lecture 6

Question 1

What is the main example of a type of protein misfolding diseases?

Answer 1

• conformational disease caused by misfolding of proteins into Beta-sheet aggregated structures
• normally buried fragments of the protein are abnormally exposed to the solvent and become sticky and stack together

Question 2

What is an amyloid?

Answer 2

• protein misfolding results in beta-sheets forming intermolecular aggregates
• stack together to form oligomers
• wind together to form protofibrils > fibrils > amyloid
• amyloid gets deposited in the brain as amyloid plaques e.g. in Alzheimer’s

Question 3

Why might misfolding occur?

Answer 3

• mutation in the gene encoding the protein (quite a rare event)
• somatic mutation in the gene encoding the protein (as opposed to germ line)
• errors in transcription and translation (not the DNA per say)
• failure of folding and chaperone machinery
• mistakes in post-translational modification/trafficking (e.g. alzheimer’s: in some cases an enzyme cleaves more of the protein than usual allowing it to stack more easily)
• structural modification caused by environment
• induction of misfolding by seeding

Question 4

What are some examples of protein misfolding diseases and the proteins by which they are caused?

Answer 4

• Alzheimer’s disease: ABeta and tau
• Prion diseases: prion protein
• Parkinson’s disease: alpha-synuclein
• Huntington’s disease: huntingtin
• ALS (Amyotrophic lateral sclerosis): SOD (superoxide dismutase)
• Tauopathies: tau
• Type 2 Diabetes: IAPP (Islet Amyloid Polypeptide)
• 2º amyloidosis: SAA (serum amyloid A)
• predominately identified in the nervous system

Question 5

What is prion disease?

Answer 5

• neurodegenerative disorders: cause a degeneration of neurons in the brain
• long incubation period: tend to affect older people
• rapid clinical progression: months to a year
• invariably fatal: if somebody shows the clinical symptoms of this disease, they will die from this disease
• common pathogenic process: degeneration and death of neurons
• accumulation of abnormal conformation of host encoded prion protein (PrP)
• Transmissible in the absence of a conventional infectious agent: no virus, bacteria or fungus associated e.g. Kuru

Question 6

What is the spectrum of prion diseases?

Answer 6

• affect both animals and humans

Animals:

• scrapie: sheep and goats
• bovine spongiform encephalopathy (BSE): cows (mad cow disease)
• chronic wasting disease: deer and elk
• exotic ungulate, feline, mink

Humans:

• sporadic: no known etiology (most common form)
• familial: mutation of prion protein gene
• Acquired: iatrogenic (medical exposure), zoonotic (eat etc)

Question 7

What are the etiologies and phenotypes of human prion diseases?

Answer 7

Sporadic (85%)

• CJD, GSS, FFI
• strains of sporadic CJD

Inherited (15%)

• Gerstmann-Straussler-Scheinker (GSS): Cerebellar ataxia (a more unsteady gait)
• Creutzfeldt Jakob Disease (CJD): rapidly progressive dementia (affecting cognition as opposed to motor skills)
• Fatal familial insomnia (FFI): intractable insomnia

Acquired (rare)

• Variant CJD
• Kuru
• Iatrogenic

All together about 1 case per million head of population per year e.g. about 20 cases a year in Australia

Question 8

Describe the history of Scrapie.

Answer 8

Scrapie in 1732:
- a shepherd wrote in his diary that an animal that looks like it has itchy skin/scrapes its wool off must be isolated from healthy stock because it is infectious and will cause serious harm to flock

Scrapie in 1930s:

• Vaccine for Louping ill virus made from the brains of affected sheep: antigen present, formalin fixed to kill conventional infectious agents
• 2.5 years later - scrapie detected in vaccinated flocks:
• • the infective agent of scrapie was present in the brain, spinal cord and or spleen of infected sheep:
• it could withstand a concentration of formalin of 0-35%, which inactivated the virus of louping-ill:
• it could be transmitted by subcutaneous inoculation;
• it had an incubation period of two years or longer
• but what caused it?

