1. 1 ALZHEIMER’S DISEASE Treatment and Prevention Ben Best President/CEO Cryonics Institute

2. 2 What is Alzheimer’s Disease (AD)? Only confirmed on autopsy Only confirmed on autopsy Characterized by Characterized by Amyloid plaque Amyloid plaque Neurofibrillary tangles Neurofibrillary tangles Neuronal loss Neuronal loss Synapse loss Synapse loss Inflammation and oxidative damage Inflammation and oxidative damage Tangles without plaque is FrontoTemporal Dementia Tangles without plaque is FrontoTemporal Dementia

3. 3 Empirical versus Mechanistic Empirical treatment and prevention Empirical treatment and prevention What appears to work What appears to work Mechanistic treatment and prevention Mechanistic treatment and prevention Understand the mechanisms Understand the mechanisms Devise strategies based on mechanisms Devise strategies based on mechanisms Evaluate what may work or not to understand the mechanisms and improve strategies. Evaluate what may work or not to understand the mechanisms and improve strategies.

4. 4 Amyloid Cascade Hypothesis Amyloid peptide Amyloid peptide Called Aß (amyloid beta) Called Aß (amyloid beta) Because aggregates in ß (beta) sheets Because aggregates in ß (beta) sheets Simplified view Aß formation Aß formation ⇒ amyloid plaques ⇒ amyloid plaques ⇒ neuron death ⇒ neuron death ⇒ dementia ⇒ dementia

5. 5 Amyloid cascade begins APP (Amyloid Precursor Protein) APP (Amyloid Precursor Protein) Cleaved by secretase enzymes Cleaved by secretase enzymes Cleavage of APP forms shorter peptides Cleavage of APP forms shorter peptides Cleavage by alpha or beta secretase Cleavage by alpha or beta secretase Only cleavage by beta secretase forms Aß Only cleavage by beta secretase forms Aß Two forms of Aß Two forms of Aß After cleavage by gamma-secretase After cleavage by gamma-secretase 40 amino acid amyloid beta peptide (Aß 40 ) 40 amino acid amyloid beta peptide (Aß 40 ) OR OR 42 amino acid amyloid beta peptide (Aß 42 ) STICKY! 42 amino acid amyloid beta peptide (Aß 42 ) STICKY! ⇒ amyloid plaques ⇒ amyloid plaques

6. 6 Amyloid cascade begins

7. 7 Damaging effects of Aß 42 Activation of microglia Activation of microglia Brain version of macrophages Brain version of macrophages Produce inflammatory cytokines Produce inflammatory cytokines InterLeukin-1ß (IL-1ß) InterLeukin-1ß (IL-1ß) Tumor Necrosis Factor alpha (TNF−α) Tumor Necrosis Factor alpha (TNF−α) Generate peroxynitrite and oxidative stress Generate peroxynitrite and oxidative stress Hyperphosphorylation of tau Hyperphosphorylation of tau Tau protein stablizes cell microtubules Tau protein stablizes cell microtubules Phosphorylated tau forms NFTs Phosphorylated tau forms NFTs NeuroFibrillary Tangles NeuroFibrillary Tangles

8. 8 Damaging effects of NFTs Amyloid plaque does not cause neuron death Amyloid plaque does not cause neuron death NFTs are always associated with neuron death NFTs are always associated with neuron death Without microtubules neurons cannot function Without microtubules neurons cannot function NFTs become glycated NFTs become glycated Intimately associated with AGEs (Advanced Glycation End-products) Intimately associated with AGEs (Advanced Glycation End-products) Generate free radicals contributing to neurodegeneration Generate free radicals contributing to neurodegeneration

