19Neuro - Prion Diseases

Question 1

Name 5 (non-human) species in which the prion protein has been found.

Answer 1

Cats, sheep, cows, hamsters and mice

Question 2

Name 4 types of prion diseases identified in humans.

Answer 2

Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), Fatal Familial Insomnia, Kuru

Question 3

What is the name of the prion disease found in cattle?

Answer 3

Bovine Spongiform Encephalopathy (BSE)

Question 4

What is the name of the prion disease found in sheep?

Answer 4

Scrapie

Question 5

What is Scrapie?

Answer 5

Scrapie is a prion disease affecting sheep and goats of middle-age.

Question 6

What are the symptoms of Scrapie?

Answer 6

Scrapie causes sheep to scrape against posts/fences. It also causes coordination problems.

Question 7

What causes the neurological symptoms observed in Scrapie sheep?

Answer 7

Gross vacuolation of the brain (spongy) due to amyloid deposits.

Question 8

Why is the amyloid found in Scrapie difficult to degrade?

Answer 8

It is highly insoluble and protease resistant.

Question 9

What is the incubation period of the Scrapie prion?

Answer 9

The incubation period is very long - it can take many months or even years.

Question 10

Scientists inoculated hamsters’ brains with extracts from Scrapie sheep’s brains. What was the key finding of this experiment?

Answer 10

That inoculation caused “Scrapie” like symptoms in the hamsters. It was therefore TRANSMISSIBLE.

Question 11

What were the three key findings of the “hamster assay”?

Answer 11

1) “Scrapie” symptoms were transmissible via scrapie-infected tissue.
2) Infectious particle is capable of some form of replication.
3) No bacteria or viruses appeared to be present.

Question 12

In the “hamster assay”, what evidence is there to support the idea that the prion can replicate?

Answer 12

Brain fractions from the inoculated hamsters were found to have 7000x greater infectivity titre than the hamsters had been infected with.

Question 13

In the “hamster assay”, what evidence is there to support the absence of bacteria and viruses in the infectious material?

Answer 13

Destruction of nucleic acids (with UV, chemicals) did NOT destroy infectivity. Treatment with protein denaturants did destroy the infectivity.

Question 14

What is a prion?

Answer 14

A proteinaceous infectious particle.

Question 15

The discovery of prions was achieved via an assay using hamsters. Describe the process used.

Answer 15

1) Scrapie-infected sheep brains were homogenised, extracted with detergents and treated with proteinase K.
2) Intracerebral injection of prions into hamster - this was followed by a long incubation period.
3) More prions were recovered than were used in the inoculation.

Question 16

What do prions look like?

Answer 16

Prions are rod-like particles.

Question 17

Who is credited as the scientist who conceived of the concept of an infectious protein?

Answer 17

Stanley Prusiner in his paper “Novel Infectious Proteinaceous particles cause Scrapie”. At the time the idea was highly controversial.

Question 18

What is the name of the polypeptide isolated from prions using Proteinase K?

Answer 18

PrPSc

Question 19

What is the size of the PrPSc polypeptide?

Answer 19

27-30 kDa

Question 20

Give five key features of PrPSc.

Answer 20

1) Resistant to proteases and highly insoluble
2) Co-purifies with prions
3) Most abundant macromolecule in prions
4) Concentration proportional to infectivity titre
5) Absent from uninfected animals

Question 21

How was PrPc discovered?

Answer 21

Two bio-molecular techniques were used in the discovery of PrPc;
1) From Scrapie infected brain tissue, mRNA was isolated. This was used to generate a cDNA library.
2) From Scrapie infected brain tissue, PrPSc polypeptide was isolated. N-terminal sequencing was then used and an oligonucleotide probe was created.
From these techniques it was possible to: screen the library, sequence clone, identify “normal” gene and protein product. PrPc sequence is identical to PrPSc

Question 22

What is PrPc?

Answer 22

PrPc is the normal cellular form of the prion protein. It is found on the membranes of cells.

Question 23

What are some of the key features of the PrPc gene?

Answer 23

It is a single gene with single exon = isoforms cannot arise by alternate splicing.
Probably a “housekeeping” gene.
Candidate PrP genes identified in human, mouse, hamster, sheep, goat, rabbit, nematode, drosophila
On human chromosome 20 (homologous mouse chromosome 2)
Familial spongiform encephalopathies associated with mutations in PrPc gene (PRNP)

Question 24

What is the structure of the PrPSc protein from N-terminal domain to C-terminal domain?

Answer 24

At the N-terminal domain, there is a signal peptide followed by an octapeptide repeat region. This is followed by the Proteinase K resistant core of the protein which contains a hydrophobic region. At the C-terminus there is a GPI-anchor signal. The C-terminal domain is structured whilst the N-terminal domain is unstructured.

Question 25

To what extent is the PrPc gene present and expressed in infected and non-infected brain tissues?

Answer 25

Gene present and expressed in normal brains. The levels of mRNA in normal and infected brains are similar.

Question 26

How are PrPSc and PrPc digested by Proteinase K?

