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

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 http://content.karger.com/ProdukteDB/produkte.asp?Aktion=JournalHome&ProduktNr=229093

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.2000. p14.

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: 341-346.

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

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

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++

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

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

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

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

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

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

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

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

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-/-

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

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

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

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

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 6 weeks 11 weeks 14 weeks Spinal Cord Cerebral Cortex Cerebellum Lung

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

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

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

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

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

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

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

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

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)

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

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

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-

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

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

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

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

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)

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

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:

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-/-

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

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

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

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-/-

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

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

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-/-

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

Acknowledgements Dr. Shubhik DebBurman Nijee Sharma D’Anne Duncan