9. 9 Damaging forms of Aß 42 Aß 42 is a 42-amino acid monomer Aß 42 is a 42-amino acid monomer Aß 42 monomers can aggregate into Aß 42 monomers can aggregate into Oligomers (many monomers) Oligomers (many monomers) Fibrils which can aggregate into plaques Fibrils which can aggregate into plaques Soluble oligomers are the most toxic form of Aß 42 Soluble oligomers are the most toxic form of Aß 42 “Halos” of Aß 42 oligomers around plaques damage or destroy synapses “Halos” of Aß 42 oligomers around plaques damage or destroy synapses PNAS;Koffie,RM;106:4012 (2009) PNAS;Koffie,RM;106:4012 (2009) JOURNAL OF NEUROSCIENCE; Lacor,PN;27:796(2007) JOURNAL OF NEUROSCIENCE; Lacor,PN;27:796(2007)

10. 10 Amyloid aggregation/disaggregation

11. 11 Amyloid Cascade Hypothesis APP ⇒ Aß 42 (peptide, monomer) ⇒ fibrillar Aß or oligomers ⇒ fibrillar Aß or oligomers ⇒ amyloid plaques ⇒ amyloid plaques ⇒ inflammation/NFTs ⇒ neuron death / synapse loss ⇒ neuron death / synapse loss

12. 12 Amyloid Cascade Evidence Genetic pre-disposition to AD Genetic pre-disposition to AD Onset at an early age Onset at an early age Most often increases gamma-secretase cleavage Most often increases gamma-secretase cleavage Produces increased Aß 42 Produces increased Aß 42 Down’s Syndrome victims produce more APP Down’s Syndrome victims produce more APP 3 chromosomes with gene for APP 3 chromosomes with gene for APP Cleaved to Aß 42 Cleaved to Aß 42 Early AD Early AD

13. 13 Discussion Choose a partner Choose a partner Tell your partner what you understood and think while your partner silently listens – talk for a couple of minutes Tell your partner what you understood and think while your partner silently listens – talk for a couple of minutes Switch roles – partner that listened becomes the speaker for a couple of minutes Switch roles – partner that listened becomes the speaker for a couple of minutes Short discussion Short discussion

14. 14 Amyloid and NFTs in AD

15. 15 Cholesterol and the amyloid cascade Human brain high in myelin to facilitate axon conduction speed Human brain high in myelin to facilitate axon conduction speed Myelin produced by oligodendrocytes (glia) Myelin produced by oligodendrocytes (glia) Brain contains 25% of body’s membrane cholesterol Brain contains 25% of body’s membrane cholesterol 80% of brain cholesterol is in myelin 80% of brain cholesterol is in myelin Cholesterol allows tight membrane packing in myelin Cholesterol allows tight membrane packing in myelin Cholesterol recycling and transport depends on APOlipoprotein E (APOE) Cholesterol recycling and transport depends on APOlipoprotein E (APOE)

16. 16 APOE and AD APOE exists in 3 alleles APOE exists in 3 alleles APOE2, APOE3 and APOE4 APOE2, APOE3 and APOE4 APOE4 is the only allele in non-human primates APOE4 is the only allele in non-human primates APOE4 is less efficient than APOE3 in transporting cholesterol and clearing the brain of Aß APOE4 is less efficient than APOE3 in transporting cholesterol and clearing the brain of Aß APOE3 is less efficient than APOE2 APOE3 is less efficient than APOE2 14% of Caucasians have one APOE4 allele, but 40% of AD victims have at least one APOE4 allele 14% of Caucasians have one APOE4 allele, but 40% of AD victims have at least one APOE4 allele Aß 42 rises markedly after head injury Aß 42 rises markedly after head injury Risk of AD increases many times with head injury, but increases much more so for those with APOE4 Risk of AD increases many times with head injury, but increases much more so for those with APOE4 APOE4 less efficient at myelin repair and Aß 42 clearance after injury APOE4 less efficient at myelin repair and Aß 42 clearance after injury

