Genetic Dissection of Aging in Drosophila

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Genetic Dissection of Aging in Drosophila

• Goals
• Identify the genes involved in aging and
  longevity
• Use knowledge of these genes to determine the
  physiological systems important in aging and
  longevity
• How?
• Mutagenesis
• What phenotype to use?

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Logistical problems using Life span as a
phenotype in a mutagenesis

• Longest lived member of a population is less
  likely to breed

• Population based measure
• cannot do F1 screen
• need to set up lines

• Takes a long time
• Normal max life span 80-100 days

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How do we measure aging?

• Demographic measures of aging
• Physiological measures of aging

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Aging populations versus individuals

• Measurement of life expectancy in a population.
• Measurement of life expectancy in individuals.
• Is chronological age the same as physiological
  age?

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Life span of fruit flies at three temperatures
Dissociation of chronological age and
physiological age
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Visualize expression of a single gene at the
level of individual cells.
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Validity of use of beta-gal as a marker of gene
expression during adult life.

• Little change in somatic cell populations in the
  adult fly.
• Protein synthesis is preserved in many cells of
  the adult fly throughout life.
• Rate of degradation of beta-gal is on the order
  of several hours.

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Environmental manipulation of life span
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Life span of fruit flies at three temperatures
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Changing life span changes the chronological
timing of 1085 gene expression
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1085 gene expression scales to life span
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Genetic manipulation of life span
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25C
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18C
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Data from 18, 25 and 29C
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Biomarkers of aging
In principle age-dependent and age-independent
gene expression should be present in any
organism including humans.
Examination of the temporal pattern of gene
expression of age-dependent and age-independent
genes should provide the much needed biomarkers
of aging allowing for the measurement of aging
on an individual basis.
Important for both understanding aging and for
the more practically based testing of
interventions that extend life span in a shorter
period of time then having to wait for age at
death.

• The ability to measure gene expression for many
  different
• genes simultaneously, as the microarray system
  affords,
• should permit this approach to be readily used in
  mammals.

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Wg (wingless) expression changes over time
Rogina and Helfand 1997
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Wg expression scales with lifespanusing ambient
temperature
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Hyperkinetic and Shaker are single gene mutations
that accelerate the rate of aging.

• Sex-linked neurological mutants.
• Die earlier than normal animals.
• Slope of mortality curve indicates an
  acceleration of the aging process.
• Oxygen consumption is increased over normal.
• Physical activity is increased over normal.

Trout and Kaplan, Genetics 1970
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Shaker
control
Hyperkinetic
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Wg expression scales with lifespanusing Shaker
mutation
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Wg expression scales with lifespanusing
Hyperkinetic mutation
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Loss of SOD function shortens life span
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drop dead (drd)

• Mutations in drd lead to adult onset
  neurodegeneration and early death. (Hotta and
  Benzer, Proc Natl Acad Sci U S A. 1970
  Nov67(3)1156-63)
• Drop dead is X-linked recessive.
• Mosaic analysis suggests it is non cell
  autonomous
• The age of death appears stochastic and is
  effected by ambient temperature.

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Idealized biomarker expression scales to life span
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Detection of biomarker threshold

• Reporter genes
• ß-gal
• GFP
• luciferase

• Selection process that removes flies with a
  normal biomarker profile
• Lethal Toxin

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Biomarker threshold detection by using a deadly
toxin
control
Rel. Biomarker Activity
long lived
Age

• Coupling biomarker activity to the expression of
  a lethal toxin enriches for long-lived mutant
  flies delayed in accumulating the lethal toxin.

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Age-dependent expression of Tetanus Toxin
X
DJ-651 or Dj-634 GAL4 Driver
UAS-tetanus toxin
GAL4
DJ-651 or DJ-634
Tetanus toxin
5X UAS
DJ-65 or DJ-634-dependent Expression of tetanus
toxin
Tetanus toxin
5X UAS
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Death is Driver Dependent
females
males
DJ651 (avg. 8 days max 15 days) DJ634 (avg. 18
days max 30 days)
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Is the toxin induced death age-dependent?

• Examine how interventions that alter life span
  and rate of aging affect rate of toxin induced
  death?

• Ambient temperature

• Reproductive status

• Calorie restriction

• Genetic alterations

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Temperature
63 increase in average survival time in females
74 increase in average survival time in males
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Reproductive Status
74 increase in average survival time in females
22 increase in average survival time in males
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Diet
43 increase in average survival time in females
41 increase in average survival time in males
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INDY Transporter involved in intermediary
metabolism. Hypomorphic mutation which results in
a doubling of mean life span in flies.
50 increase in average survival time in males
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Rpd3 A histone deacetylase, which has been shown
to extend life span in both yeast and fruit flies
when expression is abolished
Females (null) rpd3def24 34 increase
(hypomorph) rpd3P-UTR 45 increase.
Males (null) rpd3def24 63 increase (hypomorph)
rpd3P-UTR 40 increase.
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Screen for genes or drugs that change
biomarker/gene trajectory
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Lipoic Acid
Screen with rapid assay
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Validate with life span test
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Resveratrol
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Logistical problems with the tetanus toxin
mutagenesis screen

• not much time to select long-lived candidates,
  before they are also overcome by the toxin and
  die or are infertile.

