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Stanley B. Prusiner, MD The Nobel Prize in Physiology or Medicine 1997 Presented by Shannon S. Rickner-Schmidt. Proteins as Pathogens. Definition:  Proteinaceous infectious particle, devoid of nucleic acids Affect primarily the nervous system

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Stanley B. Prusiner, MD The 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)

Prions: Causative Agents of Disease

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

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

Sporadic (spontaneous)

Infectious (transmissible)

Genetic (familial)

Prions: Causative Agents of Disease

common hydrophobic segment with distinct epitopes glycosylation and size

PrPc

  • Highly conserved brain glycoprotein
  • Normal cellular component, 35kDa
  • Unique biogenesis in ER

Common Hydrophobic Segmentwith Distinct Epitopes, Glycosylation and Size

Transmembrane Forms

Secreted Form

(SecPrP)

Translocated into

ER Lumen

Prions: Causative Agents of Disease

  • 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

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: 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

Function Follows Form: Isoforms

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

Designing the Experiment

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

Measuring PrP produced by Tg mice

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 Homogenate

    • Stained using monoclonal antibody (mAB) that recognizes C-terminal epitope
    • Confirmed different lines express different levels of CtmPrP/ SecPrP

Measuring C-terminal Fragments

Homogenate Amount

Figure 1b: Stained with a 13A5, a monoclonal antibody that recognizes C-terminal PrP fragments.

correlating ctm prp to disease

Onset of Disease Symptoms in Tg Mice Without Exposure to PrPSc

    • Wild Type remains asymptomatic for longer time than strains overexpressing CtmPrP

Correlating CtmPrP to Disease

Figure 1c: Onset of disease in un-inoculated Tg mice.

correlating ctm prp to disease1

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

Correlating CtmPrP to Disease

Figure 1d: Stained with a R073, a polyclonal antibody that recognizes all SHa PrP.

susceptibility of tg mice to prp sc

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

Susceptibility of Tg Mice to PrPSc

susceptibility of tg mice to prp sc1

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

Susceptibility of Tg Mice to PrPSc

  • 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 prp sc2
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

Establishing a Causal Relationship

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 relationship1

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.

Establishing a Causal Relationship

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

Formation and accumulation of PrPSc

    • Inoculation
    • Spontaneous Conversion of Mutated PrPc

Prion Disease: Model of Pathogenesis

  • 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
prion disease future study

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Prion Disease: Future Study

CtmPrP Biosynthesis & Trafficking

CtmPrP Metabolism

}

Neurodegeneration Mechanism

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.

Prion Disease: Suggested Readings

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