17. 17 Metal toxicity and AD Role of aluminum is controversial Role of aluminum is controversial Zinc, copper and iron cause Aß aggregation Zinc, copper and iron cause Aß aggregation Copper particularly mediates Aß toxicity Copper particularly mediates Aß toxicity Aß is not toxic in the absence of Cu 2+ Aß is not toxic in the absence of Cu 2+ Zinc inhibits Aß toxicity Zinc inhibits Aß toxicity Copper enhances Aß fibril formation, an effect potentiated by APOE4 Copper enhances Aß fibril formation, an effect potentiated by APOE4 Copper is a greater catalyst for free radicals than the other metals Copper is a greater catalyst for free radicals than the other metals Aß binds to both copper and cholesterol causing oxidation of cholesterol to compounds that are extremely toxic to neurons Aß binds to both copper and cholesterol causing oxidation of cholesterol to compounds that are extremely toxic to neurons

18. 18 Clioquinol therapy for AD Clioquinol binds zinc & copper (chelator) Clioquinol binds zinc & copper (chelator) Crosses the blood-brain barrier (BBB) Crosses the blood-brain barrier (BBB) Transgenic AD-prone mice treated with clioquinol showed a 49% decrease in Aß Transgenic AD-prone mice treated with clioquinol showed a 49% decrease in Aß AD patients treated with clioquinol for 12 weeks AD patients treated with clioquinol for 12 weeks Severely demented showed slowed cognitive decline Severely demented showed slowed cognitive decline Moderately demented showed no effect Moderately demented showed no effect ARCHIVES OF NEUROLOGY;Richie,CW;60:1685 (2003) ARCHIVES OF NEUROLOGY;Richie,CW;60:1685 (2003) PBT2 is a clioquinol analog that has greater BBB permeability, and more effectively strips copper from Aß, without chelation, which could remove essential metals PBT2 is a clioquinol analog that has greater BBB permeability, and more effectively strips copper from Aß, without chelation, which could remove essential metals No adverse effects seen No adverse effects seen Currently in clinical trials Currently in clinical trials

19. 19 Copper in the diet Diets high in copper and saturated fats are associated with increased risk of AD Diets high in copper and saturated fats are associated with increased risk of AD Saturated fats increase cholesterol Saturated fats increase cholesterol Foods highest in copper content are beef liver, oysters, molluscs, almonds, and cocoa Foods highest in copper content are beef liver, oysters, molluscs, almonds, and cocoa BRITISH JOURNAL OF NUTRITION; Loef,M;107:7 (2011) BRITISH JOURNAL OF NUTRITION; Loef,M;107:7 (2011)

20. 20 Discussion Tell your partner what you understood and think while your partner silently listens – talk for a couple of minutes Tell your partner what you understood and think while your partner silently listens – talk for a couple of minutes Switch roles – partner that listened becomes the speaker for a couple of minutes Switch roles – partner that listened becomes the speaker for a couple of minutes Short discussion Short discussion

21. 21NSAIDs Persons taking Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) for more than two years show a 50% greater protection from AD than those not taking NSAIDs. Persons taking Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) for more than two years show a 50% greater protection from AD than those not taking NSAIDs. The same effect is not seen for cortisol, which has a more profound anti-inflammatory effect. The same effect is not seen for cortisol, which has a more profound anti-inflammatory effect. NEUROBIOLOGY OF AGING 22:799 (2001) NEUROBIOLOGY OF AGING 22:799 (2001) Older NSAIDs block both COX-1 and COX-2 enzymes Older NSAIDs block both COX-1 and COX-2 enzymes Newer NSAIDs block COX-2 (pain) Newer NSAIDs block COX-2 (pain) COX-1 inhibition can cause GI bleeding COX-1 inhibition can cause GI bleeding Microglia express COX-1 Microglia express COX-1 NSAIDs (ibuprofen & indomethicin) inhibit cytokine activation of beta-secretase (reduce Aß formation) NSAIDs (ibuprofen & indomethicin) inhibit cytokine activation of beta-secretase (reduce Aß formation) JOURNAL OF NEUROSCIENCE;Sastre,M;23:9796 (2003) JOURNAL OF NEUROSCIENCE;Sastre,M;23:9796 (2003)

