regeneration 12_b

Axon Regeneration Why do injured adult CNS axons fail to regrow?

1) Axon regeneration is age-dependent

2) Severed adult axons can grow in vitro

3) Scar tissue prevents growth

Glial cells

ECM

cut axons

fibroblasts

reactive astrocytes

macrophages

ECM collagen

4) Permissive influence of peripheral glial cells

Traumatic brain injury statistics

Thurman et al., 1999

Gender Cause

Age (years)

30

20

10

0 0-4 20-24 40-44 60-64 80-84

Transportation

Firearms

Falls

40

female male

Hospitalization & death (7 states, 1994)

Rate (per 105)

Age (years)

Rate (per 105)

120

80

40

0 0-4 20-24 40-44 60-64 80-84

female male

http://www.cdc.gov/braininjuryinseniors/

Response Time: 45 mins Helicopter or Ambulance 69% of population

Level I/II Trauma Center Coverage (2008)

Response Time: 60 mins Ambulance 56% of population

http://www.spinalcord.uab.edu/show.asp?durki=21446

Spinal cord injury •  11,000 new cases per year in the U.S.

•  Approx. 250,000 people alive

•  Most injuries occur between 16 to 30 years

•  Approx. 80% of injuries occur among males

•  Car accidents account for about 50%

•  Average cost for C1-4 injury: year one: $741,425 subsequent years: $132,807

What regenerates?

Cold-blooded vertebrate RGCs (rest of CNS is not good)

Mammalian peripheral nerves

Limited regeneration in neonatal mammals

Local sprouting (<500 µm) in adult mammals

juan-carlosandaluz

Sticky Note

Retinal ganglion cells can regenerate;

Kalil and Reh, 1979

lesion

Adult lesion Infant lesion (PND1 to 7)

lesion

Axons can grow around lesion Control

Age-dependence

juan-carlosandaluz

Sticky Note

In the adult, the cell will die if the axon is cut. In the infant, the cut axon will regrow around the lesion.

Age-dependent regeneration in vitro


P12 control

P6 midline section

P12 midline section

Hafidi et al., 1995, 1999

Hafidi et al., 2004


P6 P12

Club endings

Jorge Francisco Tello y Muñóz (Cajalʼs student)

Cajal in his lab

Kerschensteiner et al., 2005

In vivo imaging of cut axons

GFP-labeled axons in mouse spinal cord (Thy-1 promotor)

GFP-labeled DRG Axon is imaged

DRG

Dorsal view of mouse spinal cord

DRG axon: pre-lesion

DRG axon: post-lesion (200µm needle)



Proximal cut end: die back Distal cut end: Wallerian degeneration

GFP labeled DRG neuron axons running in the dorsal column of the spinal cord


axonal fragmentation

retraction

bulb formation


Attempted regrowth at 24 hours


Adult axons can grow

Thanos et al., 1989

Adult retinal axons growing in vitro with BDNF

Scar tissue limits regeneration

Davies et al.1997

1 mm

MIDLINE 100 µm

No injury: regeneration

INJURY SITE

axons

Transplant DRG neurons in the corpus callosum

Injury: no regeneration

juan-carlosandaluz

Sticky Note

one way to test whether the environment is good enough to grow is to take an area of the brain that contains a ton of axons. Then transplant cells from another brain region into this new environment (without any injuries) What they found is that the axons grow normally

juan-carlosandaluz

Sticky Note

On the other hand, when they transplant the cells into the new environment which contains an injury, there is no axonal regeneration. This shows that scar tissue limits axonal regeneration.

