Autophagy-related gene 7 is downstream of heat shock protein 27 in the regulation of eye morphology, polyglutamine toxicity, and lifespan in Drosophila
Journal of Biomedical Science
Autophagy-related gene 7 is downstream of heat shock protein 27 in the regulation of eye morphology, polyglutamine toxicity, and lifespan in Drosophila
Shih-Fen Chen 3
Ming-Lun Kang 3
Yi-Chun Chen 3
Hong-Wen Tang 1
Cheng-Wen Huang 0 3 5
Wan-Hua Li 3
Chun-Pu Lin 2
Chao-Yung Wang 4
Pei-Yu Wang 0 5
Guang-Chao Chen 1
Horng-Dar Wang 2 3
0 Institute of Neuroscience, National Chengchi University , 64, Section 2, Zhi- Nan Road, Taipei, 11605 , Taiwan
1 Institute of Biological Chemistry , 128, Section 2, Academia Road, Nankang, Taipei, 115 , Taiwan
2 Department of Life Science, National Tsing Hua University , 101, Section 2, Kuang-Fu Road, HsinChu, 30013 , Taiwan
3 Institute of Biotechnology, National Tsing Hua University , 101, Section 2, Kuang-Fu Road, HsinChu, 30013 , Taiwan
4 Second Section of Cardiology, Department of Internal Medicine, Chang Gung Memorial Hospital at Linkou, Chang Gung University College of Medicine , Taoyuan , Taiwan
5 Institute of Neuroscience, National Chengchi University , 64, Section 2, Zhi-Nan Road, Taipei, 11605 , Taiwan
Background: Autophagy and molecular chaperones both regulate protein homeostasis and maintain important physiological functions. Atg7 (autophagy-related gene 7) and Hsp27 (heat shock protein 27) are involved in the regulation of neurodegeneration and aging. However, the genetic connection between Atg7 and Hsp27 is not known. Methods: The appearances of the fly eyes from the different genetic interactions with or without polyglutamine toxicity were examined by light microscopy and scanning electronic microscopy. Immunofluorescence was used to check the effect of Atg7 and Hsp27 knockdown on the formation of autophagosomes. The lifespan of altered expression of Hsp27 or Atg7 and that of the combination of the two different gene expression were measured. Results: We used the Drosophila eye as a model system to examine the epistatic relationship between Hsp27 and Atg7. We found that both genes are involved in normal eye development, and that overexpression of Atg7 could eliminate the need for Hsp27 but Hsp27 could not rescue Atg7 deficient phenotypes. Using a polyglutamine toxicity assay (41Q) to model neurodegeneration, we showed that both Atg7 and Hsp27 can suppress weak, toxic effect by 41Q, and that overexpression of Atg7 improves the worsened mosaic eyes by the knockdown of Hsp27 under 41Q. We also showed that overexpression of Atg7 extends lifespan and the knockdown of Atg7 or Hsp27 by RNAi reduces lifespan. RNAiknockdown of Atg7 expression can block the extended lifespan phenotype by Hsp27 overexpression, and overexpression of Atg7 can extend lifespan even under Hsp27 knockdown by RNAi. Conclusions: We propose that Atg7 acts downstream of Hsp27 in the regulation of eye morphology, polyglutamine toxicity, and lifespan in Drosophila.
Atg7; Hsp27; Neurodegeneration; Lifespan; Drosophila
The aging process results from imbalanced homeostasis
combined with accumulating macromolecular damage
due to different intrinsic and environmental stresses [1-3].
Protein homeostasis is important in maintaining
physiological function to protect against cellular degeneration
. Autophagy and molecular chaperones are two
defensive systems utilized to uphold cellular protein quality
and homeostasis [5, 6].
Macroautophagy (herein called autophagy) is a cellular,
catabolic process that breaks down and recycles
macromolecules and organelles under starvation conditions.
