Bryan Yamamoto

Addiction Therapy-2014Chicago, USA

August 4 - 6, 2014

Bryan Yamamoto, Ph.D.Department of Neurosciences

University of Toledo College of Medicine and Life SciencesAugust 4, 2014

Volkow et al, Am. J. Psychiatry, 2001

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

Methamphetamine Neurotoxicity in Humans

Overarching Hypothesis

Excitotoxicity and oxidation of proteins and lipids are mechanisms underlying the neurotoxicity of Methamphetamine

Glutamate Release

Calcium

NOS

NO

ONOO-

(peroxynitrite)

O2-

Protein oxidation, Mitochondrial Dysfunction

Lipid Peroxidation

H2O2

Calpain

Proteolysis

Identified Events Leading to a Loss of Dopamine and 5HT

Glu Receptor Activation

Dopamine Release

Fe++

X

X

X

X

XX

MAO

OH*

But, there are caveats to the proposition that the

neurotoxicity/excitotoxicity of METH is a result of its direct action

on the brain……..

Local administration of METH does not increase glutamate

0

50

100

150

200

-1 0 1 2 3 4 5 6 7 8

Do

pa

min

e (p

g/2

0 u

l)

Time (hours)

Systemic MA

Local MA

Systemic and Local Administration of METH:

Burrows

Glu

tam

ate

(pg/

20

l)

0100020003000400050006000700080009000

10000

-1 0 1 2 3 4 5 6 7 8

PerfusionGlu

tam

ate

(pg

/20

l

Dop

amin

e (p

g/g

pro

tein

)

Without Glutamate With Glutamate

Glutamate Synergizes with Local METHTo Deplete Dopamine Content

No Meth Meth 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 liverLiver damage can produce encephalopathy

(i.e. hepatic encephalopathy) Tremor, movement disorders Delirium, stupor, confusion, and coma

Thinking Outside the Brain……

METH and Evidence of Hepatotoxicity

Halpin

ALT(IU/L)

Saline METH

*

AST(IU/L)

Saline METH

*

Alanine Aminotransferase Aspartate Aminotransferase

Methamphetamine Increases Liver Enzymes in Plasma

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 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)

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

Hyperammonemia

Methamphetamine

Neurotoxicity

LactuloseX

Saline Lactulose METH METH + Lactulose

Plas

ma

Amm

onia

(M

)

*

#

Methamphetamine Increases Ammonia in Plasma

Halpin

400

300

200

500

100

Methamphetamine Increases Ammonia in Striatum:Blocked by Lactulose

Halpin

2

3

1

METH METH + Lactulose

0

Lactulose Does Not Affect METH Concentrations in the Brain

Halpin

(pg/

g p

rote

in)

Vehicle VehicleVehicleVehicleLactulose Lactulose LactuloseLactulose

Saline Saline SalineSaline METHMETHMETH METH

(pg/

g p

rote

in)

* *

* #

#

Lactulose Attenuates METH-induced Long-term Depletions of Dopamine and 5HT Content

Dopamine Serotonin

Halpin

1.2

1.0

0.8

0.6

0.4

0.2

Vehicle VehicleLactulose Lactulose

Saline Saline METHMETH

Dopamine Transporter Immunoreactivity

*

% C

ontr

olLactulose Blocks METH-induced Decreases

in Dopamine Transporter Immmunoreactivity

Halpin

Glu

tam

ate

(% B

asel

ine)

200

400

500

600

100

300

B

Time (hrs)

Vehicle-METH

Vehicle-Saline

Lac-METH

Lac-Saline

METH-Induced Increases in Striatal Extracellular Glutamate:

Blocked by Lactulose

Halpin, Northrop

Lactulose Blocks METH-Induced Increases in Striatal Spectrin Proteolysis

Vehicle Lactulose Vehicle LactuloseSaline Saline METH METH

100

40

20

160

140

120

60

80

180 *

Perc

ent C

ontr

ol

Halpin, Northrop

METH-Induced Astroglyosis (GFAP) is Blocked by Lactulose

0

20

40

60

80

100

120

140

160

180

200

Vehicle Lactulose Vehicle LactuloseSaline Saline METH METH

Perc

ent C

ontr

ol

*

Halpin, Northrop

Glu

tam

ate

(% B

asel

ine)

Time (hrs)

TBOA or aCSF

NH3 or aCSF

Local Perfusion of Ammonia on Glutamate:Efflux Through Reversal of the Glutamate Transporter

*

100

300

250

200

150

50

Halpin, Northrop

* *

Vehicle VehicleNH3

Saline Saline METHMETHVehicle Vehicle

Saline Saline METHMETH

(pg/

g p

rote

in)

(pg/

g p

rote

in)

NH3 NH3 NH3

Combined Local Perfusions of Ammonia and METHDopamine and 5HT Content in the Striatum

Dopamine 5HT

Halpin, Northrop

METHGYKI

NH3

METHGYKI

NH3

Are there other targets of ammonia?

Amphetamines

The Blood-Brain Barrier: A Protective Barrier of the Brain

BBB Function in Cortex After Meth

VehicleSaline

LactuloseSaline

LactuloseMeth

VehicleMeth

*

#

Quantification of FITC-Dextran Extravasation

Vehicle+Saline Lactulose+Saline Vehicle+Meth Lactulose+Meth

Northrop

Blood-Brain BarrierTight Junctions

OccludinClaudin 3, 5, 12JAM

*

#

Occludin

VehicleSaline

LactuloseSaline

VehicleMeth

LactuloseMeth

Claudin-5

*

#

VehicleSaline

LactuloseSaline

VehicleMeth

LactuloseMeth

Meth-Induced Disruption of BBB Structure in Cortex

* *

Northrop

Isolated Brain Capillaries

Representative Blot for Nitrotyrosine

Ammonia as a Pro-Oxidant?

Representative Blot for Tight Junction Proteins

Northrop

Isolated Brain Capillaries from Cortex

Cyclooxygenase-2Ammonia as a Pro-Inflammatory Agent?

Northrop

VehicleSaline

VehicleMeth

LactuloseMeth

LactuloseSaline

*

&

CO

X-2

Imm

uno

rea

ctiv

ity(%

Veh

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

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

Effects of METH:

Jeffrey Brown, Ph.D.

Kristan Burrows Cline, Ph.D.

David Eyerman, Ph.D.

Amy Ferng, M.S.

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

Amanda Blaker

Veronica Chiu, Ph.D.

Stuart Collins

Nicole Harless

Katelyn Marchal

Carmen Mitchell

Reka Natarajan, Ph.D.

Allen Schroering, M.S.

Erin Semple

Branden Stansley

CollaboratorsMethamphetamine Project Past and Current

Other Current Lab Members

NIH grants DA007606, DA016866, DA035499

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Addiction Therapy-2015Florida, USA

August 3 - 5, 2015