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Addiction Therapy-2014Chicago, USA
August 4 - 6, 2014
Bryan Yamamoto
Bryan Yamamoto, Ph.D.
Department of Neurosciences
University of Toledo College of Medicine and Life Sciences
August 4, 2014
Volkow et al, Am. J. Psychiatry, 2001
Methamphetamine Neurotoxicity in Humans
Callaghan et al., Drug and Alcohol Dependence, 2011
Meth abuse increases risk of Parkinson’s Disease by 76% compared to age-
matched controls and cocaine-abusers
Overarching Hypothesis
Excitotoxicity and oxidation of proteins and lipids are mechanisms underlying the neurotoxicity of Methamphetaminethe neurotoxicity of Methamphetamine
Glutamate Release
Calcium
NOS
NO
H2O2
Calpain
Identified Events Leading to a Loss of Dopamine and 5HT
Glu Receptor Activation
Dopamine Release
X
XX
XX
MAO
NO
ONOO-
(peroxynitrite)
O2-
Protein oxidation,
Mitochondrial Dysfunction
Lipid Peroxidation
Calpain
Proteolysis
Fe++
XOH*
But, there are caveats to the
proposition that the
neurotoxicity/excitotoxicity of
METH is a result of its direct action METH is a result of its direct action
on the brain……..
Local administration of METH does not increase glutamate
0
50
100
150
200
Do
pa
min
e (p
g/2
0 u
l)Systemic MA
Local MA
Systemic and Local Administration of METH:
-1 0 1 2 3 4 5 6 7 8
Time (hours)
Burrows
Glu
tam
ate
(p
g/2
0 µµ µµ
l)
01000
2000300040005000
600070008000
900010000
-1 0 1 2 3 4 5 6 7 8
PerfusionGlu
tam
ate
(p
g/2
0 µµ µµ
l
Do
pa
min
e (
pg
/ µµ µµg
pro
tein
)
Glutamate Synergizes with Local METH
To Deplete Dopamine Content
*
#
Do
pa
min
e (
pg
/
Without Glutamate With Glutamate
No MethMeth Meth/Heat No Meth Meth Meth/Heat
#
Burrows
�If it is not the direct effect of METH alone on
the brain (striatum), what other systems could
be responsible for the neurotoxicity?
�Another target of METH is the liver
Thinking Outside the Brain……
�Another target of METH is the liver
�Liver damage can produce encephalopathy
(i.e. hepatic encephalopathy)
� Tremor, movement disorders
� Delirium, stupor, confusion, and coma
METH and Evidence of Hepatotoxicity
Halpin
80
100
120
ALT
*
400
500
600
AST
*
Alanine Aminotransferase Aspartate Aminotransferase
Methamphetamine Increases Liver Enzymes in Plasma
0
20
40
60
ALT
(IU/L)
Saline METH
0
100
200
300
AST
(IU/L)
Saline METH
Halpin
Liver Damage and Ammonia
� The liver normally removes excess ammonia from the
blood via the urea cycle.
� This is important because ammonia is a neurotoxic
byproduct of protein metabolism and other byproduct of protein metabolism and other
metabolic reactions.
� If the liver damaged, there is an accumulation of
ammonia that is thought to cause hepatic
encephalopathy
Ammonia Toxicity
� Excitotoxicity (Fan and Szerb, 1993, Hermenegildo et
al., 1996)
� Oxidative stress (Kosenko et al., 1997 and Kosenko et
al., 1995)
� Metabolic compromise (Hawkins et
al., 1973, McCandless and Schenker, 1981)al., 1973, McCandless and Schenker, 1981)
∴∴∴∴Similar mechanisms that mediate the toxicity to
ammonia and the neurotoxicity to METH
� Unknown whether peripheral organ (e.g. liver)
damage is produced by METH and if it contributes to
METH neurotoxicity
Overarching Hypothesis
Liver Damage
Methamphetamine
Hyperammonemia
Neurotoxicity
LactuloseX
120
140
160
180
200
Pla
sma
Am
mo
nia
(µµ µµ
M)
*
#
Methamphetamine Increases Ammonia in Plasma
0
20
40
60
80
100
Saline Lactulose METH METH +
Lactulose
Pla
sma
Am
mo
nia
(
Halpin
Bra
in A
mm
on
ia
(Pe
rce
nt
Ba
seli
ne
)
Veh+METH Veh+Sal
Lac+Sal Lac+METH
400
300
500
Methamphetamine Increases Ammonia in Striatum:
Blocked by Lactulose
0 1 2 3 4 5 6 7 8
Bra
in A
mm
on
ia
(Pe
rce
nt
Ba
seli
ne
)
Time (Hr)
300
200
100
Halpin
ME
TH
g p
rote
in X
10
-2)
2
3
Lactulose Does Not Affect METH Concentrations in the Brain
(pp
m/ µµ µµ
g p
rote
in X
10
1
METH METH + Lactulose
0
Halpin
80
100
120
g p
rote
in)
5
6
7
8
g p
rote
in)
*#
#
Lactulose Attenuates METH-induced
Long-term Depletions of Dopamine and 5HT Content
Dopamine Serotonin
0
20
40
60
1
(pg
/ µµ µµg
pro
tein
)
0
1
2
3
4
5
1Vehicle VehicleVehicleVehicleLactulose Lactulose LactuloseLactulose
Saline Saline SalineSaline METHMETHMETH METH
(pg
/ µµ µµg
pro
tein
)
* *
Halpin
1.2
1.0
0.8
0.6
Dopamine Transporter Immunoreactivity
*
% C
on
tro
lLactulose Blocks METH-induced Decreases
in Dopamine Transporter Immmunoreactivity
0.6
0.4
0.2
Vehicle VehicleLactulose Lactulose
Saline Saline METHMETH
% C
on
tro
l
Halpin
Glu
tam
ate
(%
Ba
seli
ne
)
400
500
600
300
Vehicle-METH
METH-Induced Increases
in Striatal Extracellular Glutamate:
Blocked by Lactulose
1 2 3 4 5 6 7 8
Glu
tam
ate
(%
Ba
seli
ne
)
200
100
300
B
Time (hrs)
Vehicle-Saline
Lac-METH
Lac-Saline
Halpin, Northrop
Lactulose Blocks METH-Induced Increases
in Striatal Spectrin Proteolysis
100
160
140
120
180 *
Pe
rce
nt
Co
ntr
ol
Vehicle Lactulose Vehicle LactuloseSaline Saline METH METH
100
40
20
60
80
Pe
rce
nt
Co
ntr
ol
Halpin, Northrop
METH-Induced Astroglyosis (GFAP) is Blocked by Lactulose
100
120
140
160
180
200
Pe
rce
nt
Co
ntr
ol
*
0
20
40
60
80
Vehicle Lactulose Vehicle LactuloseSaline Saline METH METH
Pe
rce
nt
Co
ntr
ol
Halpin, Northrop
aCSF TBOA
NH3 NH3+TBOA
Glu
tam
ate
(%
Baseli
ne)
Local Perfusion of Ammonia on Glutamate:Efflux Through Reversal of the Glutamate Transporter
*
300
250
200
150
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Glu
tam
ate
(%
Baseli
ne)
Time (hrs)
TBOA or aCSF
NH3 or aCSF
*
100
150
50
Halpin, Northrop
60
80
100
120
3
4
5
6
* *
(pg
/ µµ µµg
pro
tein
)
(pg
/ µµ µµg
pro
tein
)
Combined Local Perfusions of Ammonia and METH
Dopamine and 5HT Content in the Striatum
Dopamine 5HT
0
20
40
0
1
2
Vehicle VehicleNH3
Saline Saline METHMETH
Vehicle Vehicle
Saline Saline METHMETH
(pg
/
(pg
/
NH3 NH3 NH3
Halpin, Northrop
METH
GYKI
NH3
METH
GYKI
NH3
Are there other targets of ammonia?
Amphetamines
The Blood-Brain Barrier: A Protective Barrier of the Brain
BBB Function in Cortex After Meth
Vehicle+Saline Lactulose+Saline Vehicle+Meth Lactulose+Meth
Vehicle
Saline
Lactulose
Saline
Lactulose
Meth
Vehicle
Meth
*
#
Quantification of FITC-Dextran Extravasation
Northrop
Blood-Brain Barrier
Tight Junctions
Occludin
Claudin 3, 5, 12
JAM
*
#
Occludin Claudin-5
*
#
Meth-Induced Disruption of BBB Structure in Cortex
* *
Isolated Brain Capillaries
*
Vehicle
Saline
Lactulose
Saline
Vehicle
Meth
Lactulose
Meth
*
Vehicle
Saline
Lactulose
Saline
Vehicle
Meth
Lactulose
Meth
* *
Northrop
Representative Blot for
Nitrotyrosine
Ammonia as a Pro-Oxidant?
Representative Blot for
Tight Junction Proteins
Northrop
Isolated Brain Capillaries from Cortex
Cyclooxygenase-2
Ammonia as a Pro-Inflammatory Agent?
*
&
2 Im
mu
no
rea
ctivity
Ve
hic
le +
Sa
line
)
Northrop
Vehicle
Saline
Vehicle
Meth
Lactulose
Meth
Lactulose
Saline
&
CO
X-2
Im
mu
no
rea
ctivity
(%V
eh
icle
+ S
alin
e)
Summary and Conclusions
� Increases in extracellular glutamate and evidence of excitotoxicity
and oxidative degradation of proteins
� Causes hepatotoxicity and increases ammonia concentrations in
peripheral plasma and brain to mediate excitotoxicity
Effects of METH:
� Damages the BBB via ammonia, oxidative stress, and
inflammation (COX2-dependent prostaglandin synthesis?)
� Opening of the BBB can render the brain vulnerable to toxins by
that would otherwise by restricted to the periphery.
� Peripheral organ effects should be considered as possible causes
of the neurotoxicity associated with psychostimulant drug use
� Jeffrey Brown, Ph.D.
� Kristan Burrows Cline, Ph.D.
� David Eyerman, Ph.D.
� Amy Ferng, M.S.
� Laura Halpin, Ph.D.
� Amanda Blaker
� Veronica Chiu, Ph.D.
� Stuart Collins
� Nicole Harless
� Katelyn Marchal
Collaborators
Methamphetamine ProjectPast and Current
Other Current Lab Members
� Laura Halpin, Ph.D.
� John Irlam, D.O.
� J. F. Nash, Ph.D.
� Arunan Nadarajah, Ph.D.
� Nicole Northrop, Ph.D.
� Robert Staszewski, M.S.
� Despina Tata, Ph.D
� Katelyn Marchal
� Carmen Mitchell
� Reka Natarajan, Ph.D.
� Allen Schroering, M.S.
� Erin Semple
� Branden Stansley
NIH grants DA007606, DA016866, DA035499
Meet the eminent gathering once again at
Addiction Therapy-2015Florida, USA
August 3 - 5, 2015
Addiction Therapy – 2015 Website:
addictiontherapy.conferenceseries.com
August 3 - 5, 2015