Luigi Aurelio Nasto, M.D.

D i v i s i o n o f S p i n e S u r g e r y

D e p a r t m e n t o f O r t h o p a e d i c s a n d T r a u m a t o l o g y

" A . G e m e l l i " U n i v e r s i t y H o s p i t a l

C a t h o l i c U n i v e r s i t y o f R o m e , I t a l y

Treating intervertebral disc degeneration in an

accelerated aging model with novel therapeutics

Talk Outline

2 / 45

1.The Ercc1-/Δ mouse model, a new model for studying intervertebral disc degeneration

2.Targeting the NF-κB pathway for slowing down intervertebral disc degeneration

3.Targeting mitochondrial-generated reactive oxygen species (ROS) in intervertebral disc degeneration

1.The Ercc1-/Δ mouse model, a new model for studying intervertebral disc degeneration

2.Targeting the NF-κB pathway for slowing down intervertebral disc degeneration

3.Targeting mitochondrial-generated reactive oxygen species (ROS) in intervertebral disc degeneration

Talk Outline

3 / 45

Grade I: normal juvenile disc • nucleus pulposus and anulus fibrosus can clearly be distinguished • the nucleus pulposus has a gel-like appearance and is highly hydrated • anulus fibrosus consist of discrete fibrous lamellae • cartilage endplates are uniformly thick and consist of hyaline cartilage Grade II: normal adult disc • perpheral appearance of white, fibrous tissue in the nucleus pulposus • mucinous material is found between the lamellae of the anulus fibrosus • thickness of the cartilage endplate is irregulare Grade III: early stage of degeneration • consolidated fibrous tissue in the whole nucleus pulposus • demarcation between nucleus pulposus and anulus fibrosus is lost and extensive mucinous infiltration in the anulus fibrosus is observed • cartilage endplated show focal defects

1

2

3

Stages of Intervertebral Disc Degeneration

4 / 45

Grade IV: advanced stage degeneration • clefts in the nucleus pulposus appear, usually parallel to the end- plate • focal disruptions are found in the anulus fibrosus • hyaline cartilage of the endplate is replaced by fibrocartilage; irregularities and focal sclerosis are found in the subchondral bone

Grade V: end stage • typical disc structure may be lost completely • clefts extend through nucleus pulposus and anulus fibrosus • endplates display diffuse sclerosis

Thompson JP, Pearce RH, Schechter MT, Adams ME, Tsang IK, Bishop PB (1990) Preliminary evaluation of a scheme for grading the gross morphology of the human intervertebral disc.

Spine 15:411-415

4

5

Stages of Intervertebral Disc Degeneration

5 / 45

0,00

0,30

0,60

0,90

1,20

1,50

1,80

2,10

2,40

2,70

fetal 0-1 mo 1 mo-2yrs 3-10 yrs 11-16 yrs 17-20 yrs 21-30 yrs 31-50 yrs 51-70 yrs >71 yrs

De

gen

era

tio

n S

core

Time

notocordal cells

cell death

mucous degeneration

granular changes

concentric tears

radial tears

rim lesions

edge neovascularity

scar formation

tissue defect

Boos N, Weissbach S, Rohrbach H, et al (2002) Classification of age-related changes in lumbar intervertebral discs: 2002 Volvo Award in basic science.

Spine; 27(23):2631-2644

A continuous process

annulus fibrosus & nucleus pulposus

6 / 45

Break Make

Young

Balanced (No net loss)

Disc PG homeostasis

Loss of functional cells

PG expression

Core PG, GAG… MMP expression

Break

Make

Old

Perturbed (net PG loss)

Pro-inflammatory cytokines

Anti-inflammatory cytokines

The "central hypothesis" in disc biology

7 / 45

Aging is a major risk factor for IDD

Normal

Degenerated

Construction

quality &

maintenance

GENETICS

(faulty)

MECHANICAL

(trauma)

Excessive loads (sudden)

ENVIRONMENTAL Smoking

occupation

nutrition

Assaults radiation

wind

current

Time-dependent

wears and tears (slow)

AGING (time-dep damage of

macromolecules)

8 / 45

ENDONUCLEASE1

Repairs DNA lesions & interstrand X-links

XPF ERCC1

22

wks

2

5 w

ks

~ 2-3yr wt 1. Niedernhofer L.J., et al Nature 2006; 2. Gregg S.Q., et al DNA Repair 2011;

5 w

ks

14

wks

9

wks

Ercc1-/Δ

The Ercc1-/Δ mouse model

DNA damage

Base change

Pyrimidine Dimer

H2-bond breakage Base loss

Single-Strand Break

Kyphosis

Cachexia

Muscle degeneration

Loss of vision and hearing

Reduced renal & liver function

Neurodegeneration (dystonia, ataxia...)

