Addiction Therapy-2014 Chicago, USA August 4 - 6, 2014

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

2. Peripheral and Central Mechanisms of Methamphetamine Neurotoxicity: Connecting the Dots Bryan Yamamoto, Ph.D. Department of Neurosciences University of Toledo College of Medicine and Life Sciences August 4, 2014

3. Methamphetamine Neurotoxicity in Humans 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

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

5. Identified Events Leading to a Loss of Dopamine and 5HT X X Glutamate Release Dopamine Release X Glu Receptor Activation Calcium MAO X NOS X H2O2 Calpain NO Fe++ X ONOO- (peroxynitrite) OH* O2- Protein oxidation, Mitochondrial Dysfunction Lipid Peroxidation Proteolysis

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

8. Glutamate Synergizes with Local METH To Deplete Dopamine Content * Dopamine (pg/mg protein) # Meth No Meth Meth/Heat No Meth Meth Meth/Heat Without Glutamate With Glutamate Burrows

9. Thinking Outside the Brain…… • 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

10. METH and Evidence of Hepatotoxicity Halpin

11. Methamphetamine Increases Liver Enzymes in Plasma * Alanine Aminotransferase Aspartate Aminotransferase ALT (IU/L) * AST (IU/L) Saline METH Saline METH 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) \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

14. Overarching Hypothesis Methamphetamine Liver Damage Hyperammonemia X Lactulose Neurotoxicity

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

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

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

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

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

20. METH-Induced Increases in Striatal Extracellular Glutamate: Blocked by Lactulose 600 500 Vehicle-METH 400 Glutamate (% Baseline) 300 200 Vehicle-Saline 100 Lac-METH Lac-Saline B Time (hrs) Halpin, Northrop

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

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

23. Local Perfusion of Ammonia on Glutamate: Efflux Through Reversal of the Glutamate Transporter 300 250 200 Glutamate (% Baseline) * 150 100 50 NH3 or aCSF TBOA or aCSF Time (hrs) Halpin, Northrop

24. Combined Local Perfusions of Ammonia and METH Dopamine and 5HT Content in the Striatum 5HT Dopamine * * (pg/mg protein) (pg/mg protein) NH3 Vehicle NH3 Vehicle NH3 Vehicle Vehicle NH3 NH3 NH3 METH GYKI Saline Saline METH METH Saline Saline METH METH METH GYKI Halpin, Northrop

25. Are there other targets of ammonia?

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

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

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

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

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

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

32. Summary and Conclusions Effects of METH: • 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

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

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