Question 9

How did they try to determine the cause of Scrapie?

Answer 9

• could blend the brain of a sheep that had died from the disease and inoculate a healthy animal: it would later die from the disease
• to try and work out what the transmissible agent was you take the brain of an infected animal and you treat it with different things that will narrow down the possible transmissible agents and go from there:
• treatment with alkali pH in the hopes of denaturing/hydrolysing proteins and nucleic acids: transmission of disease did not occur
  if it had been a conventional agent this treatment would have hydrolised the RNA of the genome or denature the ds DNA of genome
• treatment w/ nucelase: digest nucleic acids: didn’t affect the disease
  potentially the nuclease would be unable to penetrate protein shell of a conventional agent
• treatment with UV irradiation: damage nucleic acids
  perhaps shielded by a protein shell or no critical nucleotide dimers formed if conventional agent
• protease: digest proteins: disease no longer transmissible
  CA: perhaps digestion of nucleocapsid
• strong denaturants: denature proteins: disease no longer transmissible
  CA: denaturation of nucleocapsid

IF NOT a CA then it is something without nucleic acid and is just a protein: proposed for prion diseases

Question 10

Who won the Nobel Prize for the discovery of Prions?

Answer 10

Stanley B. Prusiner (1997):
- Prions are small PROteinaceous INfectious particles which are resistant to inactivation by most procedures that modify nucleic acids… and underscores the requirement of a protein for infection

Question 10

What is the prion protein?

Answer 10

• a 27-30kDa protease resistant protein which co-purifies with infectivity-PrP^Sc (i.e. associated with the infectivity of the disease)
• the idea was that there was this protein floating around that when you acquire it you get the disease
• however Chesebro showed that the mRNA present in infected and uninfected animals i.e. an endogenous protein that was misfolding to cause the disease
• Therefore post-translational modification of a normal cellular protein-PrP^C

Question 11

What is the difference between PrP^C and PrP^Sc?

Answer 11

PrP^C

• 230 aa protein
• GPI anchor (localises to the membrane)
• protease sensitive
• alpha-helix
• many tissues (highest level in the brain)
• required for infection

PrP^Sc

• Protease resistant (about 70aa are digested)
• insoluble
• Beta-sheet
• Disease specific
• Infectious
• depending on the shape of the protein you might have more or less of the N-terminus sticking out so you might have different mobilities of the protein depending on how much is chewed away
• has a polymorphism at codon 129: all humans are either methionine or valine homozygous, or heterozygous and this affects your susceptibility to disease
• has two possible N-linked glycosylation sites (i.e. it can have sugars added) but the protein is either post translationally modified with either no sugars, one sugar or two sugars: the number of sugars affects its electrophoretic mobility

Question 12

What are methods for studying prion disease?

Answer 12

• Epidemiology of human disease: surveillance
• In vivo animal models: Natural (sheep, cattle) or Experimental (rodent) (In australia we use the brains of humans who have the disease because we have NO scrapie in australia: the department of agriculture does not want scrapie in australia therefore we cannot have it)
• In vitro cell culture models
• Biochemical systems: cell free assays or recombinant protein

Question 13

What is the neuropathology of prions?

Answer 13

• they get holes in their brain (spongiform change)
• neuronal loss
• gliosis (monocytes of the brain are activated)
• PrP^Sc deposition
• Caspase activation (i.e. induction of apoptosis)
• Tau agg

Question 14

What is the evidence that PrP^Sc is the major structural component of the infectious unit?

Answer 14

• Physical association with preclinical and clinical disease
• The major macromolecule which co-purifies with infectivity
• Concentration of PrP^Sc is generally proportional to infectivity
• Biophysical state correlates with infectivity profile (protease resistance, chemical modification)
• Mutations in PRNP linked to disease
• Gene dosage and species specificity accounted for by PRNP (transgenic animals)
• Absolute requirement of PrP for transmission (gene ablation) and pathogenesis

Question 15

Proving the protein only hypothesis: can a misfolded protein really be infectious?

