Costing et al., Ann Rev Neurosc. 2009

None Non painful Painful

DNA methylation and histone acetylation are two critical epigenetic mechanisms controlling chromatin structure and function

in mammalian neurons

Or methylation

Korzus Nat. Neurosc. 13,405,2010

His

MOR gene

opiate rec. (MOR)

Nerve injury down-regulates MOR opioid receptors via an epigenetic mechanism (Uchida et al., J. Neurosci. 31, 2010)

Top-down control of pain:

an ambiguous role for serotonin

a2, 5-HT7

Analgesic

5-HT3, 5-HT2b

Hyperalgesic

Targeting the discending analgesic pathways

Epigenetic suppression of inhibitory transmission

In the raphe nucleus of the RVM (Zhang et al., Nat. Med. 2011)

Serotonergic pathways desceding from RVM are hyperalgesic in

chronic pain

Hyperalgesic role of serotonin during the development of persistent pain (Wei,et al., J. Neurosci. 30, 2010)

Top-down control of pain: “Noradrenaline more important than serotonin

In chronic pain”

a2, 5-HT7

Analgesic

5-HT3, 5-HT2b

Hyperalgesic

Targeting the discending analgesic pathways

SNRI Duloxetine Vanlafaxine Milnacipran

TCA

Amytryptiline

MOR+NARI Tapentadol

WDR neurons

Pre-synaptic

mGlu5

mGlu4,7,8

mGlu2/3

ATP cAMP

Gi

-

CGRP L-GLU SP/NKA

NK-1R

Ca2+ Na+

Gq Gq +

Gi

Post-synaptic

NMDA AMPA

Presynaptic mGlu2/3 receptors as targets for

analgesic drug: keep the break on

Group-II mGlu receptors

AC Gi/o - ATP

cAMP -

Glutamate N and P/Q

Ca2+channel

bg

+

MAPK PI-3-K AC-II

mGlu2 - 872 aa Gene:Grm2/GRM2

mGlu3 – 877 aa Gene: Grm3/GRM3

LY354740 or LY379268

Backbone of orthosteric agonists

Increased second phase of the formalin test (nociceptive

sensitization) in mice lacking mGlu2 receptors

0 5 10 15 20 25 30 35 40 45 50 55 600

25

50

75

100

125 mGlu2 WT

mGlu2 KO

min after formalin injection

Lik

ing

beh

avio

r (s

ec)

Phase I (0-10) Phase II (20-55)0

50

100

150

200

250

300mGlu2 WT

mGlu2 KO

Lik

ing

behavio

r (s

ec)

Xct/

b-act

in

Reduced expression of the cystine/glutamate antiporter in the spinal cord

in the CCI model of neuropathic pain

Generalyzed Anxiety Disorder and Panic attack

Schizophrenia and drug-induced psychosis

Drug addiction

Chronic pain

Neurodegeneration?

LY2144023

Phase II NCT00149292 trial

28 days vs. olanzapine vs. placebo

PANSS for schizophrenia Nature Medicine September 2007

LY544344

Efficacy and tolerability in GAD

16 mg b.i.d. HRS-A and CGII

but…convulsions in preclinical studies (Danayevich et al., 2007)

Orthosteric mGlu2/3 agonists

Tolerance to the analgesic activity of mGluR2 agonists

L-Acetylcarnitine: carnitine or the acetyl group?

Mechanical allodynia in CCI rats

A single injection of LY341495 (1mg/kg, ip) reverses the analgesic activity of L-Acetylcarnitine (100 mg/kg, sc twice daily) in the CCI

model of neuropathic pain

-1.5

-1.0

-0.5

0

*

° Me

ch

an

ical

allo

dyn

ia

Ctrl LY34 LAC LAC single 24 d + LY34

LY341495 = mGlu2/3 antagonist

saline 7 days

LAC 7 days3

2.5

2

1.5

1

0.5

0

*

*

*

mG

lu2/

3 re

cept

or e

xpre

ssio

n

(ar

bitr

ary

units

)

I II-out II-in III IV________________________________

spinal cord laminae

Saline LAC

Repeated injections of L-Acetylcarnitine induce upregulation of mGlu2/3 receptors in the rat dorsal horn and in the DRG

Chiechio et al., 2002

Real-Time PCR shows that L-Acetylcarnitine Treatment Selectively

Upregulates mGluR2 mRNA Expression in Mouse DRG Cultures

30 min 60 min 120 min240 min 1 day 4 days

0

200

400

600

800

1000

mG

lu3 m

RN

A e

xp

ressio

n (

% c

han

ge)

30 min 60 min 120 min240 min 1 Day 4 Days

0

200

400

600

800

1000

mG

luR

2 m

RN

A e

xp

ressio

n (

% c

hang

e)

30 60 120 240 1 4 _______________________ ____________ min days -

30 60 120 240 1 4 ________________________ __________

min days

*

mGluR2 mRNA expression mGluR3 mRNA expression

LAC, 250 µM (every 12 hours)

