Lecture 21 - Alzheimer's Disease 2 Flashcards
What is most likely the initiating factor in AD?
Which form is most toxic to neurons?
Accumulation of Aβ oligomers
• Oligomeric forms
• (and possibly protofibrils)
derived from Aβ peptide are most toxic
List the possible neurotoxic mechanisms of Aβ
- Direct generation of free radicals
- Indirect oxidative stress
• Aβ peptide interacts with receptors on the neurons that themselves induce neurotoxic pathways - Accumulation of Aβ inside cells
- Synaptic toxicity
- Aberrant cell signalling
- ER stress
- Inhibition of glutamate uptake by astrocytes
- Decreased energy production
- Decreased trophic support
- Inflammation
What is the normal function of NMDA?
- Glutamate binds to the receptor
- Receptor opens
- Na+ and Ca2+ influx into the cell
Describe the effect of Aβ on the synapse
- Impairment of vesicle release
- Inhibition of vesicle trafficking to synapse
- Inhibition of endocytosis
- Modulation of extracellular environment
Describe the effect of Aβ intracellularly
- Inhibition of cell metabolism
* Alteration of mitochondrial metabolism
Describe how Aβ can directly lead to the production of free radicals
• Cu binds to the peptide
• Oxidation & reduction of Cu
→ Formation of the hydroxyl radical
Describe how Aβ can lead to apoptosis of neurons; i.e. ‘Indirect oxidative stress’
- Binds to NMDA receptors
- Overactive NMDA
- Ca2+ influx into cell
- Triggering of intracellular pathways; esp. apoptosis
Describe aberrant cell signalling due to Aβ
Which neuronal processes can be affected?
Many different receptors on the neurons are bound by Aβ
This triggers aberrant cell signalling
Processes affected: • Synaptic function • Transcription • Secretion of proteins • Intracellular movement of proteins • Cell energy levels • Apoptosis
Which receptors on neurons can amyloid bind?
- NMDA glutamate receptor
- Low density lipoprotein receptor
- Acetylcholine receptor
- Scavenger receptor
- Fyn kinase receptor
Describe inhibition of axonal transport by amyloid
Axon is long, a long way for stuff to be trafficked
Quite unique to neurons
Quite easily disrupted by amyloid
Describe the effect of ER stress
ER: packaging of proteins
- Any problems in cells leads to a build up of proteins in the ER
- Amyloid protein can induce ER stress in neurons
• ER releases signals:
- Down-regulate translation
- Upregulation of chaperones
- Ultimately, triggering of apoptosis
Describe glutamate neurotoxicity
Astrocytes:
• Regulation of extracellular glutamate levels
• Protect neurons from too much glutamate
- Too much glutamate → overstimulation → apoptosis
- Astrocytes take up glutamate to make sure this doesn’t happen
Amyloid:
• Impair ability of astrocytes to take up glutamate
• Overstimulation of glutamate receptors
• Overactive cell death pathways
Why is energy production particularly important to neurons?
Describe the impairment of energy production
What are the ramifications of this?
Energy is very important in neurons:
• Maintenance of electric membrane potential
• Axonal transport
Causes of energy depletion
• Aβ interacts directly w/ mitochondria: loss of cytochrome c → impaired energy production
• Impaired glucose delivery to cell
Ramifications of loss of ATP:
• Alteration of transcription
• Can’t fight oxidative stress
• Impaired maintenance of membrane potential
Describe the role of glia in neurotoxicity
Start by explaining what it is that glia do
Glia: • Protect neurons from oxidative stress w/ their high level of antioxidants • Provide nutrients • Provide antioxidant precursors • Regulate levels of metals • Secrete GFs that maintain neurons
Changes to glia function:
• Loss of this support (listed above)
Describe the tau-dependence of Aβ toxicity through Fyn kinase
Activation of fyn kinase dependent on Aβ as well as Tau:
- Aβ stimulates NDMA receptor
- Signal transduction pathway, in which Tau plays a role
- Apoptosis
Tau KO, this pathway is inhibited, and there is no apoptosis:
- W/o Tau, Fyn cannot associate with the NMDA receptor
- No neuronal death through activation of the NMDA receptor
Describe the role of inflammation in impairment of the AD brain
Triggering:
• Aβ aggregation
• Neuronal degeneration
Response:
• Activation of resident microglia
• Hypertrophy of astrocytes
• Infiltration of monocytes
→ Production of cytokines & ROS
Result:
• Impaired / dying neurons
• Loss of trophic support for neurons
Why do we need to understand the molecular and cellular biology of AD?