Autophagy function is executed by a series of autophagy
related genes (Atg) which are evolutionarily conserved
from yeast to mammals . Autophagy participates in
many physiological functions including aging and
neurodegeneration [8, 9], and mounting evidence
demonstrates that autophagy participates in the regulation of
lifespan in different species [10-12]. In C. elegans,
lossof-function of bec-1/Atg6 or RNA interference-mediated
depletion of Atg-7 or Atg-12 inhibits the extended
lifespan in daf-2 mutants [13, 14], and the knockdown of
bec-1 or Atg7 by RNAi abolishes dietary
restrictionmediated longevity in eat-2 mutants . In addition,
mutations in Atg1, Atg7, Atg18, and bec-1 reduce
lifespan in C. elegans . In Drosophila, Atg7-null mutants
are short-lived and hypersensitive to starvation and
oxidative stress , and the neuronal overexpression of
Atg8a regulates lifespan and tolerance to oxidative stress
. Atg7 is an E1-like enzyme and is important for the
membrane elongation of the autophagosome . Atg7
deficient mice exhibit polyubiquitinated protein
accumulation and neurodegeneration  and higher levels of
polyubiquitinated proteins have been detected in the
aging Atg7 mutant fly head . Autophagy also protects
against neurodegeneration  and the induction of
autophagy by the reduction of TOR (target of
rapamycin) activity reduces polyglutamine toxicity in both fly
and mouse . Suppression of basal autophagy in the
central nervous system causes neurodegeneration in
Atg7 conditional knockout mice [19, 22].
Molecular chaperones modulate protein re-folding and
facilitate the degradation of denatured proteins.
Molecular chaperones are also implicated in several
physiological functions: autophagy, neurodegeneration, stress
tolerance, and aging [23-25]. Heat shock protein 27
(Hsp27) is a member of the ATP-independent, small
heat shock protein family. Hsp27 null mutants exhibit
decreased lifespan and reduced starvation tolerance ,
while the overexpression of Hsp27 increases lifespan and
enhances stress resistance in Drosophila [27, 28].
Overexpression of Hsp27 prevents cellular polyglutamine
toxicity and rescues the mosaic eyes induced by mild
polyglutamine toxicity [27, 29].
Both Hsp27 and Atg7 are involved in maintaining
protein quality and modulating lifespan and
neurodegeneration. However, the interaction between Hsp27 and Atg7
is unknown. We report here that Atg7 is downstream of
Hsp27 in the regulation of eye morphology,
polyglutamine toxicity, and lifespan in Drosophila. The levels of
Hsp27 and Atg7 both regulate eye morphology and the
polyglutamine toxicity of 41Q. The overexpression of
Atg7 rescues both the rough eye phenotype resulting
from knockdown of Hsp27 as well as the more severe
mosaic eye phenotype induced by the knockdown of
Hsp27 under 41Q toxicity. In addition, the expression of
Atg7 regulates lifespan in Drosophila and the enhanced
lifespan seen with the overexpression of Hsp27 requires
the expression of Atg7. Together we provide several lines
of genetic evidence linking Hsp27 to Atg7 in the
modulation of eye morphology, polyglutamine toxicity, and
Fly strains and maintenance
The RNAi lines were obtained from Vienna Drosophila
RNAi Center (VDRC), UAS-hsp27RNAi (#40530),
UAShsp22RNAi (#43632), UAS-atg1RNAi (#16133), UAS-atg4RNAi
(#107317), UAS-atg5RNAi (#104461), UAS-atg7RNAi
(#45560), UAS-atg8aRNAi (#43096), UAS-atg8aRNAi
(#43097), UAS-atg9RNAi (#10045), UAS-atg12RNAi
(#102362), UAS-atg18RNAi (#105366). GMR-Gal4;
UAS41Q and GMR-Gal4/Cyo; UAS-63Q were provided by Dr.
Parsa Kazemi-Esfarjani. To generate UAS-Atg7
transgenic flies, the EST clone RE27292 containing the
fulllength Atg7 was used to amplify the coding sequence
by the primers (forward: 5-GTACTCGAGAAGCAA
AACATGAGCACGG-3 and reverse:
5-CATAGATCTATCCTCGTCGCT ATCGGA-3) and subcloned into
the XhoI and BglII sites of the transgenic vector, pINDY6
. The resultant construct was verified by DNA
sequencing to confirm that no mutations derived from
PCR amplification were made, and injected into w1118
eggs for the generation of UAS-Atg7 transgenic flies. All
flies were maintained on standard fly food as described
in Liu et al.  and incubated at 25C, 65% humidity,
in a 12 h/12 h light/dark-cycle fly incubator.
Fly eye image
Two-day-old flies of the different types were anaesthetized
by carbon dioxide on a porous platform and the eye
images were taken by light microscopy (SMZ1500, Nikon).