Potential regulatory elements identified by the TFSEARCH Database

• NF-kB (p50/p65)

• p300

• sp1

• Oct-1

• GATA-1

• GATA-2

• c-Rel

• Ap-1

• sp1

• Oct-1

• GATA

• GATA-1

GRM2 GRM3

many

ATG

Exon 2 Exon 1

TIS

p300 p50

p65

NF-kB

binding sites

LAC

Acetyl groups

Chronic treatment with L-acetylcarnitine but not carnitine

induces p65 acetylation in DRG culture

ATG

Exon 2 Exon 1

TIS

p300 p50

p65

NFk-B

binding sites

Induction of mGlu2 receptors is under the control of acetylation mechanisms

L-Acetylcarnitine

Acetyl groups

mGluR2

Me

cha

nic

al

th

resh

old

(g

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Sham None LAC PREG AMYT TRAM

100 30 10 100 mg/kg

CCI (neuropathic pain)

1 hour of withdrawal 1 week of withdrawal 2 months of withdrawal

*

Long-lasting analgesic effect of L-acetylcarnitine in a mouse model

of neuropathic pain

Drug treatment:14 days

< 200 kDa

< 42 kDa

< 100 kDa

0

0.5

1.0

1.5

2.0

2.5

mG

luR

2/3

/b-a

cti

n

* mGluR 2/3

b-actin

CCI CCI + LAC

CCI CCI +LAC

Prolonged hyperexpression of mGlu2 receptors in

LAC-treated mice: 2-months of withdrawal

Long-lasting analgesic effect of L-acetylcarnitine in the CFA model of chronic inflammatory pain

The integration of negative affect, pain, and cognitive control in the cingulate cortex (Shackman et al., Nat. Rev. Neur. 2011)

L-Acetylcarnitine

Rapid and long-lasting antidepressant-like effect of

L-acetylcarnitine in FSL rats

LAC up-regulates mGlu2 receptor expression in the

hippocampus and frontal cortex

LAC treatment enhances histone acetylation at the

mGlu2 receptor promoter

L-Acetylcarnitina

Nuovo farmaco “epigenetico” per il trattamento del dolore

cronico

Valore aggiunto: azione antidepressiva

Dept. of Pharm. Sci., University of Catania Santina Chiechio, Agata Copani

IRCCS Neuromed, Pozzilli Matteo Bernabucci, Serena Notartomaso, Cristina Zappulla, Angela Trabucco, Ludovico Ciolli, Francesco Fazio, Milena, Cannella, Marta Motolese, Roberto Gradini, Giuseppe Battaglia, Valeria Bruno

University of Rome, Sapienza, Anesthesiology Angela Macaluso, Fabiana Troisi, Saul Collini

University of Rome Sapienza, Neurology Andrea Truini, Erica Pasquale, Giorgio Cruccu

Wahington University, Pain Center, St. Louis Rob Gereau 4th

Peter Kalivas, 2009

p50

I-kB

NF-kB

responsive gene

p65

p65

p50

p50

I-kB

p65

p

p

I-kB

p

p

IKK

The NF-kB / Ik-B pathway

Pain Matrix

Perception

Emotion

Cognition

Behavior

L-Acetylcarnitina

Nuovo farmaco “epigenetico” per il trattamento del dolore

cronico e dei disturbi depressivi

LAC è efficace nel trattamento della distimia

(Zanardi e Smeraldi, Eur. Neuropsychopharm., 2006 Bersani et al., Eur. Neuropsychopharm., 2013)

Promoter transition from an active to an inactive chromatin state

Acetylation of histones or trancription factors:

opening of chromatin and gene expression

HDACs (histone deacetylases): suppression of

gene expression

LAC in dysthimic disorders in elderly patients: double-blind multicenter, control, randomized stady vs. fluoxetine

Bersani et al., Europ. Neuropsychopharmacol. 23, 2013

Fluoxetine

LAC

Touluse-Pieron’s test

CTRL LAC CAPE CAPELAC0

2

4

6

8

10

12

14

16

18

20

mG

lu2

mR

NA

Qu

an

tity

(co

pie

s)

*

CTRL LAC CAPE CAPE+LAC

(250M) (2.5g/ml) 1 hour 30 min

CAPE Real-Time PCR LAC 30 min 1 hour

Induction of mGlu2 receptors by LAC in cultured DRG neurons is prevented by the NFkB inhibitor, CAPE

Linking molecules to mood: new insight into the neurobiology

of depression (Krishnan and Nestler, Am J. Psych. 2010)

The antidepressant effect of LAC is mediated by mGlu2

receptors

HDACs (histone deacetylases)

p65 (RelA) is deacetylated by histone deacetylase

HDAC3 – Chen et al., Science, 2001

HDAC

inhibitors

Vehicle

MS-275 (3 mg/kg, sc) for 5 days

MS-275 + LY34195 (1 mg/kg ip, 30 min before formalin)

