1. AD is a PROGRESSIVE IRREVERSIBLE neurodegenerative disease, for which there is no effective treatment yet Epidemiology 1-AD is the most common form of dementia, accounting for >60% of all the cases. 2-Most of the cases of AD ARE SPORADIC. The prevalence of inherited forms of AD is <0.1%. 3-Risk factors for sporadic AD: -aging, head injuries, hormonal changes, vascular diseases, inflammation, ApoE e4 allele polymorphism, exposure to metals, like Cu ++ and Zn ++ Alzheimer’s disease Numbers about AD Increased life expectancy, more AD patients Worldwide: 2001: 24 million. Expected to double in 20 years 2040: 81 million U.S.: 2011: > 5 million AD patients, 10 million caregivers U.S. government spends ~ $200 billion/year Projection for U.S. only 2040: 11.5 million AD patients, 23 million caregivers; > 400 billion/year

2. http://scienceblogs.com/neurophilosophy/Alzheimer A nissl stain tissue section (left) and a Bielkhowsky’s stain section from Augustine Deter’s brain (right) First description of tangle and plaque pathology by Alois Alzheimer (1901)

3. Alzheimer’s disease AD is a progressive neurodegenerative disorder affecting the elderly population. Once it starts, it progresses with aging. It is characterized by the presence of lesions both at an extracellular level (the  -amyloid plaques), and at an intracellular levels (the Neurofibrillary tangles, NFT). The presence of the lesions correlates with both reduction in the volume of the brain (as a consequence of the neurodegenerative phenomena), and with cognitive decline associated to loss of memory. AD is a form of Dementia.

4. The loss of intellectual functions (such as thinking, remembering, and reasoning) of sufficient severity to interfere with a person’s daily functioning. Dementia is not a disease itself but rather a group of symptoms that may accompany certain diseases or conditions. Symptoms may also include changes in personality, mood, and behavior. Dementia is irreversible when caused by disease or injury but may be reversible when caused by drugs, alcohol, hormone or vitamin imbalances, or depression. Definition of Dementia

5. Increasing and persistent forgetfulness Difficulty performing familiar tasks Problems with finding the right words to express your thoughts Disorientation with time and place Poor or impaired judgment Problems with abstract thinking Putting everyday items in illogical places Mood, behavior or personality changes. Symptoms in AD

6. Comparison between AD signs and age-related memory changes

7. Normal AD MRI www.elements4health.com PET Anatomy of the Alzheimer’s disease brain

8. Alzheimer’s disease: characterized by extracellular depositions, the  -amyloid plaque, and intracellular depositions, the Neurofibrillary Tangles (NFT) comprised of Paired Helical Filaments (PHF), aggregates of hyperphosphorylated protein tau. Deposition of fibrillar proteinacious material in Alzheimer’s disease

9. Origin of NFTs and the mechanism of tau pathology 1- Tau is a microtubule-associated protein that regulates cytoskeleton structure. 2- Tau is hyperphosphorylated in AD. 3- When highly phosphorylated, tau is sequestered into aggregates (PHF and NFT) and causes disruption of microtubules, that ultimately leads to cell death. www.emdbiosciences.com

10. The  -Amyloid Plaque A   proteolysis APP TMD NH2 COOH amyloidogenic processing pathway Mechanisms of A  pathology: Plaque: unknown A  : oligomers affect synaptic activity and neurotransmitter release A  oligomers Origin of the  -amyloid plaque

11. The  -amyloid plaque 1-  -amyloid is DIFFERENT from amyloid.  -amyloid contains specifically the  - amyloid peptide, an approximately 4kDa peptide (A  40, A  42) deriving from the amyloid precursor protein APP when it undergoes the so called amyloidogenic pathway.  -amyloid plaque contains also ubiquitin and other proteins coming from degenerative neurons and glial cells. 2- The  -amyloid peptide is insoluble in water. When released from the precursor protein it assumes a  -sheet conformation that makes it hydrophobic. 3-  -amyloid peptides forms oligomers that may affect neurotransmitter release and synaptic plasticity. Oligomers will further aggregates in larger structures called fibrils, that will form the core of the  -amyloid plaque, depositing in the extracellular matrix. 4- The size of the plaque will increase following the progression of the disease. Scientific clear evidences are still missing in order to understand whether the plaque is a consequence or a cause of AD. Indeed, A  and  plaques formation are associated to neuronal death. Inherited forms of AD lead to substantial increase of A  production.

