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and Subramaniam Ganesh
Parihar, Vibha Dwivedi, Subhash C. Lakhotia
Amit Kumar, Pankaj Kumar Singh, Rashmi
exon1 protein fragment
from cytotoxicity mediated by huntingtin
Decreased O-linked GlcNAcylation protects
Molecular Bases of Disease:
published online March 19, 2014J. Biol. Chem.
10.1074/jbc.M114.553321Access the most updated version of this article at doi:
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Decreasing O-linked GlcNAcylation increases basal autophagic flux
1
Decreased O-linked GlcNAcylation Protects from Cytotoxicity Mediated by Huntingtin
Exon1 Protein Fragment*
Amit Kumar1,3,4, Pankaj Kumar Singh1,3,5 Rashmi Parihar1,3, Vibha Dwivedi2,3, Subhash C.
Lakhotia2 and Subramaniam Ganesh1,6
From the 1Department of Biological Sciences and Bioengineering, Indian Institute of
Technology, Kanpur, India;
2Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi,
India
3These authors contributed equally and should be considered as joint first authors
Running Title: Decreasing O-linked GlcNAcylation increases basal autophagic flux
6To whom correspondence should be addressed: Subramaniam Ganesh, Department of
Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 208016,
India; Tel (+91) 512 259 4040; Fax (+91) 512 259 4010; Email: sganesh@iitk.ac.in
Key words: O-GlcNAcylation; Autophagy; Huntington’s disease
Background: Earlier reports indicate that
O-GlcNAcylation might be protective in
neurodegenerative disorders.
Results: Suppressing O-GlcNAcylation
modulates autophagy to enhance the
viability of neuronal cells expressing
cytotoxic mutant huntingtin exon 1 protein
(mHtt).
Conclusion: O-GlcNAcylation regulates
the clearance of mHtt by modulating the
fusion of autophagosomes with lysosomes.
Significance: This regulatory mechanism
emerges as a novel therapeutic strategy for
Huntington’s disease.
ABSTRACT
O-GlcNAcylation is an important
post-translational modification of
proteins and is known to regulate a
number of pathways involved in cellular
homeostasis. This involves dynamic and
reversible modification of
serine/threonine residues of different
cellular proteins catalyzed by O-linked
N-acetylglucosaminyltransferase (OGT)
and O-linked N-acetylglucosaminidase
(OGA) in antagonistic manner. We
report here that decreasing O-
GlcNAcylation enhances viability of
neuronal cells expressing polyglutamine
expanded huntingtin exon 1 protein
fragment (mHtt). We further show that
O-GlcNAcylation regulates the basal
autophagic process and that suppression
of O-GlcNAcylation significantly
increases autophagic flux by enhancing
the fusion of autophagosome with
lysosome. This regulation considerably
reduces toxic mHtt aggregates in eye
imaginal discs, and partially restores
rhabdomere morphology and vision in a
fly model for Huntington’s disease. The
present study is significant in
unravelling O-GlcNAcylation-dependent
regulation of autophagic process in
mediating mHtt toxicity. Therefore,
targeting autophagic process through
the suppression of O-GlcNAcylation
may prove to be an important
therapeutic target in Huntington
disease.
INTRODUCTION
O-GlcNAcylation is a glucose
dependent post-translational modification
(PTM). When glucose enters the cell,
approximately 5% of it enters into the
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Decreasing O-linked GlcNAcylation increases basal autophagic flux
2
Hexosamine biosynthetic pathway (HBP)
through a series of metabolic
transformation and finally gets
transformed into uridine 5’-diphospho-N-
acetyl glucosamine (UDP-GlcNAc) (1).
This final product of HBP, amongst other
functions, acts as a substrate of O-linked
GlcNAcylation and is utilized by two
antagonistic enzymes, O-linked N-
acetylglucosaminyltransferase (OGT) and
O-linked N-acetylglucosaminidase (OGA),
which regulate the dynamic modification
of different nucleo-cytoplasmic proteins.
OGT catalyzes the addition of a single
GlcNAc moiety to serine and/or threonine
sites of various proteins while OGA
removes the same (2). O-GlcNAcylation
has been shown to regulate many vital
biological processes such as replication,
transcription, translation, stress response,
nutrient response, unfolded protein
response, and intra-cellular protein
trafficking (3-5). It is also emerging as an
important regulatory mechanism in a
number of complex diseases such as
diabetes, cancer, cardiovascular diseases,
and in ageing (5). OGT activity has been
reported to be about 10 times more
enriched in brain as compared to other
tissues like liver, muscle, adipose and heart
(6) and has been shown to glycosylate
many proteins linked to neurodegenerative
diseases such as, amyloid precursor
protein, β amyloid associated protein (7),
microtubule associated tau protein (8),
synapsin (9) and neurofilament proteins
(10). Recently, OGT was reported to play
a protective role in Alzheimer’s disease
(11) and is speculated to be protective in
other neurodegenerative disorders such as
Huntington’s disease, Parkinson’s disease
and Amyotrophic Lateral Sclerosis (ALS).
Huntington’s disease is a
neurodegenerative disorder characterized
by the formation of intracellular
aggregates of mutant huntingtin (mHtt)
(12, 13). The normal huntingtin gene codes
for the huntingtin protein, which usually
has up to 34 glutamine coding (CAG)
repeat (12, 13). The huntingtin protein
having up to 34 glutamine repeats
represents a normal functional protein,
while expansion of CAG repeats coding
for >40 glutamine as a repeat track results
in a dominant mutation, as a consequence
of which the mutant Htt loses its proper
folding state, tends to aggregate and
becomes cytotoxic (12, 13). The wild-type
huntingtin protein plays important roles in
normal functioning of brain such as
vesicular transport, neuronal gene
transcription, BDNF production (14), and
may also function as an anti-apoptotic
protein (15). The mHtt aggregates interfere
with normal synaptic transmission (16),
impair axonal transport of mitochondria
(17), sequester crucial transcription factors
(18) and hamper their functioning.
