1. © TND 2005 Oxidative Stress and Inflammation in Neurodegenerative Diseases George Perry and Mark A. Smith Case Western Reserve University Cleveland, Ohio

2. © TND 2005 I. Alzheimer’s Disease Background © TND 2004

3. © TND 2005 Alzheimer’s Disease 4 million Americans have Alzheimer’s Disease; by 2050, 14 million will have AD. 1 AD is one of the top 10 leading causes of death in Americans over 65 years of age. 2 AD is the third most costly disease after heart disease and cancer. 1 Federal funding for AD is 4 to 7 times lower than for heart disease, cancer or AIDS. 1

4. © TND 2005 PET (positron emission tomography) scans show differences in brain activity between a normal brain and a brain affected by Alzheimer’s disease. Blue and black in the images above denote inactive areas. Normal Alzheimer Metabolism is the primary source of oxidants.

5. © TND 2005 Brain Inflammation in Alzheimer’s Disease

6. © TND 2005 Proposed Etiologies of Alzheimer Disease Aluminum or other toxins Infectious agents Synaptic abnormalities Vascular/immune abnormalities Lack of growth factor Lipoproteins Protein abnormalities Oxidative stress

7. © TND 2005 A leading hypothesis of the biological basis of aging is oxidative stress. The prevalence of AD is strictly age-dependent

8. © TND 2005 Proposed Chronology of Changes in AD Tauist and BAPtist Amyloid-  Deposition [Senile Plaque] Tau Phosphorylation [Neurofibrillary Tangles] Neuronal death/Dysfunction: Dementia Tau Phosphorylation [Neurofibrillary Tangles] Amyloid-  Deposition [Senile Plaque]

9. © TND 2005 Proposed Chronology of Changes in AD Tauist and BAPtist Amyloid-  Deposition [Senile Plaque] Tau Phosphorylation [Neurofibrillary tangles] Neuronal death: Dementia Tau phosphorylation [Neurofibrillary tangles] Amyloid-b deposition [Senile plaque] Causes Consequences Age-Related Neuronal dysfunction Senile plaques & neurofibrillary tangles

10. © TND 2005 II. Oxidative Stress Role in AD

11. © TND 2005 Oxidative Stress 1.Classic definition: The production of reactive oxygen in excess of antioxidant mechanisms 2.Modern definition: Altered homeostatic balance resulting from oxidant insult.

12. © TND 2005 Oxidative Modifications Affect All Cellular Macromolecules ControlAlzheimer Lipid Peroxidation/Protein Adduction (4-HNE) Protein Oxidation (Free Carbonyl Groups) Nucleic Acids (8-OH-Guanosine) Alzheimer Control AlzheimerControl Glycoxidation (Carboxymethyllysine)

13. © TND 2005 Is oxidative stress an early event in AD? In the proposed sequence of degenerative events, it occurs earlier than cytoskeletal alterations. ?......  Glycation Normal Neuron ?Pre-NFTI-NFTE-NFT FREE CARBONYLS 80HG HNE

14. © TND 2005 Causes of Reactive Oxygen Species Generation in Alzheimer’s Disease Active microglia Redox active metals Amyloid-  Advanced glycation endproducts Mitochondria

15. © TND 2005 Partial reduction of oxygen generates ROS

16. © TND 2005 Consequences OxidationInflammation  Superoxide dismutase  NSAIDS confer protection  Complement pathway activation  Microglial activation and association with amyloid plaques  Heme-oxygenase- 1 induction  Induction of “adaptive” gene responses and repair enzymes  Apoptosis pathway

17. © TND 2005 III. Mitochondrial and microtubule abnormalities are found in Alzheimer’s Disease.

18. © TND 2005 Mitochondrial DNA is increased in Alzheimer’s pyramidal neurons. * * * * Deleted Wild type Alzheimer Control mtDNA In situ hybridization of mtDNA

19. © TND 2005 mtDNA 8OHG Nitrotyrosine The distribution of increased neuronal levels of mtDNA (A), 8OHG (B) and nitrotryosine (C) in Alzheimer’s Disease completely overlaps. The same neurons in adjacent serial sections are numbered.

