1. Pathogenesis of Alzheimer’s disease approached by multi- photon microscope Pathogenesis of Alzheimer’s disease approached by multi- photon microscope Dept. of Pathology Kyung-Chan Choi

2. Alzheimer’s disease

7. CAA? Deposition of amyloid β(Aβ) in cortical Deposition of amyloid β(Aβ) in cortical and leptomeningeal vessel walls and leptomeningeal vessel walls

8. Amyloid Amyloid-end product of a protein misfolding disorder Amyloid-end product of a protein misfolding disorder Soluble native protein-protofibrillary intermediates-8~10 nm fibrils Soluble native protein-protofibrillary intermediates-8~10 nm fibrils

9. Conversion process Post-translational-modification Of protein Mutations Protein concentration pH and tissue factors Metal Ions Amyloid-associated proteins or chaperons Soluble native protein Prefibrillar intermediate Amyloid fibrils

10. Amyloid-associated proteins Serum amyloid-p component Serum amyloid-p component Apolipoprotein E(apoE) Apolipoprotein E(apoE) Apolipoprotein J(apoJ) Apolipoprotein J(apoJ) Vitronectin Vitronectin A1-antichymotrypsin A1-antichymotrypsin Complement proteins Complement proteins Glycosaminoglycans Glycosaminoglycans Extracellular matrix proteins Extracellular matrix proteins

11. Amyloid Peptides Causing CAA in Humans

12. Pathological process during amyloid protein progressively deposits in blood vessel walls with subsequent degenerative vascular changes Pathological process during amyloid protein progressively deposits in blood vessel walls with subsequent degenerative vascular changes By Divry, amyloid deposition in senile plaques in Alzheimer’s disease(AD) By Divry, amyloid deposition in senile plaques in Alzheimer’s disease(AD) By Scholz, amyloid in cerebral vessel By Scholz, amyloid in cerebral vessel in brains of elderly patients in brains of elderly patients

13. Congo red Morphology in LM

14. Worster-Drought et al(1940), CAA in familial British dementia(FBD)- hyaline degeneration Worster-Drought et al(1940), CAA in familial British dementia(FBD)- hyaline degeneration In 1970s, often cause of cerebral hemorrhge in elderly, nonhypertensive individuals In 1970s, often cause of cerebral hemorrhge in elderly, nonhypertensive individuals

15. Multi-step process Basement membrane around sm cells Basement membrane around sm cells in abluminal area of media and adventitia– progressive loss of sm cells & vascular degenerative changes, preservation of endothelial cells in abluminal area of media and adventitia– progressive loss of sm cells & vascular degenerative changes, preservation of endothelial cells

16. Grading system 1991, Vonsattel et al 1991, Vonsattel et al “mild”: in media without significant destruction of sm cells “mild”: in media without significant destruction of sm cells “moderate” sm cells mainly absent “moderate” sm cells mainly absent “severe” loss of sm cells and degenerative changes(disruption of architect of affected vs, double barrel microaneurysm form, fibrinoid nec & leakage of blood) “severe” loss of sm cells and degenerative changes(disruption of architect of affected vs, double barrel microaneurysm form, fibrinoid nec & leakage of blood)

17. EM 8-10 nm amyloid fibrils first laid down in abluminal aspect of basal lamina around sm cells- spread towards internal elastic lamina of arteries and endothelium of arteries-sm cells degenerated 8-10 nm amyloid fibrils first laid down in abluminal aspect of basal lamina around sm cells- spread towards internal elastic lamina of arteries and endothelium of arteries-sm cells degenerated

18. Deposition of ABri amyloid fibrils Scale bar:700 nm

19. Sporadic CAA In elderly individuals with or without AD In elderly individuals with or without AD Be associated with cases of lobar cerebral hemorrhage-12~15% of all cerbral hemorrhage in the elderly; either apoE e2 or e4 allele (Revesz et al, Brain Pathol 2002) Be associated with cases of lobar cerebral hemorrhage-12~15% of all cerbral hemorrhage in the elderly; either apoE e2 or e4 allele (Revesz et al, Brain Pathol 2002)

20. Major clinicopathological manifestations Major clinicopathological manifestations - Cerbral hemorrhage - Cerbral hemorrhage - Ischemic lesion - Ischemic lesion - Dementia - Dementia

21. From transgenic models of AD From transgenic models of AD - physiology of cbr microvasculature - physiology of cbr microvasculature alterations in vessel tone & rx alterations in vessel tone & rx - inflammatory components promoting altered vessel function - inflammatory components promoting altered vessel function (In pathogenesis of CAA-related ischemia) (In pathogenesis of CAA-related ischemia)

22. Sporadic CAA and AD Overlapping biology share risk factors Overlapping biology share risk factors - incidence increase with age; - incidence increase with age; CAA, 46% in elderly individuals>70Ys CAA, 46% in elderly individuals>70Ys - With AD increase frequency & severity of CAA pathology and vascular amyloid deposition>80% of all AD cases(25% is mod to severe) (Ellis et al, Neurology 1996) - With AD increase frequency & severity of CAA pathology and vascular amyloid deposition>80% of all AD cases(25% is mod to severe) (Ellis et al, Neurology 1996)