Intervertebral Disc Degeneration

Progressive aging symptoms2

Genotoxic Stress

9 / 45

Vo N, et al Accelerated Aging of Intervertebral Discs in a Mouse Model of Progeria. J Orthop Res 2010

Ercc1 -/Δ Wt

Agc (green), Actin (red), Nuclei (blue)

Wt (3wks)

Ercc1 -/Δ (18wks)

Wt (2yrs)

X-ray images

Wt (3wks) Ercc1 -/Δ (18wks) Wt (2yrs)

Pe

rce

nt

Dis

c H

eig

ht

0

2

4

6

8

10

12

14

40

45

50

55

60

65

70

75

80

85

wt(4wk) mut(4wk) wt(20wk) mut(20wk) Wt(2.5 yrs)

Agg

reca

n f

luo

resc

en

ce

Wt Ercc1 -/D

3-5 wks

Wt Ercc1 -/D

18-20 wks

Wt

2.5 yrs

*

*

Intervertebral disc degeneration in Ercc1-/Δ

10 / 45

1.The Ercc1-/Δ mouse model, a new model for studying intervertebral disc degeneration

2.Targeting the NF-κB pathway for slowing down intervertebral disc degeneration

3.Targeting mitochondrial-generated reactive oxygen species (ROS) in intervertebral disc degeneration

Talk Outline

11 / 45

The NF-κB transcription factor

NF-κB is the transcription factor most associated with

mammalian aging3

NF-κB is up-regulated in a variety of aged tissues and age-

associated diseases4, 5

Inflammatory diseases

(Rheumatoid arthritis, diabetes, etc.)

Non-inflammatory diseases

(Atherosclerosis, osteoporosis, etc.)

3. Adler AS, et al Genes & Dev 2007; 4. Tilstra JS, et al Aging & Disease 2011; 5. Gosselin D, et al Gerontol 2003; 12 / 45

IκB

Nucleus

Extracellular

DNA

Cytoplasm

P

NF-κB DNA damage

ATM

NEMO

IKK

complex IκB

Nucleus

Extracellular

DNA

Cytoplasm

TNFR-1

TNFα

IKK

complex P

NF-κB

NF-κB is a family of trascription factors inolved

in cellular response to damage.

p50/p65 heterodimer is the most common

factor of the NF-κB family.6

NF-κB is activated in cells by many cellular

stressors; inflammatory, oxidative, genotoxic,

mechanical, and chemical stress are among

them.6

Two separate activation pathways have been

described:

1. Canonical pathway

2. DNA damage pathway

6. Hayden et al., Cell 2008

The NF-κB transcription factor

13 / 45

NF-κB activation in aged human disc nucleus pulpopus7

Aging activates NF-κB transcription factor

7. Nerlich et al., ANYAS 2007;

annulus

nucleus pulposus

14 / 45

NF-kB Promoter element

eGFP

Green cells = NF-kB activation

NF-kB–eGFP reporter mice

Activated NF-kB

• NF-κB activation in aged human disc nucleus pulpopus7

• NF-κB activation in aged mouse discs8

NF-κB is activated in aging mouse discs

7. Nerlich et al., ANYAS 2007; 8. Nasto LA et al., Spine 2012; 15 / 45

NF-kB element

NF-kB gene targets

IL-1β, IL-6, iNOS MMP-1b, MMP-3

qRT-PCR mouse disc RNA

NF-κB is activated in aging mouse discs

7. Nerlich et al., ANYAS 2007; 8. Nasto LA et al., Spine 2012;

• NF-κB activation in aged human disc nucleus pulpopus7

• NF-κB activation in aged mouse discs8

16 / 45

Young Wt (5 mths) Old Wt (2 yrs) Progeroid Ercc1-/D (5mths)

NF-κB is activated in aging mouse discs

7. Nerlich et al., ANYAS 2007; 8. Nasto LA et al., Spine 2012;

• NF-κB activation in aged human disc nucleus pulpopus7

• NF-κB activation in aged mouse discs8

17 / 45

Chronic activation of NF-kB mediates

age-associated disc degeneration.

NF-kB

Prediction

NF-kB

Hypothesis and Prediction

18 / 45

1. Pharmacological inhibition

IKK IkB NF-kB x NBD

Ercc1-/∆ Mice

mNBD / NBD peptide

Approach: Blocking NF-κB activity

19 / 45

-C Transduction Domain GG IKKg Blocking Peptide N- Peptide

8K-NBD: KKKKKKKK-------- GG-------TALDWSWLQTE

8K-mNBD: KKKKKKKK-------- GG-------TALDASALQTE

Bioactivity of 8K-NBD (NEMO Binding Domain) peptide

- Inhibits IL-1β and TNFα induced NF-κB activation in vitro

- Efficacious in animal models of

Inflammatory bowel disease (Dave et al., J Immunology 2007)

