The circadian clock gene period extends healthspan in aging Drosophila melanogaster
Abstract
There is increasing evidence that aging is affected by biological (circadian) clocks - the internal mechanisms that coordinate daily changes in gene expression, physiological functions and behavior with external day/night cycles. Recent data suggest that disruption of the mammalian circadian clock results in accelerated aging and increased age-related pathologies such as cancer; however, the links between loss of daily rhythms and aging are not understood. We sought to determine whether disruption of the circadian clock affects lifespan and healthspan in the model organism Drosophila melanogaster. We examined effects of a null mutation in the circadian clock gene period (per01) on the fly healthspan by challenging aging flies with short-term oxidative stress (24h hyperoxia) and investigating their response in terms of mortality hazard, levels of oxidative damage, and functional senescence. Exposure to 24h hyperoxia during middle age significantly shortened the life expectancy in per01 but not in control flies. This homeostatic challenge also led to significantly higher accumulation of oxidative damage in per01 flies compared to controls. In addition, aging per01 flies showed accelerated functional decline, such as lower climbing ability and increased neuronal degeneration compared to age-matched controls. Together, these data suggest that impaired stress defense pathways may contribute to accelerated aging in the per mutant. In addition, we show that the expression of per gene declines in old wild type flies, suggesting that the circadian regulatory network becomes impaired with age.
Introduction
Circadian clocks generate daily endogenous rhythms in behavior,
physiological functions, and cellular activities, which are coordinated with
external day/night cycles [1,2].
Circadian rhythms become impaired with age as evidenced by the dampening of
daily oscillations in melatonin and other hormones and the disruption of
night-time sleep in aged rodents and humans [3,4,5].
Remarkably, age-associated sleep fragmentation was also reported in Drosophila
melanogaster[6], suggesting
that effects of aging on circadian systems may
be evolutionarily conserved. While aging impairs the circadian systems, there is also evidence that
loss of circadian rhythms may, in turn, contribute to aging. Genetic disruption
of circadian rhythms by knockout of specific clock genes leads to various age
related pathologies and visible signs of premature aging in mice [7,8]. In
addition, chronic jet-lag which disrupts the circadian clock, increases
mortality in aged mice [9]. As extension of healthspan is of critical importance in
aging human population, there is a need to elucidate how strong circadian
clocks may support healthy aging.
The mechanisms linking circadian rhythms to the rate
of aging and healthspan are not well understood. To address these mechanisms,
we investigated whether disruption of the circadian clock affects response to
homeostatic challenge and aggravates selected aging biomarkers in the model
organism Drosophila melanogaster. We used a null mutation in the
circadian clock gene period (per01) [10]; this gene
is one of the four core clock genes that act in a negative auto-regulatory
feedback loop generating daily endogenous rhythms [11,12]. The
loss of per function disrupts behavioral and molecular rhythms in flies [10,11,13].
To compare lifespan and healthspan in flies with
normal or disrupted circadian clock, we measured their ability to maintain ROS
homeostasis during aging. We probed the health status of aging flies by
exposing them to mild oxidative stress of 24h hyperoxia
at increasingchronological ages, followed by assessment of the resulting
oxidative damage and mortality hazards. Hyperoxia was chosen as a homeostatic
challenge, because it directly leads to ROS production irrespective of
age-related changes in food consumption and other physiological parameters [14].
Figure 1. Lifespan of per01and CSpD. melanogaster in normoxia and following 24h
hyperoxia at different ages (marked by arrow in B-D). (A)
In normoxia, there was no significant difference in mean survival curves
(p=0.23) (B) Hyperoxia on day 5 did not significantly affect
longevity or survival curves (p=0.12) (C) Hyperoxia on day 20
resulted in a significant reduction (p<0.05) in average survival of per01flies compared to CSp with significant (p<0.0001)
difference in survival curves. (D) Hyperoxia on day 35 resulted in
more significant reduction (p<0.001) in average lifespan in per01flies compared to CSp and significant difference in survival
curves (p<0.0001). Males with rescued per function (per01{per+}) treated with hyperoxia on day 35 had average
lifespan similar to CSp but significantly different (p<0.001)
from per01mutants.
We report that per01 flies have
shortened healthspan as evidenced by their increased mortality hazard in
response to homeostatic challenge during aging. This conclusion is also
supported by accelerated functional senescence, and increased signs of
neurodegeneration in per mutants compared to age-matched controls with
an intact circadian clock. In addition, we show that the expression of per
gene declines with age leading to disruption of the circadian regulatory
network in old wild type flies.
