Leah hotchkiss asr junior november 9 2012
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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

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


What is a prion

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


Prion diseases

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


Prp c and prp sc of equal importance

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


A new type of prion

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


The unknowns of sho

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


The pi hypothesis

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


Key terms

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.


Key terms continued

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


Review of literature

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.


Review of literature1

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.


Review of literature2

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.


Problem

Problem

How do different

Prnp/Sprn knockout combinations affect embryonic lethality?


Hypothesis

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.


Leah hotchkiss asr junior november 9 2012

questions?


Creating knockout mice

methods and materials

Creating Knockout mice

  • “Turned off” specific genes in embryonic stem cells


Animal husbandry and prion inoculation

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


Western blot analyses

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


Sub cellular fractionation

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


Histology

methods and materials

Histology

  • Fixing of samples in Carnoy’s fixative

  • Dehydration, processing, and staining to examine brain tissue

  • Different dyes use specific antigens that correspond with protein

http://www.cancerdiagnostics.com/UploadDocuments/

http://www.apn-histopathology.unimelb.edu.au/


Overview of experiment

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


Verifying knockout mice

results

Verifying knockout mice

Method: Western blot

Result: Successful creation

Control

No Sprn Expression


Observing neuroanatomy of sho protein in comparison to prp c

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


Sho and prion infection

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


Are double knockout mice sprn 0 0 prnp 0 0 embryonic lethal

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


Double knockout histology

Double knockout histology

Method: histology

Result: Double knockout mice are viable, producing a weaker phenotype

No significant changes

=

viability


Discussion

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


Discussion1

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


Conclusion

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


Future work

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


Personal future

Personal Future

  • Communication with Nathalie Daude, University of Alberta

    -Ask about research opportunities

http://1.bp.blogspot.com/-8-NLFFwHVKo/


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