April 1, 2009 L ech Kiedrowski, Ph.D. UIC Department of Psychiatry lkiedr@psych.uic

1. April 1, 2009 Lech Kiedrowski, Ph.D. UIC Department of Psychiatry lkiedr@psych.uic.edu Neuroprotective agents

2. Objectives • Learn about the mechanisms of neurodegeneration caused by brain ischemia (stroke, heart attack). • Learn about neuroprotective agents being developed to counteract ischemic brain damage.

3. Outline • High susceptibility of brain to ischemia • Mechanisms of ischemic neuronal death and the role of Ca2+ and Zn2+ in triggering these mechanisms • Agents developed to protect the brain from ischemic damage

4. Ischemic brain damage may occur after: • Heart attack (global ischemia) • Stroke (focal ischemia) • ischemic (occlusion of a blood vessel) 87% • hemorrhagic (bleeding in the brain) 13% American Heart Association 2009

5. Heart attack and brain damage • Brain damage can start to occur just 4-6 min after the heart stops pumping blood • Survival rate is only 2% if heart is arrested for more than 12 min

6. Stroke and brain damage • About795,000 cases each year • Every 40 seconds someone in the USA has a stroke and every 3 min someone dies of it • Stroke is the third leading cause of death, after heart disease and cancer • Stroke is the leading cause of long-term disability (60% of survivors become handicapped) • The estimated direct and indirect cost of stroke for 2009 is $68.9 billion. American Heart Association 2009

7. Stroke and brain damage • Lack of effective neuroprotective therapy • The only FDA-approved therapy for stroke is intravenous injection of t-PA (Tissue Plasminogen Activator, a clot-dissolving agent) • However, t-PA can only be applied during ischemic stroke and it must be applied during the first 3 hours of stroke

8. High energy requirements of the brain The human brain constitutes only 2% of the body’s weight, yet it utilizes approximately 25% of total glucose and almost 20% of oxygen.

9. What happens when the blood supply to the brain is suddenly interrupted?

10. 126 volunteers ! This was the first (fortunately the last) controlled investigation on the effects of acute arrest of the circulation to the human brain Arch. Neurol. Psych. 50 (1943) 510-528

11. Arch. Neurol. Psych. 50 (1943) 510-528

12. The Kabat-Rossen-Anderson apparatus

13. Dramatic Symptoms Arch. Neurol. Psych. 50 (1943) 510-528

14. 7 seconds of brain ischemia will make you unconscious, but will not damage your brain Arch. Neurol. Psych. 50 (1943) 510-528

15. EEG is flat within 10 sec of global brain ischemia Ischemic depolarization (high elevation in external K+) takes place about 2 min after the onset of ischemia. Hansen, Acta Physiol. Scand. (1978) 102: 324-329.

16. Sagital section through rat brain Hippocampus

17. Selective vulnerability of CA1 neurons to ischemia CA1 CA = Cornu Ammonis (Ammon’s horn) DG = Dentate Gyrus Sham operated DG CA3 CA1 neurons die CA3 and DG neurons survive 3 days after 10-min ischemia 7 days after 10-min ischemia Yokota et al. Stroke (1995) 26: 1901-1907.

18. Ischemia has to last over 2 min to kill CA1 neurons 2 min of ischemia 3 min of ischemia Hippocampal CA1 region in gerbil brain 7 days after ischemia Kato et al. Brain Res. (1991) 553: 238-242.