- Drugs that target disease not symptoms
- Ability to diagnose & treat the disease at an earlier stage
- Potential for the drug to be useful for other neurodegenerative diseases
What are the model systems used in AD?
Why?
- Synthetic Aβ peptides:
• Aβ1-40
• Aβ1-42
This is because amyloid can’t be isolated from brains of sufferers
NB these may not be a very good model of the native species in the brain
- Cell culture models
• Neuroblastoma cell lines
• or, Primary neurone cultures - Animal models
• Transgenic mice
• KO mice
What are the inherent difficulties in isolating amyloid from brains of sufferers?
- Contamination of other toxic molecules
- Brains of health people also have Aβ
- Separation is difficult
- Aβis very sticky
What is a neuroblastoma?
A cell that has come from a neuron initially
But keeps dividing indefinitely
Where do we get Primary neurons from?
Grown from brain regions that are mostly effected in AD
e.g.
• Frontal lobes
• Hippocampus
Describe how neuron cultures are used in the lab to study AD
- Neurons are allowed to mature in culture
• In culture, they form connections that are similar to synaptic connections in animals - Addition of synthetic or purified Aβ to the culture
• Cells: healthy → show degeneration of connection between cells - Observation of changes within the cells
• Cell viability assays
Do cell cultures contain astroglia & microglia?
Yes
This is probably a better model of the brain
What sort of things can be identified in neuron cell cultures?
- Identification of specific structures:
• axons
• dendrites
• pre- & post-synaptic proteins - Identify processes
• Release of synaptic vesicles
• Electrical & chemical signalling
Describe an example of a cell viability assay
- Soluble yellow dye added to the cells
- Once in the cell, the dye is converted to insoluble purple crystals when reduced by electron donation
- Extent of conversion to purple dye assessed by spectrometry
- Extent of conversion gives an indication of the health of the cell
• High levels of conversion: healthy mitochondrial function
• Low levels: aberrant mitochondrial function
What are the advantages & disadvantages of cell viability assays?
Advantages: • Subtle measure of cell toxicity: • Small changes in cell energy can be assessed • Rapid, simple assay • Well known & reproducible
Disadvantages:
• Doesn’t tell if cells are unhealthy or dead
• If cells are replicating there will be increased measured viability in spite of death of other cells
• Assay compound is toxic to cells
• Not in real-time; only an end point
Describe some methods of identification of potential therapeutic targets
- Protein Knockout
- Inhibition of enzymes
- Analysis of protein changes; eg. Tau
- Measuring cell growth, function & viability induced by amyloid
Give an example of protein KO in identification of targets
W/ Fyn kinase
• Amyloid added to cells and they die
Fyn kinase KO:
• No amyloid toxicity
We can conclude that Fyn kinase is important in the process
Give an example of protein inhibition in identification of targets
NMDA inhibitors:
• Reduced toxicity observed
Conclusion that NMDA play an important role in pathogenesis
What is observed visually in neurons when they are treated w/ Aβ?
Reduction in the connections between neurons
What key features of the pathogenesis have been identified using cell cultures?
- Neurotoxicity of the oligomeric forms of Aβ
- Neurotoxicity of Histadine residues binding Cu
- Tyrosine residues cross linking to form oligomers
What are the possible mechanisms of drugs?
