Lysosomal enhancement

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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
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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)
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• 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

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

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• 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

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• 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

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• 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
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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?
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• 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

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Example DGGE Results from Perchlorate-Reducing,
Membrane Biofilm Reactors
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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
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Environmental decontamination in vivo
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This might just work preliminary data
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This might just work preliminary data
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• 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

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

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

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Amino Acid Chain
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Amino Acid Chain
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• Safety the main issues
• Immunogenicity?
• Tolerisation brief expression low expression
• Extralysosomal toxicity?
• Specificity coding as proenzymes try again!

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• 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

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• 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..