Lecture 20 - Molecular Pathology of Alzheimer's Disease II Flashcards Preview

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Flashcards in Lecture 20 - Molecular Pathology of Alzheimer's Disease II Deck (55):
1

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

1) Insulin
2) NMDA
3) Frizzled

2

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

1) Cu(II), bound to Abeta, is oxidised to Cu(I) by O2
2) Cu(I) + H2O2 --> Cu(II) + OH- (radical)

3

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

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

4

What does NMDA stimulation lead to?

Increased intracellular Ca2+

5

Abeta synaptic toxicity
1)
2)
3)
4)

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

6

Effects of intraneuronal Abeta accumulation
1)
2)

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

7

Why are axons particularly vulnerable to changes in intracellular transport?

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

8

ER stress
1)
2)

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

9

Effect of inhibiting glutamate uptake in astrocytes

Excitotoxicity

10

Important astrocyte function w/r/t glutamate

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

11

Astrocyte glutamate transporter

GLT1

12

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

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

13

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

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

14

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

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)

15

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

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

16

What does Abeta toxicity depend on?

Tau interaction with fyn kinase in neuronal dendrites

17

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

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

18

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

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

19

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

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

20

Causes of neurotoxic effects in AD

Involves synaptic toxicity caused by amyloid-beta oligomers

21

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

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

22

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

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

23

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

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?

24

Synthetic Abeta peptides
1)
2)

1) Abeta1 - 40
2) Abeta1 - 42

25

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

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

26

Where are primary neurons derived from for cell culture?

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

From animal brains

27

Problems with model systems for testing AD
1)
2)

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

28

Common neurotoxicity assay

MTT/MTS

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

29

Interpretation of MTT/MTS results

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.

30

Advantages of MTT/MTS
1)
2)
3

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

31

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

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)

32

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

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

33

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

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

34

What does Abeta effect on neurite growth model?

Damage to synaptic connections in vivo

35

Amino acids in Abeta that to bind Cu

His6, His13 and His14 residues

36

What do His6, His13 and His14 do in Abeta?

Bind to Cu

37

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

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

38

Effect of mutating Tyr10 in Abeta

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

39

Potential target of protein knockout

Fyn kinase

40

Potential enzyme inhibitors for treatment of AD

Calcineurin inhibitors, NMDAR inhibitors

41

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

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

42

AD-like mice
1)
2)

1) Tg2576 mice
2) APP/PS1 mice

43

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

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

44

What is the MTT/MTS test?

Tests cell metabolic activity

45

PS1

Human presenilin 1
Gene causing familial AD, early Abeta production

46

Tg2576 mice

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

47

APP/PS1 mice
1)
2)

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

48

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

1) No changes to tau or presence of neurofibrillary tangles
2) Might be too aggressive to be accurate

49

Mice with tau mutation

P301L mice

50

P301L mice
1)
2)
3)

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

51

Effects of crossing APP/tau mutant mice

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

52

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

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

53

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

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

54

APLP2

Amyloid precursor-like protein 2

55

Current aim of therapeutic drugs

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