Upload
benjamin-phillips
View
214
Download
0
Tags:
Embed Size (px)
Citation preview
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