Lecture 3- Prion Diseases

Question 1

prion diseases

Answer 1

transmissible spongiform encephalopathies

Question 2

3 pathogenic features of prion diseases

Answer 2

1. degeneration and death of neurons- spongy form, vacuolation, associated with apoptosis, activated caspases
2. accumulation of abnormal form of PrP (misfolded protein PrP*Sc)
3. astrocytosis- hypertrophy and hyperplasia of astrocytes in gray matter

Question 3

GFAP

Answer 3

detects protein present in normal astrocytes- more staining in affected brain

Question 4

mad cow disease

Answer 4

bovine spongiform encephalopathy

Question 5

how do exotic animals get prion disease?

Answer 5

feed mad cow meat to cheetahs, antelopes etc- disease easily transmissible

Question 6

sporadic Prion disease

Answer 6

85%- majority of forms in australia

Question 7

familial prion disease

Answer 7

15%- mutation of prion protein predisposes you to misfolding of protein

Question 8

3 familial prion diseases

Answer 8

GSS- cerebellar ataxia
CJD- rapidly progressive dementia
FFI- intractable insomnia

Question 9

acquired prion disease

Answer 9

iatrogenic (medical intervention) or zoonotic (eating mad cow)

Question 10

epidemiology of prion disease

Answer 10

rare; 1-2 cases per million per year

Question 11

main characteristic of prion disease

Answer 11

transmissible in the absence of conventional infectious agent e.g. zoo animals eating mad cow, urine/faeces

Question 12

proteinaceous infectious particle

Answer 12

resistant to inactivation by most procedures that modify nuclei acids i.e. prions dont require nuclei acids for infectivity

Question 13

what was the infectious agent initially thought to be?

Answer 13

exogenous agent, proteases resistant which copurifies with infectivity

Question 14

what was infectious agent later shown to be?

Answer 14

post translational modification of a normal cellular protein PrP (since mRNA present in infected and un infected)

Question 15

PrP*C

Answer 15

normal cellular form, protease sensitive (disappears under proteinase K), soluble, alpha helix, many tissues (highest in brain), required for infection

Question 16

PrP*Sc

Answer 16

misfolded protein, protease resistant, insoluble, beta sheet, disease specific, infectious
- can go back and recruit more normal cellular form

Question 17

protein only hypothesis

Answer 17

PrP from normal cellular form gets mutation–>disease associated form
- misfolded protein can go back and recruit more normal cellular form

Question 18

5 evidence for PrP*Sc- major component of disease/infectivity

Answer 18

1. physical association with clinical disease
2. co-purifies with infectivity
3. concentration of PrP*Sc proportional with infectivity
4. biophysical state correlates with infectivity profile (protease resistance, solubility)
5. mutations in PRNP (gene that encodes prion protein) linked to disease

Question 19

gene dosage effect for PrP

Answer 19

increasing amount of PrP; incubation period and clinical progression is shorter

Question 20

in the presence of PrP*Sc, where is pathology observed?

Answer 20

only observed in tissue expressing PrP*C

Question 21

evidence that pathology can be reversed

Answer 21

1. despite presence of PrPC, pathology reversed when PrPSc propagation stopped (reversed early spongiform change)- transgenic mice–>PrP 12 weeks then off
2. RNAi targeting PrP*C epxression
   - both prolong survival time, reduce pathology, improve cognitive and behavioural deficits

Question 22

why was there no clinical disease in GPI/Tg mice despite accumulation of misfolded PrP*Sc?

Answer 22

PrPSc propagation was not associated with membrane anchored PrPC

Question 23

proving the protein-only hypothesis; propagation

Answer 23

• partially purified PrPSc can induce conformational change in mammalian and recombinant PrPSc
• crude brain homogenates propagate prions
• cell-free conversion assay

Question 24

cell-free conversion assay

Answer 24

PrPC converted into protease resistant protein in presence of excess PrPSc
- proved template directed misfolding

Question 25

proving protein only hypothesis; infectivity

Answer 25

• infectivity spontaenously generated from partially purified mammalian and recombinant PrP*Sc
• RNA stimulated misfolding of mammalian PrPC when not seeded with PrPSc

Question 26

recombinant PrP forms into?

Answer 26

amyloid fibrils

Question 27

prion strains characterised by (4)

Answer 27

clinical phenotype e.g. weight loss, weight gain
location of damage in brain
incubation period
conformation of PrP*Sc

Question 28

strains of hamster scrapie

Answer 28

HY- hyper strain

DY- drowsy strain

Question 29

HY-hyper strain

Answer 29

short incubation, brain stem and cerebellar cortex, hyperactivity

Question 30

DY-drowsy strain

Answer 30

long incubation, pyramidal layer adjacent to hippocampus, lethargy

Question 31

PrP*Sc from HY and DY strains

Answer 31

different electrophoretic mobility following PK digestion, different FTIR spectroscopy profile

• suggests different conformatins of PrP*Sc associated with strain variation
• same with CJD strains

Question 32

prion strains overall

Answer 32

not due to nucleic acid variation

can be reproduced in cell-free assays of prion propagation