O-GlcNAcylation is a nutrient
sensitive protein modification. With the
emerging understanding of the important
roles of O-GlcNAcylation in various
neurodegenerative disorders along with
reports about glucose-dependent regulation
of protein clearance machineries (19, 20)
and that of protein aggregation mediated
toxicity (21, 22), we aimed to explore the
role of this glucose-dependent post-
translational modification in the regulation
of mHtt-mediated toxicity and its
clearance. We report here that suppression
of O-GlcNAcylation increases basal
autophagy flux by enhancing
autophagosome-lysosome fusion and helps
in the clearance of toxic aggregates of
mutant huntingtin exon 1 coded protein,
thereby increasing survival and
suppressing the degenerative phenotypes
in cellular and Huntington fly models,
respectively.
EXPERIMENTAL PROCEDURES
Reagents and antibodies
−
Cell culture
media and drugs (Azaserine, Glucosamine,
3-Methyladenine, and Bafilomycin A1)
were purchased from Sigma Aldrich Pvt.
Ltd., India. Polyfect transfection reagent
was from QIAGEN India Pvt. Ltd., India.
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Decreasing O-linked GlcNAcylation increases basal autophagic flux
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Antibodies were procured from following
sources: Anti-LC3 and Anti-Myc for
immunostaining (Cell Signaling
Technology, USA); Anti-p62 (Enzo Life
Sciences, USA); Anti-GFP and Anti-Myc
for immunoblots (Roche, India); Anti-HA
(used in experiments done in Neuro2A
cell) (Sigma-Aldrich Pvt. Limited, India),
Anti-O-GlcNAc and Anti-γ-tubulin
(Sigma-Aldrich Pvt. Limited, India).
Rabbit polyclonal anti-haemagglutinin
(used in experiments with Drosophila)
(Santa Cruz, USA), Secondary antibodies
were procured from Jackson
ImmunoResearch Inc., USA, except Anti-
rabbit conjugated with Cy3 (Sigma-
Aldrich, India) and Alex-Fluor 488
(Molecular Probes, USA).
Expression constructs
−
Mammalian
expression constructs were obtained from
following sources: The GFP-tagged
truncated Huntingtin Q97 expression
vector was generously provided by Dr
Lawrence Marsh (University of California,
Irvine, CA, USA). Plasmid coding for
OGA (Myc-tagged) was a kind gift from
Dr John A Hannover (NIDDK, National
Institutes of Health, USA), the HA-tagged-
OGT was gifted by Dr Gerald W. Hart
(Johns Hopkins University School of
Medicine, Baltimore, USA), the mRFP-
GFP-LC3 construct was gifted by Dr T.
Yoshimori (National Institute for Basic
Biology, Okazaki, Japan), and construct
coding for the mutant form of α-synuclein
as GFP fusion was a gift from Dr. Peter
Lansbury (Harvard Medical School, USA).
Cell culture, treatment and transfection
−
The experiments were conducted in the
murine neuroblastoma cell line Neuro2A
under normal glucose conditions (25 mM).
Neuro2A cells were grown in Dulbecco’s
modified Eagle’s medium (Sigma-Aldrich
Pvt. Limited, India) supplemented with
10% (v/v) fetal calf serum, 100 U/ml
penicillin and 100 µg/ml streptomycin.
The cells were treated with 40 µM
azaserine (inhibitor of O-GlcNAcylation),
10 mM glucosamine (inducer of O-
GlcNAcylation), 100 nM Bafilomycin A1
(inhibitor of the fusion of autophagosomes
with lysosomes) and 10 mM 3-
Methyladenine (3MA; an inhibitor of
autophagosome formation). The cells were
transiently transfected with expression
constructs at around 50% of confluence
using PolyFect Transfection Reagent
(QIAGEN India Pvt. Ltd) as recommended
by the manufacturer. Under these
conditions the transfection efficiency was
consistent and around 70% as assessed by
microscopic observation of the
fluorescence positive cells transiently
expressing the GFP-tagged protein. In all
the experiments, the cells were harvested
at 36 hrs post-transfection and wherever
required, treatment with the
pharmacological agents was given for the
last 12 hrs unless stated otherwise.
Fly stocks and rearing condition − All fly
stocks were maintained under un-crowded
condition at 240C±10C. For each
experiment, regular or azaserine
(250µg/ml) supplemented food was
prepared from the same batch. Using the
w1118; UAS-httex1p Q93/CyO (20) and
w1118; GMR-GAL4 (21) fly stocks,
appropriate genetic crosses were set to
obtain w1118; UAS-httex1p Q93/GMR-
GAL4 (GMR-GAL4>UAS-httex1p)
progeny. The GMR-GAL4 driver targets
expression of the UAS-httex1pQ93
transgene in developing eye discs (23) and
thereby induce retinal neurodegeneration
(24). In some cases, Oregon R+ stock was
used as wild type. Freshly hatched larvae
for a given experiment were derived from
a common pool of eggs of the desired
genotype and reared in parallel on regular
or azaserine supplemented food.
We also reared larvae on food
supplemented with glucosamine (1 mg/ml,
10 mg/ml or 25 mg/ml). However, in each
case, all the larvae died before reaching 3rd
instar stage and therefore, no further
studies on the effect of glucosamine on
polyglutamine (polyQ) degeneration in the
fly model could be carried out.
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Decreasing O-linked GlcNAcylation increases basal autophagic flux
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Immunostaining
−
Cells on coverslip were
fixed with 4% paraformaldehyde in 1X
PBS for 20 minutes followed by
permeabilization for 5 minutes in 1X PBS
with 0.05% Triton X-100. The expression
of Myc-OGA and HA-OGT was checked
by probing with Anti-Myc or Anti-HA
antibodies followed by FITC or TRITC
conjugated secondary antibodies,
respectively. Nuclei were counterstained
with 10µM 4’,6-diamidino-2-phenylindole
(DAPI). Images were obtained with a
Nikon (Japan) Eclipse 80i fluorescence
microscope using a 10X or 40X objective
lens.
