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

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