For the scanning electron micrograph, the fly was fixed on
a copper stage and the fly eye image was acquired by
scanning electron microscopy (TM-1000, Hitachi). For each fly
line, a total of more than 86 eye images from at least three
independent crosses were examined.
RT-PCR and real-time PCR
Total RNA was prepared from about 20 flies of each
specific allele and homogenized in 1 ml Trizol solution. Equal
amounts (1 g) of each DNase I-treated RNA were
reversetranscribed to cDNA with MMLV reverse transcriptase
(Promega). The cDNAs were used as templates for
RTPCR or real-time PCR as described in Liu et al. . The
information of the primers is available upon request.
Lifespan and starvation assays
For the lifespan assay, all the fly lines have been
outcrossed with w1118 as described previously . The
newly eclosed flies of each allele were collected by sex
with 30 flies per vial, maintained at 25C, 65% humidity
in a 12 h/12 h light/dark-cycle fly incubator and
transferred to a new vial every 3 or 4 days until all were dead.
The statistical significance was calculated by log rank
test. At least three independent measurements were
performed for each experiment.
For the starvation assay, newly eclosed flies of each
type were collected by sex with 20 flies per vial and
recovered overnight. Next day the flies were transferred
to the vials with 1% agar and transferred to new agar
vials daily. The numbers of the dead flies were recorded
every 4 hours until all were dead. The statistical
significance was calculated by students t test.
GFP-NLS-marked Atg7 or Hsp27 RNAi knockdown
clones in the larval fat body were generated by heat
shock-independent FLP/FRT induction as described
previously [32, 33]. FLP/FRT method allows to examine the
mitotic GFP-NLS-marked RNAi knockdown clones
surrounded by the control cells that do not incorporate the
RNAi knockdown in the same tissue under the same
condition . Fat bodies from early third instar larva
cultured in standard fly food with yeast paste (fed
condition) or in dishes containing 20% sucrose only
(starvation condition) for 4 hr were dissected and fixed with 4%
paraformaldehyde and then examined by confocal laser
scanning microscope (LSM510; Carl Zeiss Inc.) equipped
with a 63x Plan-Apochromat (NA1.4) objective lens.
Autophagy-related gene 7 is downstream of heat shock
protein 27 in the regulation of Drosophila eye phenotype
Protein homeostasis plays an important role in lifespan
and stress response [1, 2]. Heat shock protein 27 (Hsp27)
has been shown to regulate lifespan and response to
different stresses [26-28]. Autophagy-related gene 7 (Atg7) is
required for normal lifespan and tolerance to starvation
and oxidation . However, the genetic interaction
between Hsp27 and Atg7 is unknown. We examined the
effects of altering Hsp27 and Atg7 expression in the
Drosophila eye using the GMR-Gal4 driver followed by the
analyses of eye morphology utilizing scanning electron
microscopy and light microscopy. Overexpression of
Hsp27 or Atg7 results in a normal eye phenotype and
regular ommatidia shape as seen in the GMR-Gal4 control flies
(Figure 1, A-A, B-B, D-D). Interestingly, knockdown
expression of either Hsp27 or Atg7 by expression of
interfering RNAs using GMR-Gal4 results in similar rough eye
phenotypes with fused and enlarged ommatidia (Figure 1,
C-C, E-E). Overexpression of Atg7 in the Hsp27
knockdown background fully rescues the rough eye phenotype
of the Hsp27 knockdown (Figure 1, F-F). However,
overexpression of Hsp27 in the Atg7 knockdown background
fails to rescue the rough eye phenotype of the Atg7
knockdown (Figure 1, G-G). These results suggest that
Atg7 is located downstream of Hsp27 in the regulation of
Drosophila eye morphology. To further confirm that
Hsp27 and Atg7 function in the same pathway
controlling eye phenotype, we examined whether there is any
additive effect on fly eye morphology by either the
cooverexpression or co-knockdown of Hsp27 and Atg7.
The overexpression of both Hsp27 and Atg7 in
combination produces a normal eye phenotype, similar to the
overexpression of Hsp27 or Atg7 alone (Figure 1, B-B,
DD, H-H). The simultaneous knockdown of Hsp27 and
Atg7 does not further deteriorate the rough eye
phenotype when compared to the effects of either gene alone
(Figure 1, C-C, E-E, I-I), implying that Hsp27 and Atg7
operate in the same pathway. These data provide the first
evidence that Atg7 is downstream of Hsp27 in the
regulation of Drosophila eye morphology.
