1. Proteins as Pathogens
Stanley B. Prusiner, MD The Nobel Prize in Physiology or Medicine Presented by Shannon S. Rickner-Schmidt

2. Prions: Causative Agents of Disease
Definition: 
Proteinaceous infectious particle, devoid of nucleic acids
Affect primarily the nervous system
Spongiform degeneration of brain tissue
Protein accumulations in brain tissue (plaques)
Source: Prusiner, S.B. (1998) Prions. Proceedings of the National Academy of Sciences, USA, 95;

3. Prions: Causative Agents of Disease
Sporadic (spontaneous)
Infectious (transmissible)
Genetic (familial)
Source: Pruisner, S.B. (1998) Prions. Proceedings of the National Academy of Sciences, USA, 95;

4. Prions: Causative Agents of Disease
PrPc
Highly conserved brain glycoprotein
Normal cellular component, 35kDa
Unique biogenesis in ER
Transmembrane Forms
Secreted Form
(SecPrP)
Translocated into
ER Lumen
NtmPrP
N-trans PrP
N-terminal of protein in ER lumen
CtmPrP
C-trans PrP
C-terminal of protein in ER lumen
Overexpression
Results in Severe
Neurodegenerative
Disease
Common Hydrophobic Segment with Distinct Epitopes, Glycosylation and Size
Source: Hegde, RS, Mastrianni, JA, Scott, MR, DeFea, KA, Tremblay, P, Torchia, M, et al (1998) A transmembrane form of the prion protein in
Neurodegenerative disease. Science; 279:

5. Function Follows Form: Isoforms
PrPSc: Prion Protein Scrapie- Infectious form
When exposed to PrPSc, normal constituent of mammalian cells (CtmPrP) becomes infectious form through a structural change
Hypothesis: Ability of host to make the CtmPrP form determines effectiveness of PrPSc in causing neurodegenerative disease

6. Designing the Experiment
Mutant mice that do not produce CtmPrP (FVB/Pmp0/0)
Create transgenic (Tg) lines by introducing either mutated or normal hamster genes (SHaPrP)
Correlate neurodegeneration with the PrP form expressed
Transgenic Line
Expression
(KH→II)H
High CtmPrP
(KH→II)M
Medium CtmPrP
(A117V)H
(N108I)H
(ΔSTE)H
SecPrP only
(KH→II)L
Low CtmPrP
(A117V)L
(N108I)L

7. Measuring PrP produced by Tg mice
All Tg mice lines express PrP
WT, A117V, N108I and KH→II lines express CtmPrP
Digestion with Proteinase K results in two distinct fragments that result from CtmPrP and NtmPrP forms
ΔSTE strain is resistant to proteolysis, indicative of SecPrP
WT High Levels CtmPrP Low CtmPrP No CtmPrP
Figure 1a: Stained with a R073, a polyclonal antibody (pAB) that recognizes all PrP.

8. Measuring C-terminal Fragments
Level of PrP Expression in Brain Tissue Homogenate
Stained using monoclonal antibody (mAB) that recognizes C-terminal epitope
Confirmed different lines express different levels of CtmPrP/ SecPrP
Homogenate Amount
Figure 1b: Stained with a 13A5, a monoclonal antibody that recognizes C-terminal PrP fragments.

9. Correlating CtmPrP to Disease
Onset of Disease Symptoms in Tg Mice Without Exposure to PrPSc
Wild Type remains asymptomatic for longer time than strains overexpressing CtmPrP
Transgenic Strains
Days until 50% Mice have Disease Symptoms
■ Tg[SHaPrP(KH→II)H
~75
○ Tg[SHaPrP(N108I)H
~250
●Tg[SHaPrP(A117V)H
~500
--- Wild Type (non-transgenic hamsters)
~675
Figure 1c: Onset of disease in un-inoculated Tg mice.

10. Correlating CtmPrP to Disease
PrP species found in transgenic mice
Evidence of CtmPrP in clinically ill mice
(A117V)H
(N108I)H
No evidence of CtmPrP in unaffected mice
(A117V)L
(N108I)L
No evidence of PrPSc in any mice
Figure 1d: Stained with a R073, a polyclonal antibody that recognizes all SHa PrP.

11. Susceptibility of Tg Mice to PrPSc
Tg Mice were Inoculated with Sc237 (SHa prions)
Correlation of Disease with CtmPrP Expression
Minimum level of CtmPrP expression necessary for disease
Within strains, increased CtmPrP expression correlates with more rapid onset of disease

12. Susceptibility of Tg Mice to PrPSc
Lines ΔSTE and (A117V)H were inoculated with PrPSc
ΔSTE:
Develops neurodegeneration much later,
Accumulates more PrPSc prior to symptom onset
Lines (KH→II)L and (KH→II)M were inoculated with PrPSc
(KH→II)L :
Develops neurodegeneration much later,
Accumulates more PrPSc prior to symptom onset
Lines (A117V)L and (Av117V)H were inoculated with PrPSc
(A117V)L :
Develops neurodegeneration much later,
Accumulates more PrPSc prior to symptom onset
Figure 2a-f: Propensity of Lines to Produce CtmPrP influences disease onset and PrPSc accumulation

13. Susceptibility of Tg Mice to PrPSc
Figure 2g: Ctm-index (%Ctm in Vitro x Level PrP expression) is inversely proportional to amount of PrPSc accumulated

14. Establishing a Causal Relationship
Hypothesis: PrPSc accumulation is not the proximate cause of neurodegeneration, but increased generation of CtmPrP is.
Prediction 1:
Tg mice that produce higher levels of CtmPrP do not need PrPSc to develop neurodegenerative disease, and shouldn’t be infectious
Assess transmissibility of CtmPrP-associated disease
Figure 3: Prediction 1–inoculums from terminally ill mice with CtmPrP associated neurodegenerative disease or WT mice do not induce disease in null mice, Tg mice or Syrian Hamsters. Most animals live expected lifespan.

15. Establishing a Causal Relationship
Prediction 2: CtmPrP levels should rise during accumulation of PrPSc
Harder to measure directly due to assay interference
Figure 4a: Solution- use Doubly Transgenic mice, expose to mouse PrPSc, which will not interfere with assay for SHa CtmPrP.
Over nine weeks, doubly Tg mice were
assayed for total PrP (pAB R073)
assayed for Syrian Hamster PrP (mAB 3F4)
Samples with no PK digestion show all mouse and SHa CtmPrP and PrPSc
Harsh PK digestion leaves PrPSc only, demonstrating no SHa PrPSc present in mice.
Amount Homogenate:
Samples assayed for SHa CtmPrP, demonstrating increase over time
This increase was not seen in un-inoculated mice, indicating inoculation with PrPSc caused CtmPrP increase

16. Prion Disease: Model of Pathogenesis
Formation and accumulation of PrPSc
Inoculation
Spontaneous Conversion of Mutated PrPc
CtmPrP Generation
In-trans by accumulated PrPSc
In-cis by mutations within PrP
CtmPrP mediated neurodegeneration
Misfolded protein exits ER, avoiding typical degradation process
PrPSc not necessary
CtmPrP required

17. Prion Disease: Future Study}
CtmPrP Biosynthesis & Trafficking
CtmPrP Metabolism }
Neurodegeneration Mechanism

18. Prion Disease: Suggested Readings
Horwich, A.L. & Weissman, J.S. (1997). Deadly Conformations- Protein Misfolding in Prion Disease. Cell; 89:
Prusiner, S.B. (1998). Prions. Proceedings of the National Academy of Sciences, USA; 95: