Proteins as Pathogens Stanley B. Prusiner, MD The Nobel Prize in Physiology or Medicine 1997 Presented by Shannon S. Rickner-Schmidt

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.

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; 13363-13383.

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: 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) 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

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 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.

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.

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 disease Within 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 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

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.