Week 7: Neuroscience

Question 1

What are the normal mechanisms that protect the cell against misfolded proteins?

Answer 1

1. Chaperone molecules protect proteins from aggregation while they are folding
2. Proteosomal system ubiquinates any problematic proteins and sends them to the proteosomes which breakdown these proteins- but sometimes this system can be overwhelmed

Question 2

What different formations can proteins exist as?

Answer 2

1. Synthesised proteins can remain in an unfolded state
2. Unfolded proteins may go onto form a local secondary structure forming a stable hydrophobic core (an intermediate state) and then reach its native functional state
3. Native functional state proteins can form a number of structures including functional oligomers, fibres and crystals
4. Unfolded proteins are potentially dangerous as they can interact with other proteins (often via hydrophobic reactions) and lead to disordered aggregates forming (the formation of disordered aggregates is what chaperone molecules aim to prevent)
5. Unfolded proteins may also be degraded by proteases into degraded fragments (a irreversible process)- these degraded fragments may for disorded aggregates
6. Intermediate structured proteins that are folded improperly can form prefibrillor species which can also generate degraded fragments
7. Degraded fragments and/or improperly folded intermediates (prefibrillar species) interact to generate B-sheet type orders which are potentially toxic e.g. amyloid fibrils- the formation of amyloid fibrils from degraded fragments and improperly folded intermediates is largerly irreversible

Question 3

What was the process of Anfinsen’s experiments and what did it show us?

Answer 3

1. Anfinsen used an RNase proteins in its native state that was catalytically active
2. Anfinsen added urea (which disrupts hydrogen bonding in the protein) and mercaptoethanol (which reduces the disulphide bonds)
3. The protein was then studied and it was seen to be unfolded and completely inactive
4. Dialysis was done to remove the urea and mercaptoethanol
5. The protein then spontaneously refolded into its native, catalytically active state

• The experiment showed that the primary sequence of amino acids contains all the information required for a protein to fold into its native 3D state, therefore we should be able to predict the structure of a protein from its amino acid sequence (not always the case e.g. B-sheet rich amyloid states_

Question 4

What are the 3 classical protein folding mechanisms?

Answer 4

1. Framework model:
   - Certain parts of the unfolded protein are predisposed to forming a-helices whilst others are predisposed to forming B-sheets
   - These local secondary structures form a framework which collides to form the 3D protein
2. Nucleation model:
   - One part of the protein adopts a particular secondary structure it is predisposed to, but then propegates in an outward direction with the 3D state forming in a step-wise manner (the adjacent parts of the protein fold onto the nucleus of the structure)
3. Hydrophoblic collapse:
   - The hydrophobic residues first collapse to the centre of the molecule
   - Secondary structures then take form after that
   - The 3D form of the protein is subsequently formed

• Summary: protein folding is considered as an amalgamation of all these models

Question 5

Describe the standard thermodynamic nature of protein folding?

Answer 5

• Protein folding can be described as a free energy tunnel
• Unfolded states have a relatively high free energy and a large number of species
• As folding proceeds the intermediates form which restricts the number of species present which narrows the funnel
• At the bottom of the funnel a single native state is present that has a relatively low free energy- it will have the most hydrogen bonds and electrostatic interactions satisfied and the most internalization of hydrophobic residues
• Bumps within the funnel are intermediate structures formed- conformation traps

Question 6

What is the actual free energy protein folding tunnel like?

Answer 6

• In reality the energy landscape is rough and there can be poor folding fidelity
• This funnel has folding intermediates at a higher gibbs free energy than the native state
• Higher in energy than the native state also are partially folded states which can (for reasons partially unknown) give rise to other possible 3D confirmations
• The other possible 3D confirmations include B-sheet type fibrils and amyloid fibrils (twisted fibrils)

Question 7

What diseases are associated with protein aggregation (the formation of extracellular amyloid fibrils or intracellular inclusions)?

