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Possible biomedical applications of environmental biotechnology -- no kidding! Aubrey D.N.J. de Grey Department of Genet

Possible biomedical applications of environmental biotechnology -- no kidding! Aubrey D.N.J. de Grey Department of Genetics, University of Cambridge Email: ag24@gen.cam.ac.uk Website: http://www.sens.org/. Acknowledgements Preliminary data: John Archer (Cambridge), Ulf Brunk (Linköping)

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Possible biomedical applications of environmental biotechnology -- no kidding! Aubrey D.N.J. de Grey Department of Genet

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  1. Possible biomedical applications of environmental biotechnology -- no kidding! Aubrey D.N.J. de Grey Department of Genetics, University of Cambridge Email: ag24@gen.cam.ac.uk Website: http://www.sens.org/

  2. Acknowledgements Preliminary data: John Archer (Cambridge), Ulf Brunk (Linköping) More recent data: John Schloendorn and colleagues (ASU), Jacques Matthieu (Rice) Microbiology: Bruce Rittmann (Northwestern/ASU), Perry McCarty (Stanford), Pedro Alvarez (Rice) Enzyme delivery: Ana Maria Cuervo (Albert Einstein), Roscoe Brady (NINDS) Biomedical applications: Ralph Nixon (NYU), Jay Jerome (Vanderbilt), Janet Sparrow (Columbia)

  3. Structure of this talk • Age-related intracellular aggregates and the evidence for their pathogenicity • Bioremediation meets biomedicine • Efficacy in principle • Delivery • Safety

  4. Structure of this talk • Age-related intracellular aggregates and the evidence for their pathogenicity • Bioremediation meets biomedicine • Efficacy in principle • Delivery • Safety

  5. Aggregates: three major examples - A2E in macular degeneration - Proteins in neurodegeneration - Oxysterols in atherosclerosis

  6. Aggregates: three major examples - A2E in macular degeneration - Proteins in neurodegeneration - Oxysterols in atherosclerosis

  7. Age-related accumulation of fluorescent compounds in retinal pigment epithelium neural retina RPE

  8. Fundus autofluorescence (=RPE lipofuscin) increases with age Exc.: 550 nm normal eyes; 7° temporal to the fovea Individually corrected for lens absorption Delori et al., IOVS 42:1855. 2001 MOD FA

  9. RPE lipofuscin forms in photoreceptor outer segments as a byproduct of the retinoid cycle opsin 11-cis-retinal (vitamin A derivative) visual cycle lipofuscin fluorophores

  10. Cl - Amphiphilic compound 2 hydrophobic side-arms cationic polar head Cl - iso-A2E A2E detergent-like activity

  11. Detecting A2E-epoxides by mass spectroscopy A2E bis-epoxide A2E 624 640 A2E nona-epoxide 608 656 672 FAB-MS 592 688 16 704 720 736

  12. blue light A2E A2E + 1O2 A2E-photoxidation oxidation of DNA bases modifications of protein changes in gene expression apoptosis

  13. Aggregates: three major examples - A2E in macular degeneration - Proteins in neurodegeneration - Oxysterols in atherosclerosis

  14. Autophagy in Alzheimer’s Disease Dystrophic Neurites IEM Calnexin Cat D

  15. Autophagy in Dystrophic Neurites Autophagosomes pH = 7.4 Autophagolysosomes LEP100 pH < 6.7 Dystrophic Neurite Lysosomes

  16. Can lysosomal accumulation of anything possibly be beneficial? Model 1: aggregation of misfolded proteins is bad (prevents their digestion by cytosolic proteases or chaperone-mediated autophagy) Inference: lysosomal aggregates result from macro- or microautophagy of cytosolic aggregates followed by failure of their lysosomal proteolysis Thus: intralysosomal accumulation is bad

  17. Can lysosomal accumulation of anything possibly be beneficial? Model 2: aggregation of misfolded proteins is good, as a staging-post when their proteolysis is failing; aggregates are autophagocytosed when possible Inference: lysosomal aggregates result from CM-, macro- or microautophagy of cytosolic proteins followed by failure of their lysosomal proteolysis Thus: intralysosomal accumulation is bad

  18. Can lysosomal accumulation of anything possibly be beneficial? Model 3: aggregation of misfolded proteins is good, because aggregates nucleate more misfolded proteins and thus clear them faster Inference: lysosomal aggregates result from CM-, macro- or microautophagy of other molecules, which cause failure of lysosomal proteolysis Thus: intralysosomal accumulation is bad

  19. Can lysosomal accumulation of anything possibly be beneficial? • Nothing that gets into lysosomes gets out again • Lysosomal aggregates are biochemically inert Thus: intralysosomal accumulation is bad