Knockdown of other autophagy-related genes and heat
shock protein 22 does not result in a rough eye
phenotype in Drosophila
To determine whether the rough eye phenotype is
specific to Atg7, or whether it represents a general effect
of altering autophagy, the effects of knockdown of
additional autophagy-related genes were examined by using
GMR-Gal4 and none of these displayed the rough eye
phenotype (Figure 2, Figure 1, E-E). Knockdown of
Atg1 shows a normal eye phenotype (Figure 2, A-A,
Figure 1, A-A), while knockdown of other
autophagyrelated genes: Atg 4, 5, 8, 9, 12, 18 displayed subtle eye
color phenotypes but had no effect on the ommatidia
structure (Figure 2, B-G, B-G, B-G). These data
suggests that the rough eye phenotype resulting from Atg7
knockdown is Atg7-specific and not involved in the
alteration of other autophagy-related genes. Similarly, to
examine whether the rough eye phenotype is specific to
Hsp27 knockdown, we tested the effects of knockdown
of Hsp22, another known lifespan modulation gene ,
by GMR-Gal4 and did not observe any effects on the eye
like that of Hsp27 knockdown (Figure 1, C-C). Q-PCR
analysis confirms that there is reduced expression of Atg
and Hsp22 genes in the RNAi knockdown experiments
(data not shown). Thus the rough eye phenotype is
specific to the knockdown of either Atg7 or Hsp27.
Knockdown of Atg7 but not Hsp27 blocks
starvationinduced autophagosome formation
To verify that the knockdown of Atg7 by UAS-Atg7RNAi
from VDRC can affect starvation-induced autophagy, we
generated UAS-Atg7RNAi clones in the fat-body by using
the FLP/FRT method [32, 33] and examined the
distribution of mcherry-Atg8a puncta. The distribution of
mcherry-Atg8a is in a uniformly diffuse structure under
optimal feeding conditions (Figure 3, B, J) and becomes
localized to punctate structure under starvation conditions
(Figure 3, F, N). Under starvation conditions, the GFP-NLS
clones with the Atg7 knockdown, where the cells are circled
by dotted line, display a reduced number of mcherry-Atg8a
puncta than the surrounding control clones without Atg7
Figure 1 Atg7 Is Downstream of Hsp27 in the Regulation of Drosophila Eye Phenotype. (A-A) The GMR-Gal4/+ control fly has a normal
eye phenotype and normal shape of individual ommatidia. (B-B, D-D) Overexpression of Hsp27 or Atg7 also results in a normal eye phenotype
and regular ommatidia. (C-C, E-E) Knockdown of Hsp27 or Atg7 displays similar rough eye phenotype and enlarged and fused ommatidia. (F-F)
Overexpression of Atg7 rescues the rough eye and irregular shape of ommatidia by knockdown of Hsp27. (G-G) Overexpression of Hsp27 cannot
revert the rough eye and abnormal shape of ommatidia resulting from the knockdown of Atg7. (H-H) Co-overexpression of Hsp27 and Atg7 still
leads to normal eye phenotype and ommaditia. (I-I) Co-knockdown of Hsp27 and Atg7 causes a similar phenotype: rough eyes and irregular
shape of ommatidia like that of the individual knockdowns of Hsp27 or Atg7. Optical micrograph (A-I) and scanning electron micrograph (A-I:
300X; A-I:1500X). Genotypes: GMR-Gal4 in trans to the alleles indicated.
knockdown which have no GFP-NLS signal (Figure 3, E, F).
These results demonstrate that knockdown of Atg7 by
UAS-Atg7RNAi is able to block mcherry-Atg8a mediated
autophagosome formation under starvation. To examine
whether knockdown of Hsp27 can alter autophagosome
formation, we also generated UAS-Hsp27RNAi clones in the
fat body and inspected the distribution of mcherry-Atg8a
puncta. Under starvation, the autophagosome formation
Figure 2 Knockdown of the Other Autophagy-related Genes and Heat Shock Protein 22 Do Not Result in Any Rough Eye Phenotype in
Drosophila. RNAi knockdown of different autophagy-related genes by GMR-Gal4 show normal eye morphology and regular ommatidia (like the
control in Figure 1, A-A. (A-A) Atg1, (B-B) Atg4, (C-C) Atg5, (D-D) Atg8a, (E-E) Atg9, (F-F) Atg12, (G-G) Atg18, and (H-H) Hsp22. Optical
micrograph (A-H) and SEM (A-H:300X; A-H:1500X). Genotypes: GMR-Gal4 in trans to the alleles indicated.