Rudge and Silver, 1990

Hippocampal axons don’t growth on adult scar material

Adult optic nerve Adult optic nerve + macrophages

Cortex

nitrocellulose

Neonatal scar stuff Adult scar stuff


axon

axon

axon

cut axons

fibroblasts

reactive astrocytes

Macrophages

ECM

collagen

What’s in the scar?

myelin

Reactive astrocytes express EphA4 GFAP EphA4 Merged

Lesi

on s

ite

Goldshmit et al., 2004

… which is a poor growth substrate WT reactive astrocytes Eph4A-/- astrocytes

Better regeneration in EphA4-/- mice

Goldshmit et al., 2004

Eph

A4-

/-

Con

trol

chain of disaccharides covalently linked to a core protein

Core protein

Glycosaminoglycans (GAG)

“Average” Proteoglycan aggregate •  one hyaluronic acid •  100 core proteins •  50 keratan sulfate chains (250 disaccharide units/chain) •  100 chondroitin sulfate chains (1000 disaccharide units/chain) •  Molecular weight: tens of millions

Proteoglycan aggregate

Hyaluronic acid (also a GAG)

link protein


Davies et al.1997

1 mm

MIDLINE 100 µm

No injury: regeneration

axons

Transplant DRG neurons in the corpus callosum

CSPG: no regeneration

laminin keratin sulfate &

chondroitin sulfate proteoglycan

laminin

50% laminin +

50% keratin sulfate & chondroitin sulfate

proteoglycan

laminin DRG

Neurocan: a proteoglycan expressed at injury site

Asher et al., 2000

Control cortex Injured cortex

Neurites do not grow on Neurocan

Asher et al., 2000

Cerebellum on L1

Cerebellum on L1 + Neurocan stripes

Neurocan L1

L1

(a chondroitin sulfate proteoglycan)

juan-carlosandaluz

Sticky Note

Cerebellar neuron grows on Laminin but it does not grow in L1 + neurocan stripes. Neurocan is really good at repelling growth.

GC behavior on proteoglycan (Aggrecan)

Tom et al., 2004

Laminin

60x time lapse 60x time lapse

Laminin + Aggrecan

juan-carlosandaluz

Sticky Note

Club ending forms in axon regrowing in laminin + aggrecan.

Growth cone behavior on Aggrecan

260x time lapse

Bradbury et al., 2002

Blockade of proteoglycans restores growth in spinal cord

labeled CST fibers no label

Control Lesioned

Control Lesioned _+ vehicle Lesioned _+ chondroitinase

Lesioned _+ chondroitinase: invasion of grey matter

Fibr

e co

unts

(%)

Distance from lesion (mm)

Sham Les+Veh Les+CHase

100

80

60

40

20

0

Moon et al., 2001

Blockade of proteoglycans restores growth in nigrostriatial pathway

chondroitinase

Tyrosine hydroxylase immunoreactivity

Chondroitinase

Control

cut axons

Macrophages


Chen et al., 2000

Macrophage migrate to axon injury site in leech

5 minutes post-injury 3 hours post-injury E

xten

t of n

erve

cru

sh

Davalos et al., 2005

Microglial response to injury in Cortex

Lesion with laser ATP

David et al.

Macrophages accumulate at optic nerve lesion

Lesion site

Cut optic nerve

Obtain cryostat section of optic nerve lesion

lesion site

Do macrophages have an effect in CNS injury?

Cut optic nerve

Obtain cryostat section of optic nerve lesion

lesion site

DRG growth near lesion site

DRG growth distal to lesion site


Cortex

nitrocellulose


Injuring lens induces macrophages in retina


Leon et al., 2000; Lorber et al.,2005

nerve crush nerve crush + lens injury

brown = macrophage stain

injuring lens induces regeneration

injure lens

crush

lens retina

crush

regeneration Leon et al., 2000

100 µm


Macrophage activation mimics lens injury

Zymosan injection increases macrophages in the retina (Zymosan = a yeast cell wall suspension)

Leon et al., 2000; Lorber et al.,2005

Macrophages in retina Regeneration in Zymogen-treated retina

Macrophages produce oncomodulin, a Ca+2 binding protein

Yin et al., 2006

control

lens injury

macrophages oncomodulin

Oncomodulin enhances axon regeneration

control

oncomodulin

cut axons


myelin

juan-carlosandaluz

Sticky Note

Because myelin activates the RhoA/Rho kinase (ROCK)signaling pathway in PC12 cells and blocking of RhoA activityovercomes myelin inhibition

