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A New Mouse Model for Zellweger Spectrum Disorders: Pex1-G844D July 28, 2013

A New Mouse Model for Zellweger Spectrum Disorders: Pex1-G844D July 28, 2013. Steven Steinberg, Ph.D., F.A.C.M.G. Hugo W. Moser Research Institute at KKI & Johns Hopkins University School of Medicine. Neonatal Adreno- leukodystrophy. Infantile Refsum Disease. Zellweger Syndrome.

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A New Mouse Model for Zellweger Spectrum Disorders: Pex1-G844D July 28, 2013

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  1. A New Mouse Model for Zellweger Spectrum Disorders: Pex1-G844D July 28, 2013 Steven Steinberg, Ph.D., F.A.C.M.G. Hugo W. Moser Research Institute at KKI & Johns Hopkins University School of Medicine

  2. Neonatal Adreno- leukodystrophy Infantile Refsum Disease Zellweger Syndrome Most Severe Least Severe Zellweger Spectrum Disorders

  3. Peroxisome Assembly: Matrix Protein Receptor Recycling

  4. PEX1 Common Mutations Severity Neonatal Adreno- leukodystrophy Infantile Refsum Disease Zellweger Syndrome Most Severe I700fs & I700fs I700fs & G843D G843D & G843D Least Severe

  5. Biochemical: Summary

  6. 37 30 37 30 P thiolase M Pex5 wild type PEX1- P, thiolase precursor, 44 kD M, mature thiolase, 42 kD Why Create a Pex1-G844D Mouse Model? -A model for testing possible therapeutic interventions. e.g.PEX1-G843D is temperature sensitive, indicating it may be responsive to a chaperone-like molecule -A means of exploring aspects of pathogenesis not readily explored in humans e.g. RNA expression profiling of multiple tissues

  7. Construct Design: Pex1-G844D Construct generated → ES cells electroporation and selection → Blastocyst injection, implantation & chimera birth → Chimera breeding → Germline G844D Transmission

  8. Matings of Pex1-G844D Heterozygotes Male Female Total Wt / Wt 9 14 23 (25.3%) G844D / Wt 19 25 44 (48.4%) G844D / G844D 13 11 24 (26.4%) TOTAL: 41 (45%) 50 (55%) 91 No evidence of neonatal lethality.

  9. 3 Week Old Littermates Pex1-G844D / WT Pex1-G844D / Pex1G844D

  10. Growth Curve (n = 14) (n = 13) (n = 26) (n = 22) (n = 17) (n = 10)

  11. Survival Homozygotes as of June 2013 DIED OF NATURAL CAUSES Sex NumberRange (in days)Median Male 6 15-147 38 Female 8 7-45 16 LIVING COMFORTABLY Sex NumberRange (in days)Median Male 20 26-250 106 Female 17 69-258 119

  12. Fertility Male homozygotes can be fertile 4 Male Homozygote x Female Heterozygote Matings yielded litters Male homozygote age range at litter birth: 95-110 days Female heterozygote age range at litter birth: 89-110 days Number of days pair were together at litter birth: 25-67 days Noevidence that female homozygotes are fertile 5 Male Homozygote x Female Homozygote Matings Male homozygote age range at end of time together: 95-135 days Female heterozygote age range at end of time together:95-187 days Number of days pair were together: 52-92 days

  13. Cultured Fibroblasts Very Long Chain Fatty Acid Content Pex1-G844D AllelesC26:0 µg/mgC26:0/C22:0 Wildtype 0.023 0.053 Carrier 0.028 0.073 Homozygote 0.465 1.298

  14. Cultured Fibroblasts Plasmalogen Synthesis Pex1-G844D Alleles3H / 14CC16DMAC18DMA Wildtype 0.69 4.075 1.795 Carrier 0.66 4.097 2.041 Homozygote 0.83 2.098 0.641

  15. Cultured Fibroblasts Branched Chain Fatty Acid Metabolism pmol / 48 hours / mg protein Pex1-G844D AllelesPhytanic AcidPristanic Acid Wildtype 582 487 Carrier 406 (70%) 509 (105%) Homozygote 119 (20%) 300 (62%)

  16. Cultured Fibroblasts Catalase Solubility Pex1-G844D Alleles% Soluble Wildtype 11.6% Carrier 11.8% Homozygote 83.3%

  17. Immunocytochemistry Wildtype Pex1-G844D / Pex1G844D

  18. Chaperone Treatment of Fibroblasts

  19. Chaperone Treatment of Fibroblasts

  20. Whole Blood Lipids C26:0-Lysophosphorylcholine Pex1-G844D Alleles% C26:0-LPC Wildtype (n=42) 0.052 ± 0.016 Carrier (n=67) 0.052 ± 0.018 Homozygote (n=17) 0.558 ± 0.363

  21. Whole Blood Lipids Phosphatidylethanolamine Plasmalogens Pex1-G844D AllelesSum of Four (pmoles) Wildtype (n=42) 5.97 ± 2.34 Carrier (n=67) 5.96 ± 2.83 Homozygote (n=17) 2.82 ± 2.22

  22. Bile Acid Intermediate DHCA Pex1-G844D AllelesPlasma*Feces** Wildtype (n=3,4) 0.063±0.129 1.064±0.378 Carrier (n=5,4) 0.073±0.052 0.971±0.406 Homozygote (n=1,3) 8.7 19.115±14.529 *pmol per 10µl plasma; **pmol per mg feces

  23. Liver Histology Pex1-G844D / G844D Wildtype

  24. Tissue Lipids p = 0.007

  25. Visual Testing Electroretinograms ERG Wave Forms b-wave a-wave

  26. Visual Testing Electroretinograms Series 1 = Wildtype and Heterozygous Mice, n=4 Series 2 = Pex1-G844D Homozygous Mice, n=4

  27. Visual Testing Retinal Histology: Cone Photoreceptor Staining Homozygote, 28 days Heterozygote, 28 days inl = inner nuclear layer onl = outer nuclear layer is = inner segment of photoreceptor layer os = outer segment of photoreceptor layer Homozygote, 147 days Wildtype, 149 days

  28. Pex1-G844D Mouse Model Summary -Growth stunted - -Males can be fertile, but not female -Biochemical profile is comparable to human patients homozygous for PEX1-G843D -Compelling evidence of retinal pathology and dysfunction comparable to human patients -Preliminary evidence of liver dysfunction that is likely comparable to human patients

  29. Pex1-G844D Mouse Future Plans -Treatment with compounds identified by drug screening using a cell model system to test for in vivo efficacy -Behavioral studies -Generate Pex1 knock out and generate Pex1-KO / Pex1-G844D compound heterozygotes and tissue specific knock outs

  30. Outside KKI / JHU • Jean Bennett • Nancy Braverman • Gerald Raymond • Joseph Hacia • Michael Paine • Phyllis Faust Collaborators • Shandi Hiebler • Ann Moser • Anita Liu • Paul Watkins • Dominik Reisinger • Amanda Lauer • Tomohiro Masuda • Don Zack Sponsored by the Park-Hopkins and Woodbury research funds donations at Kennedy Krieger Institute

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