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Elizabeth M. Simpson, Ph.D.

Elizabeth M. Simpson, Ph.D. Senior Scientist, Associate Professor & Canada Research Chair Centre for Molecular Medicine & Therapeutics at Children’s & Women’s Health Centre & Department of Medical Genetics University of British Columbia.

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Elizabeth M. Simpson, Ph.D.

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  1. Elizabeth M. Simpson, Ph.D. Senior Scientist, Associate Professor& Canada Research Chair Centre for Molecular Medicine & Therapeuticsat Children’s & Women’s Health Centre& Department of Medical GeneticsUniversity of British Columbia Towards QTL Mapping & Microarrays to Identify Modifiers of Caspase 3 Induced Exencephaly

  2. Caspases • Caspases are cysteinyl proteases that mediate most events that culminate in the apoptotic phenotype.

  3. CaspaseApoptotic Pathways Nicholson, DW (2000), Nature 407:810-816

  4. Many Roles of Caspase-Mediated Apoptosis Suggests Therapeutic Potential for Inhibitors • Embryogenesis • Cancer • Neurodegenerative Disorders • Huntington’s and Alzheimer’s Diseases • Ischemia-induced injury • Stroke, Sepsis, Acute Liver Injury • Infectious Disease • HIV-medicated AIDS

  5. Unfortunately, Caspases have Not Proven Good Drug Targets • Non-specificity • Poor cell permeability • Poor efficacy

  6. Search for New Drug Targets Aim 1. QTL Aim 2. Candidates Drug Targets Aim 3. Prostate Aim 4. Microarrays

  7. 129-Casp3 – lateral view (e18.5) KO (Exencephaly) WT (Normal) brain brain brain tongue 129-Casp3 – ventral view (e18.5) { brain brain L cheek brain R cheek Caspase 3 KO Phenotype at e18.5

  8. A Congenic Resource

  9. Phenotype at e18.5 - B6 v 129

  10. B6-Casp3 129-Casp3 Resistant Sensitive FVB-Casp3 CAST-Casp3 Variable Resistant DBA-Casp3 ?

  11. Brain Phenotype

  12. DNA Fragmentation on Telencephalic Vesicle Cultures 1 day culture B6

  13. DNA Fragmentation on Telencephalic Vesicle Cultures 1 day culture 2 days culture B6 129

  14. More activated Casp7 in B6 (res) v 129 (sen) precursor neurons during apoptosis e12.5

  15. Differential Caspase 7 Levels at e12.5 but not e14.5

  16. Candidate Alternative Therapeutic Targets

  17. cDNA Microarrays Analysis of Candidate Genes in B6 (res) • AI844488 Bcl2-like 2 • (AI837675 Bcl2 homologous antagonist/killer) • (AI843083 Apoptotic protease activating factor 1) • B6-KO • B6-WT

  18. AI839583 Mus musculus ICAD-L • (AI847216 Cytochrome C oxidase, subunit VI a, polypeptide 1) • (AI849253 Tumor necrosis factor super family 3-like) • B6-WT • B6-KO

  19. AI854211 Cytochrome C oxidase, subunit VIIIa • (AI845508 Tumor necrosis factor alpha converting enzyme (TACE/ADAM17)) • B6-KO • B6-WT

  20. DNA Fragmentation Brain Caspase 7 129 B6 129 B6 129 B6 WT Nor Nor WT Nor Nor WT Less More Abnor KO Nor KO Less Less KO Less More Conclusions To Date

  21. Kathy Banks, M.Sc. Lab Manager, CMMT, UBC Caroline Houde, M.Sc. Merck Frosst Colleen Nelson, Ph.D.Assistant Prof., UBC Don Nicholson, Ph.D. Merck Frosst Sophie Roy, Ph.D. Merck Frosst Elizabeth M. Simpson, Ph.D.Associate Prof., CMMT, UBC Collaborators

  22. COMPLEX TRAITS OXFORD 030702

  23. NOT USED

  24. B6-Casp3 – ResistantHow Normal is it? • < 1% embryos show brain abnormalities • 10 – 12.5% of B6-Casp3 KO adults die with an obvious phenotype • big brain  malocclusion ± runting • 6 adult B6-Casp3 (3 WT, 3 KO) to Frimorfo • histology: brain and other tissues

