lecture 21 Flashcards
Alzheimer's II - neurotoxic mechanisms in Alzheimer's disease - approaches to understanding the molecular and cellular biology of Alzheimer's disease
1
Q
Around how many publications exist on amyloid peptide toxicity?
A
3000+
2
Q
What is the amyloid cascade hypothesis? Is this the only hypothesis re: the progression of AD?
A
- accumulation of Aβ peptide neurotoxicity is believed to be the most likely initiating factor in synaptic/neuronal dysfunction and death
- there are many alternative hypotheses, however these have not yet had the rigorous scientific support of the amyloid hypothesis
3
Q
What are two general ways in which Aβ contributes to increased oxidation?
A
- direct reactive oxygen species generation by peptide
- indirect oxidative stress e.g. through NMDA type glutamate receptor modulation
4
Q
What are the effects of accumulation of intraneuronal Aβ?
A
- inhibits cell metabolism (especially protein turnover/axonal trafficking)
- alters mitochondrial metabolism (inhibits cyt c oxidase and energy production)
5
Q
How does Aβ cause synaptic toxicity?
A
- NMDA receptor mediated
- impairment of vesicle release
- inhibition of vesicle trafficking to synapse
- inhibition of endocytosis
- modulation of extracellular environment
6
Q
How does Aβ produce aberrant cell signalling? What are the effects of this?
A
- binding of Aβ to membrane receptors and altering cell signals e.g.
- NMDA glutamate receptor
- low density lipoprotein receptor
- acetylcholine receptor
- scavenger receptor
- fyn kinase receptor
changes in intracellular signalling can affect many aspects of neuronal function:
- synaptic function
- transcription
- protein turnover
- secretion of proteins
- intracellular movement of proteins
- cell energy levels
- apoptosis
7
Q
What is a critical element of Aβ neuronal impairment?
A
- inhibition of axonal transport
- changes in neurons that we may not see in other cell types
8
Q
What is ER stress?
A
- ER is important for the packaging of proteins to be transferred throughout the cell and excreted from the cell
- build up of proteins in the ER
- signals from the ER to make changes
- reducing translation of proteins
- signals upregulation of chaperone proteins
- if things becoming overwhelming initiates apoptotic pathways
9
Q
What does the inhibition of glutamate uptake cause?
A
- excitotoxicity
- astrocytes have an important function in regulating extracellular glutamte levels and protecting neurons from too much glutamte (excitotoxicity of NMDA and/or AMPA receptors)
- impairment of astrocyte function by lack of energy (glucose) or Aβ effects can reduce expression of astrocyte glutamate transporters such as GLT1
10
Q
What is normal neuronal energy demand? How is this affected by Aβ?
A
- neurons have high energy demand to maintain electric membrane potential
- loss of ATP energy production may be due to impaired glucose delivery or uptake
- Aβ can affect mitochondrial function
- additional environmental stresses can reduce cellular energy levels (e.g. mitochondrial toxins)
loss of ATP production:
- alters transcription and ability to fight oxidative stress
- impairs ability to maintain resting potential and therefore synaptic signalling (memory)
11
Q
What is the normal function of astrocytes? What happens to astrocytes in AD?
A
normal function:
- protect neurons from oxidative stress
- provide nutrients and precursors for important molecules such as precursors of antioxidant molecules (e.g. cysteine = glutathione precursor)
- regulate levels of metals, co-factors etc.
- secrete growth factors that maintain neurons
- changes to astrocytes (e.g. activation) can result in loss of this support and neuronal dysfunction
12
Q
What is fyn kinase?
A
- Aβ toxicity depends on tau interaction with fyn kinase in neuronal dendrites
- integrating tau and Aβ is important to understanding neurotoxic processes
- in the past few years a very important mechanism that may underlie this interaction has become evident
- the activation of a kinase that is an enzyme that induces phosphorylation of a number of other proteins that lead to a number of cell processes
- activation of this kinase (fyn kinase) that occurs via Aβ is also dependent on tau
- in normal cells we have to tau protein that interacts with fyn kinase
- links to NMDA glutamate receptor
- overstimulation of this leads to a toxic process (e.g. Aβ peptide)
- signals through fyn kinase dependant on tau protein
- leads to cell death
- supported by studies where they generated animals that don’t express the tau protein – don’t get any changes occurring because there’s no tau protein to translate the signalling processes into the cell
- important interaction with Aβ toxicty interacting with the NMDAR and intracellular tau mediating these signals (and therefore the toxic processes)
13
Q
By what mechanisms does inflammation occur that leads to neuronal impairment/death?
A
- resident microglia
- invading monocytes
- activated astrocytes
- production of cytokines and reactive oxygen species that impair neurons
- loss of trophic support for neurons
- possibly a major role in secondary neurotoxic effects in AD
14
Q
So in summary, what are the neurotoxic mechanisms in AD?