Figure 3 Starvation-induced Autophagosome Formation is Inhibited by RNAi-mediated Depletion of Atg7 but not Hsp27. (A, E, I, M)
GFP-NLS labeled fat body cells circled by dotted line indicate the presence of UAS-Atg7RNAior UAS-Hsp27RNAigenerated by the FLP/FRT method.
The cells outside of the circled dotted line are used as the control cells without UAS-Atg7RNAior UAS-Hsp27RNAi. (B, F, J, N) The distribution patterns
of mcherry-Atg8a are shown under either fully-fed or starvation conditions. (C, G, K, O) The fat body cells are stained with DAPI. (D, L) The picture
D is merged from panels A, B, C and L is merged from panels I, J, K under nutrient-rich conditions. (H, P) Picture H is merged from panels E, F, G
and P is merged from M, N, O under starvation conditions. The distribution of mcherry-Atg8a puncta is dramatically altered in starved fat body
cells (F, N) compared to those under nutrient-rich conditions (B, J). GFP-labeled cells expressing Atg7-RNAi markedly suppress mCherry-Atg8a
puncta formation (F), but not in that of Hsp27 knockdown (N).
indicated by mcherry-Atg8a puncta is not altered by
comparing the GFP-NLS marked Hsp27 RNAi knockdown
clones, which are circled by dotted line, to the surrounding
control clones without GFP-NLS signal and no Hsp27
RNAi knockdown (Figure 3, M, N). The data indicate that
Hsp27 knockdown does not block the mcherry-Atg8a
mediated autophagosome formation under starvation. The
notion is consistent with the previous data since
knockdown of Atg8 does not result in the rough eye as the
knockdown of Hsp27, suggesting that Hsp27 and Atg8 do
not function in the same genetic pathway.
Atg7 and Hsp27 attenuate the mild polyglutamine toxicity
of 41Q but cannot rescue longer polyglutamine tract
toxicity by 63Q
Overexpression of Hsp27 can rescue the mosaic eye
phenotype resulting from mild polyglutamine
(41Q)induced toxicity but not the rough eye phenotype resulting
from severe polyglutamine (127Q) toxicity . Since Atg7
acts downstream of Hsp27 in the eye, we were interested
in determining whether the overexpression of Atg7 would
also only rescue mild polygutamine phenotypes. As with
Hsp27, the overexpression of Atg7 rescues the mosaic eye
phenotype caused by 41Q (Figure 4, A, B, D) but cannot
rescue the more severe, rough eye phenotypes resulting
from the longer polyglutamine tract of 63Q (Figure 4, G,
H, J). The knockdown of either Hsp27 or Atg7 enhances
the pigmentation phenotype observed in the eye of flies
expressing 41Q. Interestingly only the knockdown of
Atg7, but not that of Hsp27, enhances the eye
morphology phenotype (rough eye) in combination with 41Q
overexpression (Figure 4, C, E). The knockdown of Hsp27
or Atg7 does not exacerbate the rough eye phenotypes of
63Q (Figure 4, I). Interestingly, the overexpression of
Figure 4 Atg7 is Downstream of Hsp27 in the Attenuation of the Mild Polyglutamine Toxicity by 41Q, but the Overexpression of Both
Genes Cannot Rescue the Longer Polyglutamine Tract Toxicity by 63Q. (A) Expression of UAS-41Q by GMR-GAL4 results in mosaic eyes.
(B, D) Both the overexpression of Hsp27 and Atg7 rescue the mosaic eye by 41Q. (C, E) Under 41Q background, both the knockdown of Hsp27 and
Atg7 generate comparable worsened mosaic eyes, whereas knockdown of Atg7 leads to a rough eye surface. (F) Overexpression of Atg7 as well as
knockdown of Hsp27 improves the mosaic eye by 41Q. (G, M) The expressions of UAS-63Q by GMR-Gal4 produce similar rough eye phenotype. (H, J)
Overexpression of Hsp27 or Atg7 cannot rescue the rough eye induced by 63Q. (I, K) Knockdown of Hsp27 or Atg7 in conjunction with 63Q does not
cause further deterioration of the eyes. (L) Overexpression of Atg7 together with knockdown of Hsp27 does not alter the rough eye phenotype by
63Q. Genotypes: (A-F) GMR-Gal4; UAS-41Q in trans to the alleles indicated. (G-L) GMR-Gal4/Cyo; UAS-63Q in trans to the alleles indicated.
