Dietary Restriction and Glycolytic Inhibition
Delay Proteotoxicity in C. elegans Model of
Huntington’s Disease
Nydia Ekasumara
Mobbs Laboratory
AGS meeting - Grapevine, TX
May 3rd, 2013
Disclosures
• I do not have conflicts of interest to disclose
• The research reported during this presentation
was supported by the MSTAR grant
• The investigators retained full independence in
the conduct of this research.
Huntington’s Disease (HD)
• Genetic neurodegenerative
disorder
• Clinical aspect
– Symptoms: motor (chorea, motor
impersistence), cognitive
(dementia, affective disorder),
metabolic (problems with
glucose homeostasis, weight
loss, cachexia)
– No cure
• Polyglutamine repeats in
Huntingtin protein
– Aggregation
– Inhibit important proteins, among
which is CBP
Soto, 2001
Marcellino, 2012
CBP in Health and HD
Health
HD
TOXICITY
Regulate
metabolism
(favor β-oxidation)
Glycolysis
Oxidative Stress
Transcriptional Dysregulation
Legend
CBP
wt huntingtin
Expanded polyQ tract
CBP is sequestered in HD, leading to toxicity
HD
Poly-Q
aggregates
CBP complex proteins
(CBP, PPAR, PGC-1α)
Glucose
Glycolysis
Reactive oxygen species
Toxicity
Beta oxidation
Less reactive
oxygen species
Dietary Restriction and
glucose toxicity
• Glucose decreases lifespan in C. elegans
• Dietary Restriction (DR) was shown to:
– Increase lifespan in yeast, Drosophila, C. elegans, mice, rats,
and dogs
– Decrease the effect of neurodegenerative diseases (AD, HD, PD)
– Potential treatment of metabolic diseases and delay aging
• Mechanism of DR is unknown
– Potentially through CBP
Our aim is to find drugs that confer protective
effect of DR without food deprivation!
CBP and PPAR
• CBP interact with other proteins in a complex to
activate transcription
• Peroxisome proliferating activating factor receptors
(PPARs)
– Induced by nutritional deprivation
– Mediate switch toward beta-oxidation
– Glitazones are PPAR agonists used to treat non-insulin
dependent diabetes
CBP
PGC-1
Fatty Acid Metabolites,
Glitazones (γ)-Type II
Diabetes
Fibrates (α)-CV disease
TXN
PPAR
PPRE
Metabolic gene
Marcellino, 2012
Hypotheses
• CBP and PPAR mediate the protective
effect of DR and glycolytic inhibition in their
ability to
– Delay poly-Q proteotoxicity
– Extend lifespan
• Glitazones are protective against
proteotoxicity and are dependent on CBP
and PPAR
C. elegans as a Model Organism
• Short lifespan
– Lifespan assay
– Paralysis/ proteotoxicity assay
• Knockdown of genes by
feeding RNAi in E. coli
• N2 = wild type
• AM140 = HD model
– Q35 Huntingtin- YFP in
muscle wall
– Paralysis phenotype
Methods: DR
Day 4
Day 8 - death
Control
L4440 /
cbp-1 RNAi /
nhr-49 RNAi
L4440
L4440 /
cbp-1 RNAi /
nhr-49 RNAi
No food
DR
Paralysis
&
Death
Methods: Drug study
Day 8 - death
Control
L4440 / cbp-1 / nhr-49
Drug:
Pioglitazone
2DG
L4440 / cbp-1 / nhr-49
Paralysis
&
Death
1. CBP and PPAR mediate the protective effect of DR
DR
CBP
PPAR
Glycolysis
Toxicity
Paralysis
Lifespan
Beta oxidation
CBP knockdown reverses the protective effect of
DR on proteotoxicity and lifespan of HD worms
***
***
control vs DR p<0.0001
control vs CBP p<0.0001
***
PPAR knockdown (nhr-49) reverses the protective effect of
DR on proteotoxicity and lifespan of HD worms
***
***
control vs DR p<0.0001
***
2. CBP and PPAR mediate the protective effect of
glycolytic inhibition
CBP
CBP
PPAR
PPAR
2-DG
Glycolytic inhibition
Glycolysis
Toxicity
Paralysis
Lifespan
Beta oxidation
CBP knockdown reverses the protective effect of glycolytic
inhibition (2DG) on proteotoxicity and lifespan of HD worms
***
***
control vs 2DG p<0.0001
control vs CBP p<0.0001
***
PPAR knockdown reverses the protective effect of glycolytic
inhibition (2DG) on proteotoxicity and lifespan of HD worms
***
control vs 2DG p<0.0001
***
3. Glitazones are protective against proteotoxicity and are
dependent on CBP and PPAR
CBP
CBP
PPAR
PPAR
Glycolysis
Toxicity
Paralysis
Lifespan
Pioglitazone
Beta oxidation
PPAR agonist Pioglitazone delays paralysis and
increases lifespan on HD worms
***
control vs Pioglitazone p<0.0001
***
Conclusion
• DR & glycolytic inhibition reduce proteotoxicity and
extends lifespan
• Dependent on CBP & PPAR
• PPAR agonist pioglitazone is able to mimic DR
protective effect
• Dependent on CBP
DR, glycolytic inhibition, and pioglitazone reduce proteotoxicity and
extends lifespan dependant on CBP and PPAR
DR
CBP
PPAR
2-DG
Glycolytic inhibition
Glycolysis
Toxicity
Paralysis
Lifespan
Pioglitazone
Beta oxidation
Future directions
• Further working out the mechanism behind DR
and glycolytic inhibitors
• Assessing the role of other PPAR agonist
(fenofibrate)
• Potential drug treatments for HD: inhibitors of
glycolysis, complex I inhibitors, pyruvate mimetics,
PPAR agonists
• Viral delivery of CBP decoy as potential treatment
Acknowledgements
Charles Mobbs PhD
Bridget Marcellino PhD
Rainier Soriano MD
Melissa Carlson PhD
The Mobbs Lab
Mount Sinai Neuroscience Department
Funded by the MSTAR Grant
3. Glitazones are protective against proteotoxicity and are
dependent on CBP and PPAR
CBP
CBP
PPAR
PPAR
Glycolysis
Toxicity
Paralysis
Lifespan
Pioglitazone
Beta oxidation
PPAR agonist Pioglitazone delays paralysis on HD worms
and this protection is CBP dependent
***
***
control vs Pioglitazone p<0.0001
control vs CBP p<0.0001
***
PPAR knockdown reverses Pioglitazone protective
effect in paralysis
**
**
control vs Pioglitazone p<0.01
Marcellino, 2012
Health
HD
CBP complex proteins
(CBP, PPAR, PGC-1α)
Pioglitazone
Bind to CREB,
PPARs, etc.
Regulate
metabolism
(favor β-oxidation)
NADH
Complex II usage,
Complex I usage
Transcriptional
Regulation
PGC-1α
2-DG
glycolysis
Legend
CBP
wt huntingtin
Expanded polyQ tract
Mitochondrial
function
Oxidative stress
Toxicity