10.1 Prions and Prion disease

Question 1

What are Prion diseases and what is it alternatively known as?

What does PrP stand for and what does PrP^Sc stand for and why?

Answer 1

A fatal neurodegenerative diseases

• prion disease has a long incubation time
• asymptomatic, till disease causes rapid neurodegenration

Alternatively known as Transmissible Spongiform Encephalopathy (TSE)

PrP: proteinaceous infectious particle

PrP^Sc: scrapie form of the prion protein found in sheep (abnormal form)

• some times called PrP^Res (resistant form)

Question 2

Give 3 types of prion disease found in mammals

Answer 2

1) Scrapie (sheep)
2) Bovine Spongiform Encephalopathy- BSE (cows)
3) Chronic Wasting Disease- CWD (deer)

Question 3

Give 4 types of prion disease found in humans

Answer 3

1) Creutzfeldt-Jakob disease (CJD) and variant Creutzfeldt- Jakob disease (vCJD)
* varient form evolved from “Mad cow disease”
2) Kuru
3) Fatal Familiar Insomnia (FFI)
4) Gerstmann-Straussler-Scheinke syndrome (GSS)

Question 4

Give 5 general symptoms common to Prion disease

Answer 4

1) Anxiety and depression
2) Ataxia (loss of physical coordination)
3) Memory loss and loss of cognition
4) Dystonia (muscle spasms)
5) Incontinence (bowel & urinary)

Question 5

What is the outcome of a patient with Prion disease

What is the life expectancy of someone with CJD

Answer 5

Inevitably fatal: no cures, only treatments to ease symptom

Most people with CJD will die within a year of the symptoms starting as hit is extremely aggressive (often from infection)

Question 6

What are the characteristics of Prions disease

Answer 6

1) neuronal death leading to a spongiform appearance of the brain
2) proliferation of astrocytes and microglia
3) build up of amyloid plaques (protein aggregates)
4) evidence of oxidative stress

Question 7

What are astrocytes and microglia?

TOB: What are the other main glial cells in the CNS and PNS and what are their function

Answer 7

Astrocytes and Microglia: act as immune cells of the brain (aim to compartmentalise the problem and clear up the dead neurones)

CNS: cell bodies are “nuclei”

• astrocytes: BBB
• oligodendrocytes: myelination
• ependyma: line ventricles and spinal cord
• microglia: macrophages

PNS: cell bodies are “ganglia”

• schwann cells: myelination
• satellite cells: cover the sensory, SNA, and PNS ganglia
• microglia: macrophages

Question 8

How common is prions disease and what are the 3 ways in which they can occur?

Which age groups do each usually occur in and which are the most common?

Answer 8

very rare, can occur by:

1) sporadic (spontaneous) 85-90%
* symptoms usually develop between 60 to 65
2) genetic (familial) 10-15%
* symptoms usually develop in early 50s
3) acquired (infectious/ transmitted) 2-5%
* very rare

Prion diseases are more likely to be sporadic or inherited

Question 9

Give 3 prion disease that can be inherited

What can be said about onset of inherited vs sporadic occurrence

Answer 9

CDJ, FFI and GSS

Inherited often have a more rapid onset than sporadic diseases (start early 50s)

Question 10

Which part of the brain do the following prion disease affect most?

CJD, FFI, Scrapie, BSE, CWD and Kuru

Answer 10

Classical CJD ➞ cerebral cortex

FFI ➞ hypothalamus

Scrapie, BSE and CWD ➞ brainstem

Kuru ➞ cerebellum

Question 11

Image on the left shows a protein plaque - PrPSc, what speicifc histolgical changes can be seen in the image on the right?

Answer 11

White: the spongiform changes (gaps in brain where neurones have died)

Brown: the microglia trying to get rid of the necrosing neurones

Question 12

What is the composition of Amyloid Plaques in Prions disease?

How does this differ from amyloid plaques seen in Alzheimer’s disease?

