lecture 20

Question 1

When was Alzheimer’s ‘discovered?

Answer 1

• 1906 - German neuropathologist Alois Alzheimer studied a patient called Auguste D: wrote up the first documented case of the disease that was to be named after him
• examination of Auguste D’s brain after death by Alzheimer revealed ‘globs’ of sticky protein (amyloid) between neurons and tangled bundles of fibrils in neurons (neurofibrillary tangles)

Question 2

What are milestones in the history of Alzheimer’s disease?

Answer 2

• 1910 - 1940: belief that “senile dementia” is a normal part of aging process
• 1960s: AD believed to be a distinct disease (age-related dementia), not just aging
• 1984: Amyloid β peptide purified and sequenced from AD plaques (Colin Masters, University of Melbourne)
• 1992: amyloid cascade hypothesis developed
• 1994: oxidative stress is an important feature of AD
• 1994: role of metals in amyloid plaque formation
• 1998: amyloid oligomers shown to be more important to AD memory impairment than amyloid plaques
• 2000 onwards - development of drugs to target amyloid generation or increase its removal (clinical trials)
• unknown! - why amyloid aggregates and induces neuronal damage and death only late in life

Question 3

Why study this disease?

Answer 3

• estimated 35.6 million patients in the world (more than 500,000 in Australia)
• estimate of 114 million patients by 2050 - World population is rapidly aging - will result in dramatic increase in AD patients
• current world wide cost to health care US$604 billion/year
• chance of getting AD doubles every 5 years after 65 (1 in 4 chance of getting AD after 80)

Question 4

What is the general neuropathology of Alzheimer’s disease?

Answer 4

• gross atrophy (shrinkage) of the brain (characteristic but not specific to AD)
• extracellular neuritic (amyloid) plaques
• intraneuronal neurofibrillary tangles
• cerebrovascular amyloid (cerebral amyloid angiopathy, CAA)
• activation of microglia, hypertrophy of astrocytes
• degree of dementia/memory impairment (amnestic dementia) in AD correlates with loss of synapses
• loss of neurons as disease progresses
• amyloid plaques appear as dark circular structures throughout the grey matter
• accumulation of amyloid in the vasculature of the brain

Question 5

What are amyloid plaques?

Answer 5

• aggregated amyloid β peptide (forming fibrils)
• green-red birefringence with congo red stain
• many non-amyloid β components in plaques
• high concentration of metal ions
• readily turned over in the brain
• may be ‘end point’ of amyloid pathway
• associated with secondary inflammation

Question 6

What is amyloid?

Answer 6

• term amyloid first coined by Virchow in mid 19th century (meaning starch or cellulose)
• amyloidogenic means that protein aggregates appearing red microscopically in normal light but green when viewed in polarised light after staining with congo red dye (termed birefringence)
• distinct from amorphous protein aggregates (that have no structure)
• fibrillar nature and β pleated sheet configuration described by electron microscopy in 1959
• • form non-bracing fibrils of up to 8nm in diameter

Question 7

How do we get amyloid formation?

Answer 7

• formation of fibrils is not specific for a primary protein sequence
• amyloidogenic proteins can begin as unstructured monomers (little alpha-helix or β-sheet structure)
• with increasing concentration or under certain environmental conditions, β-sheet structure increases
• monomers begin to form parallel β-sheet structure (protofibril)
• protofibrils mature into fibrils and form plaques

monomeric –> protofibrils –> mature fibrils –> amyloid plaques

Question 8

What is the amino acid sequence of Aβ monomer?

Answer 8

DAEFRHDSGYEVHHQKL
VFFAEDVGSNKGAIIGLM
VGGVVIA

Question 9

How does oligomeric Aβ compare to fibrillar Aβ?

Answer 9

• monomeric Aβ peptide can aggregate to form fibrillar amyloid
• monomeric Aβ can form oligomeric species consisting of 2-10+ monomers packed closely together
• oligomers may become cross-linked by a specific amino-acid modifications (e.g. di-tyrosine cross-link) increases stability of oligomer
• oligomers are thought the be the primary toxic form of Aβ
• small solubule oligomers –> in vitro neuronal function –> synaptic damage and neuronal cell death
• extended fibrils also follow that path and:
• -> amyloid –> ? inert storage form
• moves backwards and forwards

Question 10

How is amyloid generated?

Answer 10

Generation of amyloid β peptide:

• amyloid β is hydrophobic 40-42 amino acid peptide
• cleaved from a larger Amyloid Precursor Protein by ‘secretases’ (proteases)
• cleavage occurs at the membrane and amyloid β (Aβ) is released into the extracellular space (but can be recycled back into the cell by endocytosis )
• amyloid deposits form between cells (brain parenchyma)

Question 11

Which peptide tends to aggregate more in Alzheimer’s disease?

Answer 11

42

40 seems to be more normal

Question 12

How do we get cleavage of Aβ from APP?