Answer 26

PrPc starts at 33-35 kDa and is completely digested by Proteinase K. PrPSc is partially digested from 33-35 kDa to 27-30 kDa by Proteinase K.

Question 27

How are the amino acid sequences of PrPc and PrPSc related?

Answer 27

The amino acid sequences of PrPc and PrPSc are identical and the product of the same gene.

Question 28

What does the gene sequence suggest the protein has?

Answer 28

The gene sequence suggests that the protein has an N-terminal signal sequence, glycosylation sites, C-terminal hydrophobic domain which could be cleaved to reveal a GPI-anchoring site.

Question 29

What is GPI?

Answer 29

Glycosylphosphatidylinositol

Question 30

What is believed to account for the differences in properties, infectivity and protease resistance between PrPc and PrPSc?

Answer 30

It is believed that post-translational modifications account for these differences. These PTMs could be simply protein folding.

Question 31

What is the result of PrPc knockout in mice and cows?

Answer 31

PrPc knockout mice and cows show little phenotypical change.

Question 32

Where is PrPc normally localised?

Answer 32

PrPc is normally localised to the synaptic boutons.

Question 33

What do PrP 0/0 mice demonstrate?

Answer 33

PrP 0/0 mice demonstrate impaired GABA-receptor mediated fast inhibition and long term potentiation. These things are associated with memory.

Question 34

What other roles is PrPc believed to carry out within the cell?

Answer 34

PrPc may have the following roles within the cell:
Neuroprotective? Anti-apoptotic activity (anti-Bax)
Signalling through ERK2 and NCAM
PrPc binds metal ions e.g. Cu2+ = Cu2+ reuptake from synaptic cleft?
Nitrosylation of MNDA glutamate receptors, reduces [Ca2+]
May regulate cell adhesion

Question 35

Are prion diseases believed to be due to a toxic gain of function or a toxic loss of function?

Answer 35

Prion diseases are believed to be due to a toxic gain of function.

Question 36

How is PrPSc different to PrPc?

Answer 36

It is partially resistant to proteolysis, it losses some of its alpha-helices and gains beta-sheet. (This may indicate that PrP is an intrinsically disordered protein which is prone to conformational change and subsequent aggregation. There is also a change in epitopes; as there are PrPSc specific antibodies.

Question 37

What do we know about PrPSc’s infectivity?

Answer 37

PrPc required for PrPSc infectivity
PrPSc replicates species specifically
The more similar PrPc and PrPSc = the “easier” for PrPSc to propagate = SPECIES BARRIER
PrP0/0 are resistant to disease = must express PrPc

Question 38

What evidence is there for the protein only hypothesis?

Answer 38

PrPSc replicates = more PrPSc collected from brain of animal than infected with
No strong evidence for virus/nucleic acid
Mice with ablated PrPc (PrP0/0) cannot be infected by PrPSc - with one copy of PrPc (+/-) longer incubation times
Partially denatured PrPc incubated with large excess of PrPSc forms PrPSc in vitro
Protein is the only requisite for transmission

Question 39

How does association of PrPSc with PrPc lead to transmission of PrPSc’s conformation?

Answer 39

Association of the normal conformer with oligomers of PrPSc may promote conformational switch (“seeding”) or the switch occurs spontaneously (“refolding”) but oligomer traps rogue conformer. Oligomers may fragment to form “propagons”, dissociate and be degraded or further aggregate to form plaques.

Question 40

Where in the cell does the PrPSc peptide replicate?

Answer 40

Cell compartments;
PrPc expression at cell surface appears to be necessary for PrPSc replication
PrPSc normally enters endosome system following internalisation from plasma membrane = some released in exosomes
PrPSc internalised with PrPc?
PrPc conversion to PrPSc in endosome (low pH)?
Co-factors required? Glycoaminoglycans (GAGs) necessary for “scaffolding” PrPc/PrPSc for conversion
Factor X?
Cell to cell: Prions released in exosomes, endocytosed by other cells

Question 41

Which specific features of the replication of PrPSc does the prion hypothesis support?

Answer 41

Prion hypothesis would explain why host must express PrPc for infection to occur and “species barrier”
Model could also explain “strain” and “strain specific replication”
Different rogue conformers possible which induce similar conformation in PrPc
Different prion isolates (strains) produce different pathology
Evolution at the protein level

Question 42

What in vitro evidence is there for the prion hypothesis?

Answer 42

In vitro replication when PrPc incubated with PrPSc
Species specificity of in vitro propagation correlated with in vivo species barriers
Cycles of sonication amplify replication (protein misfolding cycle amplification, PMCA)
Strain-specific properties propagated in vitro
Radiolabeled PrPc incubated with PrPSc
Different strains of PrPSc partially digested differently by Proteinase K

Question 43

What percentage of prion disease does the sporadic form of CJD account for? And what is the cause?

Answer 43

80% of cases and the cause is unknown.

Question 44

What factors have been hypothesised to promote the conversion of PrPc to PrPSc in sporadic cases?

Answer 44

Spontaneous conformational changes or somatic mutations.

Question 45

What are some of the main routes for transmission of CJD?

