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Using Gene Knockout and Using Gene Knockout and Transgenic Approaches to Transgenic Approaches to
Evaluate Evaluate in vivoin vivo Functions of Functions of CNS Regeneration Inhibitors: CNS Regeneration Inhibitors:
How Important is Nogo ?How Important is Nogo ?
David MannDavid Mann
Tammy HiblerTammy Hibler
Isaac HolmesIsaac Holmes
Arun George PaulArun George Paul
Road MapRoad Map
BackgroundBackground HypothesesHypotheses Specific AimsSpecific Aims Expected ResultsExpected Results ConclusionConclusion Future ResearchFuture Research
The Big PictureThe Big Picture
Alzheimer’s DiseaseAlzheimer’s Disease
Parkinson’s DiseaseParkinson’s Disease
Amyotrophic Lateral Amyotrophic Lateral Sclerosis (Lou Sclerosis (Lou Gehrig’s Disease)Gehrig’s Disease)
Huntington’s DiseaseHuntington’s Disease
http://content.karger.com/ProdukteDB/produkte.asp?Aktion=JournalHome&ProduktNr=229093
Background
Spinal Cord InjurySpinal 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.2000. p14.
Background
What are the myelin-What are the myelin-derived inhibitors?derived inhibitors?
MAG OMgpNgR Receptor
66 Amino AcidDomain
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: 341-346.
Background
Nogo ControversyNogo ControversyBackground
LabLab Knockout Knockout
MouseMouseExtent ofExtent of
RegeneratiRegenerationon
SchwabSchwab Nogo-ANogo-A-- ModestModest
StrittmattStrittmatterer
Nogo-A/BNogo-A/B-/--/-
(6-11 (6-11 weeks)weeks)
SignificantSignificant
StrittmattStrittmatterer
Nogo-A/BNogo-A/B-/--/-
(>11 (>11 weeks)weeks)
NoneNoneHypothesis 2
Hypothesis 1
Our Hypotheses Our Hypotheses
Hypothesis 1Hypothesis 1: Modest axonal regeneration in : Modest axonal regeneration in Schwab’s Nogo-ASchwab’s Nogo-A-- mice was due to the mice was due to the
presence of Nogo-Bpresence of Nogo-B
Hypothesis 2Hypothesis 2: Absence of axonal : Absence of axonal regeneration in older mice was due to increased regeneration in older mice was due to increased
age-dependant inhibition by OMgpage-dependant inhibition by OMgp
Specific AimsSpecific 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 AxonalRegeneration
Nogo-B
Schwab’s Nogo-A- mouse
Aim
Creation of the Nogo-A/BCreation of the Nogo-A/B-/--/- Knockout Mouse using a Knockout Mouse using a Gene Gene
trapping vectortrapping vector
A/B A A A/B/C
1 2 3 4-9Nogo Gene
Exon 1
Nogo-B
IRES-ß-geo PKG-NEOGene Construct
Schwab
Aim
Has Nogo-B really been Has Nogo-B really been knocked out?knocked out?
In vitroIn vitro test 1: Northern Blot test 1: Northern Blot
WT
Nogo-B mRNA
Nogo-A-
-actin
NegativeControl Nogo-A/B-/-
Expected Result
Has Nogo-B really been Has Nogo-B really been knocked out?knocked out?
In vitroIn vitro test 2: Western Blot test 2: Western Blot
Nogo-B protein
WT Nogo-A-
MAG protein
Negative Control
Nogo-A/B-/-
Expected Result
Is the absence of Nogo-B affecting Is the absence of Nogo-B affecting the general brain structure? the general brain structure?
In vivoIn vivo test: Histological Analysis test: Histological AnalysisNogo-
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-How does the absence of Nogo-B affect axonal growth?B affect axonal growth?
In vitroIn 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
VentralIpsi-Lateral
VentralIpsi-Lateral
??? ?
T6DorsalContra-Lateral
DorsalContra-Lateral
Rostral Caudal
T4 L2
How does the absence of Nogo-How does the absence of Nogo-B affect axonal regeneration B affect axonal regeneration
after SCI?after SCI?In vivoIn 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-How does the absence of Nogo-B affect axonal regeneration B affect axonal regeneration
after SCI?after SCI?In vivoIn vivo
Wild Type
Nogo-A-
Nogo-A/B-/-
Sagittal Studies:
Expected Result
SCICST
How does the absence of Nogo-How does the absence of Nogo-B affect functional recovery B affect functional recovery
after SCI?after SCI?
