of the Nogo-A/B-/- Knockout Mouse using a Gene trapping vector

  11. Expected Result Has Nogo-B really been knocked out?In vitro test 1: Northern Blot Negative Control Nogo-A/B-/- Nogo-A- WT Nogo-B mRNA -actin

  12. Expected Result Has Nogo-B really been knocked out?In vitro test 2: Western Blot Negative Control Nogo-A/B-/- WT Nogo-A- Nogo-B protein MAG protein

  13. Expected Result Is the absence of Nogo-B affecting the general brain structure? In vivo test: Histological Analysis Nogo- A/B-/- Nogo- A/B-/- Wild Type Cerebral Cortex X-gal Staining Hematoxylin & Eosin Staining Cerebellum Corpus Callosum

  14. Expected Result How does the absence of Nogo-B affect axonal growth?In vitro ControlNogo-A-Nogo-A/B-/- Neurite Outgrowth Assay Growth Cone Assay

  15. Aim Caudal Rostral Dorsal T6 Dorsal Contra- Lateral Dorsal Contra- Lateral ? ? L2 T4 ? ? Ventral Ipsi- Lateral Ventral Ipsi- Lateral Ventral Where & what are we looking for?In vivo

  16. Expected Result How does the absence of Nogo-B affect axonal regeneration after SCI?In vivo Coronal Studies: Nogo-A/B-/- Control Nogo-A- Dorsal Dorsal Dorsal CST Coronal Sections at T4 (Rostral) Ventral Ventral Ventral Dorsal Dorsal Dorsal Coronal Sections at L2 (Caudal) Ventral Ventral Ventral

  17. Wild Type Expected Result Nogo-A- Nogo-A/B-/- How does the absence of Nogo-B affect axonal regeneration after SCI?In vivo Sagittal Studies: SCI CST

  18. Expected Result 20 15 Nogo-A/B-/- Nogo-A- 10 5 0 15 10 20 5 How does the absence of Nogo-B affect functional recovery after SCI? Behavioral Study: BBB Score Days Post SCI

  19. Expected Result What we expect… SCI Nogo-B Schwab Nogo-A- mice More Axonal Regeneration

  20. Aim What we propose Adult Mice (>11 weeks) Younger Mice (6-11 weeks) Low OMgp Expression Increased OMgp Expression Aim 2:To study the expression of OMgp in wild-type mice: young (6-11 weeks) vs. adult (>11 weeks) Rationale:OMgp expression should increase with age to account for age-dependant inhibition seen in Strittmatter’s Nogo-A/B-/- mice

  21. Expected Result Does OMgp expression increase in adult mice?In vitro test 1: Northern Blot Week 6 Week 11 Week 14 OMgp mRNA ß-actin mRNA

  22. Expected Result Does OMgp expression increase in adult mice?In vitro test 2: Western Blot Week 6 Week 11 Week 14 OMgp Protein MBP

  23. Expected Result Does OMgp expression increase in adult mice?In vitro test 3: Immunohistochemistry 6 weeks 11 weeks 14 weeks Spinal Cord Cerebral Cortex Cerebellum Lung

  24. Expected Result Does OMgp expression increase in adult mice?In vitro test 4: In Situ Hybridization 6 weeks 11 weeks 14 weeks Spinal Cord Cerebral Cortex Cerebellum Lung

  25. Expected Result What we expect… Adult Mice (>11 weeks) Young Mice (6-11 weeks) Low OMgp Expression Increased OMgp Expression

  26. Aim What we propose Younger OMgp-/- Mice (6-11 weeks) Adult OMgp-/- Mice (>11 weeks) No Axonal Regeneration Axonal Regeneration Aim 3: Part 1: To study the inhibition by OMgp in wild-type mice: young (6-11 weeks) vs. adult (>11 weeks) Rationale: If OMgp is an inhibitor in adult mice, then absence of OMgp in adult mice will lead to axonal regeneration in adult OMgp-/- Mice

  27. Aim Creation of the OMgp-/- Knockout Mouse Wild Type OMgp-/-

  28. Expected Result Has OMgp really been knocked out?In vitro • Northern Blot • Western Blot • Histological Analysis Is the absence of OMgp affecting motor functions? • Rotarod Test

  29. Expected Result How does the absence of OMgp affect axonal growth? In vitro Control OMgp-/- OMgp-/- (11-14 weeks) (6-11 weeks) Growth Cone Assay Neurite Out-growth Assay

  30. Aim Where & what are we looking for?In vivo Caudal Rostral Dorsal T6 Dorsal Contra- Lateral Dorsal Contra- Lateral ? ? L2 T4 ? ? Ventral Ipsi- Lateral Ventral Ipsi- Lateral Ventral

  31. Expected Result How does the absence of OMgp affect axonal regeneration? In vivo Coronal Studies: OMgp-/- (Adult) OMgp-/- (Young) Wild Type Dorsal Dorsal Dorsal CST Coronal Sections at T4 (Rostral) Ventral Ventral Ventral Dorsal Dorsal Dorsal Coronal Sections at L2 (Caudal) Ventral Ventral Ventral

  32. Wild Type Expected Result OMgp-/- (Adult) OMgp-/- (Young) How does OMgp inhibition affect axonal regeneration?In vivo Sagittal Studies: SCI CST