Results
Short-term oxidative stress shortens the lifespan in per01 mutants
To determine how loss of per affects lifespan and healthspan, per01
were backcrossed for 6 generations to Canton S strain, and this control stock
was designated as CSp. Under normal laboratory conditions, the
longevity of per01 males was similar to CSp
controls (Figure 1A, Table 1). However, lifespan was significantly reduced in per01flies exposed to 24 h hyperoxia in middle age. Hyperoxia on day 20
shortened the average lifespan in per01 mutants by 12% while
hyperoxia on day 35 decreased average lifespan of per01 flies
by 20% compared to CSp males (Table 1); survival curves were
significantly different in both ages (Figure 1C-D). We also calculated age
specific mortality trajectories, and showed that mortality hazard significantly
increased after exposure to 24 h hyperoxia on day 20 or 35 in per01
but remained unchanged in CSp males (see Supplementary Figure 1 and Supplementary Table 1). To verify that these effects are indeed linked to the
lack of per gene function, we tested the lifespan of per01
flies transformed with a wild type copy of per, designated as per01{per+}.
When flies with rescued per function were exposed to hyperoxia on day
35, their average survival (59 ± 2.0 days) and mortality trajectories were
similar to CSp controls, but significantly different from per01
mutants (Figure 1D, Supplementary Figure 1D, and Supplementary Table 1). This
verified that shortened lifespan and
increased death-risk in per mutants are due to the loss of per
gene. Importantly, exposure to hyperoxia on day 5 did not affect the average
lifespan or mortality trajectories of per01 mutants (Figure 1B and Supplementary Figure 1B), demonstrating that hyperoxia sensitivity in these mutants is an
age dependent phenotype.
Table 1. Average lifespan of CS p and per01 males
exposed to 24h hyperoxia at indicated ages.
Values shown with SEM, n denotes the sample size. One-Way ANOVA with Tukey-Kramer
multiple comparisons test. Statistical comparison across genotypes * = p<0.05, ** = p<0.001;
within genotype, values with different superscripts are significantly different at
p<0.05.
Treatment | Genotypes |
CSp | per01 |
Normoxia |
61.5 ± 1.8a
(n= 596)
|
59.0 ± 1.02a
(n= 640)
|
Hyperoxia day 5 |
60.4 ± 0.8a
(n= 447)
|
56.9 ± 0.93b
(n= 480)
|
Hyperoxia day 20 |
58.4 ± 0.93a
(n= 415)
|
51.35 ± 1.07*c
(n= 385)
|
Hyperoxia day 35 |
59.5 ± 1.03a
(n = 328)
|
47.8 ± 1.68**c
(n= 350)
|
per01
mutants accumulate more oxidative damage in response to stress and during
normal aging
Given
the increased mortality hazard in response to hyperoxia in per01
mutants, we next assessed the levels of oxidative damage incurred after 24 h
hyperoxia exposure at the age of 5, 20, 35 and 50 days in both genotypes. Levels
of protein carbonyls (PC) and the lipid peroxidation product 4-HNE were
measured separately in heads and bodies. Exposure to hyperoxia induced
significantly higher (p<0.001) PC levels in per01 than in
CSp heads at all ages except day 5 (Figure 2A and Supplementary Table 2). Similar
as in heads hyperoxia on day 35 or 50 led to moderate PC increase in CSp
bodies and dramatic increase in the bodies of per01 flies
(Figure 2B and Supplementary Table 2). Restoring per+ function in a per01
background resulted in PC content similar as in CSp and
significantly lower than in per01males (Supplementary Table 2).
Thus, the loss of per function leads to dramatically higher
accumulation of PC in per01 flies faced with oxidative
challenge. Similar as in the case of mortality hazard this deleterious phenotype
is age dependent occurring in middle aged and old flies but not young per01mutants (Figure 1-2 and Supplementary Figure 1).
Figure 2. Oxidative damage accumulates to higher levels in aging per01flies.