Answer 7

• Most of these diseases are sporadic (85%) although hereditary forms have been documented (10%)
• There is a correlation between the disease the the amyloid structures within affected tissues (the amyloid structures did not necessarily cause the disease)

1. Parkinson’s Disease:
   - a-synuclein protein misfolds and forms amyloid structures in the form of Lewy bodies
2. Creutzfeldt-Jakob Disease:
   - Prion protein forms vCJD amyloids
3. Alzheimer’s Disease:
   - B-amyloid protein forms B-amyloid plaques
4. Huntington’s Disease:
   - Huntingtin protein forms Hungtintin inclusions

Question 8

What are Amyloids?

Answer 8

• Amyloid refers to the extracellular or intracellular fibrillar deposits associated with disease
• Amyloidoses (diseases caused by amyloids) are slow onset and degenerative
• The correlation between diseases and amyloids suggests that fibril formation is pathogenic
• Amyloidogenic proteins are distinct (different) with respect to amino acid sequence and native fold
• Fibrils are structurally similar (have very high levels of B-sheet formation) as shown by a number of tests including FT-IR, Thioflavin T and Electron microscopy
• The common properties of amyloids implies similar mechanisms of fibrillogenesis and disease pathogenesis

Question 9

What are some functional roles of amyloid fibrils in living systems?

Answer 9

• Some organisms are found to convert one or more of their endogenous proteins into amyloid fibrils that have functional rather than pathogenic properties
  1. Curlin: E.coli use curlins to colonise surfaces and bind host proteins
  2. Chaplins and Hydrophobins: help reduce water surface tension
  3. Spidron: part of spider’s silk
• In humans amyloid fibrils tend to be pathogenic

Question 10

How are amyloids formed?

Answer 10

1. An amyloidogenic intermediate (a protein that has not folded properly) folds into a B-sheet rich structure
2. These B-sheet rich structures propegate and stack upon each other to form continued B-sheet structures
3. The B-sheet rich structures then twist around eachother to form stiffer, thicker rods which are the amyloid fibrils
4. The amyloid fibrils cluster together in damaged tissue to form deposits

Question 11

What is the structure of amyloid fibrils?

Answer 11

• EM shows fibrils to be straight and 7-12 nm in diameter
• NMR and X-ray fibre diffraction indicates a cross-beta structure of 4.7A repeating structures which are perpendicular to 10A repeating structures
  i. e. strands in the sheet are hydrogen bonded 4.7A apart and layered sheets are hydrogen bonded 10A apart
• Amyloid fibres bind Congo red and thioflavin T which indicates they are B-sheet rich
• Crystal diffraction and FT-IR indicate a high B-sheet content
• Only a core part of the protein forms the B-sheet rich structure, the other parts have their native confirmation forming random loops

Question 12

Describe the kinetics of fibril formation:

Answer 12

• The time course for the conversion of a protein into a fibrillar form typically includes a lag phase which is followed by a rapid exponential growth phase
• Within the lag phase: a small proportion of the protein starts to misfold which then propegates and causes surrounding healthy proteins to adopt the misfolded B-sheet rich structure (it “seeds” the misfolding of other healthy proteins)
• Within the elongation phase: the protein rapidly propagates into a longer fibrils
• Healthy proteins have long lag phases where there is no aggregation, adding a misfolded protein “seed” into a healthy protein sample will reduce the lag phase as the “seed” acts as the nucleus
• Amyloid fibrils are formed via the nucleation model of folding whereby an initial secondary structure forms which creates the nucleus and the rest of the fibril forms in a step-wise manner

Question 13

What is the process of pathogenesis in protein deposition (fibril) diseases?

Answer 13

• Pathogenesis can be a combination of both loss of function and gain of function processes

1. Loss-of-function:
   - A specific protein may be unable to function because it is incorrectly folded
   - Misfolding may lead to incorrect protein trafficking
   - Aggregation leads to loss of protein function
2. Gain-of-function:
   - Cellular function is impaired due to interaction between the aggregate and cellular components
   - Non-neurological amyloidoses are due to large deposits of aggregated protein in/around vital organs

Question 14

What contributes to the toxicity of amyloids?