19. What kills the CA1 neurons?

20. The Pulsinelli et al. experiment

21. Denervation protected the CA1 neurons from ischemic death Pulsinelli (1985) Prog Brain Res 63: 29-37

22. ? ? ? Death

23. Extracellular glutamate during ischemia and reperfusion Baseline Ischemia Reperfusion 10 20 30 10 2010 20 30 60 120 Glutamate (µM) sampled from various brain regions of the rat subjected to 20-min ischemia. Globus et al. (1988) J Neurochem 51:1455-1464

24. Glutamate is neurotoxic Olney, J.W., Brain lesions, obesity, and other disturbances in mice treated with monosodium glutamate. Science, 1969. 164: p. 719-721. A single subcutaneous injection of glutamate (4 mg/g) produces brain lesions and kills 2 – 9 day-old mice within 1 to 48 hours

25. Receptor Glutamate ? Death

26. In cultured spinal neurons, glutamate deregulates Ca2+ homeostasis in a Ca-dependent manner Tymianski et al. J. Neurosci. 13 (1993) 2085-2104

27. Ionotropic receptors Metabotropic receptors Glutamate activates a number of receptors Glutamate AMPA (-Glu-R2) AMPA (+Glu-R2) NMDA Kainate mGluRs group 1 mGluRs group 2 and 3 out in

28. MK-801 NBQX NBQX NBQX Some of these receptors are Ca-permeable channels Glutamate AMPA (-Glu-R2) AMPA (+Glu-R2) NMDA Kainate mGluRs group 1 mGluRs group 2 and 3 Na+ Ca2+ Na+ Ca2+ Na+ Na+ out in IP3 K+ K+ K+ K+ cAMP

29. MK-801 NBQX NBQX NBQX Some of these receptors are Ca-permeable channels Glutamate AMPA (-Glu-R2) AMPA (+Glu-R2) NMDA Kainate mGluRs group 1 mGluRs group 2 and 3 Na+ Ca2+ Na+ Ca2+ Na+ Na+ out in IP3 K+ K+ K+ K+ cAMP

30. Blocking NMDA receptors prevents glutamate-induced deregulation of Ca2+ homeostasis and neuronal death Ca2+ deregulation Dead Neurons Fraction deregulated/dead APV – NMDA receptor inhibitor CNQX – AMPA/kainate receptor inhibitor NIM – voltage-gated Ca channel inhibitor Conclusion: Inhibiting NMDA receptors is sufficient to protect the neurons against glutamate-induced death Tymianski et al. J. Neurosci. 13 (1993) 2085-2104

31. Failure of clinical stroke trials with glutamate receptor antagonist Drugs Mode of action Result Selfotel competitive NMDA antagonist trial discontinued Aptiganel noncompetitive NMDA antagonist adverse effects MK-801 noncompetitive NMDA antagonist adverse effects Dextrorfan noncompetitive NMDA antagonist adverse effects GV150526 glycine site antagonist of NMDA rec. no efficacy Eliprodil polyamine site antagonist of NMDA rec. no efficacy NBQX competitive AMPA receptor antagonist trial discontinued adverse effects renal toxicity Cerebrovasc. Dis. 11, suppl 1 (2001) 60-70

32. !

33. CaEDTA but not ZnEDTA protects CA1 neurons against ischemic death The role of zinc in ischemic neuronal death Zinc-specific fluorescence in rat hippocampus before ischemia CA1 region 3 days after 10-min ischemia Zinc-specific fluorescence Fuchsin staining (pink) of degenerating neurons Koh et al. Science 272 (1996) 1013-1016

34. The data indicate that preventing zinc translocation, using CaEDTA, prevents the ischemic death of CA1 neurons What are the zinc-induced neurotoxic phenomena?

35. Activation of Poly(ADP-ribose) polymerase-1 (PARP-1) may lead to neuronal death Kauppinen and Swanson, Neuroscience 145 (2007) 1267-1272

36. PARP-1 (called also PARS)-mediated NAD- and ATP-depletion leads to cell death NO – nitric oxide PARS – Poly(ADP-ribose) synthetase NAm – nicotinamide NMN – nicotinamide mononucleotide PRPP – phosphoribosyl pyrophosphate PPi – inorganic phosphate Zhang et al. Science 263 (1994) 686 - 689