22. 22 AChE Inhibitors Most FDA drugs for AD inhibit the enzyme AcetylCholineEsterase (AChE) Most FDA drugs for AD inhibit the enzyme AcetylCholineEsterase (AChE) Was assumed to work by prolonging the existence of acetylcholine in synapses Was assumed to work by prolonging the existence of acetylcholine in synapses AChE promotes Aß aggregation, which inhibition may block AChE promotes Aß aggregation, which inhibition may block AChE inhibitors increase proportion of APP cleaved by alpha-secretase rather than beta-secretase AChE inhibitors increase proportion of APP cleaved by alpha-secretase rather than beta-secretase AChE inhibitors cause nausea, vomiting and diarrhea AChE inhibitors cause nausea, vomiting and diarrhea AChE inhibitors cause modest and transient slowing of progress of AD AChE inhibitors cause modest and transient slowing of progress of AD

23. 23Memantine Inflammation & oxidative stress can cause excessive glutamate release (excitotoxicity) Inflammation & oxidative stress can cause excessive glutamate release (excitotoxicity) Memantine protects against excitotoxicity without interfering with glutamate signalling Memantine protects against excitotoxicity without interfering with glutamate signalling Like AChE inhibitors, benefit of memantine therapy for AD is, although statistically significant, nonetheless marginal Like AChE inhibitors, benefit of memantine therapy for AD is, although statistically significant, nonetheless marginal

24. 24 Nicotine and smoking Nicotine administered to transgenic mice reduced Aß 42 plaques Nicotine administered to transgenic mice reduced Aß 42 plaques JOURNAL OF NEUROCHEMISTRY;81:655 (2002) JOURNAL OF NEUROCHEMISTRY;81:655 (2002) Nicotine administered to the rat hippocampus enhanced acetylcholine production and release Nicotine administered to the rat hippocampus enhanced acetylcholine production and release BIOLOGICAL PSYCHIATRY;49:200 (2001) BIOLOGICAL PSYCHIATRY;49:200 (2001) Transgenic mice show worsening of NFTs with nicotine Transgenic mice show worsening of NFTs with nicotine PNAS;102:3046 (2005) PNAS;102:3046 (2005) Many studies show more twice the risk of AD among smokers Many studies show more twice the risk of AD among smokers THE LANCET; 351:1840 (1998) THE LANCET; 351:1840 (1998) NEUROBIOLOGY OF AGING; 24:589 (2003) NEUROBIOLOGY OF AGING; 24:589 (2003) AMERICAN JOURNAL OF EPIDEMIOLOGY;166:367(2007) AMERICAN JOURNAL OF EPIDEMIOLOGY;166:367(2007) Tobacco smoke contains more than just nicotine Tobacco smoke contains more than just nicotine

25. 25 Insulin and Insulin sensitivity Diabetics have greater risk for AD Diabetics have greater risk for AD Insulin increases Aß degradation Insulin increases Aß degradation Chromium picolinate (which increases insulin sensitivity) improved memory performance in patients with mild AD Chromium picolinate (which increases insulin sensitivity) improved memory performance in patients with mild AD NUTRITIONAL NEUROSCIENCE;13:116 (2010) NUTRITIONAL NEUROSCIENCE;13:116 (2010) Similar benefits were seen with the insulin-sensitizing drug metformin Similar benefits were seen with the insulin-sensitizing drug metformin PNAS;Chen,Y;106:3907 (2009) PNAS;Chen,Y;106:3907 (2009)

