Lecture 20 - Molecular Pathology of Alzheimer's Disease II

Question 1

Receptors that Abeta can bind to to cause apoptosis
1)
2)
3)

Answer 1

1) Insulin
2) NMDA
3) Frizzled

Question 2

How can copper lead to reactive oxygen species?
1)
2)

Answer 2

1) Cu(II), bound to Abeta, is oxidised to Cu(I) by O2

2) Cu(I) + H2O2 –> Cu(II) + OH- (radical)

Question 3

Pathways leading to neuronal loss and cytotoxicity in AD
1)
2)
3)
4)

Answer 3

1) Direct reactive oxygen species generation by Cu bound to Abeta
2) Indirect oxidative stress through NMDA type glutamate receptor modulation
3) Accumulation on intraneuronal Abeta
4) Synaptic toxicity

Question 4

What does NMDA stimulation lead to?

Answer 4

Increased intracellular Ca2+

Question 5

Abeta synaptic toxicity
1)
2)
3)
4)

Answer 5

1) NMDA-receptor mediated
2) Impaired vesicle release
3) Inhibited vesicle trafficking to synapse
4) Inhibited endocytosis

Question 6

Effects of intraneuronal Abeta accumulation
1)
2)

Answer 6

1) Inhibits cell metabolism (protein turnover, axonal trafficking)
2) Alters mitochondrial metabolism (inhibits cytochrome C oxidase, energy production)

Question 7

Why are axons particularly vulnerable to changes in intracellular transport?

Answer 7

Axon is very long, needs functioning intracellular trafficking to work properly

Question 8

ER stress
1)
2)

Answer 8

When the ER is stressed in AD, it:

1) Tries to decrease translation, increase chaperones that bind to misfolded proteins
2) If this fails, cell undergoes apoptosis

Question 9

Effect of inhibiting glutamate uptake in astrocytes

Answer 9

Excitotoxicity

Question 10

Important astrocyte function w/r/t glutamate

Answer 10

Astrocytes regulate extracellular glutamate levels, protect neurons from too much glutamate

Question 11

Astrocyte glutamate transporter

Answer 11

GLT1

Question 12

How can neuron excitotoxicity occur in AD?
1)
2)

Answer 12

1) Abeta or a lack of glucose reduces astrocyte GLT1 function
2) This impairs the ability of astrocytes to protect neurons from excess glutamate

Question 13

How can Abeta decrease cellular energy production in AB?
1)
2)
3)

Answer 13

1) Impaired glucose delivery or uptake
2) Abeta can impair mictochondrial function
3) Additional environmental stresses can reduce cellular energy levels (EG: mitochondrial toxins)

Question 14

How does decreased energy affect neurons in AD?
1)
2)

Answer 14

1) Loss of ATP reduces transcription and ability to fight oxidative stress
2) Loss of ATP affects neurons ability to maintain a membrane potential (memory)

Question 15

Functions of astrocytes
1)
2)
3)
4)
5)

Answer 15

1) Protect neurons form oxidative stress
2) Provide nutrients
3) Provide precursors to important molecules (EG: glutathione precursor cysteine)
4) Regulate levels of metals
5) Secrete growth factors

Question 16

What does Abeta toxicity depend on?

Answer 16

Tau interaction with fyn kinase in neuronal dendrites

Question 17

Relation between Abeta, tau, fyn kinase and NMDAR in AD
1)
2)
3)

Answer 17

1) In presence of tau and fyn kinase, overstimulation of NMDAR results in apoptosis
2) Without tau and fyn kinase, stimulation of NMDAR doesn’t cause apoptosis
3) Abeta stimulates NMDAR

Question 18

Effectors of inflammation in AD brain
1)
2)
3)

Answer 18

1) Resident microglia
2) Invading monocytes
3) Activated astrocytes

Question 19

Causes of inflammation in AD brain
1)
2)
3)

Answer 19

1) Pro-inflammatory stimuli (Abeta buildup, cell stress from tau, oxidative stress)
2) Cytokines, ROS released by activated glial cells (astrocytes, microglia)
3) Inflammation probably cause of secondary neurotoxic effects in AD

Question 20

Causes of neurotoxic effects in AD

Answer 20

Involves synaptic toxicity caused by amyloid-beta oligomers

Question 21

Neurotoxic effects of amyloid-beta oligomers in AD
1)
2)
3)
4)
5)
6)
7)
8)
9)

Answer 21

1) Direct generation of reactive oxygen species
2) Increase in indirect oxidative stress through NMDAR stimulation
3) Aberrant cell signalling
4) Impaired axonal transport
5) ER stress
6) Inhibited glutamate uptake by astrocytes
7) Decreased energy levels
8) Decreased glial trophic support
9) Inflammation

Question 22

How can the molecular and cellular background of AD be tested?
1)
2)
3)

Answer 22

1) Animal models
2) Synthetic Abeta peptides
3) Identify toxic Abeta species - isolate from brain, separate into different size oligomers and monomers, test on cells in culture

Question 23

Problems with testing molecular and cellular background of AD
1)
2)
3)
4)

Answer 23

1) Contamination with other toxic molecules
2) Normal brain contains Abeta
3) Abeta is very sticky, aggregates easily
4) Abeta might change forms from what it was in the brain to something else in culture. How do you know?

Question 24

Synthetic Abeta peptides
1)
2)

Answer 24

1) Abeta1 - 40

2) Abeta1 - 42

Question 25

Example of a model system with which to test AD pathogenesis
1)
2)
3)
4)

Answer 25

1) Make a neuron cell culture from either neuroblastoma cells or primary neuron culture
2) Neurons mature in culture, form connections as they do in the brain
3) Cultures also contain astrocytes and microglia
4) Synthetic

Question 26

Where are primary neurons derived from for cell culture?