37. Does PARP play a role in ischemic neuronal death?

38. PARP plays a role in ischemic brain infarct formation in vivo PARP-1 knockout PARP-1 inhibition PBS = phosphate buffered saline (control) 3-AB = 3-aminobenzamide (PARP inhibitor) PARP knockout or PARP inhibition reduce the size of ischemic brain infarct caused by the middle cerebral artery occlusion (MCAO) Endres et al. J Cereb Blood Flow Metab 17 (1997) 1143-1151

39. Inhibition of PARP with 3-AB prevents the MCAO-induced NAD depletion Dark color represents NAD staining. This staining is decreased in the MCAO-affected territory These data implicate that the mechanism of ischemic neuronal death in vivo involves PARP activation Endres et al. J Cereb Blood Flow Metab 17 (1997) 1143-1151

40. Is there a link between zinc exposure and PARP activation?

41. Exposure of cultured cortical neurons to toxic concentrations of zinc (400 µM) for 15 min activates PARP and causes NAD and ATP depletion ABAM = 3-aminobenzamide, 3-AB (PARP inhibitor) NAM = nicotinamide (another PARP inhibitor) PAR = Poly(ADP-ribose) Kim et al. Exp Neurology 177 (2002) 407-418

42. PARP inhibitors prevent zinc-induced NAD-depletion and neuronal death ABAM = 3-aminobenzamide, 3-AB (PARP inhibitor) NAM = nicotinamide (another PARP inhibitor) LDH = lactate dehydrogenase (LDH release from cells is used as an index of cell death) Kim et al. Exp Neurology 177 (2002) 407-418

43. The effect of zinc (15 min exposure) on PARP and neuronal death is dose-dependent Kim et al. Exp Neurology 177 (2002) 407-418

44. The data presented indicate that a transient exposure of neuronal cell cultures to over 100 µM zinc activates a PARP-dependent mechanism of neuronal death What about lower zinc concentrations?

45. Prolonged exposure (24 hours) of neuronal cultures to low (<100 µM) concentrations of zinc is neurotoxic and involves p75NTR and NADE activation LDH = lactate dehydrogenase (LDH release from cells is used as an index of cell death) p75NTR = a nonselective low-affinity neurotrophin receptor belonging to TRK family; nerve growth factor (NGF) is an agonist of this receptor NADE = a 22 kDa cytosolic protein called p75NTR-associated death executor NeuN = neuron-specific nuclear protein (negative control) Park et al. J Neurosci 20 (2000) 9096 - 9103

46. Exposure of neuronal cultures to 25 µM zinc for 24 hours is neurotoxic and involves a capase-dependent mechanism AS#1 and #2 – NADE antisense #1 and #2 oligonucleotides used to knockdown NADE NS – a nonsense oligonucleotide (negative control) Note that both NADE knockdown and caspase inhibitors protect against zinc toxicity Park et al. J Neurosci 20 (2000) 9096 - 9103

47. The data show PARP, caspases, p75NTR, and NADE being involved in zinc-induced toxicity in vitro Do these mechanisms play a role in ischemic neuronal death in vivo?

48. Zinc chelation with CaEDTA prevents ischemic activation of p75NTR and NADE, and also prevents neurodegeneration TUNEL (staining of apoptotic cells) p75NTR NADE Sham operated control 3 days after 15 min ischemia 3 days after 15 min ischemia + CaEDTA Park et al. J Neurosci 20 (2000) 9096 - 9103

49. Two types on cell death Low zinc concentrations High zinc concentrations Apoptosis Programmed Cell Death Requires activation of caspases Necrosis Does not involve caspases • Cells swell • Only modest condensation of chromatin • Cytoplasmic vacuolization and breakdown of organelles • Rupture of plasma membrane followed by leakage of cellular content to the extracellular space • Cells shrink • Chromatin becomes pyknotic (condensed) • Cytoplasmic organelles remain intact • Plasma membrane remains intact • Eventually nucleus and cytoplasm break into apoptotic bodies that are phagocytized by macrophages or adjacent cells

50. How could we protect the brain against ischemic damage?