Alzheimer’s Disease Bill Lian. Alzheimer's Disease The most common cause of dementia. 5% of all persons over the age of 70 have AD. 3 to 4 million patients.

Alzheimer’s Disease

Bill Lian

Alzheimer's Disease

The most common cause of dementia.

5% of all persons over the age of 70 have AD.

3 to 4 million patients with AD in the United States, with a total health care cost of more than $50 billion a year.

Clinical Feature

Early stage: memory loss, cognitive problems.

Later stage: hyperactive tendon reflexes, may become rigid and bedridden.

Death usually results from secondary infections, such as pneumonia, and malnutrition.

Typical course of AD is 8 to 10 years.

Etiology of AD

AgeThe most important risk factors for AD. The frequency of AD increases with each decade of adult life to reach 20 to 40 percent of the population over the age of 85.

GenderStudies have suggested that female gender may also be a risk factor independent of the greater longevity of women. Genetics10% of AD cases are known to have a genetic basis. -APP gene mutations on (chromosome 21). -Mutations in the presenilin-1 gene on (chromosome 14). -Mutations in the presenilin-2 gene on (chromosome 1). -Association with the ApoE4 allele on the (chromosome 19).

Others

Neuropathology

PlaquesPlaques are extracellular aggregation of beta amyloid peptide

Neurofibrillary Tangles They are intracellular accumulation of paired helical filaments (PHF). The major component of the PHF is abnormally phosphorylated tau protein.

Neuronal loss30-40% loss of neocortical neurons.

Synaptic alterationsDecrease of synapse-related protein marker, synaptophysin.

Molecular Mechanism of Alzheimer's Disease

Cholinergic and Other Neurochemical Changes

Molecular Genetics

NeurofibrillaryTangle

Oxidative Stress and Free Radicals

Others

Amyloidβ-protein

Processing of Amyloid Precursor Protein (APP)

Intracellular

NH2+ COO-

67

1

68

7

71

3

Extracellular

Aβ

Aβ COO-NH2+COO-NH2+

A β

α-se

cret

ase

path

way β/γ-secretase pathw

ay

Beta-amyloid Plaques

Amyloid precursor protein (APP) is the precursor to amyloid plaque.

1. APP sticks through the neuron membrane.

2. Enzymes cut the APP into fragments of protein, including beta-amyloid.

3. Beta-amyloid fragments come together in clumps to form plaques.

1.

2.

3.

In AD, many of these clumps form, disrupting the work of neurons. This affects the hippocampus and other areas of the cerebral cortex.

ROS.O2-

H2O2

.RO

.OH

.RO

ONOO-

.NOO2

.NO2.O2

Exogenous SourcesUV light

RadiationToxins

ChemotherapiesInflammatory

cytokines

Impaired Physiological FunctionRandom cellular damage

Specific Signaling Pathways

Normal Growth and Metabolism

The Sources and Cellular Responses to Reactive Oxygen Species (ROS)

Endogenous SourcesMitochondriaPeroxisomes

LipoxygenasesNADPH oxidase

Cytochrome P450

Antioxidant DefensesEnzymatic Systems

Cat Sod GPxNo-enzymatic systems

GlutathioneVitamins (A, C, & E)

Ageing Disease Cell Death

TauTubulin

Polymerized microtubule (composed of and tubulin subunits & tau)

Depolymerization of microtubule into tubulin monomers & formation of paired-helical filaments

Increased phosphorylation of tau

Hyperactivekinases

Hypoactivephosphatases

AD Tau

Tubulin

Tau

NFT

Imbalance of Phosphatase/Kinase Activity and Hyperphosphorylation of NFT tau in AD

P

P

P

P

PP

Calcineurin and Alzheimer’s Disease

Inhibition of calcineurin activity by neuroleptic drugs enhances phosphorylation of tau similar to AD. (Gong , et al. 1996)

Abnormally phosphorylated tau can be dephosphorylated by protein phosphatase 2A and 2B and restores its biological activity of facilitating assembly of microtubules. (Wang, et al. 1996)

In knock-out mice, the disruption of calcineurin Aα gene leads to the accumulation of hyperphosphorylated tau in hippocampus of these mice. (Kayyali et al., 1997)

.

.

.