Answer 14

• The structures that form early in the aggregation process are the most toxic to cells (pre-fibrillar aggregates)
• As the protein becomes more aggregated (becomes mature fibrils) the cell viability actually increases

Question 15

Describe a potential therapeutic prospects for treating non-neurological amyloidoses?

Answer 15

• Non-neurological amyloidoses can cause disease by accumulating fibrils that then form deposits around organs

E.g. Transthyretin amyloid disease:

1. Transthyretin is a tertamer transport protein in serum that carries thyroid protein
2. This protein is very susceptible to forming amyloid fibrils
   - There are many single mutations which predispose transthyretin to forming amyloid
3. Amyloid fibrils are formed from a the tetramer in a process that goes from tetramer -> native monomer -> amyloiogenic (misfolded) monomer -> amyloid fibril
4. When the protein dissociates into its native monomer form (as part of the equilibrium process) it is at a danger of forming a misfolded B-sheet rich amyloidogenic monomer which can assemble to form amyloid fibrils
5. Anything that shifts the equilibrium back to the tetramer form of protein could be used as a therapeutic
   e. g. the addition of a ligand (a thyroid hormone like molecule) to the binding site that stabilised the tetramer
   - There is redundancy of the thryoid hormone transport system so even if transthyretin is bound by these ligands there are other proteins in blood that can transport thyroid hormone

Question 16

What are polyglutamine diseases?

Answer 16

• Diseases in which proteins have an excessive number of glutamine residues in a row
• They are acquired within DNA, and manifest as expanded regions of polyQ (many glutamine residues) within other normally functional regions
• There are 9 main types of proteins in which polyQ regions cause protein aggregation and disease
  e. g. huntingtin, atrophin 1 and the ataxins etc.

Question 17

What is Machado-Joseph Disease?

Answer 17

• An inherited spinocerebellar ataxia disease caused by polyQ expanion of ataxin3
• A group of people in Australia (Groot Eylandt) have a mutation in their gene for ataxin3 which predisposes them for this condition
• The disease causes neurodegeneration that occurs at about the age of 30 and people die 10-15 years later

Question 18

What are polyglutamine repeat proteins?

Answer 18

• Polyglutamine repeat proteins are proteins in which the codon CAG (that encodes glutamine) expands to form poly-Q regions that lead to fibrillogenesis
• The long glutamine tracts tend to promote protein aggregation by self-interaction
• The expansion of the polyQ region to more than 27 glutamines generally correlates with neurodegenerative disease due to the formation of nuclear inclusions in neurons which cause neuronal dysfunction and death
• The severity and age of disease onset (usually 30-50 years old) is related to polyQ region length
• There are 9 proteins affected by polyglutamine expansion:
  1. Huntingtin: causes huntington’s disease
  2. Atrophin 1: causes Dentatorubral-pallidolusysian atrophy
  3. Androgen receptor: causes Spinal bulbar muscular dystrophy
  4-8: Ataxin 1,2,3,6 and 7: causes spinocerebellar ataxia 1,2,3,6 and 7
  9. TATA binding protein: causes spinocerebellar ataxia 17

Question 19

What do polyQ proteins have in common?

Answer 19

1. A tendency to form B-sheet structures

2. A glutamine tract that expands over generations and makes those proteins even more pre-disposed to self-associate

Question 20

Why are neurons so susceptible to polyQ expansion?

Answer 20

• PolyQ expansion leads to the formation of fibrils
• Neurons are very vulnerable to the formation of fibrils
• Neurons cannot be readily replaced as they have poor regenerative capacity
• This makes neurons very susceptible to dysfunction as a result of fibril formation

Question 21

How are PolyQ length and disease onset age related?