23. Cerebral hmr related to severe CAA Cerebral hmr related to severe CAA 1) initial seeding of Aβ 42 1) initial seeding of Aβ 42 2) expansion of vascular amyloid deposits through Aβ40 to vessel wall enhanced by apoE ε4 2) expansion of vascular amyloid deposits through Aβ40 to vessel wall enhanced by apoE ε4 3) rupture of vessel wall with hmr, 3) rupture of vessel wall with hmr, apoE ε2 is a risk factor apoE ε2 is a risk factor (Alonzo et al, J Neuropathol Exp Neurol 1998) (Alonzo et al, J Neuropathol Exp Neurol 1998)

25. ββ A β PP mutations and their relationship to A β

26. Amyloid Deposition in Leptomeningeal Vessels: Thioflavin S Stain Nature of vessels Number counted Proportion of vessels positive for amyloid Total arteries 68120.3% Total veins 3524.4% Arteries <60 µ m in diameter 45824.7% Arteries >60 µ m in diameter 22311.2% Veins <60 µ m in diameter 1934.5% Veins >60 µ m in diameter 1594.2% A total of 56 blocks from seven AD brains

27. Why CAA rarely involves larger intra Why CAA rarely involves larger intra -cranial arteries -cranial arteries Why Aβ dose not accumulate in walls Why Aβ dose not accumulate in walls of extracranial vessels of extracranial vessels

28. Origin of Aβ? Mechanisim unknown Mechanisim unknown - CAA in vessel wall - CAA in vessel wall AD in senile plaques AD in senile plaques Aβ in vessel wall Aβ in vessel wall - derived from vascular smooth - derived from vascular smooth muscle cells (Wisniewski et al, NP App muscle cells (Wisniewski et al, NP App Neurobiol 1996; Vinters et al, Brain Pathol 1996) Neurobiol 1996; Vinters et al, Brain Pathol 1996)

29. Source of vascular amyloid Smooth muscle cells contain Smooth muscle cells contain β-amyloid precursor protein(APP) β-amyloid precursor protein(APP) fragments fragments ? Extracranial vessels ? Extracranial vessels ? Early deposition: not in tunica ? Early deposition: not in tunica media media ? Larger arteries ? Larger arteries

30. (Roy et al, Am J Pathol 1998) CAA of cortical vessels in AD 10% SDS Thioflavin S

31. CAA of cortical vessels in AD

32. CAA of leptomeningeal Arteries in AD Confocal microscopy Phase-contrast microscopy

33. Smooth muscle cells as source of Aβ Internalization of Aβ- ApoE(transport molecule)? Internalization of Aβ- ApoE(transport molecule)?

34. Water soluble Aβ 2.75 ng/g of cerebral cortex in control 2.75 ng/g of cerebral cortex in control 36.8 ng/g in AD 36.8 ng/g in AD - water soluble pool of non-AD brain - water soluble pool of non-AD brain tissue without visible deposits of Aβ tissue without visible deposits of Aβ (Kuo Y-M et al, J Biol Chem 1996) (Kuo Y-M et al, J Biol Chem 1996)

35. Periarterial pathways along which Periarterial pathways along which interstitial fluid drains from cerebral interstitial fluid drains from cerebral cortex cortex ISF from brain drains to regional ISF from brain drains to regional cervical LN cervical LN - injection of tracers into rat brain - injection of tracers into rat brain (Cserr et al, Brain Pathol, 1992) (Cserr et al, Brain Pathol, 1992)

37. Under normal circumstances, brain eliminates Aβ peptides via periarterial ISF drainage pathways Under normal circumstances, brain eliminates Aβ peptides via periarterial ISF drainage pathways Aβ1-42 deposited in vessel walls Aβ1-42 deposited in vessel walls 1) obstruction of ISF drainage 1) obstruction of ISF drainage 2) failure of solubility factor(SF) 2) failure of solubility factor(SF) - compensating for ApoE or other - compensating for ApoE or other solubility defects solubility defects

38. Insoluble Aβ accumulates selectively aound small arteries in brain and Insoluble Aβ accumulates selectively aound small arteries in brain and leptomeninges leptomeninges Earliest deposits of Aβ in leptomen. Earliest deposits of Aβ in leptomen. arteries- ISF drainage pathways in adventitia, later in tunica media arteries- ISF drainage pathways in adventitia, later in tunica media

39. Degree of Amyloid Angiopathy in Leptomeningeal Vessels Compared with Density of Fibrillar Amyloid Plaques in the Underlying Cortex: Thioflavin S. *No amyloid angiopathy. The mean density of plaques in the cortex underlying vessels with grade +++ amyloid angiopathy is significantly higher than plaque density with no (-), +, and ++ grades of amyloid angiopathy ( P = 0.05). Thioflavin S-stained plaques were up to 100 times less numerous than those identified by A ß immunocytochemistry. Severity of amyloid angiopathy Plaques per mm 2 cortex ±SD Plaques per mm 2 cortex ±SD -*-*-*-*0.00 + 0.23 ± 0.27 ++ 0.7 ± 0.4 +++ 1.04 ± 0.5 + 1.04 ± 0.5 + ++++ 0.33 ± 0.34