Fold increase was calculated based on day 5 values in CSp males
under normoxia (numerical values are shown in Supplementary Table 2 and Supplementary Table 3). Top:
Protein carbonyls (PC) in heads (A) and bodies (B) of CSp
(solid line) and per01(broken line) in normoxia (black)
and after hyperoxia (gray). PC levels were significantly higher in per01than in CSp fly heads on day 35 and 50, and on day 50 in
bodies under normoxia. Hyperoxia on day 35 and 50 induced significantly
higher PC levels per01head and bodies compared to CSp
age-matched controls. Bottom: Lipid peroxidation product 4-HNE in
heads (C) and bodies (D). In normoxia, per01flies
accumulated significantly more 4-HNE in heads and bodies compared to CSp
in all ages except day 5. Under hyperoxia, significant increase in 4-HNE
accumulation was observed in per01heads and bodies on
day 20, 35 and 50 compared to CSp males. For statistical
analysis of PC and HNE data refer to Supplementary Table 2 and Supplementary Table 3.
The second indicator of oxidative damage,
the lipid peroxidation product 4-HNE, was also measured in heads and bodies of
CSp and per01 flies. Exposure to hyperoxia on day
35 and 50 significantly increased HNE in per01 heads compared
to respective CSp controls (p<0.001) while exposure on day 5 or
20 had no significant effect (Figure 2C and Supplementary Table 3). Similar as in heads,
hyperoxia administered on day 35 and 50 induced significantly more HNE in per01
than in CSp bodies, however, the increase was less pronounced than
in fly heads (Figure 2C-D). These effects depend on the per gene as males
with restored per function exhibited significantly lower HNE profiles
than per01males, and similar as those observed
in CSp flies (Supplementary Table 3).
Aging
per01 mutants show greater mobility impairment and neurodegeneration
Our
data show significantly higher accumulation of oxidative damage even in
unchallenged per01 mutants under normoxia compared to age
matched controls (Figure 2, Supplementary Table 2, Supplementary Table 3). As oxidative damage is one of the
important biomarkers of aging, we asked whether other signs of aging are
advanced in per01 mutants. First, we compared age-related
locomotor performance between mutant and control flies. We used the RING assay,
which utilizes negative geotaxis in Drosophila to assess climbing
performance [15,16]. We measured climbing ability of
per01
and CSp flies aged to day 5, 20, 35
or 50. Surprisingly, 5 day old per01 flies showed
significantly higher climbing ability than control flies. In contrast, middle-aged and older per01
males showed significantly impaired climbing ability compared to age-matched
controls (Figure 3).
The difference was especially dramatic on
day 50; at this age the average climbing ability of per01
males was approximately 4 fold lower than in CSp controls. This was
partly caused by lack of vertical movement in many per01
flies at this age. The fact that young per01 mutant flies did
not show impaired climbing demonstrate that the period gene does not
affect fly geotaxis per se, but rather contributes to impaired climbing
ability in an age-dependent fashion.
Another
indicator of aging that we tested in per01 flies was the
health of their nervous system. As aging is associated with degenerative
morphological changes in the central nervous system, we examined brain
sections from 50 day old per01, CSp, and per01{per+}
males. We evaluated number of vacuoles, as they reflect the level of
neurodegenerative damage in the brain [17]. Brains of per01 males showed significantly (p<0.05) greater number
of vacuoles than control CSp and per01{per+}
flies with restored per function (Figure 4). These vacuoles, which were
found mainly in the neuropils of the optic lobes and the central brain, lead to
disrupted neuronal connections. Increased vacuolization in 50 day old per01
flies is consistent with their severely impaired mobility (Figure 3).
Figure 3. Vertical mobility deteriorates faster in per01flies,
as demonstrated by the RING assay. Bars represent mean height climbed
(with SEM) in CSp (open bars) and per01(black
bars) males at indicated age. The climbing performance of per01males on day 5 was significantly higher (p<0.001) compared to CSp.
With age, a rapid deterioration in climbing performance was noted in per01flies with mobility being significantly lower (* p<0.001) on day 20,
35, and 50 compared to age-matched CSp controls.
Expression
of per gene declines significantly with age
Since
age related functional decline is accelerated in per01 flies
compared to flies with normal clock, it was of interest to investigate daily
profiles of per expression during aging in control CSp flies.
Therefore, we used qRT-PCR to measure the expression levels of per mRNA
extracted from flies collected every 4h for 24h at age 5, 35 and 50 days. As
expected [11], per mRNA
levels showed daily cycling with lowest levels in the morning and a peak at
early night in the heads of young flies (Figure 5A). The levels of per
between peak and trough changed with a 12-fold amplitude. This amplitude
dampened significantly in 35 day old flies; however, there was still pronounced
cycling of per mRNA with 8-fold amplitude. A dramatic dampening of per
oscillation was observed on day 50 with the amplitude reduced to 2-fold.