Answer 12

• cleavage is by beta secretase (B-secretase or VACE) and then gamma secretase (γ-secretase) to release Aβ from APP
• APP can also be cleaved by α-secretase at a different site and this prevents Aβ from forming
• the remaining APP can be released as soluble APP
• cleavage is known to occur on the membrane of neurons in the grey matter of the brain (especially the cerebral cortex and hippocampus)
• the end part of the APP molecule when cut by alpha-secretases seems to have a protective effect

Question 13

Why is there a bit of a problem in the way we understand/research disease processes?

Answer 13

• tend to focus on what we can see easily
• easy to see accumulation of amyloid protein
• there may be changes that we can’t see that are even more important e.g. APP intracellular domain

Question 14

What is Amyloid Precursor Protein?

Answer 14

• integral membrane protein concentrated at synaptic connections in the brain
• gene is located on chromosome 21. Downs syndrome (extra Chr 21) over produce APP and Aβ
• APP has domains with different functions such as growth factor-like domain, protease inhibitor domain and metal binding domains
• APP undergoes extensive post-translational processing (phosphorylation, glycosylation and cleavage)
• the function of APP is unknown but a lot of activities have been described:
• • growth promotion
• • regulation of synaptic function
• • metal homeostasis
• • cell signalling

Question 15

What is amyloid turnover?

Answer 15

• origianlly thought that amyloid plaques were long lasting or permanent
• now believed that they are rapidly formed and degraded but the total number increases with age
• amyloid accumulation (plaques or oligomeric forms) depends on synthesis and degradation
• small changes in either can affect the total accumulation
• ~8% of total CSF amyloid is turned over every 36 hours
• proteases in the brain degrade amyloid peptide leading to its clearacne
• • insulin degrading enzyme
• • neprilysin
• • matrix metalloproteases
• • angiotensin converting enzyme
• reduction in protease activity has been observed in AD brain
• microglia can also remove amyloid, especially aggregated peptide and plaques
• recent studies suggest that even a 2% difference between production rate and clearance rate in AD patients may result in accumulation
• • potentially, only small decreases in production or small increases in degradation could shift the balance in favour of clearing amyloid as a therapeutic treatment

Question 16

What is the normal function of amyloid?

Answer 16

• antioxidant molecule

- modulating metal (copper, zinc) homeostasis

Question 17

What is the abnormal function of amyloid?

Answer 17

• aggregation into amyloid
• aggregation into oligomers
• neurotoxic effects
• inflammation

Question 18

What are neurofibrillary tangles?

Answer 18

• protein aggregates found within neurons in AD brain (also called paired helical filaments)
• tangles are composed of hyperphosphorylated form of a microtubule protein called tau
• the hyperphosphorylation causes tau to become insoluble and aggregated into NFTs (filaments of protein twisted together in pairs)
• also associated with tauopathies (frontotemporal dementia)

Question 19

Where is tau under normal conditions? What does it do?

Answer 19

attached to microtubules
allows normal function of cell
(structure, movement, intracellular transport)

Question 20

What happens when tau is hyperphosphorylated?

Answer 20

tau releases from microtubules resulting in abnormal cytoskeletal structure and inhibition of intracellular transport

paired helical filaments
phosphorylated by proteins such as GSK3 and cdk5

Question 21

What can further inhibit the degradation of NFTs by the cell?

Answer 21

increased oxidative stress in the neurons that can cause cross-linking, further inhibiting the degradation of NFTs by the cell

Question 22

What is the BAptist vs TAUist argument?

Answer 22

disagree as to main cause/route of progression of AD
i.e. what occurs first

cause:
tau hyperphosphorylation (NFTs) vs amyloid beta deposition (senile plaques) 

consequences:
amyloid beta deposition (senile plaques) vs tau hyperphosphorylation (NFTS)

synaptic damage and neuronal degradation

Question 23

What are contribution factors in Alzheimer’s disease?

Answer 23

• age
• • simply don’t get AD when you’re 20
• • something that is occurring across decades that we can’t see that is driving this process
• • so many neurons it could take years or decades before loss affected neuronal function
• • the changes seem to be related to slow accumulation of damage of years/damage
• • able to see earlier stages with improved technology
• oxidative stress (metals)
• loss of normal neuronal metal homeostasis
• • linked to oxidative stress
• inflammation

Question 24

What are oxidative and nitrosative stress?

Answer 24

• excessive production of oxygen/nitrogen-based radicals
• ‘leakage’ of molecular intermediates from the mitochondrial electron transport chain
• imbalance between generation and removal of radicals (reactive species)
• balance between need to use oxygen for energy and develop of free radicals
• superoxide dismutase
• peroxynitrite
• hydroxyl radical

Question 25

What are sources of oxygen radicals in AD?