Answer 21

• The longer the polyQ tract is, the earlier the age of onset
• Furthermore, the more severe the phenotype of the disease

Question 22

What is Huntington’s Disease?

Answer 22

• A neurodegenerative disease characterised by progressive motor impairment, cognitive decline and emotional disorientation
• 10% of cases exhibit symptoms before the age of 20 but most cases occur after 55
• It is a dominant genetic disorder that is transmitted autosomally
• It is due to an unstable CAG repeat in the first exon of Huntingtin protein which is present in many cell types
• The intranuclear inclusions of Huntington and Ubiquitin in neurons of the striatum and cerebral cortex leads to dramatic neuronal loss in the brain
• Even though during fibril formation the cell will try and get rid of the misfolded fibrils by tagging them with Ubiquitin, the proteosomal system becomes overwhelmed and all the ubiquinated fibrils cannot be degraded

Question 23

What affect do PolyQ aggregates have on a cell’s proteasome system?

Answer 23

• When PolyQ aggregates accumulate in the cell they overwhelm the ubiquitin-protease system
  i. e. when pathogenic polyQ proteins are present in the cell the ubiquitin-protease system will not be able to degrade other proteins as it is so overwhelmed by the pathogenic polyQ which is ubiquinated but cannot be degraded

Question 24

What are the mechanisms of PolyQ Toxicity?

Answer 24

1. Expanded polyQ disrupts gene transcription:
   - PolyQ recruits any other proteins in the cell which have glutamine tracts
   E.g. A number of polyQ proteins interact with proteins in the transcription complexes
   E.g. misfolded polyQ regions can bind proteins such as transcriptional coactivators
   - Therefore cellular toxicity occurs due to the sequestration of these factors (such as transcriptional activators) into the polyQ aggregates
2. PolyQ blocks the proteosome:
   - The protein aggregates are ubiquinated but cannot be sucessfully destroyed by the proteosome
   - This blocks the ubiquitin-protease system which leads to the build-up of many proteins in the cell

Question 25

How does fibril formation occur in CAG repeat Huntingtin protein?

Answer 25

Kinetics:

1. When the concentration of Huntingtin protein is increases, there is increased B-sheet formation/aggregation and a decreased lag time
2. When a “seed” is added there is a reduction in the lag phase
3. When polyglutamine length is increased (more CAG repeats) there is less lag time and rapid formation of B-sheet structures compared to shorter polyQ lengths where the protein can remain soluble over a range of concentrations

Structure:

1. Expanded polyQ regions do NOT cause structural changes in the surrounding protein- rather the polyQ expansion reduces protein stability
2. Fibrillisation occurs via prefibrilliar intermediates
   - Huntingtin protein is expressed as a fusion protein with MBP
   - The MBP-Htt protein can be cleaved with a protease
   - The longer polyQ region (e.g. 44Q) MBP-Htt protein forms soluble monomers but over time can form small oligomers (these may be the toxic species)
   - These small oligomers start to form single protofibrils
   - The protofibrils then twist around each other to form strong fibres (amyloid fibres_

Stability:

• Proteins have an equilibrium between their unfolded -> intermediate -> native forms
• If the equilibrium lies closer to the (folded) native form the protein is said to be stable
• When polyQ regions are longer the protein is LESS stable and the equilibrium is shifted towards the unfolded species

Question 26

What is the therapeutic concept or chaperone folding?

Answer 26

• Experiments in drosphila with polyQ proteins that normally destroy eye cells where the polyQ protein is co-expressed with heat shock protein 70 (HSP) which is a standard chaperone protein that binds to hydrophobic regions and prevents improper interactions between misfolded proteins)
• The eye cells with the polyQ protein co-expressed with HSP70 remained healthy
• It is now theorised that increasing the concentration of natural chaperones such as HSP70 in human cells could help treat patients with neurodegenerative diseases

Question 27

What is the therapeutic prospect of stabilising the native state?