Muscolar dystrophy (Acharyya et al., JCI 2007)

Arthritis (Dai et al., J Biol Chem 2004)

The NBD peptide: structure

20 / 45

NBD peptide

decreases NF-kB

expression in

Ercc1-/Δ mice

IκB

Nucleus

Extracellular

DNA

Cytoplasm

IKK

complex P

NF-κB DNA damage

ATM

NEMO

TNFR-1

TNFα

NBD peptide

The NBD peptide: functioning

21 / 45

Wt (untreated)

mtN

BD

N

BD

Ercc1 -/Δ

*

NBD treatment delays the onset of osteoporosis in Ercc1-/Δ mice

22 / 45

NBD treatment improves disc histology in Ercc1-/Δ mice

23 / 45

NBD treatment improves disc matrix content

24 / 45

1. Pharmacological inhibition

IKK IkB NF-kB x NBD

Ercc1-/∆ Mice

p65

p65

p65+/- Ercc1-/∆

Mice

x 2. Genetic reduction

NF-kB dimer

p65 p50 p65

p65 Ercc1-/∆

Mice

Non-target effects

Approach: Blocking NF-κB activity

25 / 45

20 wks

20 wks

NF-kB p65 haploinsufficiency delays the onset of age-related IDD

26 / 45

• Increased level of NF-kB activity in disc cells of natural aging (Wt)

and progeroid (Ercc1-/Δ ) mice.

• Systemic treatment of Ercc1-/Δ mice with the NBD peptide (NF-kB

inhibitor) ameliorated age-dependent degenerative changes in

discs/spine.

Disc cellularity

Disc matrix PG content

Disc PG synthesis

Vertebral bone porosity

• Similar protective effects were seen with p65 genetic

haploinsufficiency.

Conclusions

27 / 45

1.The Ercc1-/Δ mouse model, a new model for studying intervertebral disc degeneration

2.Targeting the NF-kB pathway for slowing down intervertebral disc degeneration

3.Targeting mitochondrial-generated reactive oxygen species (ROS) in intervertebral disc degeneration

Talk Outline

28 / 45

Reactive oxygen species (ROS) are chemically reactive molecules (e.g. O2

-, H2O2, OH-). ROS are a natural byproduct of normal oxidative metabolism.

O2-

H2O2

OH-

O2-

O2- H2O2

H2O2

OH-

OH-

Mitochondria are the main source of ROS

within most cells.

Oxidative Stress and ROS

29 / 45

Production of ROS is an inevitable biochemical consequence of oxygen metabolism Antioxidant systems maintain a controlled balance against oxidative stress within the cell

Oxidative Stress

Damage

Repair

ROS and oxydants Anti-oxidant mechanisms

Oxidative Stress and ROS

30 / 45

Numerous studies have shown that oxidative damage increases with

age in many tissues and various organisms.9, 10, 11, 12

9. Bokov A et al., Mech Ageing & Dev 2004; 10. Ali R et al.., Biotech Histochem 2008; 11. Ulrich JA et al., Spine 2007; 12. Kang JD et al., Spine 1997;

Role of oxidative stress in disc degeneration

Neurodegenerative diseases (Patel VP, Int J Clin Exp Pathol 2011)

Lou Gehring’s disease (ALS)

Parkinson’s disease

Alzheimer’s disease

Huntington’s disease

Cardiovascular diseases (Wang JC, Circ Res 2012)

Reperfusion injury following hypoxia

Chronic fatigue syndrome (Nijs J, Man Ther 2006) Gastric cancer (Handa O, Redox Rep 2011)

31 / 45

...but intervertebral disc is hypoxic!

Urban JP, Smith S, Fairbank JC (2004) Nutrition of the intervertebral disc.

Spine; 29 (23):2700-2709

32 / 45

0,00

0,50

1,00

1,50

2,00

fetal 0-1 mo 1 mo-2yrs 3-10 yrs 11-16 yrs 17-20 yrs 21-30 yrs 31-50 yrs 51-70 yrs >71 yrs

cell density

physiolog. vessels

obliterated vessels

cartilage disorg.

cartilage cracks

microfracture

neovascularity

new bone formation

bony sclerosis

scar formation

tissue defect

Adams MA, Clin Biomech 2010

...yes, but not when it’s degenerated!

Boos N, Weissbach S, Rohrbach H, et al (2002) Classification of age-related changes in lumbar intervertebral discs: 2002 Volvo Award in basic science.

Spine; 27(23):2631-2644

Vascular ingrowth

in degenerated

intervertebral discs

cartilage endplate

33 / 45

12000X

12000X

40000X

Wt

Ercc

1 -

/D

Disc cells from 20 wk old Ercc1-/Δ mice show greater

mitochondrial death (arrows) compared to wild-type

littermates by transmission electron microscopy.