20
15
10
5
05 10 15 20
Nogo-A-
Nogo-A/B-/-
Behavioral Study:
Expected Result
Days Post SCI
BBB
Score
What we expect…What we expect…
More AxonalRegeneration
Nogo-B
Schwab Nogo-A- mice
Expected Result
Aim 2:Aim 2:To study the expression of OMgp in To study the expression of OMgp in
wild-type mice: young (6-11 weeks) vs. wild-type mice: young (6-11 weeks) vs.
adult (>11 weeks)adult (>11 weeks)
What we proposeYounger Mice (6-11 weeks)(6-11 weeks) Adult Mice (>11 weeks) (>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 Does OMgp expression increase in adult mice?increase in adult mice?
In vitroIn vitro test 1: Northern Blot test 1: Northern Blot
OMgp mRNA
ß-actin mRNA
Week 6 Week 11 Week 14
Expected Result
Does OMgp expression Does OMgp expression increase in adult mice?increase in adult mice?
In vitroIn vitro test 2: Western Blot test 2: Western Blot Week 6 Week 11 Week 14
OMgp Protein
MBP
Expected Result
Does OMgp expression increase in Does OMgp expression increase in adult mice? adult mice?In vitroIn vitro test 3: test 3:
ImmunohistochemistryImmunohistochemistry
Cerebellum
Cerebral Cortex
Lung
Spinal Cord
6 weeks 11 weeks 14 weeks
Expected Result
Does OMgp expression increase in Does OMgp expression increase in adult mice?adult mice?
In vitroIn vitro test 4: test 4: In SituIn Situ HybridizationHybridization
Cerebellum
6 weeks
Cerebral Cortex
Lung
11 weeks 14 weeks
Spinal Cord
Expected Result
What we expect…What we expect…
Young Mice (6-11 weeks)(6-11 weeks) Adult Mice (>11 weeks) (>11 weeks)
Low OMgp Expression Increased OMgp Expression
Expected Result
Aim 3:Aim 3:Part 1: To study the inhibition by Part 1: To study the inhibition by OMgp in wild-type mice: young (6-11 OMgp in wild-type mice: young (6-11 weeks) vs. adult (>11 weeks)weeks) vs. adult (>11 weeks)
What we proposeYounger OMgp-/- Mice
(6-11 weeks)(6-11 weeks)Adult OMgp-/- Mice
(>11 weeks) (>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 adultOMgp-/- Mice
Aim
Creation of the OMgpCreation of the OMgp-/--/- Knockout MouseKnockout Mouse
Wild Type
OMgpOMgp-/--/-
Aim
Has OMgp really been knocked Has OMgp really been knocked out?out?
In vitroIn vitro Northern BlotNorthern Blot Western BlotWestern Blot Histological AnalysisHistological Analysis
Expected Result
Is the absence of OMgp affecting Is the absence of OMgp affecting motor functions? motor functions?
Rotarod TestRotarod Test
How does the absence of OMgp How does the absence of OMgp affect axonal growth? affect axonal growth?
In vitroIn vitro
Neurite Out-growth Assay
Growth Cone Assay
Control OMgpOMgp-/- -/- OMgpOMgp-/- -/-
(11-14 weeks) (6-11 weeks)
Expected Result
Where & what are we looking for? In vivo
Aim
Dorsal
Ventral
VentralIpsi-Lateral
VentralIpsi-Lateral
??? ?
T6DorsalContra-Lateral
DorsalContra-Lateral
Rostral Caudal
T4 L2
How does the absence of OMgp How does the absence of OMgp affect axonal regeneration? affect axonal regeneration?
In vivoIn 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 OMgpOMgp-/- -/- (Adult) OMgpOMgp-/- -/- (Young)
Expected Result
CST
How does OMgp inhibition How does OMgp inhibition affect axonal regeneration?affect axonal regeneration?
In vivoIn vivo
Wild Type
OMgpOMgp-/- -/-
(Adult)
OMgpOMgp-/- -/-
(Young)
Sagittal Studies:
Expected Result
SCICST
What we expect…What we expect…
Young OMgp-/- Mice (6-11 weeks)(6-11 weeks)
Adult OMgp-/- Mice (>11 weeks) (>11 weeks)
No Axonal Regeneration Axonal Regeneration
Expected Result
Part 2: To study the inhibition of Part 2: To study the inhibition of OMgp in Nogo-A/BOMgp in Nogo-A/B-/--/- mice: young (6-11 mice: young (6-11 weeks) vs adult (>11 weeks)weeks) vs adult (>11 weeks)
Aim 3:Aim 3:
Young Nogo-A/B-/--OMgp- Mice(6-11 weeks)(6-11 weeks)
Adult Nogo-A/B-/--OMgp- Mice (>11 weeks) (>11 weeks)
What we propose
Excessive Axonal Regeneration 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 Creation of the Nogo-A/B/OMgpNogo-A/B/OMgp-/-/--/-/- Knockout Knockout
MouseMouseWild Type
Nogo-A/B-/--OMgp-
Aim
Has OMgp really been knocked Has OMgp really been knocked out?out?
