Lecture 20 - Molecular Pathology of Alzheimer's Disease II Flashcards
Receptors that Abeta can bind to to cause apoptosis
1)
2)
3)
1) Insulin
2) NMDA
3) Frizzled
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)
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
What does NMDA stimulation lead to?
Increased intracellular Ca2+
Abeta synaptic toxicity 1) 2) 3) 4)
1) NMDA-receptor mediated
2) Impaired vesicle release
3) Inhibited vesicle trafficking to synapse
4) Inhibited endocytosis
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)
Why are axons particularly vulnerable to changes in intracellular transport?
Axon is very long, needs functioning intracellular trafficking to work properly
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
Effect of inhibiting glutamate uptake in astrocytes
Excitotoxicity
Important astrocyte function w/r/t glutamate
Astrocytes regulate extracellular glutamate levels, protect neurons from too much glutamate
Astrocyte glutamate transporter
GLT1
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
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)
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)
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
What does Abeta toxicity depend on?
Tau interaction with fyn kinase in neuronal dendrites
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
Effectors of inflammation in AD brain
1)
2)
3)
1) Resident microglia
2) Invading monocytes
3) Activated astrocytes
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
Causes of neurotoxic effects in AD
Involves synaptic toxicity caused by amyloid-beta oligomers
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
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
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?
Synthetic Abeta peptides
1)
2)
1) Abeta1 - 40
2) Abeta1 - 42
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
Where are primary neurons derived from for cell culture?
Grown from brain regions of primarily affected areas (hippocampus, frontal cortex)
From animal brains
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
Common neurotoxicity assay
MTT/MTS
Soluble yellow dye added, turns to purple crystals when reduced by electron donation inside cell)
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.
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
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)
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
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
What does Abeta effect on neurite growth model?
Damage to synaptic connections in vivo
Amino acids in Abeta that to bind Cu
His6, His13 and His14 residues
What do His6, His13 and His14 do in Abeta?
Bind to Cu
Effect of mutating Abeta to not have His6, His13 or His14?
Doesn’t bind copper, reduced toxicity (reduced aggregation, reduced ROS generation)
Effect of mutating Tyr10 in Abeta
Reduces ability of peptide to crosslink (dityrosine crosslink between monomers forms oligomers)
Potential target of protein knockout
Fyn kinase
Potential enzyme inhibitors for treatment of AD
Calcineurin inhibitors, NMDAR inhibitors
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
AD-like mice
1)
2)
1) Tg2576 mice
2) APP/PS1 mice
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
What is the MTT/MTS test?
Tests cell metabolic activity
PS1
Human presenilin 1
Gene causing familial AD, early Abeta production
Tg2576 mice
1) Contain human APP gene with 2 mutations that give it the ‘Swedish mutation’
2) Swedish mutation give aggressive, early-onset AD
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
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
Mice with tau mutation
P301L mice
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
Effects of crossing APP/tau mutant mice
Increased levels, faster onset of neuropathological features, but nothing different to amyloid mice
Gene knockout mice 1) 2) 3) 4) 5)
1) APP
2) Tau
3) Presenilin
4) BACE
5) ApoE
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
APLP2
Amyloid precursor-like protein 2
Current aim of therapeutic drugs
Detect disease early, treat early (40-50 years old)
