1. Bryan Yamamoto Addiction Therapy-2014 Chicago, USA August 4 - 6, 2014

2. Bryan Yamamoto, Ph.D. Department of Neurosciences University of Toledo College of Medicine and Life Sciences August 4, 2014

3. 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

4. Overarching Hypothesis Excitotoxicity and oxidation of proteins and lipids are mechanisms underlying the neurotoxicity of Methamphetamine

5. Glutamate Release Calcium NOS NO ONOO - (peroxynitrite) O 2 - Protein oxidation, Mitochondrial Dysfunction Lipid Peroxidation H2O2H2O2 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*

6. But, there are caveats to the proposition that the neurotoxicity/excitotoxicity of METH is a result of its direct action on the brain……..

7. Local administration of METH does not increase glutamate Systemic and Local Administration of METH: Burrows Glutamate (pg/20  l) -1 0 1 2 3 4 5 6 7 8 Perfusion Glutamate (pg/20  l

8. Dopamine (pg/  g protein) Without GlutamateWith Glutamate Glutamate Synergizes with Local METH To Deplete Dopamine Content No Meth Meth Meth/Heat No MethMeth Meth/Heat * # Burrows

9.  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  Liver damage can produce encephalopathy (i.e. hepatic encephalopathy)  Tremor, movement disorders  Delirium, stupor, confusion, and coma Thinking Outside the Brain……

10. METH and Evidence of Hepatotoxicity Halpin

11. ALT (IU/L) SalineMETH * AST (IU/L) SalineMETH * Alanine AminotransferaseAspartate Aminotransferase Methamphetamine Increases Liver Enzymes in Plasma Halpin

12. 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

13. 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)  Si milar 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

14. Overarching Hypothesis Liver Damage Hyperammonemia Methamphetamine Neurotoxicity Lactulose X

15. Saline Lactulose METH METH + Lactulose Plasma Ammonia (  M) * # Methamphetamine Increases Ammonia in Plasma Halpin

16. 400 300 200 500 100 Methamphetamine Increases Ammonia in Striatum: Blocked by Lactulose Halpin

17. 2 3 1 METHMETH + Lactulose 0 Lactulose Does Not Affect METH Concentrations in the Brain Halpin

18. (pg/  g protein) Vehicle Lactulose Saline METH (pg/  g protein) * * * # # Lactulose Attenuates METH-induced Long-term Depletions of Dopamine and 5HT Content DopamineSerotonin Halpin

19. 1.2 1.0 0.8 0.6 0.4 0.2 Vehicle Lactulose Saline METH Dopamine Transporter Immunoreactivity * % Control Lactulose Blocks METH-induced Decreases in Dopamine Transporter Immmunoreactivity Halpin

20. Glutamate (% Baseline) 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

21. Lactulose Blocks METH-Induced Increases in Striatal Spectrin Proteolysis Vehicle Lactulose Saline Saline METH METH 100 40 20 160 140 120 60 80 180 * Percent Control Halpin, Northrop

22. METH-Induced Astroglyosis (GFAP) is Blocked by Lactulose Vehicle Lactulose Saline Saline METH METH Percent Control * Halpin, Northrop

23. Glutamate (% Baseline) 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

24. * * Vehicle NH 3 Saline METH Vehicle Saline METH (pg/  g protein) NH 3 Combined Local Perfusions of Ammonia and METH Dopamine and 5HT Content in the Striatum Dopamine 5HT Halpin, Northrop METH GYKI NH 3 METH GYKI NH 3

25. Are there other targets of ammonia?

26. Amphetamines The Blood-Brain Barrier: A Protective Barrier of the Brain

27. BBB Function in Cortex After Meth Vehicle Saline Lactulose Saline Lactulose Meth Vehicle Meth * # Quantification of FITC-Dextran Extravasation Vehicle+SalineLactulose+SalineVehicle+MethLactulose+Meth Northrop

28. Blood-Brain Barrier Tight Junctions Occludin Claudin 3, 5, 12 JAM

29. * # Occludin Vehicle Saline Lactulose Saline Vehicle Meth Lactulose Meth Claudin-5 * # Vehicle Saline Lactulose Saline Vehicle Meth Lactulose Meth Meth-Induced Disruption of BBB Structure in Cortex * * Northrop Isolated Brain Capillaries

30. Representative Blot for Nitrotyrosine Ammonia as a Pro-Oxidant? Representative Blot for Tight Junction Proteins Northrop Isolated Brain Capillaries from Cortex

31. Cyclooxygenase-2 Ammonia as a Pro-Inflammatory Agent? Northrop Vehicle Saline Vehicle Meth Lactulose Meth Lactulose Saline * & COX-2 Immunoreactivity (% Vehicle + Saline)

32. 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:

33.  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 Collaborators Methamphetamine Project Past and Current Other Current Lab Members NIH grants DA007606, DA016866, DA035499

34. Addiction Therapy – 2015 Website: addictiontherapy.conferenceseries.com Meet the eminent gathering once again at Addiction Therapy-2015 Florida, USA August 3 - 5, 2015