Eye discs from wandering late third
instar GMR-GAL4>UAS-httex1p Q93
larvae reared on normal or azaserine
supplemented food were dissected and
immunostained as described previously
(25) with the anti-HA (1:80 dilution,
Santa-Cruze). Chromatin was
counterstained with DAPI. Immuno-
fluorescence stained eye discs were
examined with a Zeiss LSM 510 Meta
confocal microscope using appropriate
lasers, dichroics and filters.
Cell death assay − For the MTT assay
cells were treated with azaserine or
glucosamine for 12 hrs or transfected for
36 hrs and thereafter, cells were incubated
with 0.5 mg/ml MTT (thiazolyl blue
tetrazolium bromide) (Sigma-Aldrich Pvt.
Limited, India) and chased for 2 hrs. After
removal of the medium, cells were
incubated with DMSO (100%) for 10 min
to dissolve formazon crystals. The change
in optical density was recorded through
spectrophotometer at λ570nm against
background reading at λ650nm.
Alternatively, treated or transfected cells
were fixed, permeabilized and stained with
DAPI as mentioned for immunostaining,
and the apoptotic nuclei were scored in a
blinded fashion as reported earlier (26).
Quantification of LC3-positive cytoplasmic
puncta - Cells transiently expressing the
tandem mRFP-GFP-LC3 construct were
fixed, and the fluorescence images of
about 50 cells for each set were examined
using a Zeiss AxioImager 2 microscope
outfitted with an ApoTome accessory. The
green, red and yellow puncta in the
captured images were quantified using the
Colocalization Macro in ImageJ software,
as described (27)
Immunoblotting
−
Protein samples were
resolved on 6-12% SDS-PAGE as required
and transferred to nitrocellulose membrane
(MDI, India). Thereafter, the membranes
were blocked with either 5% non-fat dry
milk powder or 5% BSA in 1X TBST and
probed sequentially with the desired
primary and secondary antibodies at their
recommended dilutions followed by
detection with a chemiluminescent
detection kit (Supersignal West PICO,
Pierce, USA).
Filter-trap assay − The filter-trap assay
was carried out essentially as described by
Juenemann et al. (28). Briefly, the pellet
fraction of the cell lysate was suspended in
the benzonase buffer (1 mM MgCl2, 50
mM Tris/HCl pH 8.0), and treated with a
RNAse/DNAse cocktail (50 U each;
Fermentas) and incubated for 1 hr at 37oC.
The reaction was arrested with the addition
of 2x termination buffer (40 mM EDTA, 4
% SDS, 100 mM DTT), and 50 μg of the
sample was mixed in 2% SDS buffer (2 %
SDS, 150 mM NaCl, 10 mM Tris/HCl pH
8.0) and filtered through a 0.2 μm pore
size cellulose acetate membrane (GE
Healthcare Life Sciences, USA) using a
slot blot apparatus (Bio-Rad Indian Pvt.
Ltd., India). The filter membrane was used
for immunodetection as described for the
immunoblot.
Proteasome activity assays - Cells, that
were either transfected or treated with
indicated drugs (12 h), were harvested in
lysis buffer (1x PBS, 0.1% Triton X100,
0.5% NP40) and the cleared lysate was
used for the proteasome activity assay
using a fluorogenic proteasome substrate
(Suc-Leu-Leu-Val-Tyr-AMC;
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Decreasing O-linked GlcNAcylation increases basal autophagic flux
5
Calbiochem). Briefly, 10 µg of protein for
each sample was incubated in a reaction
buffer and the generation of fluorescent
signal was measured using a
spectrofluorometer (Parkin Elmer) as
recommended by the manufacturer.
Reactions in the presence of the
proteasomal blocker, MG132, served as
control.
Pseudopupil Analysis − Heads of 1 day old
GMR-GAL4>UAS-httex1p Q93 flies,
reared since the 1st instar larval stage on
normal or azaserine supplemented food
were decapitated and the arrangement of
photoreceptor rhabdomeres in the
ommatidia of compound eyes was
visualized by the pseudopupil technique
(29) using 63X (NA= 1.4) oil objective on
a Nikon E800 microscope and the images
were recorded with a Nikon DXM 1200
digital camera. The total number of flies
observed for each group was 50.
Phototaxis Assay − Phototaxis of adult
flies was assayed using a Y maze
consisting of a Y shaped glass tube of 12
mm internal diameter and 30 cm length of
each arm. Twenty replicates, each with 10
flies, were carried out for each feeding
regime and age of flies. Wild type Oregon
R+ flies were used as positive control. The
same sets of flies were used for phototaxis
assay on days 0, 5, 10 and 15.
Statistical analysis − Sigma Plot 11.0
software was used for statistical analysis.
For cell biology assays, data were
analyzed by two-tailed, unpaired Student’s
t-test. For assays involving flies, one-way
ANOVA was performed for comparison
between the control and formulation-fed
samples. Pooled data are expressed as
mean ± S.E. of means of the different
replicates of the experiment.
RESULTS
Global suppression of O-linked
glycosylation reduces the aggregation
propensity and cytotoxicity of mutant
Huntingtin in a cellular model
−
Based on
the previous findings (21, 22), we were
interested in exploring the role of O-
GlcNAcylation in suppressing the
cytotoxicity caused by aggregate-prone
proteins. For this, we used a mammalian
expression construct that codes for the
OGT or OGA, the two proteins which
work antagonistically to regulate the O-
linked protein glycosylation. Transient
expression of OGT in the murine
neurobalstoma cell line Neuro2A resulted
in increased global O-GlcNAcylation
while overexpression of OGA led to a
reduction in global O-GlcNAcylation (Fig.