  33. Expected Result What we expect… Young OMgp-/- Mice (6-11 weeks) Adult OMgp-/- Mice (>11 weeks) No Axonal Regeneration Axonal Regeneration

  34. Aim What we propose Adult Nogo-A/B-/--OMgp- Mice (>11 weeks) Young Nogo-A/B-/--OMgp- Mice (6-11 weeks) Excessive Axonal Regeneration Excessive Axonal Regeneration Part 2: To study the inhibition of OMgp in Nogo-A/B-/- mice: young (6-11 weeks) vs adult (>11 weeks) Aim 3: Rationale: If OMgp is an inhibitor in older mice, then the absence of OMgp in young and adult Nogo-A/B-/-mice will have axonal regeneration after SCI

  35. Aim Creation of the Nogo-A/B/OMgp-/-/- Knockout Mouse Wild Type Nogo-A/B-/-- OMgp-

  36. Expected Result Has OMgp really been knocked out?In vitro • Northern Blot • Western Blot • Histological Analysis Is the absence of OMgp affecting motor functions? • Rotarod Test

  37. Expected Result How does the absence of Nogo-B affect axonal growth in Nogo-A/B-/-mice? In vitro Control Nogo-A/B-/-Nogo-A/B-/-OMgp- (Adult) OMgp-(Younger) Growth Cone Assay Neurite Out-growth Assay

  38. Aim Where & what are we looking for?In vivo Caudal Rostral Dorsal T6 Dorsal Contra- Lateral Dorsal Contra- Lateral ? ? L2 T4 ? ? Ventral Ipsi- Lateral Ventral Ipsi- Lateral Ventral

  39. Expected Result How does the absence of Nogo-B affect axonal regeneration in Nogo-A/B-/-mice? In vivo Coronal Studies: Adult Young Nogo-A/B-/--OMgp- Wild Type Nogo-A/B-/--OMgp- Dorsal Dorsal Dorsal CST Coronal Sections at T4 (Rostral) Ventral Ventral Ventral Dorsal Dorsal Dorsal Coronal Sections at L2 (Caudal) Ventral Ventral Ventral

  40. Wild Type Expected Result Nogo-A/B-/--OMgp- (Adult) Nogo-A/B-/--OMgp- (Young) How does the absence of Nogo-B affect axonal regeneration in Nogo-A/B-/-mice? In vivo Sagittal Studies: SCI CST

  41. Expected Result What we expect… Young Nogo-A/B-/--OMgp- Mice (6-11 weeks) Adult Nogo-A/B-/--OMgp- Mice (>11 weeks) Excessive Axonal Regeneration Excessive Axonal Regeneration

  42. Aim What we propose: Overexpress OMgp Young Nogo-A/B-/- No Axonal Regeneration Aim 4: To study the effects of the overexpression of OMgp in axonal regeneration after SCI in Nogo-A/B-/- mice Rationale: Since older mice highly express OMgp and do not display axonal regeneration, then young Nogo-A/B-/- mice will not display axonal regeneration when overexpressing OMgp.

  43. Aim Creation of Transgenic Nogo-A/B-/- with overexpression of OMgp OMgp gene MBP Promoter OMgp gene P-MBP OMgp++-Nogo-A/B-/-

  44. Expected Result Has OMgp really been overexpressed in the Transgenic Nogo-A/B-/- mice?Western Blot OMgp++-Nogo-A/B-/- OMgp+-Nogo-A/B-/- WT OMgp MAG MBP

  45. Expected Result How does OMgp overexpression affects axonal growth in Nogo-A/B-/-mice? In vitro tests + -/- ++ -/- Cont r o l O Mgp -N ogo - A / B O Mgp -N ogo - A / B Neurite Outgrowth Growth Cone Collapse

  46. Expected Result How does OMgp overexpression affects axonal regeneration in Nogo-A/B-/-mice? In vivo Coronal Studies: OMgp++-Nogo-A/B-/- OMgp+-Nogo-A/B-/- Wild Type Dorsal Dorsal Dorsal Coronal Sections at T4 (Rostral) Ventral Ventral Ventral Dorsal Dorsal Dorsal Coronal Sections at L2 (Caudal) Ventral Ventral Ventral

  47. Wild Type Expected Result OMgp+- Nogo-A/B-/- OMgp++-Nogo-A/B-/- How does OMgp overexpression affects axonal regeneration in Nogo-A/B-/-mice? In vivo Sagittal Studies: SCI CST

  48. Expected Result + - /- O M gp - Nogo- A / B + + - /- O M gp - Nogo- A / B How does OMgp overexpression affect axonal regeneration in Nogo-A/B-/-mice? In vivo Behavioral Study 20 15 BBB Score 10 5 0 0 5 10 15 20 Days Post SCI

  49. Expected Result What we expect… Nogo-A/B-/- Overexpresses OMgp No Axonal Regeneration

  50. Conclusion Nogo-B is a secondary inhibitor Aim 1 Schwab Nogo-A- Mice Nogo-B Aim 2 WT OMgp Aim 3 Nogo-A/Nogo-B-/- -OMgp- OMgp is an inhibitor in adult mice Aim 4 OMgp++-Nogo-A/B-/-

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