Accelerating the clearance of mutant huntingtin protein aggregates
through autophagy induction by europium hydroxide nanorods
Peng-Fei Weia,1, Li Zhanga,c,1, Susheel Kumar Nethib, Ayan Kumar Baruib, Jun Lina,
Wei Zhoua, Yi Shena, Na Mana, Yun-Jiao Zhanga, Jing Xua, Chitta Ranjan Patrab,**,
aHefei National Laboratory for Physical Sciences at The Microscale, School of Life Sciences, University of Science and Technology of China,
230027 Hefei, PR China
bBiomaterials Group, CSIR e Indian Institute of Chemical Technology, 500007 Hyderabad, India
cDepartment of Urology, The First Affiliated Hospital of Anhui Medical University and Institute of Urology, Anhui Medical University, 230022 Hefei, PR China
a r t i c l e i n f o
Received 13 September 2013
Accepted 5 October 2013
Available online 26 October 2013
Europium hydroxide [EuIII(OH)3] nanorods
a b s t r a c t
Autophagy is one of the well-known pathways to accelerate the clearance of protein aggregates, which
contributes to the therapy of neurodegenerative diseases. Although there are numerous reports that
demonstrate the induction of autophagy with small molecules including rapamycin, trehalose and
lithium, however, there are few reports mentioning the clearance of aggregate-prone proteins through
autophagy induction by nanoparticles. In the present article, we have demonstrated that europium
hydroxide [EuIII(OH)3] nanorods can reduce huntingtin protein aggregation (EGFP-tagged huntingtin
protein with 74 polyQ repeats), responsible for neurodegenerative diseases. Again, we have found that
these nanorods induce authentic autophagy flux in different cell lines (Neuro 2a, PC12 and HeLa cells)
through the expression of higher levels of characteristic autophagy marker protein LC3-II and degra-
dation of selective autophagy substrate/cargo receptor p62/SQSTM1. Furthermore, depression of protein
aggregation clearance through the autophagy blockade has also been observed by using specific in-
hibitors (wortmannin and chloroquine), indicating that autophagy is involved in the degradation of
huntingtin protein aggregation. Since [EuIII(OH)3] nanorods can enhance the degradation of huntingtin
protein aggregation via autophagy induction, we strongly believe that these nanorods would be useful
for the development of therapeutic treatment strategies for various neurodegenerative diseases in near
future using nanomedicine approach.
? 2013 Elsevier Ltd. All rights reserved.
Recent advances in the neuroscience research demonstrate that
neurodegenerative diseases like Huntington’s disease, Parkinson’s
disease and Alzheimer’s disease, are derived from intracellular
accumulation of misfolded and altered proteins . Though these
diseases vary in their origin and evolution, they have a common
feature of deposition of protein aggregates like huntingtin, a-syn-
uclein, tau etc. [2,3]. These misfolded aggregated proteins usually
promote neuronal death when processed to the form of toxic
multimeric complexes . The ubiquitineproteasome system (UPS)
and autophagyelysosomal pathway are the two main mechanistic
pathways responsible for eukaryotic intracellular proteolysis and
protein modifications . The short-lived nuclear and cytosolic
proteins are selectively removed by ubiquitineproteasome system
(UPS), while misfolded proteins, protein aggregates, bulk intracel-
lular organelles and long half-lived cytosolic proteins are degraded
by virtue of highly regulated autophagyelysosomal pathway .
Pathogenic misfolded proteins and protein aggregates are usually
excluded from UPS and escape proteasome-mediated cellular
quality control due to their sizes [6,7]. Protein aggregates subse-
quently result in the toxic disruption of normal cellular processes
. To counterattack this, autophagy is one such protective mech-
anismwhich helps in clearance of these toxic aggregated misfolded
proteins from the cells and maintaining homeostasis .
Autophagy, also termed as cellular self-digestion, is a lysosome-
dependent, evolutionarily conserved and dynamic degradation
process in eukaryotic organisms for clearance of misfolded
proteins anddamagedorganelles, which isassociated with survival,
* Corresponding author. Tel./fax: þ86 551 63600246.
** Corresponding author. Tel.: þ91 402 7191480; fax: þ91 402 7160387.
firstname.lastname@example.org (L.-P. Wen).
1These authors equally contributed to the work.
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/biomaterials
0142-9612/$ e see front matter ? 2013 Elsevier Ltd. All rights reserved.
Biomaterials 35 (2014) 899e907
differentiation, development and homeostasis [10e14]. It helps in
safeguarding the human system against various invading microbial
pathogens and strengthening the innate defense system . Also a
few essential autophagy-related genes are reported to help in the
longevity phenotype and life span extension and thus prevent age-
related diseases . It also plays a vital role in the protection
against cardiovascular diseases and mainly against cancer [17,18].