  20. Aggregates: three major examples - A2E in macular degeneration - Proteins in neurodegeneration - Oxysterols in atherosclerosis

  21. Atherosclerosis Progression

  22. Atherosclerotic Lesion

  23. Foam Cell Formed by Receptor-Mediated internalization of Low Density Lipoproteins (LDL). LDL

  24. LDL

  25. LIPOPROTEIN Chol-E Endocytosis LYSOSOME/LATE ENDOSOME Chol-E LAL Chol-E Chol + FA FA Plasma Membrane ACAT Chol-E Chol Chol NCEH Cholesterol Efflux Promoter

  26. Atherosclerotic Lesion Composition Monocytes Endothelium Lipoprotein Macrophage FoamCell Smooth Muscle Primary lipid in lesion is Cholesterol and Cholesteryl Esters

  27. Endothelial Cells Lipid-engorged Lysosome Foam Cell

  28. OxLDL-treated Human Macrophages accumulate FC and CE in lysosomes Acid Phosphatase Stained

  29. Known effects of Oxidation of LDL on Lysosomal Function • Heavily oxidized LDL will directly inhibit cathepsins and lipases. • However, does not produce cellular cholesterol accumulation because cholesterol converted to oxysterol • Heavily oxidized LDL is cytotoxic • Modestly oxidized LDL promotes lysosomal cholesterol accumulation.

  30. Possible Lipid-Induced alterations in Lysosomes • Direct inhibition of enzymes • Inhibition of delivery of enzyme to lysosome • No inhibition of delivery of endocytosed lipoprotein • Some evidence of disruption of trafficking between lysosomes and TGN • Alteration in the lysosomal environment

  31. Structure of this talk • Age-related intracellular aggregates and the evidence for their pathogenicity • Bioremediation meets biomedicine • Efficacy in principle • Delivery • Safety

  32. Bioremediation: the concept • - Microbes, like all life, need an ecological niche • - Some get it by brawn (growing very fast) • - Some by brain (living off material than others can't) • Any abundant, energy-rich organic material that is hard to degrade thus provides selective pressure to evolve the machinery to degrade it • - That selective pressure works. Even TNT, PCBs…

  33. R1 day 20 R5 day 71 R4 day 71 R1 day 71 R1 day 36 R2 day 71 R5 day 20 R2 day 36 R5 day 36 R3 day 71 R3 day 36 R4 day 36 1 2 5 7 9 11 12 15 Example: DGGE Results from Perchlorate-Reducing, Membrane Biofilm Reactors

  34. Xenocatabolism: the concept Graveyards: - are abundant in human remains… - accumulate bones (which are not energy-rich)… - do not accumulate oxysterols, A2E etc... - so, should harbour microbes that degrade them - whose catabolic enzymes could be therapeutic

  35. Environmental decontamination in vivo

  36. Steps to biomedical application Isolate competent strains; select by starvation Identify the enzymes (mutagenesis, chemistry, genomics) Make lysosome-targeted transgenes, assay cell toxicity Assay competence in vitro (more mutagenesis/selection) Construct transgenic mice, assay toxicity in vivo Assay competence in disease mouse models Test in humans as for lysosomal storage diseases

  37. Structure of this talk • Age-related intracellular aggregates and the evidence for their pathogenicity • Bioremediation meets biomedicine • Efficacy in principle • Delivery • Safety

  38. This might just work: preliminary data

  39. This might just work: preliminary data

  40. 7-ketocholesterol degradation - a promising start

  41. Identifying the genes - first steps

  42. Other major efficacy issues - How many enzymes would we need? Maybe not many: LSDs (single-gene disorders) imply big synergy between the various enzymes - How could we make them work in mammals? LacZ does… but also, in vitro evolution; fungi • What about low-abundance lysosomal toxins? • Abundance is presumably not that low

  43. Structure of this talk • Age-related intracellular aggregates and the evidence for their pathogenicity • Bioremediation meets biomedicine • Efficacy in principle • Delivery • Safety

  44. How can we get these enzymes to lysosomes of affected cells? Gene therapy Stem cell therapy Enzyme therapy

  45. Main Trafficking Pathways Vesicular Traffic Chaperone-mediated Autophagy CVT Macroautophagy Modified from Alberts 2002

  46. How can we get these enzymes to lysosomes of affected cells? Gene therapy Stem cell therapy Enzyme therapy

  47. Amino Acid Chain

  48. Amino Acid Chain

  49. Structure of this talk • Age-related intracellular aggregates and the evidence for their pathogenicity • Bioremediation meets biomedicine • Efficacy in principle • Delivery • Safety

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