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David Mann Tammy Hibler Isaac Holmes Arun George Paul

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1 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

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

3 The Big Picture Alzheimer’s Disease Parkinson’s Disease
Background The Big Picture Alzheimer’s Disease Parkinson’s Disease Amyotrophic Lateral Sclerosis (Lou Gehrig’s Disease) Huntington’s Disease

4 Spinal Cord Injury Background 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 p14.

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

6 Nogo Controversy Schwab Nogo-A- Modest Strittmatter Nogo-A/B-/-
Background Nogo Controversy Lab Knockout Mouse Extent of Regeneration Schwab Nogo-A- Modest Strittmatter Nogo-A/B-/- (6-11 weeks) Significant (>11 weeks) None Hypothesis 1 Hypothesis 2

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 Aim 1 Hypothesis 1 Aim 2 Aim 3 Hypothesis 2 Aim 4 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 1: To study inhibitory 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 What we propose: SCI Increased Axonal Regeneration Nogo-B Schwab’s Nogo-A- mouse

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

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

12 Has Nogo-B really been knocked out? In vitro test 2: Western Blot
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 How does the absence of Nogo-B affect axonal growth? In vitro
Expected Result How does the absence of Nogo-B affect axonal growth? In vitro Control Nogo-A Nogo-A/B-/- Neurite Outgrowth Assay Growth Cone Assay

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

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

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

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

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

20 Aim 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 What we propose Younger Mice (6-11 weeks) Adult Mice (>11 weeks) Low OMgp Expression Increased OMgp Expression

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

22 Expected Result Does OMgp expression increase in adult mice? In vitro test 2: Western Blot Week Week 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 What we expect… Expected Result Young Mice (6-11 weeks)
Adult Mice (>11 weeks) Low OMgp Expression Increased OMgp Expression

26 Aim 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 What we propose Younger OMgp-/- Mice (6-11 weeks) Adult OMgp-/- Mice (>11 weeks) No Axonal Regeneration Axonal Regeneration

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

28 Has OMgp really been knocked out? In vitro
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 How does the absence of OMgp affect axonal growth? In vitro
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 Where & what are we looking for? In vivo
Aim Where & what are we looking for? In vivo Caudal Rostral Dorsal T6 Dorsal Contra- Lateral Dorsal Contra- Lateral ? ? T4 L2 ? ? Ventral Ipsi- Lateral Ventral Ipsi- Lateral Ventral

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

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

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

34 Young Nogo-A/B-/--OMgp- Mice Adult Nogo-A/B-/--OMgp- Mice
Aim Aim 3: Part 2: To study the inhibition of OMgp in Nogo-A/B-/- mice: young (6-11 weeks) vs adult (>11 weeks) 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 Young Nogo-A/B-/--OMgp- Mice (6-11 weeks) Adult Nogo-A/B-/--OMgp- Mice (>11 weeks) What we propose Excessive Axonal Regeneration

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

36 Has OMgp really been knocked out? In vitro
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 Where & what are we looking for? In vivo
Aim Where & what are we looking for? In vivo Caudal Rostral Dorsal T6 Dorsal Contra- Lateral Dorsal Contra- Lateral ? ? T4 L2 ? ? 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 Wild Type Nogo-A/B-/--OMgp- 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 Expected Result How does the absence of Nogo-B affect axonal regeneration in Nogo-A/B-/- mice? In vivo Sagittal Studies: Wild Type SCI CST Nogo-A/B-/--OMgp- (Adult) Nogo-A/B-/--OMgp- (Young)

41 Young Nogo-A/B-/--OMgp- Mice Adult Nogo-A/B-/--OMgp- Mice
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 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. Young Nogo-A/B-/- No Axonal Regeneration Overexpress OMgp What we propose:

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

44 Expected Result Has OMgp really been overexpressed in the Transgenic Nogo-A/B-/- mice? Western Blot WT OMgp+-Nogo-A/B-/- OMgp++-Nogo-A/B-/- 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: Wild Type OMgp+- Nogo-A/B-/- OMgp++-Nogo-A/B-/- Dorsal Dorsal Dorsal Coronal Sections at T4 (Rostral) Ventral Ventral Ventral Dorsal Dorsal Dorsal Coronal Sections at L2 (Caudal) Ventral Ventral Ventral

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

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

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

50 Conclusion Aim 1 Aim 2 Aim 3 Aim 4 Nogo-B is a secondary Schwab
inhibitor Schwab Nogo-A- Mice Aim 1 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-/-

51 Future Research Possible drugs that can inhibit Nogo, OMgp and MAG
Directed pathways for axonal regeneration

52 Acknowledgements Dr. Shubhik DebBurman Nijee Sharma D’Anne Duncan

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