Answer 27

• Through screening the small molecule trehalose (a sugar) was seen to stabilise the native state of Huntingtin protein
• It was shown to protect the protein against destabilisation (as seen from its maintenance with increased temperature)
• It was shown to restore a normal hanging phenotype in mice

Question 28

What is Alzheimer’s Disease?

Answer 28

• AD is the most common neurodegenerative disease, most cases are late onset (>65) and sporadic
• It is due to protein misfolding
• The hallmarks of AD are:
  1. Extracellular B-amyloid plaques
  2. Intracellular neurofibrillary tangles (made from phosphorylated tau -ptau)
• AD afflicted brains have neuronal destruction leading to decreased size and reduction in glucose usage
• AD disrupts the ability of neurons to communicate, carry out metabolism and repair themselves

Question 29

Describe the 2 abnormal structures found in the brains of people with AD:

Answer 29

1. B-amyloid plaques:
   - Dense deposits of protein and cellular material that accumulates outside and around nerve cells
   - Extracellular
   - Made up of AB peptide
2. Neurofibrillary tangles:
   - Twisted fibres of hyperphosphorylated tau protein that builds up within the nerve cell
   - Intracellular
   - Made up of ptau

Question 30

What is the mechanism of B-amyloid plaque formation?

Answer 30

1. Amyloid precursor protein (APP) is the precursor to an amyloid plaque
2. The APP is an extracellular protein that sticks through the neuron membrane
3. Enzymes (proteases known as secretases) cut the APP into fragments of protein which include B-amyloid
4. B-amyloid fragments (particularly the AB 1-40/42) aggregate to form plaques

Question 31

How does the cleavage of APP by secretases determine what form of amyloid B is produced?

Answer 31

• When APP is embedded inside the membrane there is a C terminus inside the cell and the N terminus outside the cell
• There are 3 types of secretases:
  1. a-secretases: cuts at residue 17
  2. b-secretases: cuts at residue 1
  3. y-secreases: cuts at the portion between the membrane which is somewhere between 40-42)
• The AB (17-40/42) that is formed after the action of a- and y-secretases is non-toxic
• The AB (1-40/42) that is formed after the action of b- and y-secretases is extremely toxic and predisposed to forming B-sheet like fibrillar structures

Question 32

Describe the kinetics of AB fibril formation:

Answer 32

• The AB(1-40/42) forms fibrils via a “nucleated growth mechanism”
  1. Over time (the lag phase), a small B-sheet structure forms which them goes on to form an oligomer
  2. The B-sheet then rapidly lengthens and thickens (in the elongation phase) to form the amyloid fibrils

Question 33

Describe the structure of amyloid B oligomers:

Answer 33

• Amyloid B (1-40) and (1-42) are mainly unstructured, with a small section predisposed to forming an a-helix)
• The peptides are bipolar
• AB (1-42) is more aggregation prone than AB (1-40)

1. AB Oligomers form via interactions between their C-terminal hydrophobic ends- these C-terminal ends tend to self-associate as dimers or trimers
2. These self-associated AB-peptides start to exhibit characteristics of B-sheet formation as the hydrophobic end forms a fold on itself and forms an antiparallel B-sheet structure
3. This B-sheet structure can build up upon itself to form long B-sheet based fibrils

Question 34

What are the current AD drugs in development?

Answer 34

• Current drug discovery approaches in AD are focused on:
  1. Preventing AB formation or improving ‘normal’ APP processing via the inhibition of y-secretase and B-secretase or the activaiton of a-secretase

2. Removing existing amyloid deposits using immunotherapeutic approaches e.g. antibodies or vaccines against amyloid

• There are currently drugs that are y-secretase inhibitors, a-secretase activators and antibodies against various forms of AB
• Currently the only approved therapy is Valsartan which is an angiotensin receptor blocker than reduces blood pressure
• Many drugs don’t pass clinical trials as once AD is diagnosed there is often too much neuronal damage for the drugs to be useful
• The next biggest drug in AD is Aducanumab which is shown to reduce levels of amyloid in the brain, slow rate of memory decline and is well tolerated

Question 35

What is the tau hypothesis?

