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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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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
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
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.
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.
Background Hypothesis 1 Hypothesis 2 Nogo Controversy
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 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 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
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
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
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
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
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
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
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
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
Expected Result What we expect… SCI Nogo-B Schwab Nogo-A- mice More Axonal Regeneration
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
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
Expected Result What we expect… Adult Mice (>11 weeks) Young Mice (6-11 weeks) Low OMgp Expression Increased OMgp Expression
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
Aim Creation of the OMgp-/- Knockout Mouse Wild Type OMgp-/-
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 OMgp affect axonal growth? In vitro Control OMgp-/- OMgp-/- (11-14 weeks) (6-11 weeks) Growth Cone Assay Neurite Out-growth Assay
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
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
Wild Type Expected Result OMgp-/- (Adult) OMgp-/- (Young) How does OMgp inhibition affect axonal regeneration?In vivo Sagittal Studies: SCI CST
Expected Result What we expect… Young OMgp-/- Mice (6-11 weeks) Adult OMgp-/- Mice (>11 weeks) No Axonal Regeneration Axonal Regeneration
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
Aim Creation of the Nogo-A/B/OMgp-/-/- Knockout Mouse Wild Type Nogo-A/B-/-- OMgp-
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
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
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
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
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 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.
Aim Creation of Transgenic Nogo-A/B-/- with overexpression of OMgp OMgp gene MBP Promoter OMgp gene P-MBP OMgp++-Nogo-A/B-/-
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
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: 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
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
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
Expected Result What we expect… Nogo-A/B-/- Overexpresses OMgp No Axonal Regeneration
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-/-