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 Background Hypotheses Hypotheses Specific Aims Specific Aims Expected Results Expected Results Conclusion Conclusion Future Research Future Research

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

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

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

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

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

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

Aim 1: 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: Increased Axonal Regeneration Nogo-B Schwab’s Nogo-A - mouse Aim

Creation of the Nogo-A/B -/- Knockout Mouse using a Gene trapping vector A/B A A A/B/C Nogo Gene Exon 1 Nogo-B IRES-ß-geoPKG-NEO Gene Construct Schwab Aim

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

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

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

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

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

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

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

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

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

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

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

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

Does OMgp expression increase in adult mice? In vitro test 3: Immunohistochemistry Cerebellum Cerebral Cortex Lung Spinal Cord 6 weeks11 weeks14 weeks Expected Result

Does OMgp expression increase in adult mice? In vitro test 4: In Situ Hybridization Cerebellum 6 weeks Cerebral Cortex Lung 11 weeks14 weeks Spinal Cord Expected Result

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

Aim 3: Part 1: To study the inhibition by OMgp in wild-type mice: young (6-11 weeks) vs. adult (>11 weeks) What we propose Younger OMgp -/- Mice (6-11 weeks) Adult OMgp -/- Mice (>11 weeks) No Axonal Regeneration Axonal Regeneration 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 Aim

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

How does OMgp overexpression affects axonal growth in Nogo-A/B -/- mice ? In vitro tests Control OMgp + -Nogo-A/B -/- OMgp ++ -Nogo-A/B -/- Neurite Outgrowth Growth Cone Collapse Expected Result

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

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

How does OMgp overexpression affect axonal regeneration in Nogo-A/B -/- mice ? In vivo OMgp + -Nogo-A/B -/- OMgp ++ -Nogo-A/B -/ Days Post SCI BBB Score Behavioral Study Expected Result

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

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

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

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