Phase I Phase II MS275PhaseI LY341495MS D MS2755 MS275LY

0

20

40

60

80

100

120

140

Lik

ing

beh

avio

r (s

ec)

o

#

mGlu2 →

actin →

Ctrl MS-275

CTRL MS2750.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

mG

lu2 e

xpre

ssio

n/a

ctin

(arb

itra

ry u

nits)

CTRL MS-275

*

The histone deacetylase inhibitor, MS-275, induces

analgesia by up-regulating mGlu2 receptors

ATG

Exon 2 Exon 1

TIS

p300 p50

p65

NFk-B

binding sites

Induction of mGlu2 receptors is under the control

of acetylation mechanisms

Acetyl donors

Inhibitors of histone deacetylase

Acetyl groups

mGluR2

Glutamate Receptor Families

AMPA Kainate NMDA Metabotropic

GluR1 GluR2 GluR3 GluR4

GluR5 GluR6 GluR7

KA1 KA2

NMDAR1

NMDAR2A NMDAR2B NMDAR2C NMDAR2D

mGluR1 mGluR5 mGluR2 mGluR3 mGluR4 mGluR6 mGluR7 mGluR8

Go Gi, Gq

NMDAR3

The Wind-up phenomenon as an example of homosynaptic sensitization in nociceptive neurons

“Wind-up”

Heterosynaptic sensitization:

depolarization removes the Mg2+ blockade of

NMDA receptor channels

Ca2+

Memantine Ketamine Metadone Dextropropoxyphene

-

Pre-synaptic

mGlu5

CGRP L-GLU SP/NKA

NK-1R

Ca2+ Na+

Gq Gq +

Post-synaptic

NMDA AMPA

Ca2+

Voltage-sensitive calcium channels as targets for

“disease-dependent” analgesic drugs: let off the accelerator

N-type

VSCC

Ziconotide

Voltage-

sensitive

Ca2+ channels

(VSCC)

Binding affinity

Pregabalin: 20-30 nM

Gabapentin: 40-50 nM L-leucine, L-isoleucine,

L-methionine: 50 nM

Reduced expression of mGlu2/3 receptors in the hippocampus

of rats subjected to prenatal stress (Zuena et al., PlosOne, 2008)

Epigenetic DNA and histones

modifications

Active chromatin

•Unmethylated DNA •Histone acetylation •H3-lysine trimethylation (H3K4, H3K36 and H3K79)

Inactive chromatin

•Methylated DNA •Histone deacetylation •H3/H4-lysine trimethylation (H3K9, H3K27, H4K20 )

DNA methyltransferases

DNA active demethylation

GADD45 XPG

Polymerase Ligase

NER REPAIR

APOBEC, AID, MBD4, TDG and GADD45

DRG

Pregabalin inhibits the increased trafficking of the a2d subunit

associated with chronic pain (Bauer et al., J. Neurosci., 29, 2009)

Expression of histone deacetylase (HDAC) type 1 and 2 in

the DRG and dorsal horns of the spinal cord

Dorsal Horn Dorsal root Peripheral

Laminae Neurons ganglion endings Stimulus

Nociceptive-Specific

Non-Nociceptive

Nociceptive-Specific

Wide Dynamic-Range

Non-Nociceptive

Non-Nociceptive

Non-Nociceptive

Wide Dynamic-Range

Wide Dynamic-Range

Nociceptive-Specific

Wide Dynamic-Range

Nociceptive-Specific

Wide Dynamic-Range

Nociceptive-Specific

I

IIo

IIi

III

IV

V

VI

X

Noxious

Mechanical

Small, slow

Intermediate

Large, fast

C

Ad

Ab

Afferent distribution to second order neurons

Hyperalgesia

Allodynia

Heterosynaptic facilitation between Aß and C/Ad fibers

and WDR neurons

Aß

C/Ad

HDAC inhibition up-regulates mGlu2 receptors in the DRG

Single injection Single injection

5-day administration 5-day administration

0

50

100

150

200

250

300

Col 1

AG

S3

/b-a

ctin

Sham CFA Sham CFA

3 days 7 days

p<0.01

Increased expression of AGS3 in the spinal cord of rats after Intraplantar injection of CFA

S3 S7 C3 C7 C7 S3 S7 C3 C7

AGS3 (75 kDa)

HDAC inhibition enhances p65 phosphorylation and

mGlu2 receptor expression in the DRG

ATG

Exon 2 Exon 1

TIS

p300 p50

p65

NFk-B

binding sites

Induction of mGlu2 receptors is under the control

of acetylation mechanisms

Acetyl donors

Inhibitors of histone deacetylase

Acetyl groups

mGluR2

p65 (RelA) is deacetylated by histone deacetylase HDAC3 – Chen et al., Science, 2001

HDAC inhibitors

Top-down control of pain

*

Sham CCI CCI + LAC CCI CCI + LAC

a2d-1 b-actin

Prolonged down-regulation of the a2d subunit of VSCCs

2 months after termination of chronic LAC treatment

(100 mg/kg, i.p., for 21 days) in CCI mice