Molecular Characterization of Polyglucosan Body, Cause or Consequence in the Disease
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Hasan Orhan Akman, Ph.D. William J Craigen, M.D., PhD. Salvatore DiMauro, M.D.
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Molecular Characterization of Polyglucosan Body, Cause or Consequence in the Disease
- 1. Molecular Characterization of Polyglucosan Body, Cause or Consequence in the Disease Hasan Orhan AKMAN , PhD. William J Craigen, Md PhD. Salvatore DiMauro, MD
- 2. Polyglucosan Body (PGB) • Polyglucosan (PG) is the polymer of glucose molecules, however, unlike glycogen due to none or poor branching it is insoluble showing starch like properties. • PG accumulates in the cytosol and forms large aggregates known as PGB. They are visible after diastase digestion and PAS staining.
- 3. Polyglucosan Accumulates in the Axons and Cells. Klein et al. Muscle Nerve. 2004 Feb;29(2):323-8
- 4. Molecular Mechanisms for Poor Branching; • Skewed ratio of glycogen synthase vs. branching activity (GS/GB). • PFK deficiency Normal Glycogen Polyglucosan
- 5. PGB Disease Caused By GBE Deficiency has Different Variants Based on Residual Enzyme Activity Andersen Disease Complete Loss of Enzyme Activity – Lethal in infantile – Decrease in fetal movements and hydromnios – Liver and neuromuscular problems Juvenile form with low Activity – Mainly Liver and Heart, Adult PGB Disease (APBD) 20% or Less Residual Activity – Muscle weakness, – Loss of sensation – Muscles wasting in the arms and/or legs. – Impaired bladder control (neurogenic bladder) – Mental confusion (dementia very rare component of APBD)
- 6. Targeting Vector 5’ ARM 2.1 kb Exon 7 PGK-Neomycin 3’ARM 6.5 kb Intron 6 Exon 7 Intro n 7 Exon 7 PGK-Neo Gbe1Y329S ? Exon 7 PGK-Neo Gbe1neo Cre Recombination and Excision Exon 7 Y329S TAT to TCT Tyr Ser Exon 7 FRT LoxP Flpe Recombination and Excision of Exon 7 in mouse genome Intron 6 Intron 7 Gbe1del
- 7. Effect of Selection cassette on the progress of the disease
- 8. Relative Activity of GBE in Gbe1Neo/Neo Tissue Genotype % Value % SD n=5 WT 100 2 Brain Flox/Flox 2 42 WT 100 16 Heart Flox/Flox 16 11 WT 100 6 Liver Flox/Flox 6 28 WT 100 7 Muscle Flox/Flox 7 11 WT 100 1 Kidney Flox/Flox 0 NA
- 9. Physical Activity (4 months of age) Average distance run (meter) 400.0 Distance run (meters) 300.0 200.0 * 100.0 0.0 Control GBE1
- 10. Heart
- 11. Muscle
- 12. Liver 3 weeks old liver 4 month old liver
- 13. Brain
- 14. PGB in Axons and Cell Body
- 15. Mouse Skin Fibroblasts have Polyglucosan
- 16. Heart
- 17. Gbe1neo/neo Control Gbe1neo/neo Gbe1neo/neo Skeletal Muscle
- 18. Glucose Metabolism is Altered in Gbe1neo/neo mice 450 400 Control 350 GBE1 300 Glucose Concentration 250 mg/dl 200 150 * 100 50 * * 0 0 20 40 60 80 100 120 140 160 180 200 Time (minute)
- 19. Polyglucosan Can Be Used in The Liver During Fasting * *p< 0.002
- 20. Conclusion These two models are successful mouse models of APBD These animal models can be used to study • Molecular characterization of (PGB). (ii) Determine if the PGB cause or consequence in the disease? (iii)Is it possible to digest PGB in the cell or tissue? if possible how it effects the course of the disease
Editor's Notes
- First on left; polyglucosan is visualized in the axons of neuron where the information travels from brain to muscles and from sensory cells such as proprioreceptors, taste buds on the tongue or cone cells in the eye.Second; PAS stained muscle cells carbohydrate stains pink and from the subsequent section Diastase digested and PAS stained section, Diastase digestion clears allthe GLYCOGEN HOWEVER polyglucosan remains undigested.
- Decrease in genetic dose accelerate the progress of the disease. Y329S mutation decreases the enzyme activity yet heterozygous animals looks normal. homozygous animals deteriorates in terms of appearance (middle photo) at 4 months of age their size are comparably less than heterozygotes. On the left, one of the allele has been deleted the other has the Y329S mutation, although these mice are siblings they show different progress of the disease due to the amount of enzyme produced from the gene.
- Polyglucosan body is absent in the liver, unlike other organs liver is devoid of polyglucosan which suggests that polyglucosan can be degraded in the liver cells yet we have seen this decrease only in the liver.
- In Mouse model polyglucosan also exists in peripheral nerves and the cell body
- Polyglucosan is free in the cytosol. Unlike the membrane bound glycogen accumulated in Pompe disease (GSDII)