Regulation of Calcineurin Activity

Calmodulin

Calcineurin ACalcineurin A

Calcineurin B

R

SX

X X

OP

R

SX X X

NH3

COO-

P

OH

Cyclophilin

CsA

FKBP

FK-506

NH3

COO-

AKAP79Ni2+

Mn2+

Co2+

1. Is there a decrease of calcineurin activity in AD?

2. What is the cause of the decrease of calcineurin activity in AD?

3. What is the role this decrease of calcineurin activity may play in tau phosphorylation?

Basal

*

Mn2+Ni2+

*

Control (n=5)

AD (n=7)

0

1

2

3

4

5

Co2+

*

Mic

rom

oles

ph

osp

hat

e re

leas

ed/m

in/g

pro

tein

Calcineurin Phosphatase Activity in Superior Frontal Cortex

Control (n=5)AD (n=7)

*

*

*

0

1

2

3

4

5

6

7

8

Basal Mn2+Ni2+ Co2+

Calcineurin Phosphatase Activity in Sensorimotor Cortex

0

2.5

5

7.5

10

12.5

AD (n=7)

Control (n=5)

Basal Mn2+Ni2+

Ph

osp

hat

ase

Act

ivit

y

Correlations between Calcineurin Activity and AD Pathological Changes

r = -0.770p = 0.043

 r =-0.276p = 0.549

 r = 0.026p = 0.954

r = -0.756p = 0.018

 r = -0.270p = 0.558

 r = -0.574p = 0.117

 r = -0.722p = 0.067

 r = -0.037p = 0.939

 r = -0.213p = 0.646

 r = -0.584p = 0.089

 r = -0.520p = 0.565

 r = 0.406p = 0.366

 r = -0.213p = 0.643

 r = -0.060p = 0.899

 r = -0.424p = 0.344

 NFT

 Neuronal

Plaque

 Diffuse Plaque

 Assay Condition

 Ni++ Stimulated Activity (Total Cell homogenate)

 Ni++ Stimulated Activity

(P2 Fraction) 

 Mn++ Stimulated Activity (Total Cell homogenate)

 Mn++ Stimulated Activity

(P2 Fraction) 

 Co++ Stimulated Activity (Total Cell homogenate)

 Co++ Stimulated Activity

(P2 Fraction) 

 r = -0.571p = 0.180

 r = -0.701p = 0.079

 r = -0.012p = 0.979

0.2 0.3 0.4 0.5 0.6

0

10

20

30

Calcineurin Activity

Neu

rofi

bri

llar

y T

ang

le C

ou

nts

Search for the Mechanisms of Decrease of Calcineurin Phosphatase Activity in AD

1. Protein levels of calcineurin subunits;

2. Protein levels of cyclophilin A, FKBP, and calmodulin;

3. Protein-protein interactions between calcineurin and AKAP79;

4. Oxidative damage of calcineurin;

5. Effect of oxidative stress on calcineurin activity.

Calmodulin

Calcineurin ACalcineurin A

Calcineurin B

R

S

XX

X

OP

R

S

XX

X

NH3

COO-

P

OH

Cyclophilin

CsA

FKBP

FK-506

NH3

COO-

AKAP79

ROS.O2-

H2O2

.RO

.OH

.RO

ONOO-

.NOO2

.NO2.O2

Western Blotting Analysis of Calcineurin A and B Protein Level in AD and Controls

A

B

C CCCAD CCADAD AD AD AD

C CCCAD CCADAD AD AD AD

Control AD0

1000

2000

3000

4000

Inte

grat

ed O

pti

cal D

ensi

ty

Control AD0

1000

2000

3000

Inte

grat

ed O

pti

cal D

ensi

ty

Control (n=6)

AD (n=6)

Protein Levels of Calmodulin, FKBP12 and Cyclophilin A

C ADCCAD CCADAD AD AD AD

CyP A

FKBP12

Actin

19kDa

19kDa

43kDa

CA A A A A ACCCCC

19kDaCalmodulin

A, AD; C, control

Immunoprecipitation Study of the Interaction between Calcineurin and AKAP79

79kDac c c cc cA AAAA A

IP: anti-calcineurin A;Western blot: anti-AKAP79

61kDac c c cc cA A AA AA

A.

B.