12. APP processing and formation of aggregates of beta-amyloid

13. s  APP AA  secretase  secretase C99 TMD APP  secretase  secretase Amyloidogenic processing of APP NH 2 COOH NH 2

14. Structure of the  -Amyloid peptide 25-35, the most critical region for the change of conformation from  -sheet to  -sheet

15. Genetic of AD -Depending on the kind of mutation and on the gene that carries it, the onset of the disease is SIGNIFICANTLY EARLIER than in sporadic cases (<before 65 years of age). All the mutations are autosomal dominant, and involve *APP protein *Members of the  -secretase complex, like Presenilin 1 (PS1) and Presenilin 2 (PS2)

16. Mutations on APP Swedish mutation: K670M/N671L, facilitates the amyloidogenic processing of APP by increasing the affinity for and the cleavage by  -secretase of ~100 times. Dutch Mutation: E693Q, induces cerebral amyloidosis Arctic Mutation:E693G enhances  -Amyloid protofibril formation London Mutation: V717I, Affects the cleavage of  -secretase K670M/N671L Swedish Mutation V717I London Mutation E693G Arctic Mutation

17. s  APP AA  secretase  secretase C99 TMD APP  secretase!!!  secretase!!! Amyloidogenic processing of APP NH 2 COOH NH 2 K670M/N671L V717I E693G

18. 46 y DS (Lott and Dierssen, 2010) http://www.hcplive.com/articles/Alzheimers -Promoter mutations that increase APP expression are associated with Alzheimer disease (Thenus et al., 2006). Trisomy of Chromosome 21, Down Syndrome The higher the levels of APP, the more the A  and AD pathology A  positive amyloid plaques Human AD brain

19. Mutations on Presenilins PS1 (on chromosome 14) PS2 (on chromosome 1) are ~450 aa long aspartyl proteases comprised of 7- to 8- transmembrane spanning domains. They are the catalytic part of a tetrameric protein complex called  -secretase, able to cleave APP at the end of the sequence for  -Amyloid peptides, generating  -Amyloid species. More than 70 mutations on PS1 gene account for inherited AD. Two mutations on PS2 gene account for inherited AD.

20. Hardy and Selkoe, 2002 PS1 mutations and APP

21. s  APP AA  secretase  secretase/mutantPS1 C99 TMD APP  secretase  secretase Amyloidogenic processing of APP NH 2 COOH NH 2

22. The  -Amyloid Plaque Alois Alzheimer: “miliary bodies (the plaques) and dense bundles of fibrils (NFT)”

23. Characteristic of the  -amyloid plaque -is extracellular -normally, it has a core composed of the  -amyloid peptide -it can be cored (with an intensely stained core with a weak periphery stain-halo) or diffused, which do not have a core and the immunoreactivity is uniform over the plaque. -aggregation is massive in the center, and diffused at the sides (disaggregation hypothesis?) -diameter ranges between 20  m and 90  m -is composed of dystrophic neurites,  -amyloid peptides (40/42/43), ubiquitin, tau protein and other proteins, some involved in the generation of  -amyloid, like the secretases.

24. Amyloid: definition Insoluble fibrous protein aggregations sharing specific structural traits: -Insolubility in water -  -sheet conformation -largest aggregates deposit extracellularly -positive to the staining with specific dyes, such as Congo Red -associated with tissue degeneration (cause or consequence?)  -Amyloid Are formations of amyloid characterized by a core specifically formed by  -amyloid peptides, a product of the so called amyloidgenic processing of a larger precursor, the protein APP (Amyloid Precursor Protein)

25. Congo red staining of amyloid plaques Under polarized lightUnder unpolarized light

26. How to specifically detect  -Amyloid plaques? Using antibodies that specifically recognize the different  -Amyloid peptides, A  1-40, A  1-42, A  1-43

27.  -Amyloid peptides 40/42 43

28.  -Amyloid plaques depositions follow the progression of AD

29. A  40, A  42 and A  43 are part of the  -amyloid plaque in AD

30. Levels of deposited A  40, A  42 and A  43 follow the progression of the disease

31. Diagnosis of AD At the moment, the exact diagnosis for AD can be performed only post-mortem by means of immunohistochemistry, verifying the presence of the  -amyloid plaques in the patient’s brain section using specific anti  -amyloid antibodies. However, a clinical diagnosis for AD is based on: Neuropsycologic evaluation: behavioral and cognitive tests (MMSE, CDR). Diagnosis of a possible or probable AD (“Dementia of AD type”). Imaging techniques: MRI to detect changes in the volume of the brain PET analysis to identify areas of the brain with reduced glucose utilization Progress has been done in the direction of using dyes/compounds that specifically recognize the  -amyloid structure in both MRI and PET analysis. This would improve the quality of the diagnosis, identifying the structures (  -amyloid plaques) that are specific for AD.