1A). To check if O-GlcNAcylation could
alter the aggregate forming propensity of
mutant huntingtin, we co-expressed OGA
or OGT with an expression construct
coding for the amino terminal huntingtin
protein having 97 polyglutamine repeat
tagged with green fluorescence protein
(mHtt-Q97-GFP) in Neuro2A. Cells that
expressed only the mHtt-Q97-GFP served
as control (co-transfected with the empty
vector pcDNA). As shown in Fig. 1B and
1D, co-expression of OGA resulted in a
significant reduction in the number of
transfected cells showing the mHtt-Q97-
GFP-positive aggregates as compared to
the control cells that were co-transfected
with the empty vector, pcDNA. On the
other hand, OGT co-expression resulted in
a higher proportion of cells with the mHtt-
Q97-GFP aggregates (Fig. 1B and 1D).
Co-expression of OGA or OGT did not
affect the transfection efficiency of the
mHtt-Q97-GFP coding construct (see the
inlet Fig. 1D). Similarly, there was no
significant difference in cell survival when
OGA or OGT was expressed alone as
compared to cells that were transfected
with an empty vector (Fig. 1C). To test
further whether the reduction in the global
O-GlcNAcylation helps the cell to reduce
the aggregation of mHtt-Q97-GFP, we
evaluated the total and SDS-insoluble
forms of mHtt-Q97-GFP by immunoblot
and the filter-trap assay, respectively (Fig.
2A). Consistent with our observations on
mHtt aggregates in situ, co-expression of
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Decreasing O-linked GlcNAcylation increases basal autophagic flux
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OGA led to a significant reduction in the
level of SDS-insoluble form of mHtt-Q97-
GFP when compared with that in cells co-
expressing OGT or only the mHtt-Q97-
GFP (pcDNA control; Fig. 2A). Co-
expression of OGA or OGT did not show
significant change in the total level of
mHtt-Q97-GFP in the immunoblots (Fig.
2A). We also found that co-expression of
OGA, but not of OGT, resulted in a
significant reduction in mHtt-Q97-GFP-
mediated cell death, as measured by MTT
assay (Fig. 2B), and also by scoring
apoptotic nuclei (Fig. 2D). To check
whether the protective effect of OGA was
limited to mtHtt-Q97-GFP, or whether
OGA can ameliorate the toxicity of other
disease associated cytotoxic proteins, we
expressed Parkinson’s disease associated
α-synuclein mutant A30P protein (26)
either alone or along with OGA or OGT
and measured the cell viability by MTT
assays as well as by counting the apoptotic
nuclei. As shown in Fig. 2C and 2E, OGA,
but not OGT, was able to confer protection
against the toxicity of the A30P mutant,
suggesting that the OGA-mediated
protective response could be a generic
effect of O-GlcNAcylation, and is not
specific to mtHtt-Q97-GFP. Taken
together, our results suggest a causal role
for O-GlcNAcylation in modulating the
level of insoluble, aggregated mutant
huntingtin and its cytotoxicity.
Inhibition of O-GlcNAcylation enhances
autophagy − Our next aim was to identify
the mechanism by which suppression of
O-GlcNAcylation reduces the level of
cytotoxic insoluble form of mHtt-Q97-
GFP. Since autophagic process is known
to clear the aggregated proteins (30, 31),
we were interested in testing the impact of
O-GlcNAcylation in basal autophagic
process. For this, Neuro2a cells were
transfected with the expression construct
coding for OGT or OGA or with an empty
vector (pcDNA control). At 36 hrs post-
transfection, the cells were harvested and
levels of two autophagic marker proteins,
LC3 and p62, were evaluated. As shown in
Fig. 3A, transient overexpression of OGA
led to a reduction in the level of both p62
and LC3II, suggesting that suppression of
O-GlcNAcylation resulted in an enhanced
autophagic flux. To further confirm that
the observed effect is indeed because of
the changes in global O-GlcNAcylation,
we examined the autophagic process after
treating the cells with, azaserine, which
inhibits glutamine fructose-6-phosphate
amidotransferase, one of the key enzymes
of hexosamine biosynthesis pathway and
thereby inhibits O-GlcNAcylation (32,
33). As shown in Fig. 3B, treatment of
Neuro2A cells with azaserine for 12 hrs
resulted in a significant reduction in global
O-GlcNAcylation levels. As was observed
for OGA expression, azaserine also led to
a reduction in the level of the autophagic
markers LC3II and p62 (Fig. 3C),
confirming that a reduction in the cellular
O-GlcNAcylation level correlates with
increased levels of basal autophagic flux.
To further confirm that the observed effect
of azaserine on autophagic process is
indeed through O-GlcNAcylation process,
we treated cells both with azaserine and
glucosamine and looked at the level of
LC3II and p62. Glucosamine is known to
rescue the effect of azaserine on O-
GlcNAcylation process hence the double
treatment should rescue the effect of
azaserine on the autophagic process (Fig.
3B). As shown in Fig. 3C, azaserine-
glucosamine treatment increased the level
of LC3II and p62 as compared to only
azaserine treatment, confirming that the
level of autophagic induction inversely
correlate with O-GlcNAcylation level.
Our next aim was to identify the key
step through which the O-GlcNAcylation
regulates the autophagic process. The
reduction in the level of the autophagy
marker LC3II upon depletion of
glycosylation may be because either (i) the
autophagosome formation is inhibited
(inhibition of autophagy initiation), or (ii)
enhanced degradation of LC3II (increased
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Decreasing O-linked GlcNAcylation increases basal autophagic flux
7
autophagic flux) via lysosome since LC3
itself is an autophagy substrate (34). Our
observation that the cellular level of
another autophagic substrate, p62, was
also at lower levels upon OGA
overexpression suggests that the second
possibility is more likely. Therefore, we
checked whether inhibition of fusion of
autophagosome with the lysosome would
rescue the level of LC3II and p62. For this,
the cells were transfected with expression
construct coding for OGA of OGT and
then were treated with bafilomycin A1
(BafA1), an inhibitor of autophagosome
lysosome fusion (34) for 12 hrs and the
cellular level of LC3II and p62 was
evaluated. As shown in Fig. 4A, we found
that the BafA1-mediated inhibition of
autophagosome-lysosome fusion led to an
increase in the level of both LC3II and p62
even in those cells that overexpressed
OGA or OGT. Very similar observations
were made when the glycosylation was
inhibited by azaserine treatment (Fig. 4B).