IP: anti-AKAP79;Western blot: anti-calcineurin A

C, Control (n=6)A, AD (n=6)Ctl, Omissions of IP antibody

Ctl

Ctl

Detection of Nitrotyrosine Residue in Calcineurin

61 kDa

61 kDaA

B

A C C CC CA AAAA

A C C CC CA AAAA

Atn

Atn

Ctl

Ctl

C, Control (n=6)A, AD (n=5)Ctl, Omissions of IP antibodyAtn, Anti-actin antibody as primary IP antibody

IP: anti-calcineurin A;Western blot: anti-nitrotyrosine

Western blot: anti-calcineurin A

- -5 -4 -3 -2 -10

50

100

Log Concentration of H2O2

150%

% o

f C

on

tro

l Act

ivit

y

H2O2 Induced Inactivation of Calcineurin

**

(* p<0.05)

The effect of DTT, 2-Mercaptoethanol, and Ascorbic Acid on Calcineurin Phosphatase Activity

-0.00025

0.00000

0.00025

0.00050

0.00075

- -4 -3 -2 -1

Ascorbic Acid

Act

ivit

y

*0.0000

0.0005

0.0010

0.0015

DTT- -4 -3 -2 -1

ControlAD

Act

ivit

y

*

#

0.00000

0.00025

0.00050

0.00075

- -4 -3 -2 -12-Mercaptoethanol

Act

ivit

y

*

#

NGF (9 days) FK506 (24 hours)

Antibodies Used in Western Blotting:

Phosphorylation State: Conformational Changes:

Tau-5 Alz-50 PHF-1 MC-1AT-8

Inhibition of Calcineurin Induces Phosphorylationof Tau in PC12 Cells

10 uM 5 uM Control1 uM

Tau-5

Actin

76 kD

52 kD

43 kD

10 uM 5 uM Control1 uM

Tau-5 Immunoreactivity in FK506 Treated PC 12 Cells

110 kD

76 kD

52 kD

10 uM5 uM Control1 uM

PHF-1 Immunoreactivity in FK506 Treated PC12 Cells

- -7 -6 -5 -40.0

100

200

300

FK506 Log Concentration%

of

Co

ntr

ol

43 kD

10 uM5 uM Control1 uM

*

AT-8 Immunoreactivity in FK506Treated PC12 Cells

10 uM 5 uM Control1 uM

76 kD

52 kD

Control1 uM 5 uM 10 uM0

1000

2000

3000

4000

5000

FK506In

teg

rate

d O

pti

ca

l D

en

sit

y *

43 kD

10 uM 5 uM Control1 uM

ROS

.O2-

H2O2

.RO .OH

.ROONOO-

NOO2

.NO2

.O2

Calcineurin B

RS

X X X RS

X X X

NH3

COO-

POH

NH3 COO-

Calcineurin ACalcineurin A

P

AggregationIn

activ

atio

n

IncreasedPhosphorylation

Calcineurin’s Role in the Hyperphosphorylationof Tau in AD

AD Tau

Tubulin

Tau

NFT

PP

PP

PP

Recent Developments in the Treatment of Alzheimer’s Disease

Course of Aging, MCI and AD

MCI

Clinical AD

Time (Years)

Cog

nitiv

e D

eclin

e

“Brain”ADBrain Aging Mild

Moderate

Moderately Severe

Severe

Brain Aging

Cholinesterase Inhibitors for AD

• FDA

Approved

• Current

trials

• Cognex (Parke-Davis)

• Aricept (Eisai/Pfizer)• Exelon (Novartis)• Reminyl (Janssen)

• Phenserine (Axonyx)

Memantine

• Mechanism: inhibits glutamate neurotransmitter system (NMDA receptor)

• Approved for AD by FDA

• Effective in combination with Aricept

Changefrom

Baseline(SIB)

Memantine in Advanced AD: Cognitive Benefit

Week 4 Week 12 Week 28

Wor

sen

ing

memantineplacebo

P=0.002

Reisberg, et al. NEJM, 2003

-12

-10

-8

-6

-4

-2

0

2

Vitamin E and Selegiline Delay Clinical Progression of AD

Potential Anti–Amyloid Therapies

•Secretase inhibitors (APP processing)Secretase inhibitors (APP processing)

•Activators of AActivators of Aββ degrading enzymes degrading enzymes

•Anti-Anti-ββ-sheet conformational agents-sheet conformational agents

•““Vaccination” against AVaccination” against Aββ

Amyloid Associated Proteins,Apolipoprotein E etc.

