ALZHEIMER’S DISEASE Treatment and Prevention

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

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

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

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

5. Amyloid cascade begins • APP (Amyloid Precursor Protein) • Cleaved by secretase enzymes • Cleavage of APP forms shorter peptides • Cleavage by alpha or beta secretase • Only cleavage by beta secretase forms Aß • Two forms of Aß • After cleavage by gamma-secretase • 40 amino acid amyloid beta peptide (Aß40) • OR • 42 amino acid amyloid beta peptide (Aß42)STICKY! ⇒ amyloid plaques 

6. Amyloid cascade begins

7. Damaging effects of Aß42 • Activation of microglia • Brain version of macrophages • Produce inflammatory cytokines • InterLeukin-1ß (IL-1ß) • Tumor Necrosis Factor alpha (TNF−α) • Generate peroxynitrite and oxidative stress • Hyperphosphorylation of tau • Tau protein stablizes cell microtubules • Phosphorylated tau forms NFTs • NeuroFibrillary Tangles

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

9. Damaging forms of Aß42 • Aß42 is a 42-amino acid monomer • Aß42 monomers can aggregate into • Oligomers (many monomers) • Fibrils which can aggregate into plaques • Soluble oligomers are the most toxic form of Aß42 • “Halos” of Aß42 oligomers around plaques damage or destroy synapses • PNAS;Koffie,RM;106:4012 (2009) • JOURNAL OF NEUROSCIENCE; Lacor,PN;27:796(2007)

10. Amyloid aggregation/disaggregation

11. Amyloid Cascade Hypothesis APP ⇒ Aß42  (peptide, monomer) ⇒ fibrillar Aß or oligomers  ⇒ amyloid plaques  ⇒ inflammation/NFTs  ⇒ neuron death / synapse loss

12. Amyloid Cascade Evidence • Genetic pre-disposition to AD • Onset at an early age • Most often increases gamma-secretase cleavage • Produces increased Aß42 • Down’s Syndrome victims produce more APP • 3 chromosomes with gene for APP • Cleaved to Aß42 • Early AD

13. Discussion • Choose a partner • 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 • Short discussion

14. Amyloid and NFTs in AD

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

16. APOE and AD • APOE exists in 3 alleles • APOE2, APOE3 and APOE4 • APOE4 is the only allele in non-human primates • APOE4 is less efficient than APOE3 in transporting cholesterol and clearing the brain of Aß • APOE3 is less efficient than APOE2 • 14% of Caucasians have one APOE4 allele, but 40% of AD victims have at least one APOE4 allele • Aß42 rises markedly after head injury • 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 Aß42 clearance after injury

17. Metal toxicity and AD • Role of aluminum is controversial • Zinc, copper and iron cause Aß aggregation • Copper particularly mediates Aß toxicity • Aß is not toxic in the absence of Cu2+ • Zinc inhibits Aß toxicity • Copper enhances Aß fibril formation, an effect potentiated by APOE4 • 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

18. Clioquinol therapy for AD • Clioquinol binds zinc & copper (chelator) • Crosses the blood-brain barrier (BBB) • Transgenic AD-prone mice treated with clioquinol showed a 49% decrease in Aß • AD patients treated with clioquinol for 12 weeks • Severely demented showed slowed cognitive decline • Moderately demented showed no effect • 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 • No adverse effects seen • Currently in clinical trials

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

20. Discussion • 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 • Short discussion

21. 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. • NEUROBIOLOGY OF AGING 22:799 (2001) • Older NSAIDs block both COX-1 and COX-2 enzymes • Newer NSAIDs block COX-2 (pain) • COX-1 inhibition can cause GI bleeding • Microglia express COX-1 • NSAIDs (ibuprofen & indomethicin) inhibit cytokine activation of beta-secretase (reduce Aß formation) • JOURNAL OF NEUROSCIENCE;Sastre,M;23:9796 (2003)

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

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

24. Nicotine and smoking • Nicotine administered to transgenic mice reduced Aß42 plaques • JOURNAL OF NEUROCHEMISTRY;81:655 (2002) • Nicotine administered to the rat hippocampus enhanced acetylcholine production and release • BIOLOGICAL PSYCHIATRY;49:200 (2001) • Transgenic mice show worsening of NFTs with nicotine • PNAS;102:3046 (2005) • Many studies show more twice the risk of AD among smokers • THE LANCET; 351:1840 (1998) • NEUROBIOLOGY OF AGING; 24:589 (2003) • AMERICAN JOURNAL OF EPIDEMIOLOGY;166:367(2007) • Tobacco smoke contains more than just nicotine

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

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

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 • 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 • 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. • NEUROBIOLOGY OF DISEASE; 24:506 (2006)

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 • JOURNAL OF NEUROINFLAMMATION;5:2 (2008)

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

30. Low folate and elevated homocysteine • Low serum folic acid and elevated plasma homocysteine are both independent risk factors for AD • 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 • 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) • MOLECULAR AND CELLULAR NEUROSCIENCE;28:195 (2005)

31. Exercise • A mouse model of AD showed reduced extracellular Aß with voluntary exercise • JOURNAL OF NEUROSCIENCE;25:4217 (2005) • A 14-year study showed a 29-50% reduction in AD with physical activity • JAMA; 302:627 (2009) • 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)

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

33. Environmental enrichment • A study of transgenic AD-prone mice showed that environmental enrichment worsened amyloid plaque formation • 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 • CELL; 120:701 (2005)

34. Discussion • 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 • Short discussion

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

36. Amyloid and NFTs in AD