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Temporal control over the Toxin

• Tetanus Toxin-not conditional
• Inducible/conditional expression
• Cyanide
• produced by Linamarase/Linamarin

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Cassava or manioc (Manihot esculenta also yuca
in Spanish, and mandioca, aipim, or macaxera in
Portuguese) is a woody perennial shrub of the
spurge family, that is extensively cultivated as
an annual crop for its edible starchy tuberous
root. It was originally observed in what are now
called Brazil and Paraguay, and today is widely
diffused in tropical and subtropical regions, and
may be called the "potato of the tropics".
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• Linamarase is an enzyme found in the Cassava
  plant (Manihot esculenta Crantz). The enzyme is
  a b-glucosidase that hydrolyzes the innocuous
  substrate linamarin (2-HO-isobutyronitrile-b-D-glu
  copyranoside) to acetone cyanohydrin and glucose.
  Acetone cyanohydrin is unstable and
  spontaneously breaks down into acetone and
  cyanide (Cortes, de Felipe et al. 1998). Cyanide
  inhibits cytochrome c and blocks e- transport,
  oxidative phosphorylation and ATP production
  resulting in death. (Pearce, Bominaar et al. 2003)

Schematic representation of linamarin breakdown
by linamarase into glucose and acetone
cyanohydrin and further spontaneous decomposition
of this product releasing free cyanide and
acetone. Text and figure taken from Cortes, de
Felipe et al. 1998.
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expression of Linamarase
X
Ubiquitous GAL4 Driver
Linamarase
GAL4
Ubiquitous promoter
Linamarase
5X UAS
Ubiquitous Expression of Linamarase
Linamarase
5X UAS
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Tubulin Driver 1mM linamarin
Death profile of UAS-Lin flies with and with out
Tubulin driver. Red are flies expressing
linamarase. Green are flies NOT expressing
linamarase.
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Tubulin 50mM linamarin
Death profile of UAS-Lin flies with and with out
Tubulin driver. Red are flies expressing
linamarase. Green are flies NOT expressing
linamarase.
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Actin 50mM linamarin
Death profile of UAS-Lin flies with and with out
Actin driver. Red are flies expressing
linamarase. Green are flies NOT expressing
linamarase.
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Age-dependent expression of Linamarase
X
DJ-651 or Dj-634 GAL4 Driver
UAS-Linamarase
GAL4
DJ-651 or DJ-634
Linamarase
5X UAS
DJ-65 or DJ-634-dependent Expression of Linamarase
Linamarase
5X UAS
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DJ651-Lin1.4
Red are flies expressing linamarase. Green are
flies NOT expressing linamarase
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Summary

• Gene expression during adult life continues to be
  well regulated
• Gene expression may serve as a biomarker of aging
• Age-dependent gene expression can be used to
  screen for drugs and genes that affect life span

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Acknowledgments
Gene expression and life span Blanka Rogina
(UConn Health Center) Kimberly Blake Barry
Hoopengardner Boris Naprta Alejandro Centurion

• National Institute on Aging
• National Science Foundation
• Ellison Medical Foundation
• American Federation for Aging Research
• The Glenn Foundation for Medical Research
• Donaghue Foundation

Tetanus toxin screening assay Johannes
Bauer Stephan Goupil Graham Garber Matt Bedoukian
Linamarase screening assay Stephan Goupil Matt
Bedoukian Suzanne Hosier Rob Reenan
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Feynman on aging
It is one of the most remarkable things that in
all of the biological sciences there is no clue
as to the necessity of death. If you say we want
to make perpetual motion, we have discovered
enough laws as we studied physics to see that it
is either absolutely impossible or else the laws
are wrong. But there is nothing in biology yet
found that indicates the inevitability of death.
This suggests to me that it is not at all
inevitable, and that it is only a matter of time
before the biologists discover what it is that is
causing us the trouble and that that terrible
universal disease or temporariness of the humans
body will be cured. The Role of Scientific
Culture in Modern Society, Richard P. Feynman
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Researchers at the Mayo Clinic reported that
a 30-year study has revealed that optimistic
people live 19 percent longer than pessimists.
("I knew it!" said the optimists. "Just goes to
show," muttered the pessimists.)