Pathogenesis of Alzheimer’s Disease-Approached by Multiphoton Microscopy 한림의대 병리학교실 최 경찬

Contents Brief introduction Patholgic findings Pathogenic mechanism Molecular genetics Experiments and review Immunotheraphy Clinical trials

퇴행성 질환의 병리 회백질 침범 신경세포의 지속적 손실 신경세포군의 선택적 침범 증상이 서서히 발현 대부분 병인 불명

대뇌피질 침범 Alzheimer’s disease Pick’s disease Cortex & basal ganglia degeneration

Alzheimer’s disease

Alzheimer’s disease: 치매, 운동기능은 비교적 정상, 고위 대뇌 기능 장애, 65 세 이상 약 6%( 국내 ) 여자 > 남자, 유전적 요인 (55%) 아밀로이드 축적 -- 유전자 21 번 염색체 아포리포단백 -- 유전자 19 번 염색체

Estimated number of dementia patients in Korea

Pathologic Findings

현미경 소견 1) Senile plaque: amyloid 2) Neurofibrillary tangle: fibrin-- tau protein 3) Neurovacuolar degeneration 4) Hirano body 5) Neuronal loss: frontal, temporal 6) Amyloid vasculopathy: amyloid, media & adventitia, not involve brain stem Amyloid angiopathy

Cortical atrophy

Senile(neuritic) plaque Aβ-APP Microglial cells Reactive astrocytes

Neurofibrillary tangles Tau protein

Amyloid angiopathy Immunostain for anti- Aβ Ab

Pathogenic mechanism

Proposed pathogenic mechanisms for AD 1. Amyloid cascade hypothesis 2. Oxidative stress 3. Chronic inflammation 4. Perturbed lipid metabolism 5. Impaired cellular ion homeostais 6. Cerebrovascular alteration

Proposed pathogenic mechanism by Aβ

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

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

Molecular genetics

Genetics of Alzheimer Disease

β β AβPP mutations and their relationship to Aβ

Experiments and review

Amyloid Peptides Causing CAA in Humans

Sporadic CAA and AD Overlapping biology share risk factors - CAA, 46% in elderly individuals>70Ys - With AD increase frequency & severity of CAA pathology and vascular amyloid deposition >80% of all AD cases (Ellis et al, Neurology 1996)

Amyloid Deposition in Leptomeningeal Vessels: Thioflavin S Stain Nature of vessels Number counted Proportion of vessels positive for amyloid Total arteries % Total veins3524.4% Arteries <60 µ m in diameter % Arteries >60 µ m in diameter % Veins <60 µ m in diameter % Veins >60 µ m in diameter % A total of 56 blocks from seven AD brains

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

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

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

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

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

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

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

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 -*-* ± ± ± ± 0.34

Congo red 6E10

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) P<0.001

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

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

Conclusio n 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

Multi-photon confocal microscopy

Thio S Tg2576

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

Immunotheraphy

Promising treatment for AD 1.Immunotherapy 2.Amyloid formation inhibitor 3.Enzyme modulators 4.Metal-ion attenuating compounds 5. Cholesterol lowering agents

Immunotherapy & succesive animal studies First Immunotherapy using active vaccination

Preventive effect of vaccination Preventive plaque formation in hippocampus Prevention of astrocytosis

Possible treatment effect by vaccination Reduction of cortical Aβ deposition

Background

AD Transgenic Mice PDAPP mouse: Games D et al., Nature,1995 -Platelet-derived growth factor- β promoter,V717F Tg2576(APPswe)mouse:Hsiao K, Science, K670N + M671L PS-1 mouse:Duff K, Nature, 1996 Double transgenic(APPswe + PS-1) - Borchelt DR, Neuron, 1997 TgCRND8, Tg23 - V717F & K670N + M671L

Mechanism for Aβ Vaccination 1.Anti-aggregation or disaggregation by antibodies 2.Microglial mediated phagocytosis 3.Clearance of Aβ from the brain to blood

Preclinical safety and toxicology tests Animals: Mice, rats, rabbits, guinea pigs and monkeys( ) No encephalitis

Summary of vaccine studies in transgenic mouse models of AD Pathology is defined as amyloid plaque load. NC, no change

Vaccination on non-human in primate

Clinical Trial and Outcome

Clinical Trial Phase I - 20 early-moderate AD:single dose( ) - 64 early-moderate AD:multiple doses( ) - Saponin(QS-21) used as adjuvants - No encephalitis - antibodies in some patients(25%)

Phase II(Jun Jan. 2002) early-moderate AD: multiple dosese 300 with AN 1792 and 75 with placebo Clinical Trial

Withdrawl of clinical trial Development of meningoencephalitis in 18 patients Halt in Jan.2002 and withdrawl in Mar Four autopsy cases after withdrawl

Development of meningoencephalitis

Change of brain volume 3% reduction of brain volume Removal of plaques could be the reason Recovery of some lost brain volume in the second year

Possible alternatives Passive immunization Conjugation and/or modification of Aβ protein Different route of administration Appropriate adjuvants or immunomodulators

Possible problems in passive immunization Risk of cerebral hemorrhage Formation of antibodies to injected antibodies Difficulties in passage of blood brain barrier