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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