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Gali Maor and Mia Horowitz EWGGD, Paris June 27-30, 2012 


ACTIVATION OF THE UNFOLDED PROTEIN RESPONSE IN GAUCHER DISEASE. Gali Maor and Mia Horowitz EWGGD, Paris June 27-30, 2012 
. EP Gaucher. Gaucher Disease. Due to the defective activity of lysosomal glucocerebrosidase (GCase) in Gaucher disease there is: ★ Accumulation of substrate

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Gali Maor and Mia Horowitz EWGGD, Paris June 27-30, 2012 


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  1. ACTIVATION OF THE UNFOLDED PROTEIN RESPONSE IN GAUCHER DISEASE Gali Maor and Mia Horowitz EWGGD, Paris June 27-30, 2012 


  2. EPGaucher Gaucher Disease Due to the defective activity of lysosomal glucocerebrosidase (GCase) in Gaucher disease there is: ★Accumulation of substrate ★ERAD of mutant GCase variants

  3. ERAD of mutant GCase variants ERAD has been shown for the following genotypes (mutations): N370S/N370S, N370S/L444P, N370S/V394, N370S/recTl, N370S/V394L, R120W/R120W, L444P/R120W, G202R/G202R+M362I, L444P/P415R, G202R/G202R, K157Q/D140H+E326K, R463C/?, R131C/R131C, L444P/L444P, L444P/R415P, D409H/D409H Every ER resident or passenger protein undergoes ERQC and, if needed, ERAD!!!

  4. Quantitation of ERAD There is a correlation between the severity of the disease and the ERAD level

  5. ERAD of mutant GCase variants ERAD of mutant GCase variants may lead to pathogenesis

  6. The Unfolded Protein Response 1 Douglas M. Cyr & Daniel N. Hebert, Embo Reports April 2012 2 3 1 (CHOP) • Test UPR by : • Changes in mRNA and protein levels of Bip, CHOP • Phosphorylation of eIF2α • Splicing of Xbp1 mRNA

  7. Gaucher Disease CUPR exists in GD derived cells CUPR also exists in carriers of GD mutations CUPR upregulates GBA transcription

  8. UPR in GD patients and carriers: Bip and CHOP mRNA levels * ** ** ** P < 0.01 * P < 0.05 ** ** ** * * * * * * * * *

  9. UPR in GD patients: Bip and CHOP proteins G202R/G202R L444P/R120W L444P/L415W N370S/N370S WT BiP * * β-tubulin ** P < 0.01 * P < 0.05 L444P/L415W L444P/R120W N370S/N370S G202R/G202R WT CHOP ** * ** β-tubulin * Bip and CHOP mRNA and protein levels are significantly elevated in GD fibroblasts

  10. UPR in GD patients: Xbp1 splicing Forward primer Reverse primer Forward primer Reverse primer Forward primer Reverse primer No splicing Splicing ** K157Q/D140H,E326K * L444P/R120W R131C/R131C * N370S/N370S WT ** Spliced xbp1 GAPDH Level of Xbp1 splicing is significantly elevated in GD fibroblasts

  11. UPR in GD patients: Phosphorylation of eIF2α ** ** * P < 0.01 ** P < 0.05 L444P/R120W G202R/G202R L444P/L415W N370S/N370S WT P-EIF2α Levels of phosphorylated eIF2α protein are significantly elevated in GD fibroblasts EIF2α

  12. Unfolded Protein Response in GD * * * P < 0.05 Substrate accumulation does not lead to UPR

  13. UPR in GD mice (Grabowski): Bip and CHOP proteins A * P < 0.05 * * Bip and CHOP mRNA levels are significantly elevated only in D409V homozygous mice!!!

  14. UPR in GD mice: Xbp1 splicing A Forward primer Reverse primer Forward primer Reverse primer Forward primer Reverse primer No splicing Splicing * * P < 0.05 * Level of Xbp1 splicing is significantly elevated in D409V homozygous mouse fibroblasts

  15. Gaucher Disease CUPR exists in GD derived cells CUPR also exists in carriers of GD mutations CUPR upregulates GBA transcription

  16. Gaucher Disease CUPR exists in GD derived cells CUPR also exists in carriers of GD mutations CUPR upregulates GBA transcription

  17. Gaucher Disease CUPR exists in GD derived cells CUPR also exists in carriers of GD mutations CUPR upregulates GBA transcription

  18. Unfolded Protein Response in GD carriers * **P < 0.01 * P < 0.05 ** ** There is UPR in GD carriers even without a detected protein

  19. Gaucher Disease CUPR exists in GD derived cells CUPR also exists in carriers of GD mutations CUPR upregulates GBA transcription

  20. GBA mRNA levels in GD fibroblasts ** There are elevated levels of GBA mRNA in GD derived fibroblasts Does it result from response to UPR??????

  21. GBA mRNA levels in GD fibroblasts ** * * P < 0.05 ** * ** * * * There are elevated levels of GBA mRNA in GD derived and carrier derived fibroblasts

  22. GBA mRNA levels in GD fibroblasts ** In UPR there are genes that are upregulated by the transcription factor CHOP. Does the GBA gene promoter have a UPR Responsive element?

  23. GBA mRNA levels in GD fibroblasts CHOP +1 AP-1 PEA -350 CAAT GBA Vector delivery to living cells

  24. Luciferase in HEK293 cells * * P < 0.01 Upon UPR induction (with thapsigargin) normal (but not CAAT mutated) GBA promoter activity elevates. GBA promoter binds CHOP

  25. Gaucher Disease Since UPR in general may lead to cell death, UPR in GD may lead to death of cells as well. Dopaminergic cells (Parkinson disease)?!

  26. Conclusions There is UPR in GD patients There is UPR in one animal models (out of few we have tested) There is UPR in GD carriers including the 84GG carriers There is upregulation of the GBA gene in patients in response to UPR, through CHOP binding Even without ERAD there is UPR (ER stress) that may lead to death of cells for example in the case of 84GG carriers and PD

  27. ERAD of mutant GCase variants Gaucher disease (mutant GCase) Parkinson disease ERAD of mutant GCase variants may lead to pathogenesis

  28. Thanks GaliMaor Collaborator: Dr. M. Filocamo Thank you for your attention

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