Comparison of the relative per mRNA levels at the peak showed
significant reduction by ca 70% in 50 day old flies relative to peak expression
levels in young flies. Since per encodes an essential component of
circadian clock, our data suggest that the circadian network is severely
impaired in old flies.
Discussion
This study demonstrates healthspan extending role of
the clock gene period and suggest that functional circadian clocks may
prevent premature aging in flies. Research on Drosophila has demonstrated that different genetic
manipulations and environmental interventions can extend fly lifespan
[18]. Less attention has been paid to
healthspan, despite that extension of healthspan is of critical importance in
aging human population. Here, we
show that
healthspan
can follow different trajectories in flies which have similar lifespan under
stress-free laboratory conditions. Healthspan is an important but poorly
defined concept, and there is an ongoing debate whether model organisms, such
as Drosophila, can help to characterize parameters that could detect
differences in healthspan [19]. We
demonstrate that a relatively mild exogenous stress of 24 h hyperoxia, which
revealed health impairment of per01 mutant, could be
established as a convenient method to probe fly healthspan in a search for
mechanisms supporting healthy aging.
Here, we show that healthspan, measured as the ability
to respond to homeostatic challenge is reduced in per01
flies. Exposure to mild oxidative stress in middle age significantly shortened
life expectancy in per01 flies but, importantly, not in
control flies. The lower capacity of per01 mutants to buffer
short-term oxidative challenge was linked to greatly increased accumulation of
oxidative damage during hyperoxia exposure. Thus, it appears that increased
mortality hazard in hyperoxia-exposed per01 mutants may be
caused by their impaired ability to clear the oxidative damage which is
suggested to be one of the major causes of aging [20].
The higher accrual of oxidative
damage observed in per01 flies in normoxia and especially
after hyperoxia could be influenced by a number of factors, with the primary
suspect being higher production of endogenous ROS, which has been reported to
increase in clock-disrupted flies [21] and mice [7]. Whether higher ROS is associated with decreased
activity of ROS scavenging en- zymes remains to be determined.
While microarray studies suggested that expression of superoxide dismutase and
catalase may be controlled by the circadian clock in flies [22], qRT-PCR did not confirm such
rhythm for catalase, but demonstrated that catalase activity is significantly lower in young
clock-deficient flies [21]. It is currently unknown whether
enzymes involved in protein repair are controlled by the circadian clock in
animals, although such control was reported in plants
[23]. Finally, excessive
agglomeration of oxidatively damaged proteins in per01 flies
could be related to impaired degradation as proteasome activity has been shown to decline with age in flies, and may be
inhibited by PC and HNE
[24,25]
Figure 4. Neuronal
degeneration is accelerated in per01mutants compared to
CSp and flies with restored per function (per01{per+}) on day 50. (A) Mean number of
vacuoles (with SEM) representing neuronal degeneration was significantly
higher in per01mutants compared with wild type CSp
and flies with rescued per. Bars with different superscripts are
significantly different at p<0.05, data based on 10-15 heads for each
genotype. (B-D) Photomicrographs of representative brain sections of
CSp, per01, and per01{per+}
males. Arrows point to vacuolization.
Figure 5. Expression of per mRNA declines with with age in heads of CSp
flies.
(A) Daily mRNA expression profiles of per in day 5, 35 and 50
male heads. White and black horizontal bars mark periods of light and
darkness respectively. Values were normalized to rp49 and calibrated
against ZT0 (taken as 1) for each age and represented as mean ± SEM of 3
bioreplicates. (B) The peak levels of per mRNA are
significantly reduced (* = p<0.05) in 50 day old males compared to young
control males. Values are mean ± SEM of 3 bioreplicates.
As in humans, age-related functional
declines such as disrupted sleep and decreased mobility are observed in Drosophila[6,26]. The negative geotaxis assay revealed significant impairment in
climbing ability in aging per01 flies relative to age-matched
controls suggesting that lack of per impairs physical performance during
aging. Importantly, exacerbated mobility decline in per01
flies was associated with increased neuronal degeneration in the brain.
