Using Gene Knockout and Transgenic Approaches to Evaluate in vivo Functions of CNS Regeneration Inhi

1
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
Background

• Alzheimers Disease
• Parkinsons Disease
• Amyotrophic Lateral Sclerosis (Lou Gehrigs
  Disease)
• Huntingtons Disease

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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.
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What are the myelin-derived inhibitors?
Background
CNS Neuron
Fournier et. al. (2001) Identification of a
receptor mediating Nogo-66 inhibition of axonal
regeneration. Nature 409 341-346.
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Nogo Controversy
Background
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Our Hypotheses

• Hypothesis 1 Modest axonal regeneration in
  Schwabs 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
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Specific Aims
Schwab Nogo-A/B-/- Mice
Aim 1
Hypothesis 1
Nogo-B
WT OMgp (Young vs. Adult)
Aim 2
Aim 3
Nogo-A/Nogo-B-/- -OMgp-
Hypothesis 2
Aim 4
OMgp
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Aim 1 To study inhibitory effects of Nogo-B on
axonal regeneration in Nogo-A- Mice
Aim
Rationale The presence of Nogo-B should account
for the modest axonal regeneration in Schwabs
Nogo-A- mouse
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Creation of the Nogo-A/B-/- Knockout Mouse using
a Gene trapping vector
Aim
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Has Nogo-B really been knocked out?In vitro test
1 Northern Blot
Expected Result
Negative Control
Nogo-A/B-/-
Nogo-A-
WT
Nogo-B mRNA
?-actin
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Has Nogo-B really been knocked out?In vitro test
2 Western Blot
Expected Result
Negative Control
Nogo-A/B-/-
WT
Nogo-A-
Nogo-B protein
MAG protein
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Is the absence of Nogo-B affecting the general
brain structure? In vivo test Histological
Analysis
Expected Result
Nogo- A/B-/-
Nogo- A/B-/-
Wild Type
Cerebral Cortex
X-gal Staining
Hematoxylin Eosin Staining
Cerebellum
Corpus Callosum
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How does the absence of Nogo-B affect axonal
growth?In vitro
Expected Result
Control Nogo-A-
Nogo-A/B-/-
Neurite Outgrowth Assay
Growth Cone Assay
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Where what are we looking for?In vivo
Aim
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How does the absence of Nogo-B affect axonal
regeneration after SCI?In vivo
Expected Result
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
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How does the absence of Nogo-B affect axonal
regeneration after SCI?In vivo
Expected Result
Sagittal Studies
SCI
CST
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How does the absence of Nogo-B affect functional
recovery after SCI?
Expected Result
Behavioral Study
BBB Score
Days Post SCI
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What we expect
Expected Result
SCI
Nogo-B
Schwab Nogo-A- mice
More Axonal Regeneration
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Aim 2To study the expression of OMgp in
wild-type mice young (6-11 weeks) vs. adult (gt11
weeks)
Aim
Rationale OMgp expression should increase with
age to account for age-dependant inhibition seen
in Strittmatters Nogo-A/B-/- mice
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Does OMgp expression increase in adult mice?In
vitro test 1 Northern Blot
Expected Result
Week
6 Week 11 Week 14
OMgp mRNA
ß-actin mRNA
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Does OMgp expression increase in adult mice?In
vitro test 2 Western Blot
Expected Result
Week 6
Week 11 Week 14
OMgp Protein
MBP
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Does OMgp expression increase in adult
mice?In vitro test 3 Immunohistochemistry
Expected Result
6 weeks
11 weeks
14 weeks
Spinal Cord
Cerebral Cortex
Cerebellum
Lung
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Does OMgp expression increase in adult mice?In
vitro test 4 In Situ Hybridization
Expected Result
6 weeks
11 weeks
14 weeks
Spinal Cord
Cerebral Cortex
Cerebellum
Lung
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What we expect
Expected Result
Adult Mice (gt11 weeks)
Young Mice (6-11 weeks)
Low OMgp Expression
Increased OMgp Expression
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Aim 3
Aim
Part 1 To study the inhibition by OMgp in
wild-type mice young (6-11 weeks) vs. adult (gt11
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
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Creation of the OMgp-/- Knockout Mouse
Aim
Wild Type
OMgp-/-
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Has OMgp really been knocked out?In vitro
Expected Result

• Northern Blot
• Western Blot
• Histological Analysis

Is the absence of OMgp affecting motor
functions?