Answer 35

• Neurofibrillary tangles (NFT) are intracellular deposits formed from aggregated hyperphosphorylated tau protein
• NFTs are seen in AD as well as other neurodegenerative diseases which leads to the hypothesis that they may be the PRIMARY CAUSE of neurodegeneration

Question 36

What is the mechanism of Tau action in the cell and how is this affected by hyperphosphorylation?

Answer 36

• Tau is a microtubule stabilising protein that is regulated by phosphorylation:
• There are 6 tau isoforms which vary in their number of +vely charged microtubule binding domains (R) and acidic stretches (N)
• Kinases regulate how tightly tau is bound to the microtubules- the more the tau proteins are phosphorylated, the more negatively charged they become and the less they are able to bind the negatively charged microtubules
• Neurons transport vesicles up and down dendrites and axons using microtubules
• The formation and reformation of microtubules is coordinated by the tau protein
• When tau proteins are hyperphosphorylated, due to overactive kinases or underactive phosphatases, they fall of the microtubules and form NFTs they can no longer stabilise them so microtubules degrade and become dysfunctional
• Dysfunctional microtubules disrupts axonal transport and dendrite structure leading to dendrites being starved of cargo vesciles and autophagy being peturbed
• Peturbed autophagy leads to neuronal swelling and inflammation
• Therapeutic efforts are therefore targeted at preventing tau hyperphosphorylation and stabilising microtubules

Question 37

How does synaptic function between normal neurons compare to that between AD neurons?

Answer 37

Normal synaptic function:

• Neurotransmitter is released from vesicles at the presynaptic terminal into the synpase
• APP may produce AB peptide, but this is cleared away
• Healthy microtubules within the neurons transport organelles and vesicles

AD synaptic function:

• AB is released and does not get cleared away so it aggregates into AB plaques
• The AB plaques block the synapse and prevent proper communication
• Small AB oligomers may enter cells and cause toxicity
• Hyperphosphorylation of tau causes microtubules to break apart with the aggregation of ptau causing the formation of NFTs and the disruption of the intracellular transport system

Question 38

What are the future direction of AD Therapy developments?

Answer 38

• Developing a means for early diagnosis of AD; as many clinical trails fail because the disease is already too far progressed
• Recognising amyloid plaques may not be the direct cause of the disease so future research aims to better understand the complete pathological process

Question 39

What are prion diseases?

Answer 39

• Prion diseases (originally called transmissible spongiform encephalopathies) are a slow-onset, progressive group or neurodegenerative diseases
• Prion diseases are transmissible via infected tissue
• Symptoms often involve ataxia and progressive dementia
• Human examples include: Creutzfeldt Jakob Disease (CJD) and kuru
• The first observed prion disease was Kuru which was seen in members of a tribe in PNG that injested contaminated tissue
• They can also be spread by the exposure to infected tissue through surgery e.g. via infected corneal implants

Question 40

What is the pattern of neurodegeneration in Prion Diseases?

Answer 40

• Characteristics of prion diseases are the presence of holes in the brain tissue
• Neurodegeneration is accompanied by aggregates of protease resistant fibrils and plaques
• Sporadic cases are very rare (more cases are familial)
• Prion diseases are transmissible via infected tissue

Question 41

What is the cause of prion disease neurodegeneration?

Answer 41

• The cause of Prion disease neurodegeneration is the misfolded conformation PRION protein (PrPsc) ALONE
• You cannot get prion disease withouth the PrP gene
• The PrPsc acts as a template and converts normal prion protein to the misfolded form
• PrPsc then damages cells by either gaining toxic activity (through the formation of oligomers or the aggregate structure) and/or causing loss of biological function of the protein(s) trapped in the aggregate

Question 42

What is the structure of prion protein?

