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