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Leah Hotchkiss ASR Junior November 9, 2012

Knockout of the prion protein (PrP)-like Sprn gene does not produce embryonic lethality In Combination with PrP C -deficiency Nathalie daude et al. University of ALberta. Leah Hotchkiss ASR Junior November 9, 2012. introduction. What is a prion?. • Proteinaceous infectious particle

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Leah Hotchkiss ASR Junior November 9, 2012

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  1. Knockout of the prion protein (PrP)-like Sprn gene does not produce embryonic lethality In Combination with PrPC-deficiencyNathalie daude et al.University of ALberta Leah Hotchkiss ASR Junior November 9, 2012

  2. introduction What is a prion? • Proteinaceous infectious particle • A misfolded protein • Causes others proteins to misfold •Chain reaction leading to exponential growth •PrP(prion protein) build up

  3. introduction Prion Diseases • Neurodegenerative: destruction of the nervous system • Fatal • Mad Cow Disease • Creutzfeldt-Jakob Disease • Mouse Scrapie • Can occur sporadically, through infection, or be inherited pathmicro.med.sc.edu

  4. introduction PrPc and PrPsc: Of equal importance • PrPc: cellular, already present in the body, has the potential to mutate • The root of the problem, without PrPc prion diseases cannot occur • PrPsc: scrapie, the mutated version, builds up in the affected areas • Acts as a template allowing the disease to proliferate

  5. introduction A new type of prion • Shadoo protein (Sho) • Discovered in 2007 • A PrPC-like protein was being depleted before clinical signs of prion infection occurred • The body might be trying to get rid of infection with a misdirected response • Similarities to PrPC • Share many protein binding partners • Found in mammals • Member of prion protein family

  6. introduction The unknowns of Sho • Structure • Specific Location • Purpose Possible Functions -Protein or RNA chaperone -Acting as pi, a hypothetical prion accessory protein upload.wikimedia.org/wikipedia

  7. introduction The pi Hypothesis • PrP, when functioning normally interacts with a partner protein that has two binding sites • This partner must be engaged at BOTH sites or else it will result in neuronal death • In PrP deficient mice pi is proposed to take over the role of PrP

  8. introduction Key terms Shadoo protein: recently discovered prion-like protein (Sho) Prion Protein: basic protein that causes a group of neurodegenerative diseases (PrP) Sprn: Shadooprotein gene Prnp: Prion protein gene Knockout: a mutant mouse that has had certain genes deleted and replaced by null alleles Null allele: a mutant form of the knockout gene that does not have any similar functions to the original gene. Takes the place of the removed allele.

  9. introduction Key terms continued Phenotype: the observable physical and biochemical characteristics of an organism Wild type: the form of a gene that is most commonly found in nature Embryonic Lethality: death of embryo in development Transient knockdown allele: temporarily silenced allele, decreases expression rather than eliminating it entirely Constitutive null allele: completely deleted allele, no expression

  10. Review of literature Carlson GA, et al. (1986) Linkage of prion protein and scrapie incubation time genes. Cell 46:503–511. Westaway D, et al. (1987) Distinct prion proteins in short and long scrapie incubation period mice. Cell 51:651–662. Büeler H, et al. (1993) Mice devoid of PrP are resistant to scrapie. Cell 73:1339–1347 . Tobler I, et al. (1996) Altered circadian activity rhythms and sleep in mice devoid of 
prion protein. Nature 380:639–642. Tobler I, Deboer T, Fischer M (1997) Sleep and sleep regulation in normal and prion 
protein-deficient mice. J Neurosci 17:1869–1879. Mallucci GR, et al. (2002) Post-natal knockout of prion protein alters hippocampal 
CA1 properties, but does not result in neurodegeneration. EMBO J 21:202–210.

  11. Review of literature Shmerling D, et al. (1998) Expression of amino-terminally truncated PrP in the mouse 
leading to ataxia and specific cerebellar lesions. Cell 93:203–214. Flechsig E, Weissmann C (2004) The role of PrP in health and disease. CurrMol Med 
4:337–353.