To further confirm that suppression of O-
GlcNAcylation indeed increases the
autophagy flux, we utilized the tandem
mRFP-GFP-LC3 expression construct
whose expression product is known to
show difference in pH sensitivity and has
been widely used to monitor the
autophagic process (35). For this the
Neuro2A cells were transiently transfected
with the mRFP-GFP-LC3 tandem
construct and empty vector (pcDNA), or
along with the expression vector coding
for OGA or OGT, and scored the
colocalization of green and red signals in
the cytoplasmic LC3-positive puncta, and
also the number of green and red puncta.
Here, autophagosomes are visible as
yellow puncta and autophagolysosomes
(post-lysosomal fusion) as red puncta (35).
As shown in Fig. 5, co-expression of OGA
led to a significant increase in the fraction
of red/green-positive LC3 puncta while no
such difference was noted for OGT.
Similarly, there was a significant increase
in the LC3 puncta that were positive only
for red fluorescence (Fig. 5), suggesting
that suppression of O-GlcNAcylation did
enhance the autophagy flux.
Next, we tested whether the
reduction in the level of insoluble fraction
of mutant huntingtin seen in O-
GlcNAcylation deprived-condition is due
to an enhanced autophagy flux. As shown
in Fig. 6A, BafA1 treatment led to an
increase in the level of insoluble fraction
of the mutant huntingtin even in the OGA
overexpressing cells, suggesting that
decreased O-GlcNAcylation promotes the
clearance of the aggregate-prone protein
by enhancing autophagosome-lysosome
fusion. Finally, to demonstrate that
autophagy is the mechanism through
which O-GlcNAcase is able to protect cells
from huntingtin aggregates, we treated
cells that coexpress mtHtt-Q97-GFP and
OGA with an autophagy inhibitor, 3-
Methyladenine (3-MA). As shown in Fig.
6B, 3-MA treatment led to a significant
increase in the insoluble form of mtHtt-
Q97-GFP even when OGA was co-
expressed, suggesting that the protective
effect conferred by OGA is indeed through
the autophagic process.
Having shown an indirect correlation
between O-GlcNAcylation and autophagic
flux, we checked possible effect of O-
GlcNAcylation on proteasomal activity.
For this cells that transiently expressed
OGA or OGT or that were treated with
azaserine or glucosamine were assayed for
proteasomal activity. As shown in Fig. 7A,
transient overexpression of OGT or OGA
led to a significant reduction in the
proteasomal activity. Treatment of cells
with azaserine or glucosamine did not
significantly alter the activity (Fig. 7B),
suggesting that the O-GlcNAcylation-
dependent clearance of mutant huntingtin
observed in our model could be primarily
through the autophagic process.
Azaserine feeding reduces mutant
huntingtin aggregation in the larval eye
discs of Drosophila − Having found that
azaserine treatment reduces the aggregates
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Decreasing O-linked GlcNAcylation increases basal autophagic flux
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of mutant huntingtin in the mammalian
cell line, we next tested whether similar
effect could also be seen in vivo, for which
we used the fly model of Huntington’s
disease (23). We reared wild type and
GMR-GAL4>httex1p Q93 larvae from the
1st instar stage onwards on food
supplemented with azaserine (250 µg/ml).
It is known (36, 37) that GMR-GAL4
driven expression of the mutant huntingtin
protein leads to accumulation of polyQ
inclusion bodies posterior to the
morphogenetic furrow in late 3rd instar
larval eye discs (Fig. 8A, B). We found
that azaserine feeding substantially
reduced the accumulation of mHtt protein
so that in ~57% of the eye discs (n = 30)
from azaserine-fed larvae, the aggregates
were nearly absent behind the
morphogenetic furrow (Fig. 8C, D) while
in the remaining discs immunostaining
was less than that in the eye discs (n = 29)
from larvae reared on regular diet (Fig.
6A, B). Western blotting for detection of
polyQ protein levels in heads of day 1 old
GMR-GAL4>httex1p Q93 flies further
confirmed that azaserine feeding reduced
the level of polyQ protein (Fig. 8E, F).
Azaserine feeding partially restores the
rhabdomere morphology and suppresses
the progressive loss of vision in GMR-
GAL4>UAS-httex1p Q93 expressing flies
− The external eye morphology and vision
of freshly eclosed GMR-GAL4>UAS-
httex1p Q93 flies is near normal. However,
these flies show a progressive age-
dependent degeneration, becoming almost
completely blind by 10 days (36-38). As
known from earlier studies (36-38), the
eye surface of GMR-GAL4>UAS-httex1p
Q93 flies did not show any appreciable
change with age in any of the feeding
regimes (not shown). However, as also
reported earlier (36, 38), the pseudopupil
images of rhabdomeres of 1 day old GMR-
GAL4>UAS-httex1p Q93 expressing flies
fed on normal diet showed severely
degenerated rhabdomeres so that unlike
the stereotyped pattern of rhabdomeres in
pseudopupil image of eyes of wild type
flies (Fig. 9A), no distinct rhabdomeres
were seen in their eyes (Fig. 8B).
Interestingly, GMR-GAL4>UAS-httex1p
Q93 flies reared on the azaserine
supplemented food displayed at least some
organized rhabdomere-like structures in
~60% flies (Fig. 8C).
Expression of UAS httex1p Q93 in
eye cells with GMR-GAL4 driver causes
progressive neuronal degeneration of the
photoreceptor neurons so that the flies lose
their vision as they age (36, 38). To
examine whether the azaserine mediated
restoration of rhabdomeric organization
improved the vision of flies, we tested the
functionality of vision in 5, 10 and 15 days
old flies (wild type and GMR-
GAL4>httex1p Q93) by the phototaxis
behavioral assay, which examines the
choice of flies to move between
illuminated and dark chambers. While
nearly all wild-type flies of different ages
moved to the illuminated chamber
(positive phototaxis), the GMR-
GAL4>httex1p Q93 flies reared on normal
food progressively lost their vision such
that the proportion of flies selecting the
lighted chamber declined with age (Fig.
