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Possible biomedical applications of environmental biotechnology -- no kidding! Aubrey D.N.J. de Grey Department of Genet PowerPoint Presentation
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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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slide1

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/

slide2

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)

slide3

Structure of this talk

  • Age-related intracellular aggregates and the evidence for their pathogenicity
  • Bioremediation meets biomedicine
  • Efficacy in principle
  • Delivery
  • Safety
slide4

Structure of this talk

  • Age-related intracellular aggregates and the evidence for their pathogenicity
  • Bioremediation meets biomedicine
  • Efficacy in principle
  • Delivery
  • Safety
slide5

Aggregates: three major examples

- A2E in macular degeneration

- Proteins in neurodegeneration

- Oxysterols in atherosclerosis

slide6

Aggregates: three major examples

- A2E in macular degeneration

- Proteins in neurodegeneration

- Oxysterols in atherosclerosis

slide8

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

slide9

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

slide10

Cl -

Amphiphilic compound

2 hydrophobic

side-arms

cationic

polar head

Cl -

iso-A2E

A2E

detergent-like activity

detecting a2e epoxides by mass spectroscopy
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

slide12

blue light

A2E

A2E + 1O2

A2E-photoxidation

oxidation of DNA bases

modifications of protein

changes in gene expression

apoptosis

slide13

Aggregates: three major examples

- A2E in macular degeneration

- Proteins in neurodegeneration

- Oxysterols in atherosclerosis

slide14

Autophagy in Alzheimer’s Disease

Dystrophic Neurites

IEM

Calnexin

Cat D

slide15

Autophagy in Dystrophic Neurites

Autophagosomes pH = 7.4

Autophagolysosomes

LEP100

pH < 6.7

Dystrophic Neurite

Lysosomes

slide16

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

slide17

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

slide18

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

slide19

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

slide20

Aggregates: three major examples

- A2E in macular degeneration

- Proteins in neurodegeneration

- Oxysterols in atherosclerosis

slide25

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

atherosclerotic lesion composition
Atherosclerotic Lesion Composition

Monocytes

Endothelium

Lipoprotein

Macrophage

FoamCell

Smooth Muscle

Primary lipid in lesion is Cholesterol and Cholesteryl Esters

slide27

Endothelial

Cells

Lipid-engorged

Lysosome

Foam

Cell

known effects of oxidation of ldl on lysosomal function
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.
possible lipid induced alterations in lysosomes
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
slide31

Structure of this talk

  • Age-related intracellular aggregates and the evidence for their pathogenicity
  • Bioremediation meets biomedicine
  • Efficacy in principle
  • Delivery
  • Safety
slide32

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…
slide33

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

slide34

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

slide36

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

slide37

Structure of this talk

  • Age-related intracellular aggregates and the evidence for their pathogenicity
  • Bioremediation meets biomedicine
  • Efficacy in principle
  • Delivery
  • Safety
slide42

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
slide43

Structure of this talk

  • Age-related intracellular aggregates and the evidence for their pathogenicity
  • Bioremediation meets biomedicine
  • Efficacy in principle
  • Delivery
  • Safety
slide44

How can we get these enzymes to lysosomes of affected cells?

Gene therapy

Stem cell therapy

Enzyme therapy

main trafficking pathways
Main Trafficking Pathways

Vesicular Traffic

Chaperone-mediated

Autophagy

CVT

Macroautophagy

Modified from

Alberts 2002

slide46

How can we get these enzymes to lysosomes of affected cells?

Gene therapy

Stem cell therapy

Enzyme therapy

slide50

Structure of this talk

  • Age-related intracellular aggregates and the evidence for their pathogenicity
  • Bioremediation meets biomedicine
  • Efficacy in principle
  • Delivery
  • Safety
slide51

Safety: the main issues

  • Immunogenicity?
  • Tolerisation; brief expression; low expression
  • Extralysosomal toxicity?
  • Specificity; coding as proenzymes; try again!
slide52

Conclusions

  • Disparate areas of biology can converge (if anyone has time to spot the link!)
  • We need to try radical approaches
  • This might just work……..