• Activation of enzymes
e.g. GF activator that stimulates growth & survival of the neuron
• Inhibition of enzymes
e.g. Calcineurin inhibitor
What are the features of an optimal drug?
- Small
- Cross blood brain barrier
- Non toxic
- Cleared quickly
- Highly specific
- Easy to make in large quantities
How may the structure of the drug be identified?
- High throughput screening
- Crystallography
- Computer aided design
Discuss briefly the time line and economics of drug development
- Many many drugs start off in screening, but very few make it to the later stages
- The process takes a very long time (11-15 years) and is very costly
- Developers need to be able to recoup some of the money when the it finally gets to market
- The drug must also be able to be accessed by everyone who needs it
List some of the animal models used in AD research
Transgenic mice: • Tg2576 • APP/PS1 • P301L • APP/tau mice
Knockout mice: • APP • Tau • Presenilin • BACE • ApoE
What is Tg2576?
This is a transgenic mouse
- Contains human AAP gene with 2 mutations: ‘Swedish mutation’
- Gives rise to aggressive early onset of AD in mice:
- Mice get amyloid aggregates in the brain, like in human AD
- Loss of cognitive function
• These transgenic mice have been studied as a model of disease
What is the Swedish mutation?
- A mutation in APP
- Observed in some people w/ AD
- Associated w/ aggressive, early onset of disease
- Accumulation of amyloid in the brain
What is Presenilin 1?
This is a gene that is mutated in a small group of people w/ AD
Describe APP/PS1
- Transgenic mice w/ mutation in the human gene Presenilin 1
- Used as an animal model for researching AD
- Develop many plaques in the brain
- Decline in cognition
What are the drawbacks of animal model in researching AD?
APP/PS1
• In humans, the disease is slow
• In the transgenic mice, it is fast and aggressive
• Question as to whether this is a good model
P301L tau mice:
• No amyloid deposits
APP/PS1 & Tg2576:
• No involvement of NFTs → not the same as AD
Describe P301L tau mice
- Transgenic mice w/ human Tau gene mutation
- Model for Tau pathology in AD
Observations:
• Formation of NFTs
• Increased levels of hyperphosphorylated Tau in brain
• No amyloid deposits
Describe APP/Tau mice models
• Transgenic mice w/ both mutant Tau and APP
Observations
• Increased onset of disease changes
• Not that different to amyloid mice
• This supports the evidence that amyloid changes are upstream to tau changes
Describe the rationale of use of KO mice.
What key features are observed?
Give an example
- Deletion of key proteins allows comparison with normal mice
- Determine the function of the protein
Redundancy:
• A gene considered very important is knocked out
• No effect, because there is another gene that does a similar thing
e.g. • APP-/- survive • APP-/- APLP2-/+ survive • APP-/- APLP2-/- die This indicates that even one copy of APLP2 can cover for APP in normal brain function
What subtle changes were observed in APP-/- mice?
APP KO mice
• Mice were more resistant to copper toxicity
• Due to Cu binding domain which converts Cu(II) to the toxic form Cu(I)
Which is the toxic form of copper?
Cu(I), i.e. the reduced form
Describe how ex vivo tissue can be used for disease research
- Living brain slices in culture (ex vivo)
- Electrophysiological & synaptic activity measured
- Important for understanding effect of amyloid on memory function
What are some useful animal models for investigating AD?
These animals have had their entire genomes sequenced: • C. elegans • Zebra fish • D. melanogaster → maybe not so relevant to AD
Dogs & primates
• Have a natural form of AD
• Ethical issues
• Long life span
Is inflammation in the AD brain primary or secondary?
Though to play a major secondary role in the neurotoxicity in AD
However, new research that indicates that this is happening very early on as well
What does ‘lack of trophic support’ mean?
In the AD brain, glial cells (astrocytes, microglia) are chronically stimulated.
This can lead to their dysfunction and inability to deliver trophic support to neurons, such as:
• Regulation of the extracellular environment
• Provision of nutrients
• Oxidative stress protection
• GF provision