40. Cerebral cortex as a source of amyloid Aβ in senile plaques, 1-42 species Aβ in senile plaques, 1-42 species vascular amyloid, 1-40 species vascular amyloid, 1-40 species Aβ 1-42, initial amyloid deposited in Aβ 1-42, initial amyloid deposited in vessels walls; entraps more soluble vessels walls; entraps more soluble Aβ1-40 Aβ1-40

41. Systemic Hypothesis (Zlokovic BV, Adv Drg Deliv Rev 2002) Systemic Hypothesis (Zlokovic BV, Adv Drg Deliv Rev 2002) Vascular Hypothesis (Burgermeister et al, Ann NY Acad Sci 2000) Vascular Hypothesis (Burgermeister et al, Ann NY Acad Sci 2000) Drainage Hypothesis (Weller et al, AmJ Pathol 1998) Drainage Hypothesis (Weller et al, AmJ Pathol 1998)

42. CAA-Related Inflammation 42 CAA patients 42 CAA patients 7/42 with inflammation & giant cell rx 7/42 with inflammation & giant cell rx 35/42 without inflammation 35/42 without inflammation

43. Congo red 6E10

44. Comparison of Clinical and Genetic Characteristics of Patients with Severe CAA with or without Perivascular Inflammatory Changes CharacteristicCAA with Inflammation (n = 7)CAA without Inflammation (n = 35) Age at presentation (yr ± SD)68.3 ± 9.6 a 75.8 ± 8.3 Sex (M/F)3/410/25 Primary clinical presentation, n (%) Intracerebral hemorrhage0 (0) b 33 (94) Cognitive decline3 (43)1 (3) Seizure4 (57)1 (3) APOE genotype, n (%) b n = 7 patients genotypedn = 26 patients genotyped 4/ 45 (71) b 1 (4) 4/ (2 or 3)1 (14)12 (46) (2 or 3)/ (2 or 3)1 (14)13 (50) a p = 0.04. b p < 0.001

45. Patient No./ Age/SexClinical Symptoms at Presentation Immunosuppresive Treatment Follow-up MRI: White Matter HyperintensitiesClinical Course White Matter Hyperintensity Microhemo -rrhges WBC (/ml) Protein (mg/dl) 1/49/F1 mo cognitive declinePatchyNoneNACS × 3 daysDecreasedImproved, not to baseline Independent 2/63/M3 mo cognitive declinePatchyMultiple25 a 211 a CS × 5 daysIncreasedMinimal improvement CP × 10 daysDependent 3/69/F3 weeks gait difficulty, few months cognitive decline PatchyMultiple055CS × 6 daysNADied 2.7 years after presentation 4/71/F1 yr cognitive decline, sudden onset confusionConfluentMultiple1164 a CS × 16 daysDecreasedImproved, not to baseline Recurrent seizure Independent 5/71/F2 seizures over 1 moConfluentMultipleNACS × 1 yrDecreasedImproved, not to baseline CP × 1 yr± independent 6/73/M3 seizures over 2 months, confusion, and personality changes over few months ConfluentMultiple150 a CS × 1 moDecreasedImproved, not to baseline Recurrent seizures Died 3.2 yr after presentation 7/79/M4 months cognitive decline, seizureConfluentMultiple284*CP × 6 weeksNAImproved, not to baseline, independent MRI at presentationCSF Clinical, Laboratory, and Radiographic Characteristics of Patients with CAA-Related Perivascular Inflammation

47. T lymphocytes(CD3) in perivascular infiltrate T lymphocytes(CD3) in perivascular infiltrate including low numbers of both CD8(+) and including low numbers of both CD8(+) and CD4(+) lymphocytes; present CD4(+) lymphocytes; present B lymphocytes(CD20); absent B lymphocytes(CD20); absent CD3

48. Conclusion This series of patients suggests that the inflammatory response to CAA can cause vascular dysfunction and cognitive impairment. The apparent response of some patients to immunosuppressive therapy points to the importance of identifying this potentially treatable form of CAA during life. Inflammation to vascular amyloid might also have a role in the meningoencephalitis reported in some AD patients treated with the experimental A  vaccine

49. Triple stain 6E10(Green) + KP1(Blue) + DAPI

50. Thio S Multi-photon confocal microscopy Tg2576

51. Factor VIII Thio S

52. Alzheimer’s Research Unit at MGH G.William Rebeck Ph.D Georgetown University Steven M. Greenberg M.D., Ph.D MGH, Harvard Brian J. Bacskai Ph.D MGH, Harvard Bradley T. Hyman M.D., Ph.D MGH, Harvard Matthew P. Frosch M.D., Ph.D MGH, Harvard