26. 26 Ginko biloba A large randomized trial showed ginko biloba improved cognition in AD patients A large randomized trial showed ginko biloba improved cognition in AD patients JOURNAL OF NEUROLOGICAL SCIENCES;283:224 (2009) JOURNAL OF NEUROLOGICAL SCIENCES;283:224 (2009) A review of a large number of studies also showed ginko biloba improved cognition in AD patients A review of a large number of studies also showed ginko biloba improved cognition in AD patients BMC GERIATRICS;10:14 (2010) BMC GERIATRICS;10:14 (2010) A large 6-year randomized trial showed ginko biloba is not effective in preventing AD A large 6-year randomized trial showed ginko biloba is not effective in preventing AD JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION;300:2253 (2008) JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION;300:2253 (2008) Does ginko biloba treat, but not prevent AD ? Does ginko biloba treat, but not prevent AD ?

27. 27 Transgenic AD-prone rodents Transgenic rats with gene delivery of Brain-Derived Neurotrophic Factor (BDNF) after disease onset reversed synapse loss and restored learning and memory Transgenic rats with gene delivery of Brain-Derived Neurotrophic Factor (BDNF) after disease onset reversed synapse loss and restored learning and memory NATURE MEDICINE;15:331 (2009) NATURE MEDICINE;15:331 (2009) Glycogen-Synthase Kinase 3 (GSK-3), an enzyme that phosphorylates tau protein, when inhibited by lithium in transgenic mice, substantially reduced NFT formation Glycogen-Synthase Kinase 3 (GSK-3), an enzyme that phosphorylates tau protein, when inhibited by lithium in transgenic mice, substantially reduced NFT formation PNAS;102:6990 (2005) PNAS;102:6990 (2005) Transgenic mice given pomegranate juice from 6 to 12.5 months of age showed 50% less accumulation of soluble Aß and amyloid deposits in the hippocampus associated with improved learning and memory. Transgenic mice given pomegranate juice from 6 to 12.5 months of age showed 50% less accumulation of soluble Aß and amyloid deposits in the hippocampus associated with improved learning and memory. NEUROBIOLOGY OF DISEASE; 24:506 (2006) NEUROBIOLOGY OF DISEASE; 24:506 (2006)

28. 28 Cytokine antagonist Etanercept, an antagonist of the inflammatory cytokine Tumor Necrosis Factor alpha (TNF−α) has shown effectiveness in reducing symptoms in an AD patient within hours of administration Etanercept, an antagonist of the inflammatory cytokine Tumor Necrosis Factor alpha (TNF−α) has shown effectiveness in reducing symptoms in an AD patient within hours of administration JOURNAL OF NEUROINFLAMMATION;5:2 (2008) JOURNAL OF NEUROINFLAMMATION;5:2 (2008)

29. 29Curcumin Curcumin is a phytochemical which gives curry a yellow color Curcumin is a phytochemical which gives curry a yellow color Incidence of AD is substantially lower in India Incidence of AD is substantially lower in India Curcumin is an antioxidant that can scavenge peroxynitrite Curcumin is an antioxidant that can scavenge peroxynitrite Curcumin not only inhibits Aß aggregation, it disaggregates existing plaques Curcumin not only inhibits Aß aggregation, it disaggregates existing plaques Curcumin can lower insulin sensitivity Curcumin can lower insulin sensitivity Curcumin can lower the inflammatory cytokine InterLeukin-1ß (IL-1ß) nearly 60% in transgenic mice Curcumin can lower the inflammatory cytokine InterLeukin-1ß (IL-1ß) nearly 60% in transgenic mice