32. Normal Alzheimer’s diseases MRI Computer graphic MRI in Alzheimer’s disease (AD)

33. Cortical thickness decreases with age

34. Number of cortical neurons does not decrease with age

35. Cerebral volume loss is associated with aging, but not necessarily with neuronal death and/or with AD. Need for diagnostic tools more specific than MRI.

36. Characteristics of a biomarker for diagnostic purposes -It should be already changed in the initial steps of the disease: implications for an early diagnosis of the disease, with deep impact in the success of the treatment. -The method to access the biomarker must not be invasive -The molecule used as a biomarker must be specific for that disease, and not be involved in other diseases. The mechanisms behind the specific changes in the disease must be clear. -The method of analysis must be reproducible :-) and must not be expensive -Ideally, the levels of the biomarker, once identified, should follow the progression of the disease (linear progression).

37. Possible biomarkers in AD -Levels of  -amyloid peptides in the CSF of patients. Concerns regarding: The method used to take the sample of CSF (spinal injection, which is invasive). In the CSF of AD patients, the levels of the different species of  -amyloid peptides, in particular the most aggressive A  42, do not always follow the progression of the disease. In fact, levels of A  42 raise in the early stages of the diseases, but drop in the late, advanced stages of the disease, maybe indicating increased deposition of secreted A  42 into plaques in the brain. It is very difficult to correlate the amount of biomarker (in particular A  42) to the amount of deposited plaques in the different stages of the disease. Cerebrospinal levels of hyperphoshorylated tau. However, since tau is involved also in other neurodegenerative phenomena called taupaties, it is questionable how it could be considered as a biomarker specific for AD.

39. Beta-Amyloid peptides can be detected in the ISF

40. Amyloid plaques develop in J20APPTg mice without changes in the precursor APP

41. Levels of soluble ISF Abeta decrease with disease progressing…

42. …whereas levels of insoluble Abeta increase

43. Higher levels of exogenous, NOT newly synthesized Abeta are sequestered from the ISF in plaque-rich animals

44. Demonstration that the plaque attracts soluble Abeta Explanation why levels of Abeta 42 in the CSF DO NOT follow the progression of the disease

45. The Pittsburgh Compound B PIB is specifically up-taken in AD brains

46. PIB is differentially retained in brain areas more susceptible to AD

47. Control AD Topography of PIB retention in AD brain

48. In AD brains, PIB uptake correlates with the progression of the disease

49. ? Are plaques a CAUSE or a CONSEQUENCE of neurite dysfunction?

51. Appearance of a novel plaque is a fast process that takes 1 week Meyer-Luehamnn et al., Nature, 2008, 451:720

52. Formation of a plaque can occur within 24 hours

53. Meyer-Luehamnn et al., Nature, 2008, 451:720 Plaque formation induces neurites curvature

54. Does the cell try to get rid of it? The plaque can be potentially toxic

55. Meyer-Luehamnn et al., Nature, 2008, 451:720 Plaques recruit activated microglia: a possible role of microglia limiting the size of the plaque

56. The many ways the system tries to get rid of the Abeta peptides

57. Insulin Degrading Enzyme (IDE) is crucial for Abeta degradation

58. IDE levels are reduced in brain areas affected by AD

59. Decreased A  plaque burden in animals overexpressing Insulin Degrading Enzyme (IDE) and Neprilysin Leissring et al.,

60. Is there anything we can do to help the system get rid of the plaques? Y E S ! H E A L T H Y L I F E S T Y L E !!

61. Abeta plaques are reduced in APPTg mice in enriched environment

63. Environmental enrichment reduces the amyloid load

64. Activity of IDE and neprilysin are increased with environmental enrichment: Abeta clearance is favored

65. Exercise reduces the levels of markers of AD in CFS: Reduced levels of Abeta42…

66. …and tau, phosphorylated tau