In vitroIn vitro Northern BlotNorthern Blot Western BlotWestern Blot Histological AnalysisHistological Analysis
Expected Result
Is the absence of OMgp affecting Is the absence of OMgp affecting motor functions? motor functions?
Rotarod TestRotarod Test
How does the absence of Nogo-B How does the absence of Nogo-B affect axonal growth in affect axonal growth in Nogo-A/BNogo-A/B-/--/-
micemice? ? In vitroIn vitro Control Nogo-A/BNogo-A/B-/--/- Nogo-A/BNogo-A/B-/--/-
OMgpOMgp-- (Adult) OMgpOMgp--(Younger)
Neurite Out-growth Assay
Growth Cone Assay
Expected Result
Where & what are we looking for? In vivo
Aim
Dorsal
Ventral
VentralIpsi-Lateral
VentralIpsi-Lateral
??? ?
T6DorsalContra-Lateral
DorsalContra-Lateral
Rostral Caudal
T4 L2
How does the absence of Nogo-B How does the absence of Nogo-B affect axonal regeneration in affect axonal regeneration in
Nogo-A/BNogo-A/B-/--/- micemice? ? In vivoIn 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- Nogo-A/B-/--OMgp-Wild Type
YoungAdult
Expected Result
CST
How does the absence of Nogo-B How does the absence of Nogo-B affect axonal regeneration in affect axonal regeneration in
Nogo-A/BNogo-A/B-/--/- micemice? ? In vivoIn vivo
Wild Type
Nogo-A/B-/--OMgp- (Adult)
Nogo-A/B-/--OMgp- (Young)
Sagittal Studies:
Expected Result
SCICST
What we expect…What we expect…
Young Nogo-A/B-/--OMgp- Mice (6-11 weeks)(6-11 weeks)
Adult Nogo-A/B-/--OMgp- Mice (>11 weeks) (>11 weeks)
Excessive Axonal Regeneration Excessive Axonal Regeneration
Expected Result
Aim 4: Aim 4: To study the effects of the To study the effects of the overexpression of OMgp in axonal overexpression of OMgp in axonal regeneration after SCI in regeneration after SCI in Nogo-A/BNogo-A/B-/- -/-
micemiceRationale: 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
OverexpressOMgp
What we propose:
Creation of Transgenic Nogo-Creation of Transgenic Nogo-A/BA/B-/--/- with overexpression of with overexpression of
OMgpOMgpMBP Promoter OMgp gene
P-MBP OMgp gene
OMgp++-Nogo-A/B-/-
Aim
Has OMgp really been Has OMgp really been overexpressed in the overexpressed in the
Transgenic Nogo-A/BTransgenic Nogo-A/B-/--/- mice? mice?
Western BlotWestern Blot
OMgp
MAG
MBP
Expected Result
WT OMgp+-Nogo-A/B-/- OMgp++-Nogo-A/B-/-
How does OMgp overexpression How does OMgp overexpression affects axonal growth in affects axonal growth in Nogo-A/BNogo-A/B-/--/-
micemice? ? In vitroIn vitro tests tests Control OMgp+-Nogo-A/B-/- OMgp++-Nogo-A/B-/-
Neurite
Outgrowth
Growth Cone Collapse
Expected Result
How does OMgp overexpression How does OMgp overexpression affects axonal regeneration in affects axonal regeneration in
Nogo-A/BNogo-A/B-/--/- micemice? ? In vivoIn 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 How does OMgp overexpression affects axonal regeneration in affects axonal regeneration in
Nogo-A/BNogo-A/B-/--/- micemice? ? In vivoIn vivo
Wild Type
OMgp+- Nogo-A/B-/-
OMgp++-Nogo-A/
B-/-
Sagittal Studies:
Expected Result
SCICST
How does OMgp overexpression How does OMgp overexpression affect axonal regeneration in affect axonal regeneration in
Nogo-A/BNogo-A/B-/--/- micemice? ? In vivoIn vivo
20
15 OMgp+-Nogo-A/B-/-
OMgp++-Nogo-A/B-/-
10
5
0 0 5 10 15 20
Days Post SCI
BBB
Score
Behavioral Study
Expected Result
What we expect…What we expect…
Nogo-A/B-/-
No Axonal Regeneration
Overexpresses OMgp
Expected Result
ConclusionConclusion
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 secondaryinhibitor
OMgp is an inhibitor in adult mice
Future ResearchFuture Research
Possible drugs that can inhibit Nogo, Possible drugs that can inhibit Nogo, OMgp and MAGOMgp and MAG
Directed pathways for axonal Directed pathways for axonal regeneration regeneration
AcknowledgementsAcknowledgements
Dr. Shubhik DebBurmanDr. Shubhik DebBurman Nijee SharmaNijee Sharma D’Anne DuncanD’Anne Duncan