30. 30 Low folate and elevated homocysteine Low serum folic acid and elevated plasma homocysteine are both independent risk factors for AD Low serum folic acid and elevated plasma homocysteine are both independent risk factors for AD AMERICAN JOURNAL OF CLINICAL NUTRITION;82:636 (2005) AMERICAN JOURNAL OF CLINICAL NUTRITION;82:636 (2005) Folic acid deficiency and excess homocysteine impair DNA repair, sensitizing neurons to cell death from DNA damage Folic acid deficiency and excess homocysteine impair DNA repair, sensitizing neurons to cell death from DNA damage JOURNAL OF NEUROSCIENCE;22:1752 (2002) JOURNAL OF NEUROSCIENCE;22:1752 (2002) Folic acid deficiency and excess homocysteine increase Aß production by inducing beta-secretase – an effect that can be overcome by administering S-Adenosyl Methionine (SAMe) Folic acid deficiency and excess homocysteine increase Aß production by inducing beta-secretase – an effect that can be overcome by administering S-Adenosyl Methionine (SAMe) MOLECULAR AND CELLULAR NEUROSCIENCE;28:195 (2005) MOLECULAR AND CELLULAR NEUROSCIENCE;28:195 (2005)

31. 31Exercise A mouse model of AD showed reduced extracellular Aß with voluntary exercise A mouse model of AD showed reduced extracellular Aß with voluntary exercise JOURNAL OF NEUROSCIENCE;25:4217 (2005) JOURNAL OF NEUROSCIENCE;25:4217 (2005) A 14-year study showed a 29-50% reduction in AD with physical activity A 14-year study showed a 29-50% reduction in AD with physical activity JAMA; 302:627 (2009) JAMA; 302:627 (2009) A randomized study of over 4,000 men and women over age 65 showed reduced AD with physical activity A randomized study of over 4,000 men and women over age 65 showed reduced AD with physical activity ARCHIVES OF NEUROLOGY;58:498 (2001) ARCHIVES OF NEUROLOGY;58:498 (2001)

32. 32Stress Transgenic AD-prone mice show increased Aß accumulation, which is apparently corticosteroid- related Transgenic AD-prone mice show increased Aß accumulation, which is apparently corticosteroid- related AMERICAN JOURNAL OF CLINICAL NUTRITION;82:636 (2005)i AMERICAN JOURNAL OF CLINICAL NUTRITION;82:636 (2005)i Glucocorticoid cascade hypothesis Glucocorticoid cascade hypothesis Cortisol (stress hormone) rises with age Cortisol (stress hormone) rises with age Damages hippocampus further increasing cortisol Damages hippocampus further increasing cortisol Destructive feedback cycle Destructive feedback cycle Pacific salmon use corticosteroids to self-destruct Pacific salmon use corticosteroids to self-destruct

33. 33 Environmental enrichment A study of transgenic AD-prone mice showed that environmental enrichment worsened amyloid plaque formation A study of transgenic AD-prone mice showed that environmental enrichment worsened amyloid plaque formation JOURNAL OF NEUROPATHOLOGY AND EXPERIMENTAL NEUROLOGY; 62:1220 (2003) JOURNAL OF NEUROPATHOLOGY AND EXPERIMENTAL NEUROLOGY; 62:1220 (2003) A subsequent study claimed that the former study had subjected the mice to excessive stress, and showed reduced Aß associated with environmental enrichment A subsequent study claimed that the former study had subjected the mice to excessive stress, and showed reduced Aß associated with environmental enrichment CELL; 120:701 (2005) CELL; 120:701 (2005)

34. 34 Discussion Tell your partner what you understood and think while your partner silently listens – talk for a couple of minutes Tell your partner what you understood and think while your partner silently listens – talk for a couple of minutes Switch roles – partner that listened becomes the speaker for a couple of minutes Switch roles – partner that listened becomes the speaker for a couple of minutes Short discussion Short discussion

35. 35 Empirical versus Mechanistic Empirical treatment and prevention Empirical treatment and prevention What appears to work What appears to work Mechanistic treatment and prevention Mechanistic treatment and prevention Understand the mechanisms Understand the mechanisms Devise strategies based on mechanisms Devise strategies based on mechanisms Evaluate what may work or not to understand the mechanisms and improve strategies. Evaluate what may work or not to understand the mechanisms and improve strategies.

36. 36 Amyloid and NFTs in AD