Atg7 partially rescues the more dramatic mosaic eye
phenotype induced by Hsp27 knockdown in the 41Q
background (Figure 4, C, F), supporting the idea that
Atg7 is downstream of Hsp27 in the alleviation of 41Q
toxicity. However, the combination of the overexpression
of Atg7 and knockdown of Hsp27 do not change the
rough eye phenotype of 63Q (Figure 4, L).
Atg7 regulates lifespan and is required for
Hsp27mediated extended lifespan in Drosophila
Hsp27 levels are likely to regulate Drosophila lifespan
since Hsp27 overexpression extends Drosophila lifespan
[27, 28] while the knockout Hsp27 mutant is short-lived
. The knockdown of Hsp27 by either hs-Gal4, or
daGal4 exhibits reduced Hsp27 levels and displays a 20%
(P < 0.001), and 27% (P < 0.001) decrease in mean
lifespan, respectively (Figure 5, A - D; Additional file 1:
Table S1). Since Atg7 is downstream of Hsp27 in the
regulation of eye morphology and mild polyglutamine
toxicity, and Atg7 null mutants display shortened
lifespan , we tested whether Hsp27-mediated enhanced
lifespan requires Atg7. Atg7 overexpression by hs-Gal4
shows a robust increase in Atg7 transcripts relative to
control flies and increases the mean lifespan by about
11% (P < 0.01) relative to the control flies (Figure 5, E
and G; Additional file 2: Table S2). Conversely,
knockdown of Atg7 by hs-Gal4 exhibits reduced levels of Atg7
transcripts and decreases mean lifespan by about 10%
(P < 0.01) when compared to the control flies (Figures 5F
and H; Additional file 2: Table S2). These results indicate
that like Hsp27, Atg7 levels also regulate Drosophila
It has been shown that neuronal overexpression of
Atg8a by appl-Gal4 extends Drosophila lifespan and
increases resistance to starvation . To test whether
neuronal overexpression of Atg7 enhances lifespan and
starvation resistance, Atg7 was overexpressed in neurons
using appl-Gal4, resulting in increases of 12%
(P < 0.001) in mean lifespan and 18% (P < 0.01) in
starvation resistance (Figure 5, I; Additional file 2: Table S2
and Additional file 3:Table S4). In addition, the
simultaneous overexpression of Atg7 and knockdown of
Hsp27 results in flies that exhibit a 21% (P < 0.001)
extension in mean lifespan (Figure 5, J). Conversely, the
flies possessing both knockdown of Atg7 and
overexpressing Hsp27 display a reduction of 27% (P < 0.001) in
mean lifespan relative to the control flies (Figure 5, J;
Additional file 4 : Table S3). To further demonstrate that
Atg7 functions downstream of Hsp27, we carried out the
locomotion assay to measure the climbing activity of the
flies with the different combination of overexpression
and knockdown of Atg7 and Hsp27 along with the
control flies under paraquat-induced oxidative stress. Similar
to the lifespan result, the flies with simultaneous
overexpression of Atg7 and knockdown of Hsp27 displayed
significantly better climbing activity (42%, P 0.001) than
that of the control flies (22%), and the flies with
simultaneous knockdown of Atg7 and overexpression of
Hsp27 exhibited a significantly lowered locomotion
activity (15%, P 0.01) than that of the control flies
Figure 5 (See legend on next page.)
(See figure on previous page.)
Figure 5 Atg7 Is Downstream of Hsp27 in the Regulation of Drosophila Lifespan. (A, B, E, F) RT-PCR verifies that the transcript levels of
Hsp27 and Atg7 are altered upon Gal4 induction. (C, D) RNAi knockdown of Hsp27 by hs-Gal4 and da-Gal4 both reduce Drosophila lifespan. (G)
Overexpression of Atg7 by hs-Gal4 increases Drosophila lifespan. (H) Knockdown of Atg7 by hs-Gal4 decreases Drosophila lifespan. (I) Neuronal
overexpression of Atg7 by appl-Gal4 enhances Drosophila lifespan. (J) Overexpression of Atg7 along with knockdown of Hsp27 by appl-Gal4
displays extended lifespan. On the other hand, simultaneous knockdown of Atg7 and overexpression of Hsp27 exhibits reduced lifespan.