9D). By day 10, these flies became nearly
blind since they moved randomly between
the dark and light chambers (Fig. 9D).
Significantly, a greater proportion of
GAL4>httex1p Q93 flies reared on
azaserine-supplemented food continued to
move to the illuminated chamber even on
day 15 (Fig. 9D). Thus azaserine feeding
partially restored the vision in
GAL4>httex1p Q93 flies so that the
proportion of flies selecting the
illuminated chamber was significantly
higher on each day of phototaxis assay
than in those grown on normal food (Fig.
9D).
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Decreasing O-linked GlcNAcylation increases basal autophagic flux
9
DISCUSSION
Dynamic modification of Ser/Thr residues
of proteins by O-linked N-
acetylglucosamine (O-GlcNAc) is an
important post-translational modification
for cellular signaling (1, 3, 39). More than
five hundred proteins, involved in diverse
cellular functions including the
transcription, translation, metabolism and
stress response, have been identified to
undergo this modification (1, 3, 39). It is
significant that about 270 of these proteins
are known in the brain tissue alone (40).
Therefore it is not surprising that aberrant
O-GlcNAcylation is associated with
various disorders, including the
neurodegenerative disorders (3-11).
A common pathological feature of
many neurodegenerative diseases
including Alzheimer’s, Parkinson’s and
Huntington’s diseases, is the
accumulation/aggregation of one or more
proteins in different regions of brain,
which is believed to underlie
neurodegeneration (12, 37, 41). These
proteotoxic aggregates are cleared by
coordinated action of cellular proteolysis
system (UPS and autophagy-lysosomal
pathways) and molecular chaperones (12,
30). Although the regulation of UPS by O-
GlcNAcylation is fairly understood, there
are contrasting reports about a protective
role of O-GlcNAcylation in
neurodegeneration. For example, it is
shown that while O-GlcNAcylation of
ubiquitin-activating enzyme E1 promotes
ubiquitination (42) and is thus expected to
enhance protein degradation, the same
modification in Rpt2 ATPase subunit of
the proteasome inhibits its ATPase activity
and suppresses proteasome function (43),
which would lead to the accumulation of
ubiquitinated proteins. Interestingly, it is
shown that elevated O-GlcNAcylation in
brain inhibits proteasome function and
promotes neuronal apoptosis (44). We find
that overexpression of either OGT or OGA
led to significant reduction in the
proteasome activity in our cellular model
and this corroborates well with a recent
report on proteasomal function in C.
elegans mutants for OGT or OGA (45).
However, we did not find any difference in
the proteasome activity when the cells
were treated with azaserine or glucosamine
for the duration and concentration used,
suggesting that the level and/or activity of
OGT and OGA, rather than flux through
the HBP alone, are more critical in
modulating the activity of proteasome. In
view of these observations, and existing
reports that accumulation of protein
aggregates blocks proteasome function
(46, 47), it appears that proteasome alone
might not be sufficient to clear these
aggregates. This notion is strengthened
with the emerging understanding of the
role of autophagy in degradation of such
aggregates in cell and animal models (12,
27, 48), identification of novel regulators
of autophagy that help in the clearance of
toxic protein aggregates is important.
Considering the established fact that O-
GlcNAcylation acts as nutrient sensor (3,
49), and the role of nutrients (serum amino
acid, glucose) in regulation of the
autophagic process (50, 51), we
hypothesized that changes in O-
GlcNAcylation level might modulate
autophagic process. Interestingly, we
found here that inhibition of O-
GlcNAcylation, either by overexpressing
OGA or by azaserine treatment, decreased
the polyQ aggregation by promoting their
clearance via autophagy.
In agreement with the results of our
in vitro cell culture model, our studies on
the in vivo fly model also revealed that
azaserine feeding resulted in the
improvement in Drosophila eyes
expressing mutant huntingtin at cellular,
phenotypic, as well as at functional level in
the form of reduced aggregation of mHtt,
improved rhabdomere organization and
improved vision, respectively. Since
glucosamine was highly toxic to larvae
even at a very low concentration, we could
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Decreasing O-linked GlcNAcylation increases basal autophagic flux
10
not examine the effects of elevated levels
of O-GlcNAcylation on polyQ toxicity in
the fly model. The decrease in
proteotoxicity on azaserine feeding might
involve either the inhibition of toxic
proteins synthesis or its enhanced
clearance through the degradation
machinery of cell. Several recent findings,
including our present in vitro findings,
suggest a greater role of enhanced
clearance of toxic proteins by azaserine.
Thus the reduced polyQ aggregate load
seen in azaserine fed GAL4>httex1p Q93
larval eye discs and adult heads is likely to
be due to their enhanced clearance, which
in turn results in partial restoration of eye
structure and function.
Azaserine has already been reported
to decrease the level of amyloid deposition
in pancreatic islets of mouse model of
diabetes (52) which indicates the
possibility of improvement in protein
clearance machinery and thereby reducing
the accumulation of amyloid deposits. Our
observations that inhibition O-
GlcNAcylation induces the clearance of
protein aggregates by enhancing the
autophagic process is in agreement with a
recent finding that cardiac O-
GlcNAcylation regulates autophagic
signaling in rat model of type II diabetes
(53). Interestingly, a recent report, which
appeared while this manuscript was in
preparation, by Wang et al. (54) in C.
elegans model of human
neurodegenerative diseases also indicates
that the suppression of O-GlcNAcylation
decreases neurodegeneration. Taken
together, our in vitro and in vivo findings
indicate that inhibition of O-
GlcNAcylation stimulates autophagy and
thereby reduces the load of proteotoxic
huntingtin aggregates and provides
protection from neurodegeneration.