Aggregation of Aβ in the CNS

Altered PP processing increasingA42 and/or total A productionOr decreased brain A peptide clearance

Amyloid Cascade Hypothesis

Aging, APP mutationsPS1/2 mutations, TraumaOxidative stress

sAToxic Aβ oligomersAmyloid deposits

Drugs Targeting Amyloid

“Vaccination” with A Peptides as Treatment for Alzheimer’s Disease

Transgenic “AD” mouseover-expressing APP with FADlinked codon 717 mutation

With increasing agedevelops extensiveamyloid deposits

Age 13months,cognitivedecline,neuronalpathology

Immunized at 6 weeks with A1-42

Develops antibodiesagainst A1-42

Normalold age,no amyloiddeposits

Schenk et al. Nature 400: 173-177, 1999

Amyloid Immunization

Elan AD Vaccine Clinical Trial

• Trial was suspended

• Major problem was vaccine toxicity

• 15 patients out of about 300 developed “cerebral inflammation”

• These complications were likely related to direct or indirect A1-42 toxicity

Novel, Potentially Safer Approach

Vaccination with immunogenic but non-amyloidogenic Aβ homologous peptides

that are not toxic

Properties of K6A30

• has low -sheet content

• does not form fibrils in vitro

• is not toxic in human neuronal culture

• reduces amyloid burden in Tg mice

• reduces cortical amyloid burden by 89% in 18 months Tg mice after 7 months of treatment

• soluble brain A1-42 is reduced by 57% in the vaccinated mice

Neurofibrillary Tangles

Neurons have an internal support structure partly made up of microtubules. A protein called tau helps stabilize microtubules. In AD, tau changes, causing microtubules to collapse, and tau proteins clump together to form neurofibrillary tangles.

AD and the Brain

Tangle pathogenesis and Treatment

• Tau phosphorylation: is it important for tangle pathology and could it be a target for drug therapy?

• Two kinases have been implicated in AD pathogenesis: cdk5 and GSK3b

Lipids and Amyloid

• Lowering lipids (cholesterol) is associated with decrease in CNS amyloid deposition in animals.

• Increased dietary cholesterol increases amyloid– Rabbits: beta amyloid immuno-reactivity with

dietary cholesterol– Transgenic mice on atherogenic or high fat diet: A-

, Apo-E, tau– High cholesterol diet (1.25%) A- deposition

earlier in transgenic mouse(Hsiao equivalent)

*

Mouse given 5% cholesterol diets compared to .005% cholesterol diets both with 10% fat.

Hypercholesterolemia accelerates amyloid deposit number

Evidence that Cholesterol Plays Role in Pathogenesis of AD

• Observational studies: Patients using cholesterol-lowering drugs (statins) have a reduced risk of AD. Both CNS penetrant and CNS non-penetrant statins were effective in reducing the risk of AD (Jick et al. 2001, Wolozin et al. 2001, Rockwood 2002).

• Brain Cholesterol enhance plaque formation directly via its high affinity for aggregated (extracellular effect) (Mori et al 2001)

• In vitro studies: Cellular cholesterol levels affect processing APP by secretases (intracellular effect), leading to the production of more, or less A peptides (several refs)

• In vivo studies (Duff lab and others): High cholesterol diet increases brain amyloid in PS/APP mice (Refolo et al 2000); drugs that reduce cholesterol (statin, BM15.766, Refolo et al. 2001, Petanceska et al. 2002) decrease amyloid load, acting through APP processing

Purpose: Study the effect of lowering cholesterol in the treatment of AD.

Design: Placebo-controlled double blind study to compare FDA-approved cholesterol-lowering drug to placebo)

Eligibility - Over 51 with mild-to-moderate probable or possible AD, in good health, ambulatory, with a reliable caregiver in the home. Duration: 1 year with clinical evaluations every 3 months

(study completion in several months)

LEADe Study and Lipitor Treatment Trial by Pfizer

Cholesterol Lowering Agent to Slow Progression of Alzheimer’s Disease (CLASP)Sponsored by the National Institute on Aging (NIA)

18 month double-blind placebo-controlled trial.AD patients:MMSE 12 - 26

Stable standard of careExclusion of those with CHD risk factors

No lower limit exclusion for lipidsN = 400

Treatment of Alzheimer’s Disease

Improving Symptoms• Cholinesterase inhibitors

– Aricept, Exelon, Reminyl• Memantine NMDA antagnist• Psychotropic drugs for

behavior• Behavioral management• Family support

Slowing Progression• Anti-oxidants (vitamin E)• Anti-inflammatories • Neuroprotective agents (NGF)• Reducing vascular risk

– statins, homocysteine reduction

Delaying AD in MCI• Cholinesterase inhibitors

– Aricept, Exelon, Reminyl

• Antioxidants (Vitamin E)

• Anti-inflammatory drugs

Prevention• Antioxidants (Ginkgo biloba)

• Anti-inflammatories -amyloid antagonists

– secretase inhibitors– anti-aggregation compounds– amyloid vaccines

• Anti-neurofibrillar drugs• Genetic engineering