Answer 42

• Prion protein is a surface glycoprotein that binds metals (copper) and may have a role in neurotransmission (but the normal function is unknown) and it is anchored to the cell membrane by the GPI anchor
• Prion protein is 208 residues long and contains in its native form has a long flexible random coil sequence towards the N-terminal end and is mainly a-helical toward the C-terminal end (PrRc)
• The PrP protein is high in glutamine content and low in complexity meaning it has a high propensity to form a self aggregating amyloid
• Prion protein can become misfolded (PrPsc) where it has a higher beta-sheet content, is thermodynamically more stable and is more resistant to proteases

Question 43

How is native conformation prion protein converted into misfolded prion protein?

Answer 43

• The conversion of PrPc (native prion protein) to PrPsc (misfolded prion protein) does not happen readily- but once some PrPsc forms it can act as a TEMPLATE and converts other PrPc into PrPsc
• This is why exposure to converted PrPsc in infected tissue can trigger disease

Question 44

Why are glutamine rich regions found in some proteins (such as PrP)?

Answer 44

• Q-rich low complexity domains are important in RNA biology
• Q-rich domains underlie the ability of proteins to form transient subcellular structures such as “stress granules”
• Cells often respond to stress by changing where their proteins and RNA are inside the cell- this occurs not only through organelles but also through the self-association of Q-rich regions of proteins
• When cells are exposed to stress, granules appear inside the cell- these granules are the result of the aggregation of Q-rich protein domains
• These stress granules help keep molecules like mRNA safe inside them during the time the cell is under stress (as a kind of ‘membrane-less organelle’)

E.g. TIA-1 protein is an RNA recruitment protein, when a cell is stressed, it wants to focus on dealing with the stress (by overexpressing heat shock proteins) and reduces the focus on other cellular functions such as RNA recruitment

• The formaiton of TIA-1 granules during stress allows for the down-regulation of RNA recruitment
• This occurs through the self-association of the TIA-1 proteins via their prion related domains (which have the same distribution of glutamines as seen in the prion protein)
• When stress subsides, the granules dissipate (important: the formaiton of stress granules is REVERSIBLE) and the proteins are released and able to resume function

Question 45

How similar is TIA-1 prion related domain to the prion protein?

Answer 45

• The prion related domain of TIA-1 (a protein that self-aggregates into stress granules in a reversible manner) is very similar to the glutamine-rich region near the N-terminal of the PrP protein
• TIA-1 proteins do display amyloid-like properties in vitro when they are forced to be in high concentration

Question 46

What other RNA binding proteins are involved in disease?

Answer 46

• There are other proteins that are able to bind RNA and self-associate (due to Q-rich domains) in times of stress to form stress granules
• These include TDP-43 and FUS proteins
• Mutant forms of these proteins underlie ALS

Question 47

What is ALS?

Answer 47

• Amytrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that affects the neurons that control voluntary muscles
• The disease is characterised by still muscles, muscle twitching and progressive weakness
• In ALS, protein aggregates are most common in spinal cord neurons
• The cause is mostly unknown

Question 48

What mutations can cause familial ALS?

Answer 48

• 5% of cases of ALS are genetic and due to mutations in TDP-43 or FUS
• 1% of cases of ALS are due to sporadic formation of inclusions by TDP-43 or FUS
• TDP-43 and FUS co-localise in FUS-positive inclusions and ubiquitin is also seen in this aggregate
• Mutations causing ALS in the TDP-43 and FUS genes mainly occur in the prion related domain regions which are glutamine-rich regions: therefore ALS causative mutations indicate that the disease is due to increased aggregation, leading to fibril formaiton
• Mutations in FUS also occur at the nuclear localisation signal causing FUS to accumulate in the cytoplasm
• ALS mutations impair the function of “membraneless organelles” due to the risk of B-sheet formation events