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Acknowledgments
−
We thank Dr Lawrence Marsh (University of California, Irvine, USA) for
the mHtt-Q97-GFP expression construct, Dr T. Yoshimori (National Institute for Basic
Biology, Okazaki, Japan) for the mRFP-GFP-LC3 construct, Dr John A Hannover (National
Institutes of Health, USA) and Dr Gerald W. Hart (Johns Hopkins University School of
Medicine, USA) for the expression constructs coding for OGA and OGT, respectively. We
would also like to thank the anonymous reviewers for their suggestions and comments which
greatly helped in improving the manuscript.
FOOTNOTES
*This work was supported by research grant from the Department of Atomic Energy (Govt.
of India) to SG. AK received a post-doctoral fellowship from the Department of
Biotechnology (Govt. of India). SG is a Ramanna Fellow and Gill-Joy Chair Professor at IIT
Kanpur and SCL is Professor Emeritus and a DAE-Raja Ramanna Fellow at Banaras Hindu
University, Varanasi.
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Decreasing O-linked GlcNAcylation increases basal autophagic flux
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4Current address: Burke Medical Research Institute, New York, USA.
5Current address: Institute of Genetics and Molecular and Cellular Biology, Strasbourg,
France.
6To whom correspondence should be addressed: Department of Biological Sciences and
Bioengineering, Indian Institute of Technology, Kanpur, 208016, India; Tel: (91) 512-259-
4040; Fax (91) 512-159-4010; Email: sganesh@iitk.ac.in
The abbreviations used are: DAPI, 4’,6-diamidino-2-phenylindole dihydrochloride; mHtt,
polyglutamine expanded huntingtin exon 1 protein fragment; OGT, O-linked N-
acetylglycosyl transferase; OGA, O-linked N-acetylglucosaminidase; PTM, post-translational
modification; HBP, hexosamine biosynthetic pathway; GFP, green fluorescence protein;
BafA1, bafilomycin A1; polyQ, polyglutamine; SDS, sodium dodecyl sulfate.
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Figure 1: Suppression of O-GlcNAcylation significantly reduces mHtt-Q97 aggregates.
(A) Neuro2A cells transfected with an empty vector (pcDNA) or expression construct for
OGA-Myc and OGT-HA were evaluated for changes in global O-glycosylation level by
immunoblotting. Expression of OGA and OGT was confirmed by probing with the tag
antibodies. Probing with γ-tubulin served as loading control. The bar diagram shown above
represent the fold change in the signal intensity of O-glycosylated proteins (normalized to γ-
tubulin in the immunoblot) as measured by densitometric analysis (N=3; ***, p<0.001; *,
p<0.1). (B) Bar diagram representing percent transfected cells showing the aggregation of
mHtt-Q97-GFP when expressed alone (pcDNA) or with an expression construct coding for
OGA-Myc or OGT-HA, as indicated. Note the significant reduction in the transfected cells
positive for mHtt-Q97-GFP aggregates when OGA was co-expressed but a significant
increase in their frequency when OGT was co-expressed (N=3; ***, p<0.001). (C) Bar
diagram showing fold change in survival of cells transiently expressing OGA or OGT as
compared with cells transfected with an empty vector (pcDNA), as measured by MTT assay
(N=3; ***, p<0.001). (D) Representative fluorescence microscopic images (first four
columns with a 10X objective) showing aggregation patterns of mHtt-Q97-GFP in Neuro2A
cells when expressed alone (pcDNA), and when co-expressed with OGA-Myc or OGT-HA.
The intense green signals in “mHtt-Q97-GFP” column represent mHtt-Q97 aggregates. The
red signal reveals the expression of OGA-Myc or OGT-HA. Nuclei were stained with DAPI
(blue). Areas boxed in the “merged” column are enlarged in the last column to more clearly
show the GFP-positives cells with or without aggregates.
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Figure 2: O-GlcNAcylation inhibition reduces mHtt-Q97-mediated cytotoxicity (A)
Western blot images of insoluble, aggregated form of mHtt-Q97-GFP in filter-trap assay
using a slot-blot apparatus (top) or its total form resolved by immunoblotting (bottom) when
expressed with either pcDNA (empty vector control), OGT or OGA, as indicated in middle.
Expression of OGT and OGA was established by probing them with anti-HA and anti-Myc
antibodies, respectively levels. The bar diagram above shows the fold changes in signal
intensities, based on densitometric analysis, of SDS insoluble, aggregated form of mHtt-Q97-
GFP (normalized to total level detected in the immunoblot; N=3; ***, p<0.001; *, p<0.1).
(B, C) Bar diagram representing the fold change in the viability of cells expressing mHtt-
Q97-GFP (B) or the α-synuclein mutant A40P (C) as measured by an MTT assay. Cells
transfected with indicated constructs were processed for the measurement, and in each set the
value obtained for the GFP transfected cells was considered and as 1, and the relative values
obtained for indicated combinations were plotted. (D, E) Bar diagram showing the percentage
of cells expressing mHtt-Q97-GFP (D) or the α-synuclein mutant A40P (E) with abnormal
(apoptotic) nuclei (as shown in F) as compared with cells that expressed GFP (control) when
co-transfected with OGA or OGT coding constructs (in B-E, N=3; **, P<0.05; ***, P<0.005
on Student's t-test). (F) Representative images showing a normal (left) and an abnormal
(apoptotic; right) nuclei as judged by DPAI staining (scale, 5 µM).
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Decreasing O-linked GlcNAcylation increases basal autophagic flux
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Figure 3: O-GlcNAcylation modulates autophagy. (A) Immunoblots (bottom panel) of
Neuro2A cells, transiently expressing pcDNA empty vector alone or OGA-Myc or OGT-HA
for 36 hrs, to show levels of the autophagic markers, LC3II and p62. Probing with anti-γ-
tubulin served as loading control. Note the change in the level of LC3II band (identified by
an arrow) in cells that expressed OGA. Co-expression of OGT did not show such an effect.
Bar diagrams above show the fold changes in signal intensities of the LC3II and p62 (both
normalized to γ-tubulin signal) bands when compared with the control (pcDNA transfected
cells). (B) Neuro2A cells were grown in a medium with or without azaserine and or
glucosamine for 12 hrs as indicated, and the changes in the global glycosylation level were
evaluated. The bar diagrams above show the fold changes in the glycosylation levels
compared to cells that were fed with glucose. (C) Samples shown in B were tested for the
level of autophagy markers LC3 and p62 as indicated. Note the reduction in the intensity of
the band for LC3II (identified by an arrow) and p62 in the azaserine treated cells and their
restoration in the azaserine/glucosamine double treated cells. Bar diagrams above represent
the fold changes in the signal intensities for LC3II and p62 (both normalized to γ-tubulin
signal) bands compared to the control (glucose fed cells) (in A-C, N=3; *, P<0.5; **, P<0.05;
***, P<0.005 on Student's t-test).
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Figure 4: Suppression of O-GlcNAcylation increases autophagy flux. (A) Neuro2A cells
at 24 h post- transient transfection with an empty vector (pcDNA) or with a construct coding
for OGA or OGT were either left untreated or treated with BafA1 for 12 h as indicated and
the levels of autophagy markers LC3 and p62 were evaluated by immunoblotting. Note the
increase in the signal intensities of LC3II (arrow) and p62 in all samples treated with BafA1.
The blot was probed with anti-Myc and Anti-HA antibodies to show the expression of OGA
and OGT, respectively; probing with anti-γ-tubulin served as the loading a control. (B)
Immunoblot to show levels of LC3II (arrow) and p62 in Neuro2A cells, as in A, untreated or
treated with azaserine, alone or in combination with BafA1 as indicated; γ-ubulin served as
loading control. Bar diagrams above represent the fold changes in the signal intensities for
LC3II and p62 (both normalized to γ-tubulin signal) bands compared to the control (N=3; **,
P<0.05; ***, P<0.005 on Student's t-test).
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Figure 5: Suppression of O-GlcNAcylation increases autophagy flux. (A) Representative
images of cells showing LC3 positive puncta in cells that were transiently transfected with
mRFP–GFP–LC3 expression construct along with an empty vector (pcDNA) or an
expression construct coding for OGA or OGT as indicated. Puncta that are positive both for
red and green fluorescence represent autophagosomes while those positive only for red
represent autolysosomes (bar = 10 µM). (B) Bar diagram showing the fraction of puncta
positive for both RFP and GFP (yellow) or only the RFP (red) in transiently transfected cells
coexpressing mRFP–GFP–LC3 and pcDNA or OGA or OGT, as indicated. N=3; *, P<0.5;
**, P<0.05 on Student's t-test).
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Figure 6: Suppression of O-GlcNAcylation increases autophagy flux. Western blots
showing changes in the levels of insoluble, aggregated form of mHtt-Q97-GFP (filter-trap
assay; top) or its total form (immunoblotting; bottom) when expressed with OGA and treated
or not treated with BafA1 (A) or 3-MA (B) as indicated. The bar diagrams, shown above,
represent fold changes in the signal intensity of SDS insoluble, aggregated form of mHtt-
Q97-GFP (normalized to total level detected in the immunoblot) as measured by
densitometric analysis (N=3; ***, p<0.001; *, p<0.1).
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Figure 7: Effect of O-GlcNAcylation on proteasomal activity. Bar diagram showing fold
change in the proteasomal activity in cells transiently transfected with a construct coding for
OGA, OGT or an empty vector (pcDAN) (A) or with the drug azaserine or glucosamine in
the presence or absence proteasomal blocker MG132, as indicated (N=3; *, P<0.5; **,
P<0.05 on Student's t-test).
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Figure 8: Azaserine feeding reduces accumulation of mutant Huntingtin protein in fly
model. (A-D) Confocal projection images (projections of 4 consecutive optical sections
which show the morphogenetic furrow) of eye imaginal discs of late 3rd instar GMR-
GAL4>UAS-httex1p Q93 Drosophila larvae, reared from the 1st instar stage onwards on
normal (A, B) or azaserine supplemented food (C, D), immunostained for HA-tagged mutant
Htt (green, A-D, identified as “PolyQ”); nuclei are counterstained with DAPI (blue, B and D).
The insets in A and C are higher magnification images of a part of the eye discs in A and C,
respectively, to more clearly show the polyQ aggregates, which are very abundant in A but
nearly absent in C. Arrows in B and D indicate position of the morphogenetic furrow. Scale
bar in A represents 20 μm and applies to A to D. (E) Immunoblot of total proteins from heads
of one day old GMR-GAL4/UAS-htt-ex1p Q93 flies, reared on normal (Aza -) or azaserine
supplemented (Aza +) food since the 1st instar stage, probed with anti-HA antibody to detect
Htt-Q93 protein. (F) Histograms show mean relative levels of HA-tagged polyQ protein
(mean ratios of Htt-Q93 and γ-tubulin densities) determined from triplicate immunoblots as
in E; the mean ratio of HttQ93 and γ-tubulin densities in Aza- food was taken as 1. (*
p<0.001).
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Figure 9: Azaserine feeding suppresses mHttQ93-induced neurodegeneration in adult
Drosophila eyes and reduces the age-dependent loss of vision. (A-C) Pseudopupil images
of eyes of 1 day old wild type (A), or GMR-GAL4>UAS-httex1p Q93 flies grown on control
(B) or on azaserine-containing food (C). Arrow in C indicates presence of two distinct
rhabdomeres in one of the ommatidial units; these are not seen in any ommatidial unit in
control flies. Scale bar in A indicates 20 μm and applies to A – C. (D) Histograms showing
phototaxis (percent flies moving to illuminated chamber, Y-axis) of wild type and GMR-
GAL4>UAS-httex1p Q93 flies reared on control or azaserine supplemented food on different
days (X – axis) after emergence. Each value in bar diagram is mean of 20 replicates with 10
flies in each set. The * in bar diagrams indicates the p-value to be <0.05when comparing the
mean phototaxis of GMR-GAL4>UAS-httex1p Q93 flies reared on control and azaserine
supplemented food, respectively, on days 5, 10 and 15 .
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