Neurodegenerative effects in the form of vacuoles in the neuropil region were
observed with higher frequency in 50-day old per01 mutants
than in CSp or per01{per+}
flies with restored per function. The formation of vacuoles was
previously linked to oxidative damage and accelerated aging in Drosophila
with impaired carbonyl reductase gene [27], and in
flies with Alzheimer-like phenotypes [28].
Our
study suggests that functional circadian rhythms support healthy aging in
flies. PER protein is the essential element of circadian clock and its absence
disrupts molecular and cellular rhythms. We reported previously that young wild
type flies have daily rhythms in ROS and PC levels, while in per01
flies levels of these deleterious compounds are significantly higher and
arrhythmic [21]. We
hypothesize that the circadian clock slows down the accumulation of oxidative
damage in aging organisms by synchronizing the activities of enzymes involved
in protein homeostasis. For example, microarray studies reported synchronous
upregulation of several GST enzymes in flies [29], and it is
known that glutathione participates in the conjugation of oxidized proteins [30]. In the
absence of circadian clock, enzymes working in a specific pathway may become
dysregulated leading to impaired removal of oxidative damage. However, we
cannot exclude the possibility that per could affect efficiency of
anti-oxidative defense systems independent of its role as a clock component, by
acting in a pleiotropic non-circadian manner.
While
loss of the circadian rhythms by disruption of the gene period
accelerates aging, organisms with normal clocks also age. Our data demonstrate
that at middle age per01 mutant shows aging phenotypes
normally observed in chronologically older wild type flies, suggesting that
clock gene activities may decline with age. Indeed, we demonstrate the
amplitude of per mRNA oscillation is severely dampened in 50 day old
flies and levels of per mRNA are significantly reduced at late night,
when PER acts as essential element of clock negative feedback loop [11]. This
suggests that circadian clocks and, consequently circadian rhythms are severely
impaired in individuals of advanced age, which is consistent with declining
strength of behavioral rhythms reported in aging flies [6]. While
factors contributing to the decline of circadian rhythms in flies remain to be
elucidated, oxidative stress is likely to be involved. We show here that
oxidative damage accumulates to high levels even in wild type aging flies, and
a previous report demonstrated that paraquat-induced oxidative stress, or
decrease in FOXO expression, led to dampened per expression in Drosophila[31]. Decline in
clock genes with age has been reported in zebrafish [32], rats [33] and most
recently in rhesus monkey [34]. The
intriguing similarities in the behavior of clock genes during aging between
mammals, zebrafish, and flies warrants investigations of the mechanisms causing
disruption of the circadian networks. Understanding these mechanisms will help
to determine in future whether strong circadian clocks add water to the
fountain of youth.
Materials and Methods
Fly
rearing and l
ife span analysis.
Drosophila
melanogaster were reared on
yeast-cornmeal-molasses-agar diet (35g yeast/l) at 25°C in a 12-hour
light/12-hour dark cycles; all experiments were performed 4-8 h after lights-on.
The per01 mutant flies [10] were
backcrossed 6 times to the Canton-S (CS) flies designated as CSP.
To rescue per-function, we used transgenic flies carrying a wild-type
copy of per (designated as perG) in a per01
background [35]. Males with
two copies of perG (y wper01;{per+:32.1};+
were crossed with per01;+;+ females, and F1 males containing
one copy of rescue construct designated per01{per+}
were used. We confirmed their rhythmic locomotor activity indicating rescue of
circadian clock function.
To
determine lifespan, 3-4 cohorts of 100 flies of each genotype were housed in 16
oz transparent plastic bottles inverted over 60 mm Petri-dishes containing 15
ml of diet. Diet was replaced on alternate days without anesthesia, and
mortality was recorded daily. For hyperoxia exposure, males were transferred
from cages to narrow vials with diet in groups of 25, and placed in a Plexiglas
chamber filled with oxygen (100% medical grade) flowing at a constant rate
(300ml/min) for 24 h. Control flies were transferred to narrow vials as above
and kept under normoxia. Hyperoxia-treated and control flies were then either
frozen for oxidative damage analysis or returned to cages and monitored for
mortality.
Oxidative
damage assays.
The amount
of protein carbonyls was assayed separately in 25 heads and bodies. Carbonyls
were quantified after reaction with 2,4-dinitrophenylhydrazine (DNPH) as
described previously [21] at 370 nm in a
BioTek Synergy 2 plate reader. Results were expressed as nmol.mg-1
protein using an extinction coefficient of 22,000 M-1cm-1.
The lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE) was assayed in heads
and bodies by competitive enzyme-linked immunosorbent assay (ELISA) as
described [36, 37]. Briefly, free
HNE (Alpha Diagnostic, San Antonio, TX, USA) was conjugated to
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein [38]. Wells in a
96-well plate were coated with 500 ng of HNE-GAPDH protein for 24h at 4°C,
washed in PBS-Tween, and blocked with 1% BSA. A standard dose-response curve
was developed from serial dilutions of HNE-GAPDH with polyclonal anti-HNE
antibody (1:1000; Alpha Diagnostic). For samples, 10 μ;g of protein lysate was
mixed with 1:1000 polyclonal rabbit anti-HNE antibody and added to wells in
triplicate. Plates were incubated for 1 h, washed with buffer, incubated with
1:5000 secondary anti-rabbit antibody conjugated with horseradish peroxidase,
washed, mixed with detection buffer TMB (Alpha Diagnostic), and read at OD
450nm in a BioTek plate reader.
Rapid
iterative negative geotaxis (RING) assay.
Vertical mobility was assayed using the RING method [15]. Briefly, 3
groups of 25 CSp or per01 flies were transferred
into empty narrow vials, which were loaded into the RING apparatus. After 3
minutes rest, the apparatus was rapped sharply on the table three times in
rapid succession to initiate a negative geotaxis response. The flies' movements
in tubes were videotaped and digital images captured 4 s after initiating the
behavior. Five consecutive trials were interspersed with a 30s rest. The
climbing performance was calculated and expressed as average height climbed in
the 4 s interval. The performance of flies in a single vial was calculated as
the average of 5 consecutive trials to generate n = 1.
Neuronal degeneration.
Paraffin-embedded
sections of heads were used to examine neurodegenerative defects. Fly heads of
all genotypes were processed
and sectioned in parallel, and microscopic pictures taken at the same level of
the brain and the number and volume of vacuoles counted in double-blind
experiments using described methods [39, 40].
Quantitative
Real-Time PCR.
25 male heads were
collected for each time point in triplicate, homogenized in TriReagent (Sigma),
and RNA was isolated following manufacturer protocol. Samples were purified
using the RNeasy mini kit (Qiagen) with on-column DNAse digestion (Qiagen).
Synthesis of cDNA was achieved with Sprint RT Complete kit (Clontech) or
iScript cDNA synthesis kit (Biorad). Real-time PCR was performed on Step-One
Plus real-time machine (Applied Biosystems) in triplicate under default thermal
cycling conditions with a dissociation curve step. Each reaction contained
iTaq SYBR Green Supermix with ROX (Biorad), 0.6-1ng cDNA, 80nM primers (IDT
Technologies). Primers sequences are available upon request. Data were analyzed
using the standard 2-∆∆CT method normalized to the gene rp49
and expressed relative to control samples at ZT0.
Statistical
analyses.
Life span and survival
curves were plotted following Kaplan Meier survival analysis and statistical
significance of curves assessed using the Log-Rank (Mantel-Cox) and
Gehan-Breslow-Wilcoxon test (GraphPad Prism v 5.0).Age-specific
mortality was calculated using the Gompertz's model of population aging. Ln
values of instantaneous mortality (μx) were plotted against
chronological time. Mortality calculations and Gompertz-Makeham maximum
likelihood estimates were done using WinModest V1.0.2 [41] and plotted
on GraphPad Prism. For statistical analysis of biochemical results three-way
ANOVA with post-hoc tests were performed using OpenStat (William G. Miller ©
2009). Statistical analysis of locomotor assays was done with one and two-way
ANOVA for comparison between ages and genotypes.
Acknowledgments
We
thank Dr. M. Grotewiel for sharing RING protocols, Dr. P. Hardin for flies, and
Drs. S. Pletcher and C. Davis for help with mortality hazard calculations. We
thank Megan Mathes, Nick Meermeier, and Katie Sherman for excellent laboratory
assistance, and Drs. L. Hooven, A. Sehgal, and P. Taghert for helpful comments
on the manuscript. This work was supported in part by the NIH-NINDS NS047663 to
DK, NIH-NIGMS GM073792, NRI, CSREES, USDA 2007-04617, and The Oregon
Partnership for Alzheimer Research grants to JMG.
Conflicts of Interest
The
authors have no conflict of interests to declare.
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