  25. Gross Morphology Cell Death ELISA 1. Embryonic dissectionPhotographic record Written description 1. Embryonic Dissection Telencephalic vesiclesCulture (2 days)ELISA at Merck? 2. Preliminary data? 4/4 parental strains1/2 F1s 2. Preliminary data?2/4 parental strains0/2 F1s 3. Relevance?Questionable 3. Relevance?High Alternative Phenotyping Strategy

  26. e12.5 telencephalic percussor neurons fragment their DNA in the absence of Casp-3 in B6 mice, but not in the 129 strain. 1 day culture 2 days culture Bl6 129

  27. Less Casp-7 is activated in 129 precursor neurons compare to B6 during apoptosis.

  28. Experimental Design

  29. 129-Casp3 –Sensitive How Abnormal is it? • Is there a pre-symptomatic age in the 129-Casp3? • Phenotype visible at e12.5 (gross and histological) • > 85% perinatal 129-Casp3 KO show brain abnormalities • Examine brains of B6 and 129 by gross morphology & histology • e8.5, e9.5, e10.5, e11.5 Compare “pre-symptomatic” timepoints between‘resistant’ (B6) and ‘sensitive’ (129) strains using Affy.

  30. Modifiers vs. Compensation • Compensatory factors • Make up for a deficiency or loss • eg. factors that compensate for Casp3-/- on the B6 background. (Casp7 ?) • Modifiers • Affect the expression of another gene • eg. factors that make B6 resistant, but 129 sensitive to Casp3-/-. (Casp7 ?)

  31. Using Microarrays for QTL • Use Microarray data on F2 offspring as a quantitative phenotype prior to QTL • Or • Use Microarray data to phenotype parents after the QTL, as a candidate gene approach

  32. Modifier Mapping Resource B6-Casp3 129-Casp3 Resistant Sensitive FVB-Casp3 CAST-Casp3 ?Sensitive Resistant DBA-Casp3 ?

  33. Simpson Lab CMMT: Present Elizabeth M. Simpson, Ph.D.Senior Scientist 9904 Brett Abrahams, B.Sc.H. Grad. Student 9905 Ravinesh Kumar, B.Sc.Grad. Student 0002 Tracey Weir, B.M.L.Sc.Res. Writer 0004 Kathy Banks, M.Sc.Lab Manager 0004 Sazzad Hossain, Ph.D.Res. Assistant 0006 Dora PakAdmin. Assistant 0009 Rebecca Devon, Ph.D.Post Doc. Fellow 0009 Bibiana Wong, B.Sc.Graduate Student 0106 Slavita Bohacec, B.Sc. Res. Assistant 0109 Melvin KwokSFU Co-op Student –Volunteer 0209 Saeed BudaghzadehUBCVolunteer 0305 Daniel Yokum UBC Volunteer 0305 Lauren Seeley Summer Student 0305 YuanYun (Robert) Xie, M.D., Ph.D. Post Doc. Fellow 0305

  34. B6129F2 + 129B6F2 -- 400 embryos DBACASTF2 + CASTDBAF1 -- 400 embryos 800 embryos x 200 markers (~10 cM) = 160,000 PCR reactions = 40,000 multiplexed (4) lanes High-throughput genotyping ABI3100 Genetic analyzer = 2,500 machine runs @ 16 capillaries = 1,200 days @ 2 runs/day = 3.3 years Genotyping

  35. Strain Generation B6-Casp3 Resistant 15 (full congenic) 129-Casp3 Sensitive 11 (full congenic) CAST-Casp3 8 (incipient congenic) Resistant DBA-Casp3 8 (incipient congenic) Sensitive Congenic Resource Phenotype

  36. F1 X 50% B6 129Casp3/+(Intermediate) 50%B6 129Casp3/+(Intermediate) F2 ±50% B6 129 ±50% B6 129 ±50% B6 129 ±50% B6 129 ±50% B6 129 Casp3/Casp3 Casp3/Casp3 Casp3/Casp3 Casp3/Casp3 Casp3/Casp3 Intermediate Intermediate Resistant Intermediate Sensitive Mapping Modifiers of Casp3 Parental X 100% 129Casp3/+(Sensitive) 100% B6Casp3/+ (Resistant)

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