A
- most likely to involve synaptic toxicity induced by oligomeric amyloid peptide
- may involve:
- direct generation of reaction oxygen species
- indirect increases in oxidative stress through NMDA glutamate receptor activation
- aberrant cell signalling
- impaired axonal transport
- ER stress
- inhibition of glutamate uptake by astrocytes
- decreased energy levels
- decreased trophic support
- inflammation
- still don’t really understand how these all fit together
- what occurs first? what occurs later?
15
Q
Why do we need to understand the molecular and cellular biology of Alzheimer’s disease?
A
- failure of current drugs in therapeutic trials
- need to diagnose and treat disease at an earlier stage (success increases with early intervention as with any disease)
- disease modifying therapies are needed, rather than ‘masking’ symptoms (e.g. panadol may stop pain but won’t stop the injury)
- potential to treat other forms of neurodegeneration
- these advances are likely to come in the next generation
16
Q
What model systems have been developed to test theories and potential treatments? What are the problems with these?
A
- synthetic Aβ peptides (Aβ1- 40 and Aβ1-42) used to examine the neurotoxicity activity of the peptide
- attempts to identify the neurotoxic species of Aβ from AD brain - purification of Aβ from brain into monomer, dimer, trimer, tetramer, and larger oligomeric species - testing on cells in culture
many inherent problems:
- contamination with other toxic molecules
- normal brain also contains Aβ
- separation of different species
- Aβ is very ‘sticky’ and aggregates easily
- how do you keep it in the form in which it was purified?
- cell culture models
- synthetic or purified Aβ added to neurons in culture
- neurons are either neuroblastoma cell lines or primary neuron cultures
- primary neurons can be grown from brain regions that are mostly affected in AD, e.g. frontal cortex and hippocampus (compared to cerebellum)
- neurons are allowed to mature in culture and form connections that are similar to synaptic connections in animals and humans
- axons and dendrites can be identified based on morphology and expression of specific proteins
- can identify pre and post-synaptic proteins, release of synaptic like vesicles, measure electrical and chemical signalling between cells
- even relatively pure cultures contain astroglia and microglia
- true ‘mixed’ glia/neuronal cultures are probably a better model of the brain
17
Q
What are assays for neurotoxicity?
A
- cell viability assays - MTT/MTS (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) - soluble yellow dye (insoluble purple crystals when reduced by electron donation inside cell
18
Q
What are advantages for assays for neurotoxicity?
A
- subtle measure of cell toxicity (small changes in cell energy levels can be assessed - more relevant to amyloid toxicity than overt cell death)
- rapid, simple assay (few steps) - easy to perform on large numbers of experiments
- well known and reproducible
19
Q
What are disadvantages for assays for neurotoxicity?
A
- doesn’t tell if cells are just unhealthy (low energy levels) or dead
- if cells are replicating (e.g. astrocytes) this will increase the measured viability in spite of death of other cells
- assay compounds are toxic to cells (can’t use them for anything else)
- not real-time (end-point assay only)
20
Q
How can we identify potential therapeutic targets?
A
Range of approaches to identify pathway and protein targets
- knockdown of proteins - effect on amyloid toxicity (e.g. Fyn kinase)
- inhibition of enzymes - effect of amyloid toxicity (e.g. calcineurin inhibitors, NMDAR inhibitors)
- analysis of protein changes induced by amyloid (e.g. antioxidant proteins, tau-phosphorylation)
- measuring cell growth, function and viability induced by amyloid
- cell viability assays (measuring mitochondrial respiration)
- lactate dehydrogenase release assay (LDH)
- propidium iodide assay
- measuring synaptic protein and gene expression (immunoblot, RNA levels, immunohistochemistry)
- electrophysiological testing on brain slices treated with Aβ (LTP assays)
- effects of Aβ on neurite outgrowth (model of damage to synaptic connections in vivo)
- measuring synaptic vesicle release and turnover
21
Q
What has been identified using MTT/MTS assay of amyloid peptide toxicity?
A
- these assays have been used to identify key amino acid residues and oligomeric structure involved in neurotoxicity of amyloid beta peptide
- oligomeric forms of amyloid beta peptide are neurotoxic
- mutation of histidine residues (His6, His13 and His14 within Aβ sequence) affect Cu binding and reduce neurotoxicity of peptide (reduced aggregation and free radical generation)
- mutation of Tyr10 also reduces toxicity - tyrosine is important in di-tyrosine cross-linking between peptides to promote oligomer formation and toxicity
22
Q
How do we use chemistry and biochemistry to develop potential drugs?
