Therapeutic Interventions Tested in APBD Models

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Or Kakhlon, Ph.D. …

Or Kakhlon, Ph.D.
Alexander Lossos, M.D.
H. Orhan Akman, Ph.D.
Salvatore DiMauro, M.D.

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  • nt = non targeting (control) lentviral vector. shGBE1 = a lentiviral vector containing short helical RNA sequence directed against GBE1. All lentiviruses also express Green Fluorescence Protein (GFP) for identifying virus-infected cells.
  • Apoptosis is programmed cell death: A process by which cells (or neurons in this case) commit suicide in a very organized fashion so as not to spill their potentially toxic content. The cell membrane of apoptotic cells undergoes changes which can be detected by the dye Annexin V, shown here in blue. Necrotic cells dye in a non-programmed fashion where their confining membrane is ruptured and content spilled. This is detected by the nuclear stain 7-AAD shown here in red.

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  • 1. Therapeutic Interventions Tested in APBD ModelsOr Kakhlon, Alexander Lossos, H. Orhan Akman, Salvatore DiMauro Department of Neurology Hadassah University Hospital 06 July 2011
  • 2. Glycogen build up is suppressed in neurons by a Nevertheless, overwell-regulated system time glycogen could precipitate as polyglucosan bodies if chain elongation is not adequately balanced by its branching.Vilchez et al (2007) Nat Neurosc APBD Striano et al (2008) Nat ClinWierzba-Bobrowicz et al (2008) Pholia Neuropathol Pract Neurol
  • 3. Main objectives•Establishing a neuronal model of APBD in which GBE1 is repressed and PB areobserved.•Using the model to test pharmacological and biochemical methods for correctingadverse phenotypes associated with GBE1 deficiency.•Testing the methods also in brain cells derived from APBD mouse model and inAPBD patient-derived cells.
  • 4. Neuronal Model validation:1) Reduced GBE1 levels: GFP GBE1 merge GBE1 nt (RT-PCR data pending)shGBE1
  • 5. Neuronal Model validation (2):2) Glycogen accumulation: GFP Glycogen merge nt shGBE1
  • 6. GBE1 knockdown increases apoptosis in the neuronal model NecroticUndergoing apoptosis End stage apoptosis
  • 7. Using the model to test therapeutic approaches: Induction of autophagyInclusion bodies (PBs) formed. Caninduction of autophagy facilitate theirclearance? Can autophagy be cytoprotective against apoptosis? Maiuri et al (2007) Nat Rev Mol Cell BiolSarkar et al (2009) Cell Death DifferTest autophagy enhancers as a therapeutic strategy against APBD
  • 8. Autophagy can be stimulated and inhibited in neurons by rapamycin and 3-methyl adenine, respectively Jaeger & Wyss-Coray (2009) Mol Neurodegen
  • 9. Stimulation of autophagy can reversibly rescue PB accumulation in GBE1 knocked down neurons shGBE1 shGBE1/Rap shGBE1/Rap/3-MAGFPLC3Glycogen
  • 10. Stimulation of autophagy can reduce apoptosis in GBE1 knocked down neurons
  • 11. Vinblastin reduces autophagosome maturation into autolysosmes and increasesautophagosome biogenesis. Vinblastin’s effects on autophagy were confirmed inGBE1 knocked down neurons Untr. Rap Rap+Vin LC3 -LC3 I -LC3 IIStill need to confirm that vinblastin reduces sensitivity of LC3degradation to lysosomal protease inhibitors
  • 12. PB clearance and antiapoptotic effects of rapamycin in GBE1 knocked downneurons are unaffected by vinblastine Rap+Vin
  • 13. 8 ? P PRapamycin active GSK3β GS 5 mTOR 4 7 ? DGKα IM Autolysosome Autoph. MVB R59949 3 1 2 Amphisome 3’ 6 =LC, autoph. marker Vin1. PB Engulfment by the isolation membrane (IM)2. Amphisome formation from Autophagosome and Multivesicular Body3. Autophagosome maturation to Autolysosome4. Inhibition of autophagosome biogenesis5. Rap. inhibits mTOR and induces autophagy. Rap was able to reduce PB and apoptosis.6. Vinblastin inhibits autophagosome maturation into Autolysosome, but doesn’t counteract rapamycin’s effects.7. Exosome release clears PB?8. Rapamycin works by GS inhibition
  • 14. The effect of rapamycin and other mTOR inhibitors (new combinations affecting mTOR, PI3 Kinase and Akt together) are now being tested in the APBD mouse model in close collaboration between Dr. Akman and Dr. Kakhlon.
  • 15. Fortuitously it wasfound that cobaltreduces PB levelsin the hearts of ControlGBE1 KD mice.Cobalt and ironchelation canactivate Hypoxia GBE1 KDInducible Factor(HIF) – a GBE1inducer. GBE1 KD/CoCl2Test ironchelation as a GBE1 KD/Rap.therapeuticstrategy GBE1 KD/LiCl
  • 16. Preliminary data: Iron chelators 60 % of PG positive 50(especially DFP) reduce PB fibroblasts 40accumulation in patient-derived 30fibroblasts 20 10 0 Ctrl Y329S Y329S Y329S Y329S Rap DFO DFPY329S Y329S Ctrl Y329S DFO Y329S Y329S DFP Rap
  • 17. Summary• We have generated a neuronal model of APBD, recapitulating the clinical findingof glycogen (possibly polyglucosan bodies) accumulation and demonstratingincreased apoptosis.•Treating model neurons with rapamycin was able to reverse the adversephenotypes associated with GBE1 deficiency.•These effects of rapamycin are possibly attributable toautophagosome/mulivesicular body-driven exosome release.•Another possible mechanism explaining rapamycin effects is inhibition ofglycogen synthase. This mode of action will be assessed.•All possible therapeutic modes (for now, especially mTOR inhibitors and ironchelators) are now being tested in the neuronal model, the mouse model andAPBD patient-derived cells in close collaboration between Dr. Akman and Dr.Kakhlon.