Leah hotchkiss asr junior november 9 2012
This presentation is the property of its rightful owner.
Sponsored Links
1 / 31

Leah Hotchkiss ASR Junior November 9, 2012 PowerPoint PPT Presentation


  • 148 Views
  • Uploaded on
  • Presentation posted in: General

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

Download Presentation

Leah Hotchkiss ASR Junior November 9, 2012

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


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


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


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


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


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


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


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


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.


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

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 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 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

How do different

Prnp/Sprn knockout combinations affect embryonic lethality?


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.


questions?


methods and materials

Creating Knockout mice

  • “Turned off” specific genes in embryonic stem cells


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


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


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


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/


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


results

Verifying knockout mice

Method: Western blot

Result: Successful creation

Control

No Sprn Expression


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

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


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

Method: histology

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

No significant changes

=

viability


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


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

  • 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

  • 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

  • Communication with Nathalie Daude, University of Alberta

    -Ask about research opportunities

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


  • Login