  12. Review of literature Premzl M, et al. (2003) Shadoo, a new protein highly conserved from fish to mammals 
and with similarity to prion protein. Gene 314:89–102. Watts JC, et al. (2007) The CNS glycoprotein Shadoo has PrP(C)-like protective prop- 
erties and displays reduced levels in prion infections. EMBO J 26:4038–4050. Westaway D, et al. (2011) Down-regulation of Shadoo in prion infections traces a pre- 
clinical event inversely related to PrP(Sc) accumulation. PLoSPathog 7:e1002391. Watts JC, et al. (2011) Protease-resistant prions selectively decrease Shadoo protein. 
PLoSPathog 7:e1002382. Miyazawa K, Manuelidis L (2010) Agent-specific Shadoo responses in transmissible 
encephalopathies. J NeuroimmunePharmacol 5:155–163. Watts JC, et al. (2009) Interactome analyses identify ties of PrP and its mammalian 
paralogs to oligomannosidic N-glycans and endoplasmic reticulum-derived chaper- ones. PLoSPathog 5 Young R, et al. (2009) The prion or the related Shadoo protein is required for early mouse embryogenesis. FEBS Lett 583:3296–3300.

  13. Problem How do different Prnp/Sprn knockout combinations affect embryonic lethality?

  14. hypothesis If the Sprn0/0/Prnp0/0 null allele is expressed then it will have a stronger phenotype than the Sprn0/wt/Prnp0/wttransient allele because it will stop gene expression entirely, producing more obvious effects.

  15. questions?

  16. methods and materials Creating Knockout mice • “Turned off” specific genes in embryonic stem cells

  17. methods and materials Animal husbandry and prion inoculation • Mice were contained in groups of up to 5 • 12 hour light/dark cycle • Living conditions and inoculation all in check with the Canadian Council on Animal Care Inoculation -Intracerebral -Intraperitoneal -Oral http://i.telegraph.co.uk/multimedia/archive/01632/labMice_1632902c.jpg

  18. methods and materials Western Blot analyses • Used to determine protein concentrations • Proteins are separated by gels and homogenate is ground and then blotted to extract protein Determines WHAT proteins are present http://www.molecularstation.com/images/southern-blot.jpg

  19. methods and materials Sub-Cellular Fractionation • Brain samples are homogenized and fractionated to see where specific proteins are located • Determining exact location can provide key information for function Determines WHERE specific proteins are located http://www.gbiosciences.com/EducationalUploads

  20. methods and materials Histology • Fixing of samples in Carnoy’s fixative • Dehydration, processing, and staining to examine brain tissue • Different dies use specific antigens that correspond with protein http://www.cancerdiagnostics.com/UploadDocuments/ http://www.apn-histopathology.unimelb.edu.au/

  21. methods and materials Overview of Experiment • Testing to see if creation of knockout mice was successful • Observing the neuroanatomy of Sho protein • Testing to see is Sho is necessary for prion infection • Testing embryonic lethality Observing a complicated topic in a simple way

  22. results Verifying knockout mice Method: Western blot Result: Successful creation Control No Sprn Expression

  23. results Observing Neuroanatomy of Sho Protein in comparison to PrPC Method: Using histology to observe coincidental expression of PrPC and Sho Result: Sho and PrPC aren’t always expressed in the same place Dark Area: Protein expression Sho being expressed in Prnp0/0 PrP being expressed in Sprn0/0

  24. Sho and Prion infection Method: western blot Result: PrPSC is visible in all variations whether Sprn is expressed or not Prion infection is not dependent on the presence of Sho protein

  25. results Are double knockout mice (Sprn0/0/Prnp0/0) embryonic lethal? Method: generation of Sprn0/0/Prnp0/0 and Sprn0/0/Prnp0/wt mice Result: Double knockout mice (homozygous pairing) were viable and fertile and continued to reproduce successfully Wild type knockout mice (heterozygous pairing) resulted in embryonic lethality Stronger phenotype: embryonic lethality

  26. Double knockout histology Method: histology Result: Double knockout mice are viable, producing a weaker phenotype No significant changes = viability

  27. discussion If the Sprn0/0/Prnp0/0 null allele is expressed then it will have a stronger phenotype than the Sprn0/wt/Prnp0/wttransient allele because it will stop gene expression entirely, producing more obvious effects. • HYPOTHESIS NOT SUPPORTED • Results were completely unprecedented

  28. Discussion Possible Explanation -Null allele deletion is expressed from early embryogenesis so the body might express a protein with similar function PrPC to allow development to continue normally http://tanyagrove.files.wordpress.com/2011

  29. Conclusion • If prion infection subsists without Sho protein, then the pi theory is unlikely • Sho protein expression does not affect PrP expression • Sho is more likely to reveal a degradative effect towards PrPSC rather than helping activity towards PrPC

  30. Future work • Determine what other proteins could be pi • Further examine pi plausibility • Look into embryonic development • Look into Sho as an agent in neuroprotection and maintenance as PrPC is speculated to be

  31. Personal Future • Communication with Nathalie Daude, University of Alberta -Ask about research opportunities http://1.bp.blogspot.com/-8-NLFFwHVKo/

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