A
- drug compounds may be designed to target and inhibit an enzyme (antagonist/inhibitor) (e.g. calcineurin inhibitor)
- drugs/molecules may also activate enzymes (growth factors and peptides derived from growth factors)
- can be based on protein structure (identified through crystallography etc)
- may be computer designed
- optimum drugs are:
- small
- cross blood brain barrier
- non toxic
- cleared quickly
- highly specific
- easy to make in large quantities
23
Q
How many drugs make it through clinical trials compared to how many are tested?
A
100+/(~250) in preclinical
1 - 2 products make it through
very convoluted
investment in trying to make a drug work can be around $1 billion
24
Q
What knockout mice have been used to model AD? What has been shown using these mice?
A
- APP, tau, presenilin, BACE, ApoE etc
- deletion of key proteins allows comparison with normal mice to determine the function of the protein
- in reality, many (most?) proteins are redundant (as proteins do the same thing), so knocking out one has only subtle or no effect on the animal
> e.g. APP -/- mice survive, APP-/- APLP-/+ survive but APP-/- APLP2 -/- die at embryonic stage (so even one copy of APLP2 can cover for APP in normal brain function) - normally need subtle tests to determine the differences in knockout mice
> e.g. we found that APP -/- neurons in culture were more resistant to copper toxicity (due to the loss of the APP copper binding domain that can convert Cu(II) to Cu(I) (toxic)
25
Q
How has ex vivo tissue been used to study AD?
A
- brain slices can be prepared fresh and used to measure electrophysiological changes and synaptic activity
- latter is critical for subsequent disease effects
26
Q
What other animals have been considered as models for AD?
A
- C. elegans (nematode worm)
- zebra fish
- drosophila melanogaster (fruit fly)
- dogs? (ethical issues)
- primates (ethical issues/cost)
27
Q
What was the Tg2576 mouse? What did it show?
A
- transgenic mouse that contained human APP gene with two mutations (known as swedish mutation)
- K595N/M569L
- aggressive early onset form of AD
- AD ‘like’ in Tg mouse
- cognition is normal at 3 months –> declines at 5.6
- models synaptic loss in early stages of human AD
- corresponds with Aβ levels
- rise from 3 months and increase rapidly from 7
- Aβ deposits form in brain at 8-9 months
- cerebrovascular deposits at 15 months
- neuronal cell death and activation (inflammation) of glia occurs towards the latter stages
28
Q
What was the APP/PS1 transgenic mouse? What did it show?
A
- contained the APP double mutation and a gene for human presenilin 1
- PS1: causes familial PD by accelerating Aβ production
- PS1 gene also contained a mutation from human familial AD: M146L
- reveal heavy plaque load @ 3-4 months
- cognitive decline after 5-6 months
- more aggressive model –> earlier onset
- good because faster to obtain results but does this reflect the slow nature of the human form of the disease?
- are mice the best models of human disease?
- over-expressed compared to the level seen in humans?
29
Q
Why make tau transgenice mice? What models have been made based on tau? What did they show?
A
Neither Tg2576 or APP/PS1 mice have any changes to tau or presence of NFTs so not the same as AD.
P301L tau mice:
- human Tau gene mutation
- model of frontotemporal dementia (involves tau) and also tau pathology in AD
- levels of hyperphosphorylated tau are increased in the brain
- formation of NFTs
- no amyloid deposits
APP/Tau models:
- a number of crossed mice exist (cross breeding APP mutant mice with Tau mutant mice)
- induces increased levels and more rapid onset of neuropathological features: but rarely anything different to amyloid mice
- studies support that changes to amyloid in AD are upstream to changes to tau (but the latter is critical for subsequent disease effects)
30
Q
In summary, what are the neurotoxic mechanisms in AD?
A
- most likely to involve synaptic toxicity induced by oligomeric amyloid peptide
- may involve:
- direct generation of reactive oxygen species
- indirect increases in oxidative stress through NMDA receptor
- aberrant cell signalling
- impaired axonal transport
- ER stress
- inhibition of glutamate uptake by astrocytes
- decreased energy levels
- decreased trophic support
- inflammation
- Aβ mediated toxicity to neurons is believed to underlie the process of neurodegeneration
- research mostly performed with synthetic or cell-derived Aβ
- cell cultures of neurons and assays for celll viability/survival have elucidated much on Aβ toxicity
- animal models of AD alos useful to determine how Aβ affects neurons in vivo
31
Q
In summary, what are approaches to understanding the molecular and cellular biology of Alzheimer’s disease?
A
- identify targets
- design and develop drugs or screen libraries of compounds
- testing in cells and animals (range of animal models but none fully captures AD)
- current aim is small drugs that target the brain and stop the early stages of the disease. Will be critical to have better early diagnosis (40-50 year old ‘patients’)