• Rotarod Test

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How does the absence of OMgp affect axonal
growth? In vitro
Expected Result
Control OMgp-/-
OMgp-/- (11-14 weeks)
(6-11 weeks)
Growth Cone Assay
Neurite Out-growth Assay
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Where what are we looking for? In vivo
Aim
Caudal
Rostral
Dorsal
T6
Dorsal Contra- Lateral
Dorsal Contra- Lateral
?
?
T4
L2
?
?
Ventral Ipsi- Lateral
Ventral Ipsi- Lateral
Ventral
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How does the absence of OMgp affect axonal
regeneration? In vivo
Expected Result
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
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How does OMgp inhibition affect axonal
regeneration? In vivo
Expected Result
Sagittal Studies
SCI
CST
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What we expect
Expected Result
Young OMgp-/- Mice (6-11 weeks)
Adult OMgp-/- Mice (gt11 weeks)
No Axonal Regeneration
Axonal Regeneration
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Part 2 To study the inhibition of OMgp in
Nogo-A/B-/- mice young (6-11 weeks) vs adult
(gt11 weeks)
Aim
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
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Aim
Creation of the Nogo-A/B/OMgp-/-/- Knockout Mouse
Wild Type
Nogo-A/B-/-- OMgp-
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Has OMgp really been knocked out?In vitro
Expected Result

• 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
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Where what are we looking for? In vivo
Aim
Caudal
Rostral
Dorsal
T6
Dorsal Contra- Lateral
Dorsal Contra- Lateral
?
?
T4
L2
?
?
Ventral Ipsi- Lateral
Ventral Ipsi- Lateral
Ventral
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How does the absence of Nogo-B affect axonal
regeneration in Nogo-A/B-/- mice? In vivo
Expected Result
Coronal Studies
Young
Adult
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
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How does the absence of Nogo-B affect axonal
regeneration in Nogo-A/B-/- mice? In vivo
Expected Result
Sagittal Studies
SCI
CST
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What we expect
Expected Result
Young Nogo-A/B-/--OMgp- Mice (6-11 weeks)
Adult Nogo-A/B-/--OMgp- Mice (gt11 weeks)
Excessive Axonal Regeneration
Excessive Axonal Regeneration
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Aim 4 To study the effects of the
overexpression of OMgp in axonal regeneration
after SCI in Nogo-A/B-/- mice
Aim
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.
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Creation of Transgenic Nogo-A/B-/- with
overexpression of OMgp
Aim
OMgp gene
MBP Promoter
P-MBP
OMgp gene
OMgp-Nogo-A/B-/-
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Has OMgp really been overexpressed in the
Transgenic Nogo-A/B-/- mice?Western Blot
Expected Result

OMgp-Nogo-A/B-/-

OMgp-Nogo-A/B-/-





WT








OMgp
MAG
MBP
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How does OMgp overexpression affects axonal
growth in Nogo-A/B-/- mice? In vitro tests
Expected Result

-/-





-/-




Cont
r
o
l
O
Mgp
-N
ogo
-
A
/
B
O
Mgp
-N
ogo
-
A
/
B


















Neurite Outgrowth
Growth Cone Collapse
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How does OMgp overexpression affects axonal
regeneration in Nogo-A/B-/- mice? In vivo
Expected Result
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
47
How does OMgp overexpression affects axonal
regeneration in Nogo-A/B-/- mice? In vivo
Expected Result
Sagittal Studies
SCI
CST
48
How does OMgp overexpression affect axonal
regeneration in Nogo-A/B-/- mice? In vivo
Expected Result
Behavioral Study






20






15
BBB Score






10








5
0




0





5





10





15






20

Days Post SCI
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What we expect
Expected Result
Nogo-A/B-/-
Overexpresses OMgp
No Axonal Regeneration
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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-/-
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Future Research

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

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Acknowledgements

• Dr. Shubhik DebBurman
• Nijee Sharma
• DAnne Duncan