20. © TND 2005 Mitochondria components are in autophagosomes.

21. © TND 2005 Normal Alzheimer Could the mitochondrial problem be related to microtubules?

22. © TND 2005 Study of biopsy samples shows microtubules are reduced specifically in AD pyramidal neurons. Pyramidal neurons Non-pyramidal neurons p=0.000004 p=0.90 Numbers of microtubules decrease with normal aging Microtubule Density Microtubule Length Control AD -PHF AD +PHF Control AD -PHF AD +PHF Control AD -PHF AD +PHF Control AD -PHF AD +PHF

23. © TND 2005 Microtubules (arrowheads) remain intact even in close proximity to paired helical filaments (*).

24. © TND 2005 IV. Interplay of pathological lesions and oxidative stress

25. © TND 2005  Accumulation is Associated with a Reduction in Oxidative Stress  /80HG  HO-1 Heme Oxygenase Alz50

26. © TND 2005

27. Increased amyloid -  (brown) correlates with decreased oxidative damage (8OHG, blue). Down Syndrome 17 yr. 61 yr. 31 yr.

28. © TND 2005 How do lesions protect? Oxidatively damaged Metal binding sites Reduce oxidative stress

29. © TND 2005 Is amyloid-  protective against a cauldron of oxidative stressors in Alzheimer’s Disease? Are there signs of established antioxidant responses in AD?

30. © TND 2005 V. Phosphorylation of Cytoskeletal Proteins Drives Oxidative Modifications.

31. © TND 2005 p-ERKp-JNK/SAPKp-p38 Stress response kinases are induced tau assembly induced by HNE is dependent on phosphorylation Effect of HNE on tau assembly. Phospho-tau polymers following 1mM HNE.

32. © TND 2005 In vitro modification of NFH and NFM by HNE is dependent upon lysine residues… Levels of HNE do not accumulate with age in the mouse’s sciatic nerve …and phosphorylation state. NFH NFM A B C D Antibody recognition of NFH and NFM (A) is abolished by HNE-lysine (B) but not cyteine (C) or histidine(D).

33. © TND 2005 VI. Therapeutics

34. © TND 2005 Progression/Incidence Estrogen Acetylsalicylic acid (Aspirin) (-) Deprenyl (selegiline) Ibuprofen Dapsone Acetyl-L-Carnitine (ALCAR) Vitamin E Tenilsetam Antioxidants and anti- inflammatories are protective Diet Lipoic Acid Fruits and vegetables

35. © TND 2005 Nutrients per 1000 kilocalories Alzheimer Disease Cases Controls p Value n=104 n=223 Vitamin A (RE) 855 9830.001  Carotene (mcg) 294 3890.001  Carotene (mcg) 1921 23700.003 Pro-A Carotene (mcg) 2231 28090.001 Lutein (mcg) 972 12140.015 Lycopene (mcg) 666 9270.001 Vitamin C (mg) 74.6 86.70.007 Vitamin E (a TE) 5.6 5.9NS Servings per day Yellow/green vegetables 2.0 2.30.022 Vitamin C fruits, vegetables 2.4 2.6NS Antioxidant diet is protective

36. © TND 2005 VII. Summary

37. © TND 2005 Primary etiology Cellular response Oxidative stress and inflammation A  and  AD phenotype Current therapeutic targets

38. © TND 2005 Mitochondria Nitric Oxide Mutated genes (SOD,  PP,  synuclein) Phospholipid metabolism Proteolysis Redox Active Metals Advanced Glycation Endproducts Microglia Proteins Lipids Nucleic Acid Apoptosis Alzheimer Disease Parkinson Disease ALS Stroke Multiple Sclerosis Vitamin E Lipoic Acid Metal Chelation STOP Amyloid  Neurofilament protein tau Glutathione HemeOxygenase-1 Superoxide dismutase Classic Cellular Protein-based STOP

39. © TND 2005 Conclusions Metal catalyzed oxidative damage to all categories of macromolecules is increased. Antioxidant pathways and inflammatory responses are induced. “Pathological changes” may be compensations that are critical to maintaining oxidative homeostasis.