Answer 25

- mitochondrial respiratory chain - brain has high oxygen consumption and low antioxidant levels (compared to the amount of oxygen it's consuming) - non-dividing cells build up oxidatively damaged molecules - amyloid beta can induce radicals - metals (copper and iron) can catalyse free radicals - inflammation (macrophages release radicals)

Question 26

What are short term consequences of excessive oxygen radicals?

Answer 26

- lipid peroxidation (malondialdehyde and hydroxynonenal) - protein oxidation (carbonyl modified proteins) - DNA oxidation and strand breaks

Question 27

What are downstream effects of oxidative stress?

Answer 27

- neurotoxic actions of altered lipids - apoptosis - accumulation of aggregated protein - disruption to normal protein turnover and axonal transport - DNA damaged - altered transcription

Question 28

What is the altered biometal homeostasis in AD?

Answer 28

- early studies found aluminium in plaques (minute quantities and unlikely to have any role in disease) - strong evidence for altered zinc and copper metabolism in AD brain - high levels of copper and zinc found in AD plaques - high extracellular levels of copper and zinc in AD brain regions where neuronal degeneration is highest - evidence for localised extracellular copper and zinc accumulation in neurodegeneration - concurrent decrease in intracellular or bioavailable biometals in diseased brain - metals are the main source of free radical generation in the brain - amyloid beta binds copper and zinc at several sites involving histidine and tyrosine amino acids - binding of metals by Aβ - - promotes aggregation of the peptide - - copper induces formation of neurotoxic oligomers - - copper causes cross-linking between Aβ monomers to form stable neurotoxic dimers

Question 29

What are the main source of free radical generation in any biological system?

Answer 29

biometals e.g. Cu, Fe

Question 30

What are amyloid metal interactions?

Answer 30

- amyloid deposits contain large amounts of copper and zinc - Aβ has binding sites for copper and zinc - binding sites involve metal co-ordination by histidine and methionine/tyrosine residues - zinc binding to Aβ may prevent formation of neurotoxic oligomers by displacing copper - Cu(II) can be reduced to Cu(I) by Aβ - resulting in formation of free radicals (damage proteins, lipids etc = neurotoxicity) - the free radicals can also further enhance Aβ aggregation and cross-linking to enhance toxicity of the peptide - metals are excreted from the neuron (through exocytosis) and are trapped by Aβ - enhances aggregation into oligomers (toxic)

Question 31

What are sources of inflammation in AD?

Answer 31

- resting resident brain macrophages (microglia) become activated - infiltration of peripheral monocytes

Question 32

What initiates infiltration of inflammation in AD?

Answer 32

- likely response to aggregated amyloid | - response to degenerating synapses and neurons

Question 33

What are consequences of inflammation in AD?

Answer 33

- release of neurotoxic cytokines - increased free radical production - further damage to neurons and synapses

Question 34

So in summary, what is alzheimer's?

Answer 34

- age related disease affecting over 65 (except genetic forms) - characterised by: - - loss of memory and higher brain function - - brain atrophy - - extracellular amyloid plaques - - intracellular neurofibrillary tangles - - gliosis (activated microglia and hypertrophic/swollen astrocytes)

Question 35

What are amyloid plaques? (summary)

Answer 35

- composed of amyloid beta peptide - amyloid is bi-refringent (red under normal light and green/yellow under polarising light when staind with the dye Congo red) - amyloid peptide: - - undergoes conversion from predominantly alpha-helix to high β-sheet content - - aggregates - monomers align/stack - cross-linked - - forms neurotoxic oligomers (dimer, trimer etc) - - forms fibrils (probably inert and make up amyloid plaques)

Question 36

What is amyloid beta? (summary)

Answer 36

- derived from larger Amyloid Precursor protein (APP) by cleavage with secretases (BACE and gamma secretases) - released into extracellular space - APP is a large transmembrane protein with several domains and a number of potential functions (potential synaptic function) - role in AD (other than source of amyloid peptide is unknown)

Question 37

What is tau? (summary)

Answer 37

- intracellular microtubule associated protein - becomes hyperphosphorylated on a number of amino acids in AD (by several kinases) - hyperphosphorylation causes breakdown of microtubules and loss of cytoskeletal integrity - aggregation of phosphorylated tau into fibrils results in formation of NFTs - neurotoxic (can kill neurons)

Question 38

What are contributing factors in AD? (summary)

Answer 38

oxidative stress - free radical (reactive oxygen species) mediated damage to proteins, lipids and DNA (probably from biometals/Aβ) - higher in non-dividing cells (neurons in brain) which accumulate oxidatively damaged molecules and have lower antioxidant levels and high oxygen use (oxygen required for ox stress) - oxidatively altered molecules disrupt normal cell processes biometals - accumulate in amyloid plaques to high levels - copper, zinc, and iron - bind to Aβ - promote aggregation and neurotoxicity - generate free radicals and oxidative stress neuroinflammation