Answer 12

• comprised of the prion protein
• β-sheet rich which allows proteins to stack up and make fibrils (fibres)
• these fibres to clump together and make a plaque

In Alzheimer’s it is the amyloid β peptide that aggregates whereas, in prion disease it is the actual PrP (prion protein) that is accumulating and is able to aggregate due to the increased β-sheets

Question 13

List 5 other neurodegenerative diseases that cause amyloid plaque build up and state how these differ from prion disease

List one other similarity between these and prions

Answer 13

The amyloid plaques in prion diseases (TSEs) are caused by the actual PrP (prion protein) that is accumulating and aggregating due to the increased β-sheets

Amyloid plaques in other ND diseases are caused by a build up of other substances:

1. Alzheimer’s disease - amyloid-β protein
2. Parkinson’s disease - α-synuclein protein
3. Huntington’s disease – huntingtin protein
4. Wilson’s disease – defects in copper metabolism
5. Amyotrophic lateral sclerosis ALS – defects in superoxide dismutase

Similarity: most of these diseases also show high levels of oxidative stress (particularly in ALS)

Question 14

Give 2 diseases that proved prions disease was transmissible from person-person

Give the age of onset to the age of death of the disease that was prevelant in the UK

Answer 14

1) Kuru
2) BSE (mad cow disease)

• to variant Creutzfeldt-Jakob disease (vCJD)
• median age of onset 26.5 years, age of death 28 years

Question 15

What specifically was done to prove Prion disease to show it was transmittable?

What did this suggest BUT why was this idea not supported?

What does the evidence therefore indicate about prions disease?

Answer 15

Diseased brain tissue of species with prions disease was injected into another animal of the same species and was shown to transmit the disease

Suggested an infectious agent such as a virus BUT there was no evidence of a virus found in the brain extracts.

Also when the extracts were treated with agents (e.g. ultraviolet light/nucleases) that would normally destroy nucleic acids, this did not reduce their infectiousness

The evidence therefore indicates that the infectious agent in the TSEs is a protein

Question 16

What is PrP^Res and is it always infectious?

Answer 16

This form of the protein is resistant to conditions that would normally break down proteins e.g. protease

It was found to be the protein in the amyloid plaques

The PrP^Sc (scrapie form) of the prion protein is some times called PrP^Res but the ‘resistant’ form may not always be infectious

Question 17

What are the 2 main routes of infection in transmittable prion disease?

Give examples

Answer 17

1) Ingestion

• Kuru: ritualistic cannabilism (ate brains of dead relatives)
• vCJD: varient form of CJD from consumption of cow meat

2) Iatrogenic
* Iatrogenic CJD usually a result of a medical procedure

Question 18

Give 4 ways in which prion disease can be transmitted Iatrogenically

Answer 18

1) corneal grafts
2) dura mater grafts
3) Human derived growth hormone injection (hGH)
4) experimental transmission (in animal models)

Question 19

If Ingested, how can Prions spread from mouth to brain?

Answer 19

1) Prions enter the body through ingestion
2) They are detected by the immune system however, as they as resistant to proteolysis they do not illicit a secondary immune response
3) via Peyers patches in the gut they are able to enter lymphoid tissue
4) The prion replicates in lymphoid tissue, especially within the follicular dendritic cells
5) as the lymphoid tissue contains a vast amount of innervation from the ANS, the prions can enter the CNS via nerves of the ANS

Question 20

What form of prions is normally found in our body?

What gene/chromosome is it located on?

Answer 20

Yes, there is a normal cellular form of the prion protein PrP^C that we ALL have in our brain (its function is unknown)

In humans this is coded by the PRNP gene on chromosome 20

Features include:

• GPI anchored to the plasma membrane
• monomeric
• protease sensitive
• binds copper
• alpha helices present
• located in the neuronal synapses and on the follicular dendritic cells

Question 21

How does the PrP^Sc (abnormal) replicate within our brain?

How was this proven?

Answer 21

PrP^C (normal) and PrP^Sc (abnormal) share the same amino acid sequence. PrP^C to PrP^Sc conversion is a key event in disease

• if PrP^{Sc i}s present it can catalase the normal cellular form PrP^C into more of the abnormal PrP^Sc form.
• It is this form that is responsible for causing aggregation of amyloid plaques and development of Prion disease

This was proven through mice trials:

• the PRNP gene was removed from the mouse (gene knock out) and when infected with the PrP^Sc form, was not able to be infected
• showing that PrP expression is essential for disease pathogenesis

However, the downside was the the knockout phenotype showed disturbed sleep patterns and sensitivity to oxidative stress which may be potential functions of the PrP normal form in humans…

Question 22

What are the characteristics of the abnormal PrP^Sc?

What is important to remember when dealing with a patient who has PrP^Sc when performing medical procedures/surgery ?

Answer 22

It is associated with infectivity due to the following:

• extracellular
• aggregated
• protease resistant
• no specific copper bound
• high beta sheet content

It is not easily decontaminated so… there is a risk that any instruments used for the person may have the prions on it. Make sure to thoroughly sterilise equiment or dispose of it appropriatly

Question 23

Compare the conformation of PrP^C and PrP^Sc and what specifically allows formation of the amyloid plaques

Answer 23

PrP^C

• 3 α–helices (α–helice-rich)
• 2 β-strands
• can be deteted using NMR & X-ray

PrP^Sc

• Secondary structure
• β-strand rich - this allows the proteins to stack up and make fibres which aggregate to form amyloid plaque
• diagnosis is difficult (can usually only be done post-mortem)

Remember: PrP^c and PrP^Sc have the same primary structure but PrP^Sc secondary structure is what is rich in beta-pleated sheets- harmful

Question 24

What are the 2 proposed mechanisms of PrP^Sc catalysed conversion?

Explain each

Answer 24

Mechanism of convention is still not 100% but both models suggest that there is a conformational change in the amino acid sequence

1) Template direct refolding model

• this model suggests there is a high energy barrier to overcome to covert PrP^C into PrP^Sc
• suggests that PrP^Sc is able to recruit PrP^C and form a heterodimer with it
• this acts as a template to catalyse its conversion to the PrP^Sc form which can then recruit more PrP^C to form more PrP^Sc OR polymerise to form amyloid fibrils

2) Nucleation dependent polymerisation model

• suggests that there is a low energy barrier to overcome (favours a more sporadic cause)
• proposes that PrP^c and PrP^Sc are in an equilibrium and only once many monomeric PrP^Sc molecules are assembled can there be further PrP^Sc recruitment
• occurs more slowly over time as the PrP^Sc builds up and eventually aggregates to amyloid plaques

Question 25

How does PrP^Sc lead to neurodegeneration?

Answer 25

PrP^Sc is cytotoxic to neurons hence, when they accumulate and cause protein plaque formation the neurones undergo apoptosis

When the apoptotic neurones are cleared by microglia they leave ‘gaps’ (sponge like)

Also, PrP^C is thought to have anti-oxidant functions and hence, loss of this normal protein may result in increased oxidative damage

Question 26

What causes initial formation of PrPSc ?

Answer 26

Unknown

Question 27

How can Prion disease be inherited?

Answer 27

There are Mutations that destabilise PrP^C and in turn make PrP^Sc more energetically favourable

Hence, more likely to be converted into the PrP^Sc form

Question 28

What is Sporadic Prion Disease and give a common example of one

What causes it to occur?

What specific mutation was common in cases of vCJD and how was this eliminated in sheep?

Answer 28

Sporadic means it occurs randomly eg. CJD occurs with:

• no family history of the disease
• no known exposure to infectious prions
• late onset

Sporadic cause is uncertain but could possibly be due to;

• a spontaneous somatic mutation that has occurred in one of the PRNP genes in a cell
• normal PrPC protein may have spontaneously been converted into the PrPSc form

There tends to be a greater susceptibility polymorphism in their PRNP genes

• In vCJD nearly all cases was Met/Met homozygous at codon 129 which was shown to increase susptibilty of prion disease development
• There was selective breeding of sheep to remove susceptible alleles

Question 29

List 4 precautions that must be taken during surgical procedures on patients with/at risk of CJD

Answer 29

1) perform the intervention in a sterile operating theatre
2) schedule at the end of the list, to allow cleaning
3) involve the minimum number of personnel required
4) PPE must be worn eg.

• liquid repellent operating gown over a plastic apron, gloves, mask and goggles or full-face visor
• for symptomatic patients, this protective clothing should be single-use

5) single-use disposable surgical instruments and equipment should be used where possible, and subsequently destroyed by incineration or sent to the instrument store
6) effective tracking of reusable instruments should be in place, so that instruments can be related to use on a particular patient if they develop an infection

Question 30

Give 4 safety measures to reduce any possible risk of spreading vCJD through blood

Answer 30

1) withdrawal and recall of any blood components donated by:

• anyone who develops vCJD
• importing plasma from the USA

2) removing white blood cells from all blood used for transfusions (leucodepletion)
3) not accepting donations from people who may have received a blood transfusion since 1980 (BSE)
4) promoting the appropriate use of blood, tissues and alternatives throughout the NHS
* this has so far led to a reduced amount of blood transfused during and following surgery

Question 31

There are no treatments for prion diseases but there are some potential methods, give 4

Answer 31

1) eliminate production of normal PrP^C protein (this would remove the required susbtate: like the knock out mice)
2) stabilising either the normal protein OR the infectious prion to prevent the conversion
3) find a mechanism to prevent aggreagation of prions to stop the formation of the fibrils and hence the amyloid plaque
4) stabilise the fibrils so they cannot form amyloid fibres and dont have a pathogenic affect