9 w

ks

25 w

ks

Ercc1-/∆ mice (DNA repair defect)

Accelerated aging

Mitochondria Dysfunction in Ercc1-/Δ mice

34 / 45

A 5% O2 20% O2 20% O2 + H2O2

Growing human NP

(nucleus pulposus) cells

in vitro increases

ROS production

Effects of oxygen on human disc cells

35 / 45

The XJB-5-131 radical scavenger

XJB-5-131 is a mitochondria-targeted ROS scavenger

XJB-5-131 has been previously shown to be

therapeutic in rodent models of hemorrhagic

shock and sepsis.13

13. Fink MP. Biochem Pharmacol 2007

Dr. Peter Wipf Professor of Pharmaceutical Sciences

Department of Chemistry University of Pittsburgh, USA

36 / 45

Bottom-to-Top Approach

Assessing the effects of oxidative stress in vitro on human disc cell cultures Assessing the effects of oxidative stress in vivo on Ercc1-/Δ mice testing the XJB-5-131 radical scavenger

37 / 45

A B

0

20

40

60

80

100

120

5% O2 20% O2

Re

lati

ve P

G s

ynth

esi

s (%

)

0

100

200

300

400

500

600

700

800

900

Agc ADAMTS4 ADAMTS5 MMP1 MMP3

Re

lati

ve g

en

e e

xpre

ssio

n (

%)

5% 20%

*

Effects of oxygen on human disc cells

High oxygen levels have

catabolic effects on

human disc cells in vitro

catabolic genes

reduced matrix synthesis

38 / 45

A B

0

20

40

60

80

100

120

5% O2 20% O2

Re

lati

ve P

G s

ynth

esi

s (%

)

0

100

200

300

400

500

600

700

800

900

Agc ADAMTS4 ADAMTS5 MMP1 MMP3

Re

lati

ve g

en

e e

xpre

ssio

n (

%)

5% 20%

*

XJB-5-131 rescues in vitro effects of high O2

0

20

40

60

80

100

120

140

20% O2 20% O2 + XJB

Re

lati

ve P

G s

ynth

esi

s (%

) *

0

25

50

75

100

125

150

175

200

20% O2 20% O2 + XJB

Re

lati

ve a

gc e

xpre

ssio

n (

%) *

XJB

XJB

39 / 45

9 w

ks

25 w

ks

Ercc1-/∆ mice (DNA repair defect)

Systemic treatment with XJB in Ercc1-/Δ mice

Accelerated aging

0 4 8 10 12 14 20 Age (weeks)

Treatment start

Treatment end

XJB-5-131 by IP injection 2mg/Kg, 3x/week

Systemic anti-oxidant therapy in Ercc1-/Δ mice to slow down IDD

40 / 45

XJB

-5-1

31

N

eg.

co

ntr

ol

H&E Safranin O

XJB treatment improves disc histology in Ercc1-/Δ mice

41 / 45

XJB treatment improves disc matrix content

0

100

200

300

400

500

600

700

800

900

1000

Tota

l GA

G c

on

ten

t (µ

gGA

G/n

g D

NA

)

NT Oil XJB

Wt Ercc1-/Δ

*

*

0

2

4

6

8

10

12

14

16

18

20

NT Oil XJB

Wt Ercc1-/Δ

PG

Syn

the

sis

(fm

ole

s su

lfat

e/n

g D

NA

) * p < 0.07

42 / 45

• Oxidative stress has been well established as a key driver of aging.

• We showed that ROS were produced in disc cells at high oxygen

tension and this correlates with:

Reduced PG (new matrix) synthesis

Enhanced expression of catabolic factors

• Depleting ROS in disc cells using XJB-5-131 rescued matrix

homeostatic imbalance

• Mitochondria-targeted free radical scavengers could be therapeutic

in delaying the onset of age-related IDD

Conclusions

43 / 45

Acknowledgements

University of Pittsburgh Hillman Cancer Institute

Laura Niedernhofer Paul Robbins Jeremy Tilstra Andria Robison Cheryl Clauson NBD peptide

Ferguson Laboratory of Orthopaedics Research

James Kang (Director) Gwen Sowa (Co-director) Nam Vo (PI) Rebecca Studer, Joon Lee

Hyoung-Yeon Seo Kevin Ngo, Qing Dong Hong Joo Moon, Takashi Yurube, Robert Hartman, Wan Huang, Paulo Coelho, Kevin Bell, Lou

Duerring.

Funding Sources

The Pittsburgh Foundation UPMC Department of Orthopaedics Surgery

Orthopaedic Research and Education Foundation AOSpine North America Young Investigators Research Grant

“BioSpina” Italian Spine Society Research Grant NIH (R21 AG033046), NIH (ES016114)

44 / 45

Thank you

45 / 45