Answer 45

Transmission of CJD via corneal transplants, growth hormone from cadavers, contaminated surgical instruments (iatrogenic, iCJD) and infected beef (vCJD)

Question 46

How is Kuru transmitted?

Answer 46

Kuru is transmitted via cannibalism.

Question 47

What are the mutations found in Gerstmann-Straussler-Scheinker?

Answer 47

Point mutations at 102, 117, 198, 217 co-segregate with Syndrome (GSS)
Transgenic mice expressing GSS PrP develop pathology

Question 48

What are the mutations found in familial CJD?

Answer 48

Octa-repeat inserts and point mutations at 178, 200, 208, 210
Mutations promote formation of abnormal conformation of PrP (PrPSc)
Familial CJD prions ARE INFECTIOUS

Question 49

What mutations in the PrPc gene can cause disease but does not from the PrPSc prion particle?

Answer 49

Some mutations (e.g. A117V) lead to disease (GSS) but not PrPSc
These mutations lead to aberrant processing of PrP
Remains “stuck” in ER membrane - PrPCtm

Question 50

How does toxic gain of function affect patients with PrPSc?

Answer 50

Toxic gain of function
Synthetic peptide of residues 106-126 is neurotoxic in vitro = but requires expression of PrPc
Suggested PrPSc may disturb calcium homeostasis
PrPSc may inhibit proteasome = tends to aggregate

Question 51

How does toxic loss of function affect patients with PrPSc?

Answer 51

Possible toxic loss of PrPc function:
Neuroprotection/anti-apoptotic
Cu2+ binding, nitrosylation of glutamate receptors (MNDA)

Question 52

How is the BSE crisis believed to have started in the United Kingdom?

Answer 52

Late 1980s epidemic of BSE in UK cattle
Carcasses from farm animals rendered and fed to cattle
Changes in rendering practice may have allowed Scrapie prions from infected sheep to survive
Sheep scrapie may have crossed species barrier into cattle
OR BSE epidemic may have started from a few cases of cattle carcasses with sporadic BSE entering food chain

Question 53

What evidence was there that vCJD was happening due to zoonotic transmission from cattle?

Answer 53

Could BSE prions replicate in human brain and cause variant CJD?
Cases of aggressive CJD among young people in UK cannot be ascribed to inherited CJD or any known routes of transmission
Suggested that this was a new variant of CJD (vCJD) with symptoms similar to BSE
Transgenic (Hu/Hu) mice infected with prion containing extracts from BSE cattle will develop disease

Question 54

How does Polymorphism 129 affect a person’s chances of developing a prion disease when exposed to PrPSc?

Answer 54

Polymorphisms at residue 129 of human PrP
39% homozygous for Met
11% homozygous for Val
50% heterozygous (Met/Val)
To date vCJD found ONLY in Met homozygotes
Heterozygosity may impede dimerisation and therefore proliferation of PrPSc
Homozygotes (Met) have increased risk of early onset Alzheimer’s disease
Links to other diseases and age-related changes

Question 55

What evidence is there in the virus vs prion battle?

Answer 55

Difficult to eliminate low copy number virus from prion isolates
But none found
Viral-like particles observed in TSE brains (Manuelidis)
Not shown they can transmit disease, could contain prions
Recombinant mutant PrP injected into mice overexpressing truncated form of PrPc appears to cause disease (slow)
Synthetic fragment of mutant PrP appears to cause disease in Tg mice
Induced to form fibrils in vivo
Injected into Tg mice expressing high levels of PrPc
High levels of PrPc expression alone may induce disease
Very large amounts of synthetic mutant PrP injected

Question 56

What other types of prions have been identified?

Answer 56

Prion-like proteins found in yeast
Ure2, Sup35, Rnq1, Het-s, Swil
Recombinant proteins form “amyloid” fibrils which propagate when introduced into yeast aided by Hsp104
Altered phenotype, inheritable, strain variability and species barriers
Formation of prion conformation not always deleterious
Proteins with prion properties found in bacteria C. botulinum - may have a role in response to stress
Induce changes in gene expression

Question 57

Could there be another vCJD epidemic?

Answer 57

Decline in cases since mid ‘90s but:
Possible longer incubation period in residue 129 heterozygotes (Met/Val)
Difficult to devise sensitive and reliable blood test for screening
May be contaminated blood used in transfusions
Babies may have been infected
BSE may have been transmitted to sheep and goats and could re-enter food chain
Prions found in hindlimb muscles of infected mice so pure beef muscle could be infectious (even at over 30 months)
Cases (2?) of infection following blood transfusion

Question 58

What treatments are there for prion diseases?

Answer 58

Modifiers of PrPSc structure or propagation may be effective therapies
Porphyrin and phthalocyanine delays disease onset in transgenic mice. Congo red derivatives, amphotericin B and 4’-iodo-4’-deoxy-doxorubicin have similar results = also contain planar polarizable hydrophobic ring - may increase degradation
Pentosan sulphate - interferes with interaction with complex carbohydrates?
Acridine and phenothiazine derivatives (e.g. quinacrine and chlorpromazine) reduces PrPSc conversion in vitro
Β-sheet breaking peptides may inhibit conversion
Vaccine?