Prions

Question 1

What are prions?

Answer 1

• Prons are misfolded forms of cellular prion protein (PrP^C),
  
  • ​PrP^C = a normal glycoprotein expressed on the neuronal cell surface and encoded by the PRNP gene

Question 2

What is the primary function of PrP^C?

Answer 2

• Myelin maintenance
• Other roles:
  
  • ​Signal transduction
  • Apoptosis regulation
  • ECM adhesion
  • Synapse formation

Question 3

Outline an experiment illustrating the requirement for cellular prion protein (PrP^C)

Answer 3

• Bueler at al 1993
  
  • ​Gene knockout experiment
    
    • Gene encoding PrP^C (PRNP) was inactivated
  • ​​Both the knockout and control groups were inoculated with scrapie prions
  • Observed that the KO mice remained symptomless while the wildtype controls all died within 6 months.

Question 4

Describe the molecular changes that form a prion

Answer 4

• Generated from PrP^C by a conformational change
  
  • Converts the native a-helix rich isoform to the beta-sheet rich PrP^Sc (Sc for scrapie), which is responsible for disease
• Interestingly, Prions are an exception to Anfinsen’s dogma
  
  • ​Anfinsen’s dogma = aminop acid sequence determines primary, secondary and tertiary structure

Question 5

Describe Creutzfeldt-Jakob disease (CJD)

Answer 5

• Creutzfeldt-Jakob disease (CJD) is the most common prion disease
• Initial symptoms
  
  • Abnormalities of memory and behaviour
• Later symptoms
  
  • Rapidly progressive dementia
    
    • Associated with startle myoclonus
      
      • Startle myoclonus = involuntary jerking movements in response to sudden and unexpected stimuli
• Inevitably fatal with an average survival time of 7 months after the onset of symptoms;
• Peak incidence = 70 to 80 years.

Question 6

Describe the genetic component of Creutzfeldt-Jakob disease

Answer 6

• Single nucleotide polymorphism (SNP) at codon 129 of the PNRP gene that encodes either methionine (ATG) or valine (GTG)
• Predominance of homozygotes amongst suffers for CJD with heterozygosity having a protective effect
• It is thought that the different protein structure alters the probability of conversion for PrP^Sc to PrP^Sc but the exact mechanism is unclear

Question 7

Explain how PrP^Sc mediates protein-mediated transmission

Answer 7

• Able to propagate by physical interactions with PrP^C,
  
  • PrP^C is converted into the misfolded state
• PrP^Sc acts as a molecular template,
  
  • Provoking conformational changes in PrP^C that produce the same abnormal structure
• Because PrP is a normal host protein, PrP^Sc is not recognised as foreign by the immune system and does not induce an immune response.

Question 8

Explain the evidence suggesting that prions are not transmitted via nucleic acids

Answer 8

• Alper et al in 1967
  
  • Brains of mice infected with scrapie were extracted and made into solutions
  • The solutions were subjected to different UV doses including at a wavelength effective at inactivating nucleic acids
  • These treated solutions were then injected back into normal mice
  • The experimenters observed that all mice developed scrapie regardless of the UV dose
    
    • ​Suggesting that the infectious agent did not rely on nucleic acid.

Question 9

List examples of prion diseases affecting animals

Answer 9

• Scrapie in sheep
• Transmissible mink encephalopathy (TME)
• Chronic wasting disease of elk (CWD)
• Bovine spongiform encephalopathy (BSE) in cattle

Question 10

List examples of prion diseases affecting humans

Answer 10

• Familial Creutzfeldt-Jakob disease (fCJD) ​
• Variant Creutzfeldt-Jakob disease (vCJD) ​
• Kuru

Question 11

What is the differecne between variant Creutzfeldt-Jakob disease and familial Creutzfeldt-Jakob disease?

Answer 11

• Mainly affects young adults
• Behavioural disorders are prominent in the early stage of the disease’
• Neurological syndrome progress more slowly
• vCJD is characterised histologically by fibrillary PrP depositions surrounded by halo of spongiform vesicles as shown by the diagram.

Question 12

Explain the factors that contributed to the Kuru disease endemic. Explain the full pathophysiology of kuru disease.

Answer 12

• Cannablism & prion’s long incubation period
• Kuru = transmissible spongiform encephalopath
  • Caused by bovine spongiform encephalopathy (BSE) AKA mad cow disease.
    