Bandtlow et al.

tubulin = yellow actin = red

GC collapse after contact with oligodendrocyte

Contact Collapse

ethidium bromide knife cut

CNS regeneration following depletion of glia

Moon et al., 2000

myelin stain

Saline

Ethidium bromide

Dopaminergic fibers regenerate past lesion (but not into striatum)

anterior posterior

lesion

Antibody to oligodendrocyte protein: Nogo-A

vehicle-treated

anterior posterior

lesion

Antibody-treated

Yiu & He, 2006

RhoA activity leads to growth cone collapse

juan-carlosandaluz

Sticky Note

RhoA activation has been shown to correlate with signals that induce growth cone collapse and axon guidance repulsion81. Evidence suggests that this pathway also mediates myelin inhibition

juan-carlosandaluz

Sticky Note

myelin-based inhibitory signals might trigger the activation of RhoA and ROCK, leading to the phosphorylation of cofilin by LIM kinase to stabilize the growth cone cytoskeleton of damaged axons, restricting regenerative outgrowth

juan-carlosandaluz

Sticky Note

In the central nervous system (CNS), RHOA GTPase signaling through Rho kinase (ROK) promotes growth cone collapse and inhibits regrowth (Luo et al., 1997). Consequently RHOA activation has been associated with impaired regeneration of spinal cord axons

juan-carlosandaluz

Sticky Note

http://people.ucalgary.ca/~mdnguyen/pdf/20081.pdf

juan-carlosandaluz

Sticky Note

RHO is associated with the Rho kinase (ROK), in turn thought to activate LIM kinase that phosphorylates cofilin. Phosphorylated cofilin, in turn, has a loss of function that interferes with actin turnover and growth cone advance (Ng and Luo, 2004). RHOA is also linked to the inhibition of myosin phosphatase activity that in turn leads to increased myosin ATPase activity and growth cone retraction (Luo et al., 1997). An additional route to retract growth cones is through direct ROK phosphorylation of myosin (Giniger, 2002). Therefore, the net result of ROK activation is an increase in actin arc and central actin bundle contractility and stability with secondary actions on microtubular organization (Zhang et al., 2003). These actions collapse growth cones and mediate active repulsion

Gaillard et al., 2007

Blocking Rho permits RGC axon regeneration Control Rho inhibitor (C3) Protocol

juan-carlosandaluz

Sticky Note

Inhibiting RhoA using C3 transferase or a dominant-negative approach also promotes axon outgrowth on inhibitory substrates

juan-carlosandaluz

Sticky Note

y inhibiting the RHOA-ROK intracellular signaling pathway we observed an increase in axon outgrowth. HA- 1077 is a relatively specific inhibitor of ROK

Sciatic nerve graft in cortex

Tello, F (1911) La influencia del neurotropismo en la regeneration de los centros nerviosos. Trab Lab Invest Univ Madrid 9:123-159

David and Aguayo, 1981

Sciatic nerve bridge in spinal cord

David and Aguayo, 1981

Fibers entering medulla Fibers entering spinal cord

200 µm

Sciatic nerve bridge in spinal cord

Olfactory bulb ensheathing cells promote regeneration

Thuret et al., 2006

Olfactory bulb ensheathing cells promote regeneration

Li et al., 1997

Lesion of corticospinal tract

0.5 mm

Green = axons Red = ensheathing cells

rost

ral

caud

al

200 µm

Spinal cord injury patients: olfactory mucosa autografts

Lima et al., 2006

7 patients, ages 18-32, treated 6 mos to 6 yrs after injury

scar tissue removed from injury site before graft nerve bundle

collagen is stained green

GFAP is stained brown

*

Lima et al., 2006

Spinal cord injury patients: olfactory mucosa autografts

Gaillard et al., 2007

Growth of embryonic rat cortex in lesioned adult

E14 tissue

GFP+ fibers were found in:

Busch and Silver, 2007

regeneration 12_b

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