Stanley B. Prusiner, MDThe Nobel Prize in Physiology or Medicine 1997 Presented by Shannon S. Rickner-Schmidt

Proteins as Pathogens

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; 13363-13383.

Source:Pruisner, S.B. (1998) Prions. Proceedings of the National Academy of Sciences, USA, 95; 13363-13383.

Prions: Causative Agents of Disease

Sporadic (spontaneous)Infectious (transmissible)Genetic (familial)

Common Hydrophobic Segmentwith Distinct Epitopes, Glycosylation

and Size

Prions: Causative Agents of Disease

PrPc

•Highly conserved brain glycoprotein•Normal cellular component, 35kDa

•Unique biogenesis in ER

Transmembrane FormsSecreted Form

(SecPrP)Translocated into

ER Lumen

CtmPrP•C-trans PrP

•C-terminal of protein in ER lumen

NtmPrP•N-trans PrP

•N-terminal of protein in ER lumen

Overexpression Results in Severe Neurodegenerative

DiseaseSource: Hegde, RS, Mastrianni, JA, Scott, MR, DeFea, KA, Tremblay, P, Torchia, M, et al (1998) A transmembrane form of the prion protein inNeurodegenerative disease. Science; 279: 827-834.

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

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)

Transgenic Line

Expression

(KH→II)H High CtmPrP

(KH→II)M Medium CtmPrP

(A117V)H High CtmPrP

(N108I)H High CtmPrP

(ΔSTE)HSecPrP only

(KH→II)L Low CtmPrP

(A117V)L Low CtmPrP

(N108I)L Low CtmPrP

Correlate neurodegeneration with the PrP form expressed

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.

Measuring C-terminal Fragments

Level of PrP Expression in Brain Tissue HomogenateStained using monoclonal antibody (mAB) that recognizes C-terminal epitopeConfirmed 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.

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 StrainsDays 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.

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.

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 diseaseWithin strains, increased CtmPrP expression correlates with more rapid onset of disease

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

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

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.

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: 1 .25 .1 .1 .25 1

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

Prion Disease: Model of Pathogenesis

CtmPrP mediated neurodegeneration

Misfolded protein exits ER, avoiding typical degradation process PrPSc not necessary CtmPrP required

Formation and accumulation of PrPSc

Inoculation Spontaneous Conversion of Mutated PrPc

CtmPrP Generation In-trans by accumulated PrPSc

In-cis by mutations within PrP

Prion Disease: Future Study

Neurodegeneration Mechanism

CtmPrP Biosynthesis & TraffickingCtmPrP Metabolism }

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Prion Disease: Suggested Readings

Horwich, A.L. & Weissman, J.S. (1997). Deadly Conformations- Protein Misfolding in Prion Disease. Cell; 89: 499-510.

Prusiner, S.B. (1998). Prions. Proceedings of the National Academy of Sciences, USA; 95: 13363-13383.