    • Cattle are believed to be infected by being fed meat-and-bone meal
      
      • ​Contained the remains of cattle who spontaneously developed the disease or from scrapie-infected sheep products
  • Spread of vCJD can also occur via blood products or contaminated surgical equipment.
• Very rare, incurable and fatal neurodegenerative disorder.
• • Targets the cerebellum
• Symptoms
  
  • Body tremors
  • Random outbursts of laughter
  • Gradual loss of co-ordination
• Formely common among the Fore people in Papua New Guinea
  • They ritualistically cooked and consumed body parts of their family members following their deaths
  • This led to an epidemic from 1957 to 1960
    
    • ​Resulting in over 200 deaths per year.
• Even though the cannibalistic practice ended in the early 1960s , the disease lingered due to kuru’s long incubation period
  
  • Anywhere from 10 to 50 year
  • From 2005 onwards, the deaths from kuru are practically zero

Question 13

How can vCJD be spread? What measures are in place to ensure they are destroyed?

Answer 13

• Blood products or contaminated surgical equipment
• Given the resistance of prions to inactivation by treatments that modify nucleic acids such as UV, proteolytic degradation and heat, the normal sterilisation procedures for medical equipment are not protective against prions.
• Infection can therefore be iatrogenic by the introduction of contaminated medical materials into host tissues, such as:
  
  • Surgical tools
  • Electrodes for deep brain stimulation
  • Supplementary hormone preparations
• However, they can be deactivated in a steam autoclave in the following conditions:
  
  • 132°C
  • 21 psi
  • 90 minutes
• Although many misfolding diseases are characterised by the protein-level transmission of abnormal structure, only prion diseases are transmissible at the level of the organism

Question 14

Explain how prions can result in neurotoxicity

Answer 14

• PrP^Sc can aggregate into oligomers and eventually amyloid fibrils that commonly surround neuronal bodies and processes.
  
  • This causes neuronal death
  • ​ However, prion diseases are not considered amyloidoses because the amyloid deposits do not cause the disease.
• Symptoms
  
  • Rapid progressive dementia
  • Cerebellar ataxia
  • Akinetic mutism
• Histology
  
  • CNS neuronal loss without replacement by glial cells
    
    • Spongiform appearance
      
      • Spongiform appearance = s large acellular areas that appear vacuolated in tissue section,
      • Commonly occurs in the deep cortical layers, cerebellar cortex or subcortical grey matter

Question 15

Outline how PrP^Sc can be detected in biopsies or post-mortem samples

Answer 15

• Immunohistochemical detection of PrP^Sc involves the incubation of brain tissue sections in a solution containing antibodies specific to the protein.
• The antibodies are attached to a peroxidase enzyme,
• Hydrogen peroxide added to the sample
  
  • Hydrogen peroxide colours the antibodies (attached to peroxidase) and thus the areas containing containing the PrP^Sc compound
  • Areas that have reacted are bleached white
  • Non-reactive areas remaisn light blue
    
    • Light blue = colour of hydrogen peroxide
• Light microscopy can then be used to visualise the PrP^Sc and its distribution.

Question 16

Describe the treatments for Creutzfeldt-Jakob disease

Answer 16

• At present, there is no proven cure for prion diseases such as Creutzfeldt-Jakob disease
• Treatments
  
  • Sedatives
    
    • For anxiety
  • Antidepressants
    
    • For depression
  • Clonazepam
    
    • Positively modulates GABA
    • Jerks
  • Sodium valproate
    
    • Increasd levels of GABA
    • Tremors
• Progressive symptoms significantly reduce patient quality of life
  
  • Ataxia
    
    • ​Loss of physical co-ordination)
  • Urinary and bowel incontinence
  • Dysphasia
  • Dystonia
    
    • Muscle spasms and stiffness)
• The ideal treatment would be to address the underlying protein misfolding and propagation
  
  • Experimental methods
    
    • Cationic tetrapyrrole compound [Fe³⁺-TMPyP].
      
      • It works by stabilising the native conformation of PrP^C thereby lowering the misfolding rate which resulted in the prolonged survival of prion-infected mice.
    • RNA interference
      
      • ​To reduce the production of PrP^C.
      • The proposed model would be through administering short interfering RNA (siRNA) complementary to the PrP^C mRNA which would then degrade PrP^C mRNA and reduce PrP^C translation
      • This lowers the amount of PrP^C available for misfolding.