24 yo with severe cardiac and skeletal myopathy

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Unformatted text preview: ls Long QT iPS The Future? 24 yo With Severe Cardiac and Skeletal Myopathy. Pathologic Studies: Glycogen Storage Disease $ A ! Lysosomal accumula1on *' (') &' &'+, AV and glycogen deposits " # % % % % % Autophagy • “Autophagy” (auto (Gk, self) + phagy • • (Gk, digesting, eating) = digestion of cellular constituents by enzymes of the same cell Lysosomal process by which cells recycle proteins as a means of energy conservation and dispose of excess or defective organelles A key mechanism of cellular homeostasis 31 Danon Disease: A Mutation in LAMP2 Leads to Impaired Autophagy Macroautophagy LAMP2 © GenomeSciencesCorp 32 Danon Disease: A Mutation in LAMP2 Leads to Impaired Autophagy Macroautophagy LAMP2 LAMP2 Danon Disease X linked cardiac & skeletal myopathy © GenomeSciencesCorp 32 LAMP ­2 Muta1on  ­ No Expression of LAMP Protein Muta1on in LAMP ­2 WT WT Danon kDa 150 100 75 20uM LAMP ­2/Phalloidin/DAPI LAMP ­2/Phalloidin/DAPI 50 Patient 2 Danon Mutation Resulted in a Premature Stop Codon and Nonsense Mediated Decay •Demonstrated absence of LAMP2 RNA by mechanism of Nonsense Mediated Decay (NMD) using cultured patient fibroblasts •RNA rescue with the protein synthesis inhibitor cyclohexamide Danon Fibroblast Electon Microscopy !"#$%"&' ()#"#' ()#"#' 35 • Danon Expression Profile Consistent with Impaired Autophagy Inducible LAMP ­2 rescue in Danon Fibroblasts Autophagic flux Late endosome Amphisome mCherry-GFP-LC3B Phagophore mCherry Autophagosome GFP LC3B Yellow fluorescence Lysosome Autolysosome mCherry GFP LC3B Red fluorescence Autolysosome mCherry GFP LC3B No fluorescence 38 Danon Fibroblasts express more LC3-II: A Marker of Autophagy WT Unt Danon Rapa Unt Actin Rapa * 2! LC3-II/Actin ! LC3-I LC3-II P<0.05 1.8! P<0.005! 1.6! ** 1.4! 1.2! 1! 0.8! 0.6! 0.4! 0.2! 0! WT+UNT! Patient Fibroblasts 4 hour treatment Rapamycin: 5uM WT+Rapa! Danon+UNT! Danon+Rapa! Fibroblasts from Danon Pa1ent Display Dysfunc1onal Matura1on of Autophagic Vacuoles No Compensa1on by LAMP ­1 in Danon fibroblasts ** Characteriza1on of Danon hiPSC clones H9 EB Dissocia1on and staining CTnT/DAPI Ac1nin/DAPI CTnT/DAPI RVC20/DAPI Danon hiPSC Recapitulate Cri1cal Features of Danon Disease in Vitro WT WT Danon UNT Starve UNT Starve LC3 ­I LC3 ­II Ac1n N Danon EB Danon hiPSC Recapitulate Cri1cal Features of...
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This document was uploaded on 04/09/2014.

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