(Additional file 5: Figure S1). The climbing activity data
in accordance with the lifespan data supports our
hypothesis that Atg7 acts downstream of Hsp27. Taken
together, these results indicate that as seen with Drosophila
eye morphology and polyglutamine toxicity, Atg7 also
acts downstream of Hsp27 in regulating lifespan.
Hsp27 and Atg7 are both implicated in the processes of
aging and neurodegeneration. In this report, we provide
several lines of evidence to show that Atg7 is
downstream of Hsp27 in the regulation of eye morphology,
polyglutamine toxicity, and lifespan. Autophagy-related
genes are conserved among different species [7, 35].
Each of the identified Atgs has a role in autophagy, but
their roles in other processes remains largely unclear.
In the examination of eye phenotype, we observed that
the knockdown of either Hsp27 or Atg7 exhibited similar
rough eye phenotypes. These effects appear to be
specific to these particular molecules since the knockdown
of other Atgs (Atg1, Atg4, Atg5, Atg8a, Atg9, Atg12, and
Atg18) or Hsp22 does not produce a similar, rough eye
phenotype. The ability of Atg7 to rescue the phenotype
induced by Hsp27 knockdown also suggests that a
unique interaction exists between Hsp27 and Atg7. A
recent study indicates that knockdown of Atg7 by
GMRGal4 on X chromosome causes retinal degeneration
. In addition, the rhabdomeres were shown
degenerated in the aged atg7d77 mutant flies . Both support
our finding that RNAi knockdown of Atg7 results in
rough eye in Drosophila.
Autophagy serves to protect against neurodegenerative
diseases  and aberrations in autophagy have been
implicated in neurodegeneration . In both fly and
mouse models, induction of autophagy by inhibiting
mTOR ameliorates polyglutamine toxicity . And in
humans, a polymorphism study of more than 900
European Huntingtons disease patients revealed that one
variant of Atg7 (Atg7V471A) is statistically correlated with
early onset of Huntingtons disease . These findings
suggest that a specific function of Atg7 is to attenuate
polyglutamine toxicity and support our findings that
Atg7 rescues polyglutamine toxicity by 41Q in
Drosophila. Hsp27 has also been shown to reduce cellular
polyglutamine toxicity  and the overexpression of Hsp27
in Drosophila rescues the pigmentation defects induced
by 41Q . Several lines of evidence suggest that heat
shock proteins may rely upon autophagy to reduce
polyglutamine toxicity. For example, the
anti-polyglutamineaggregation activity of HspB7, one of the human small
heat shock proteins, was substantially diminished in
Atg5-deficient cells . In addition, it is possible that
the small heat shock protein HspB8-Bag3 complex
enhance Htt43Q degradation via autophagy since the
treatment of the Htt43Q transfected HEK-293T and COS1
cells with an autophagy inhibitor significantly reduced
HspB8-Bag3-mediated Htt43Q degradation .
Furthermore, it was recently suggested that the small heat
shock protein HspB7 assists in the loading of misfolded
proteins or aggregates in autophagosomes .
Together, these findings indicate that autophagy is
downstream of small heat shock proteins and support our
results that Atg7 is downstream of Hsp27.
The inhibition of autophagy results in decreased
lifespan. Atg7 activity is essential for the longevity resulting
from either reduced insulin signaling or caloric
restriction in which depletion of Atg7 was found to block the
longevity phenotypes of both daf-2 and eat-2 mutants
[13, 15]. Our data showed that RNAi knockdown of
Atg7 by hs-Gal4, starting from embryonic to adulthood
stage, results in a shortened lifespan similar to that of
the Drosophila Atg7 null mutant . Loss-of-function
mutations in Atg7 as well as Atg1, Atg18, and Beclin-1
shorten lifespan in C. elegans . Several autophagy
mutants including Atg7 were identified chronologically
short-lived in a yeast genetic screen . However, it
should be noted that not all autophagy genes are linked
to aging and Atg7 is one of the conserved Atg genes that
is involved in the regulation of aging in most species .