Answer 26

Grown from brain regions of primarily affected areas (hippocampus, frontal cortex)

From animal brains

Question 27

Problems with model systems for testing AD
1)
2)

Answer 27

1) Neuroblastoma cells mightn’t behave in the same way as primary neurons
2) Synthetic Abeta mightn’t be accurate

Question 28

Common neurotoxicity assay

Answer 28

MTT/MTS

Soluble yellow dye added, turns to purple crystals when reduced by electron donation inside cell)

Question 29

Interpretation of MTT/MTS results

Answer 29

Reduction from yellow to putple colour indicates cell metabolic activity.

If no colour change, either no metabolic activity or very slow. Indicates cell death or sickness.

Question 30

Advantages of MTT/MTS
1)
2)
3

Answer 30

1) Subtly measures cell toxicity. Can detect small changes in cell energy levels
2) Rapid, simple, performable on a large number of cells
3) Well-known, reproducible

Question 31

Disadvantage of MTT/MTS
1)
2)
3)
4)

Answer 31

1) Doesn’t tell if cells are unhealthy or dead
2) If cells are replicating, this will increase the measured viability, even if other cells are dying (EG: dividing astrocytes)
3) Assay compounds are toxic to cells - cells can’t be used for anything else
4) End-point assay (not real-time)

Question 32

Ways to identify potential therapeutic targets
1)
2)
3)
4)

Answer 32

1) Protein knockout, test effect on amyloid toxicity
2) Inhibit enzymes, test effect on amyloid toxicity
3) Analysis of protein changes induced by amyloid
4) Measure cell growth, function and viability in presence of amyloid

Question 33

Ways to measure cell growth, function and viability in presence of amyloid
1)
2)
3)
4)
5)
6)

Answer 33

1) Cell viability assays measuring mitochondrial activity
2) Lactate dehydrogenase release assay
3) Propidium iodide assay
4) Measure synaptic protein and gene expression
5) Electrophysiological assays on brain slices treated with Abeta
6) Effects of Abeta on neurite outgrowth

Question 34

What does Abeta effect on neurite growth model?

Answer 34

Damage to synaptic connections in vivo

Question 35

Amino acids in Abeta that to bind Cu

Answer 35

His6, His13 and His14 residues

Question 36

What do His6, His13 and His14 do in Abeta?

Answer 36

Bind to Cu

Question 37

Effect of mutating Abeta to not have His6, His13 or His14?

Answer 37

Doesn’t bind copper, reduced toxicity (reduced aggregation, reduced ROS generation)

Question 38

Effect of mutating Tyr10 in Abeta

Answer 38

Reduces ability of peptide to crosslink (dityrosine crosslink between monomers forms oligomers)

Question 39

Potential target of protein knockout

Answer 39

Fyn kinase

Question 40

Potential enzyme inhibitors for treatment of AD

Answer 40

Calcineurin inhibitors, NMDAR inhibitors

Question 41

Optimum drugs for AD
1)
2)
3)
4)
5)
6)

Answer 41

1) Small
2) Cross blood brain barrier
3) Non-toxic
4) Cleared quickly
5) Highly-specific
6) Can be produced in large quantities

Question 42

AD-like mice
1)
2)

Answer 42

1) Tg2576 mice

2) APP/PS1 mice

Question 43

Effects of Swedish mutation on Tg2576 mice
1)
2)
3)
4)
5)
6)

Answer 43

1) 3 months - Cognition normal, Abeta levels begin to rise
2) 5 - 6 months - Cognition begins to decline
3) 7 months - Abeta levels begin rising rapidly
4) 8 - 9 months - Abeta deposits form in the brain
5) 15 months - Abeta cerebrovascular deposits
6) Neuronal death and inflammation (glial activation) occurs at later stages

Question 44

What is the MTT/MTS test?

Answer 44

Tests cell metabolic activity

Question 45

PS1

Answer 45

Human presenilin 1

Gene causing familial AD, early Abeta production

Question 46

Tg2576 mice

Answer 46

1) Contain human APP gene with 2 mutations that give it the ‘Swedish mutation’
2) Swedish mutation give aggressive, early-onset AD

Question 47

APP/PS1 mice
1)
2)

Answer 47

1) Have both APP double mutation (Swedish mutation) and human presenilin 1 gene
2) More aggressive model than Tg2576 mice

Question 48

Problem with Tg2576 and APP/PS1 mice
1)
2)

Answer 48

1) No changes to tau or presence of neurofibrillary tangles

2) Might be too aggressive to be accurate

Question 49

Mice with tau mutation

Answer 49

P301L mice

Question 50

P301L mice
1)
2)
3)

Answer 50

1) Model of frontotemporal dementia and tau pathology in AD
2) Presence of hyperphosphorylated tau, NFT
3) No Abeta plaques

Question 51

Effects of crossing APP/tau mutant mice

Answer 51

Increased levels, faster onset of neuropathological features, but nothing different to amyloid mice

Question 52

Gene knockout mice
1)
2)
3)
4)
5)

Answer 52

1) APP
2) Tau
3) Presenilin
4) BACE
5) ApoE

Question 53

Effect of knocking out AD genes in mice
1)
2)
3)

Answer 53

1) Most proteins are redundant, so no effect
2) APP-/- survive, APP-/- APLP2-/+ survive
3) APP-/- APLP-/- die

Question 54

APLP2

Answer 54

Amyloid precursor-like protein 2

Question 55

Current aim of therapeutic drugs

Answer 55

Detect disease early, treat early (40-50 years old)