Answer 15

• have not been able to purify the protein all on its own
  Ultimate proof of the Protein only hypothesis would be :
• I took recombinant prion protein all by itself that I made in a bacteria somewhere nowhere near a sheep with scrapie and I misfolded it in a test tube and I inoculated it into a mouse and the mouse got the disease: this has been very difficult to do

Cell-free assays of prion propagation

• De novo synthesis of protease resistant PrP
• • Cell free conversion assay (CFC) (proved that PrP could propagate itself)
• • Protein misfolding cyclic amplification assay (PMCA)
• • Conversion activity assay (CAA)
• • Quaking inducing conversion (QUIC)
• • Refolding of recombinant PrP

Question 16

What are the important features of past research into the protein-only hypothesis?

Answer 16

Propagation:

• partially purified PrP^Sc can induce a conformational change in mammalian and recombinant PrP^C
• Crude brain homogenates efficiently propagate prions suggesting a role for cofactors

Infectivity:

• Infectivity can be spontaneously generated from partially purified mammalian and recombinant PrP^C
• Infectivity can be spontaneously generated from recombinant protein in the presence of cofactors (much better infectivity)

Efficient prion propagation requires PrP and host derived ‘factors’

Question 17

How do strains of prions exist in the absence of nucleic acid genome?

Answer 17

Prion strains are characterised by:

• clinical phenotype
• where in the brain the damage is caused
• incubation period

(in hamsters infected with a form of scrapie)
Hyper strain (HY):
- short incubation
- brain stem and cerebellar cortex
- hyperactivity
Drowsy strain (DY):
- long incubation
- pyramidal layer adjacent to the hippocampus
- lethargy
PrP^Sc isolated from HY and DY strains of hamster scrapie:
- different electrophoretic mobility after PK digestion
- different FTIR spectroscopy profile
- Suggests different conformation of PrP^Sc associated with strain variation

(in humans)

• PrP^Sc isolated from patients with CJD
• • have different electrophoretic mobility following PK digestion
• • Have different glycosylation patters
• indicative of strains of CJD
• Used diagnostically to identify vCJD
• in the absence of a nucleic acid genome the features of prion strains are proposed to be encoded by the biochemical and biophysical features of PrP^Sc
• • electrophoretic mobility following PK digestion and differences in Beta-sheet content
• – conformation (this is indicative of a different conformation but not absolute)
• • Degree of glycosylation
• – Glycoform
• • Polymorphism in prion protein gene
• • Region of the brain affected
• – Region specific cofactors

Question 18

What is kuru?

Answer 18

• an acquired prion disease
• recognised in late 1950s
• kuru = the shiver or be afraid
• cerebellar ataxia; cognitive decline late
• transmission through cannibalistic feasts - mouring rites of deceased relatives
• no vertical transmission
• no domestic transmission
• long incubation period
• susceptibility codon 129 MM > VV > MV
• depends which part of the body you ate

Question 19

How is CJD transmitted iatrogenically?

Answer 19

• Surgical transmission from contaminated surgical equipment
• Contamination of biological material
• • hormones
• • dura mater
• • organs (cornea)

Question 20

How do we control prion disease infection?

Answer 20

• sterilisation is a process that kills microbial life
• how do you kill something which is not living but is transmissible?
• disinfection renders transmissible agents inert
• WHO recommends chemical disinfection with 1M NaOh ( > 4 logs reduction in infectivity) + steam sterilisation (gravity cycle) at 121ºC for 30 minutes where possible.

Question 21

What are the features of acquired prion disease?

Answer 21

CJD epidemics:

• susceptibility to infection
• • genetic predisposition
• • amount of infectivity
• Routes of infection
• • CNS vs peripheral exposure
• • not community acquired
• Resistant to decontamination

Question 22

What other proteins demonstrate ‘prion-like’ transmission?

Answer 22

• ABeta
• Tau
• Alpha-synuclein
• Htt
• SAA