Conversely, the induction of autophagy increases
lifespan. The induction of autophagy by caloric restriction
or reducing target of rapamycin (TOR) activity enhances
lifespan  and the neuronal overexpression of Atg8a
increases Drosophila lifespan . We have found that
the overexpression of Atg7 extends lifespan in
Drosophila and that the neuronal overexpression of Atg7 is
sufficient to reverse Hsp27-knockdown-mediated, shortened
lifespan. Knockdown of Atg7 blocks Hsp27-mediated
extended lifespan, again supporting the model that Atg7
acts downstream of Hsp27 in the regulation of lifespan.
It has been reported that in adult flies, RNAi knockdown
of Atg7 by Geneswitch-Actin-Gal4 did not show reduced
lifespan . This discrepancy may be due to the
different Gal4 drivers used and that the knockdown of Atg7
occurring only during adulthood is insufficient to cause
shortened lifespan since autophagy activity is known to
be tightly regulated during development.
Yet we cannot exclude that chaperone-mediated
autophagy (CMA) is involved in the connection between
Hsp27 and Atg7. CMA is a specific cargo delivery
process to the lumen of the lysosome, mediated by
Hsc70, Hsp90, and the lysosome-associated membrane
protein type 2A (LAMP-2A) [45, 46]. However, a recent
study in Drosophila shows that the co-chaperone Starvin
assists in the coordination of Hsc70 and HspB8 through
chaperone-assisted selective autophagy, which is distinct
from CMA, to depose damaged filamin for muscle
maintenance . It is possible that Hsp27 may function
through chaperone-assisted selective autophagy linking
In summary, our finding sheds new insight in the linkage
of Hsp27 to Atg7 in the regulation of eye morphology,
polyglutamine toxicity, and lifespan. The information
provides a new aspect in the understanding how Hsp27
may connect to Atg7 to modulate certain physiological
Additional file 2: Table S2. A summary of lifespan by the
overexpression or knockdown of Atg7 in Drosophila.
Additional file 3: Table S4. A summary of starvation stress response by
overexpression of Atg7 in Drosophila.
Additional file 4: Table S3. A summary of lifespan resulting from
simultaneous overexpression and knockdown of different combinations
of Atg7 and Hsp27 in Drosophila.
Additional file 5: Figure S1. The flies with simultaneous overexpression
of Atg7 and knockdown of Hsp27 display better climbing activity than
those with overexpression of Hsp27 and knockdown of Atg7 under
paraquat-induced oxidative stress. The climbing index for each strain:
appl-Gal4/+(the control fly): 21.8 0.02% (n = 195); UAS-hsp27/+;
applGal4/UAS-atg7RNAi: 14.7 0.02% (n = 123); UAS-atg7/+;
appl-Gal4/UAShsp27RNAi: 42.4 0.01% (n = 175). (n is the total fly number from the four
independent assays.). (**p < 0.01, ***p < 0.001).
We thank Drs. Theodore Brummel, Micheline Laurent, William Ja, Pankaj
Kapahi, and Chiou-Hwa Yuh for the critical reading and suggestions for the
manuscript. We thank the fly lines from VDRC and Bloomington stock center,
GMR-Gal4; UAS-41Q and GMR-Gal4/Cyo; UAS-63Q from Dr. Parsa
KazemiEsfarjani. We thank the funding support from National Tsing Hua University/
Chang Gung Memorial Hospital collaboration grant (101N2754E1), NTHU/
McKay Hospital collaboration grant (99N2903E1), and National Science
Council (100-2311-B-007-006-) to Dr. Horng-Dar Wang, National Science
Council grant (100-2311-B-004-001-MY3) to Dr. Pei-Yu Wang, and Academia
Sinica (AS-99-TP-B09) to Dr. Guang-Chao Chen. We are indebted for the
funding support from Brain Research Center (101N2060E1) by Dr. Ann-Shyn
Chiang at NTHU, the fly import assistance from Fly Core Taiwan by
Dr. Chau-Ti Ting, and the transgenic fly support by Dr. Y. Henry Sun at
Academia Sinica, Taipei, Taiwan.
S-F Chen, M-L Kang, Y-C Chen, H-W Tang, C-W Huang, W-H Li, and C-P Lin
carried out the experiments and analyzed the data; P-Y Wang, G-C Chen and
H-D Wang designed the experiments, analyzed the data, and together with
C-Y Wang discussed the data and wrote the manuscript. All authors read
and approved the final manuscript.
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