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Lysosomal enhancement

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Lysosomal enhancement. A safe bet for eliminating misfolded proteins in ... Nixon (Nathan Kline Institute), Jay Jerome (Vanderbilt), Janet Sparrow (Columbia) ... – PowerPoint PPT presentation

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Title: Lysosomal enhancement


1
Lysosomal enhancement A safe bet for eliminating
misfolded proteins in the brain Aubrey D.N.J. de
Grey Department of Genetics, University of
Cambridge Email ag24_at_gen.cam.ac.uk
2
Acknowledgements Preliminary data John Archer
(Cambridge), Ulf Brunk (Linkoping) Workshop
funding NIA Gene discovery Bruce Rittmann
(Northwestern), Perry McCarty (Stanford), Pedro
Alvarez (Rice) Enzyme delivery Ana Maria Cuervo
(Albert Einstein), Roscoe Brady
(NINDS) Biomedical applications Ralph Nixon
(Nathan Kline Institute), Jay Jerome
(Vanderbilt), Janet Sparrow (Columbia)
3
  • Structure of this talk
  • Are aggregates and/or aggregation good or bad?
    Four answers
  • The central anti-aging fallacy of our day
  • Bioremediation meets biomedicine
  • Efficacy in principle
  • Delivery
  • Safety

4
Autophagy in Alzheimers Disease
Dystrophic Neurites
IEM
Calnexin
Cat D
5
Autophagy in Dystrophic Neurites
Autophagosomes pH 7.4
Autophagolysosomes LEP100 pH lt 6.7
Dystrophic Neurite
Lysosomes
6
  • 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

7
  • 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

8
  • 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

9
  • Can lysosomal accumulation of anything possibly
    be beneficial?
  • Nothing that gets into lysosomes gets out again
  • Lysosomal aggregates are biochemically inert
    (unless they potentiate catabolism??! Surely not)

Thus intralysosomal accumulation is bad
10
The gerontological fallacy reversal is far
harder than retardation Many gerontologists are
unwilling even to consider ideas to repair
accumulated age-related damage, because we cant
yet even appreciably slow down its
accumulation Keeping a leaking boat afloat is
plugging the hole always so much easier than
bailing?
11
  • 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

12
Example DGGE Results from Perchlorate-Reducing,
Membrane Biofilm Reactors
13
Xenohydrolysis the concept Graveyards -
are abundant in human remains - accumulate
bones (which are not energy-rich) - do not
accumulate misfolded neural proteins... -
so, should harbour microbes that degrade them
- whose catabolic enzymes could be therapeutic
14
Environmental decontamination in vivo
15
This might just work preliminary data
16
This might just work preliminary data
17
  • Other major efficacy issues
  • - How many enzymes would we need?
  • Maybe not many LSDs (single-gene disorders)
    imply big synergy between the various proteases
  • - 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

18
  • How can we get these enzymes to lysosomes of
    neurons?
  • Gene therapy
  • Stem cell therapy
  • Enzyme replacement therapy

19
Main Trafficking Pathways
Vesicular Traffic
Chaperone-mediated Autophagy
CVT Macroautophagy
Modified from Alberts 2002
20
  • How can we get these enzymes to lysosomes of
    neurons?
  • Gene therapy
  • Stem cell therapy
  • Enzyme replacement therapy

21
Amino Acid Chain
22
(No Transcript)
23
Amino Acid Chain
24
  • Safety the main issues
  • Immunogenicity?
  • Tolerisation brief expression low expression
  • Extralysosomal toxicity?
  • Specificity coding as proenzymes try again!

25
  • 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

26
  • Conclusions
  • Disparate areas of biology can converge (if
    anyone has time to spot the link!)
  • Repair can be as feasible as retardation
  • We need to try radical approaches to AD
  • This might just work..
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