lecture 21

Question 1

Around how many publications exist on amyloid peptide toxicity?

Answer 1

3000+

Question 2

What is the amyloid cascade hypothesis? Is this the only hypothesis re: the progression of AD?

Answer 2

• 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

Question 3

What are two general ways in which Aβ contributes to increased oxidation?

Answer 3

• direct reactive oxygen species generation by peptide

- indirect oxidative stress e.g. through NMDA type glutamate receptor modulation

Question 4

What are the effects of accumulation of intraneuronal Aβ?

Answer 4

• inhibits cell metabolism (especially protein turnover/axonal trafficking)
• alters mitochondrial metabolism (inhibits cyt c oxidase and energy production)

Question 5

How does Aβ cause synaptic toxicity?

Answer 5

• NMDA receptor mediated
• impairment of vesicle release
• inhibition of vesicle trafficking to synapse
• inhibition of endocytosis
• modulation of extracellular environment

Question 6

How does Aβ produce aberrant cell signalling? What are the effects of this?

Answer 6

• 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

Question 7

What is a critical element of Aβ neuronal impairment?

Answer 7

• inhibition of axonal transport

- changes in neurons that we may not see in other cell types

Question 8

What is ER stress?

Answer 8

• 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

Question 9

What does the inhibition of glutamate uptake cause?

Answer 9

• 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

Question 10

What is normal neuronal energy demand? How is this affected by Aβ?

Answer 10

• 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)

Question 11

What is the normal function of astrocytes? What happens to astrocytes in AD?

Answer 11

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

Question 12

What is fyn kinase?

Answer 12

• 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)

Question 13

By what mechanisms does inflammation occur that leads to neuronal impairment/death?

Answer 13

• 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

Question 14

So in summary, what are the neurotoxic mechanisms in AD?

Answer 14

• 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?

Question 15

Why do we need to understand the molecular and cellular biology of Alzheimer’s disease?

Answer 15

• 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

Question 16

What model systems have been developed to test theories and potential treatments? What are the problems with these?

Answer 16

• 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

Question 17

What are assays for neurotoxicity?

Answer 17

• 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

Question 18

What are advantages for assays for neurotoxicity?

Answer 18

• 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

Question 19

What are disadvantages for assays for neurotoxicity?

Answer 19

• 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)

Question 20

How can we identify potential therapeutic targets?

Answer 20

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

Question 21

What has been identified using MTT/MTS assay of amyloid peptide toxicity?

Answer 21

• 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

Question 22

How do we use chemistry and biochemistry to develop potential drugs?

Answer 22

• 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

Question 23

How many drugs make it through clinical trials compared to how many are tested?

Answer 23

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

Question 24

What knockout mice have been used to model AD? What has been shown using these mice?

Answer 24

• 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)

Question 25

How has ex vivo tissue been used to study AD?

Answer 25

• brain slices can be prepared fresh and used to measure electrophysiological changes and synaptic activity
• latter is critical for subsequent disease effects

Question 26

What other animals have been considered as models for AD?

Answer 26

• C. elegans (nematode worm)
• zebra fish
• drosophila melanogaster (fruit fly)
• dogs? (ethical issues)
• primates (ethical issues/cost)

Question 27

What was the Tg2576 mouse? What did it show?

Answer 27

• 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

Question 28

What was the APP/PS1 transgenic mouse? What did it show?

Answer 28

• 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?

Question 29

Why make tau transgenice mice? What models have been made based on tau? What did they show?

Answer 29

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)

Question 30

In summary, what are the neurotoxic mechanisms in AD?

Answer 30

• 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

Question 31

In summary, what are approaches to understanding the molecular and cellular biology of Alzheimer’s disease?

Answer 31

• 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’)