Earlier reports have shown the degradation of some long-lived
aggregate-prone proteins, including mutant huntingtin , A53T
related clearance of mutant huntingtin and some other misfolded
proteins has also been validated in transgenic Drosophila and
mouse models of neurodegenerative diseases [21,22]. Some small
molecular agents such as rapamycin, trehalose, lithium etc. are
helpful for the treatment purposes of neurodegenerative diseases
by upregulating the autophagy flux. Autophagy inducers e rapa-
mycin and its analogs can reduce the amount of misfolded proteins
associated with neurodegenerative disorders by inhibiting the
mammalian target of rapamycin (mTOR) [22,23]. Trehalose, an
mTOR-independent autophagy inducer, enhances the degradation
molecules, such as lithium , N10-substituted phenoxazine ,
rilmenidine , methylene blue  etc. can also induce auto-
of neurodegenerative diseases. But these chemical autophagy in-
ducers have their own limitations. For example, rapamycin, a
chemical autophagy inducer, is reported to effectively target the
Alzheimer’s disease only in the early stage of disease progression
but not after the formation of plaques and tangles . An imida-
zoline receptor 1 agonist called rilmenidine is reported to enhance
the mutant huntingtin clearance in a mouse model of Huntington’s
disease . As rilmenidine belongs to the class of centrally-acting
side effects. Hence, identification of autophagy inducers is urgently
needed to combat against the neurodegenerative diseases.
In this context, nanotechnology has been introduced to play a
pivotal role in the therapy of these neurodegenerative diseases.
Therefore, researchers including our group have continuously
revealed the nanoparticles including rare earth oxides [29e32],
quantum dots , fullerene and its derivatives [34,35], CNTs ,
MnO nanocrystals , iron core-gold shell nanoparticles , iron
oxide nanoparticles , graphene oxide  and graphene
quantum dots  as autophagy inducers. However, there is no
systematic and detailed study for both the induction of autophagy
and acceleration of the clearance of aggregated and misfolded
proteins such as huntingtin protein by nanoparticles. In this
circumstance, we have hypothesized that the non-toxic and espe-
cially pro-angiogenic europium hydroxide nanorods established by
our group [42e45] may facilitate the clearance of pathogenic and
misfolded proteins by inducing autophagic responses. Therefore, in
this present study, we have synthesized europium hydroxide
autophagy-inducing activities and mechanisms involved. Subse-
quently, we attempted to introduce the neuronal cell lines stably
expressing EGFP-tagged mutant huntingtin protein aggregates
with 74 polyQ repeats as the test models for evaluating the clear-
ance ability of EuIII(OH)3nanorods. Finally, we intended to explore
the association between the clearance of mutant huntingtin with
the autophagy induction by EuIII(OH)3nanorods.
2. Materials and methods
Europium nitrate hydrate [Eu(NO3)3$H2O] and aqueous ammonium hydroxide
[aq.NH4OH, 28e30%] were purchased from Aldrich, USA and used without any
further purification for the synthesis of EuIII(OH)3nanorods. Microtubule-associated
light chain 3 (LC3) plasmid was received from generous N. Mizushima (The Tokyo
Metropolitan Institute of Medical Science, Tokyo, Japan). Trehalose (Tre, T9531),
Hoechst 33342 (B2261), monodansylcadaverine (MDC, D4008), propidium iodide
(PI, P4864) and chloroquine (C6628) were purchased from SigmaeAldrich (St. Louis,
TB0799) was purchased from Sangon Biotechnology (PR China). Wortmannin
(s1952) was purchased from Beyotime Institute of Biotechnology (PR China) and cell
culture reagents unless otherwise noted and Lyso Tracker Red (L7528) were all
purchased from Invitrogen (Carlsbad, CA). LC3 antibodies (NB100-2220) were pur-
chased from Novus (Littleton, CO). GFP (sc-101536) and HRP-conjugated anti-mouse
antibodies were purchased from Santa Cruz Biotechnology and glyceraldehyde
phosphate dehydrogenase (GAPDH) antibodies (MAB374) from Millipore. HRP-
conjugated anti-rabbit antibodies (W4011) were purchased from Promega (Wis-
consin, USA). Enhanced chemiluminescence (ECL) kits were purchased from Bio-
logical Industries (Kibbutz Beit Haemek, Israel). Geneticin/G418 sulfate (Gibco,
11811-031) is dissolved in sterile water to prepare a 100 mg/mL stock and stored
2.2. Preparation and characterization of EuIII(OH)3nanorods
EuIII(OH)3nanorods were synthesized by using hydrothermal method through
the interaction of aqueous europium(III) nitrate solution and aqueous NH4OH at
atmospheric pressure in an open reflux system. In a typical synthesis, 1 mL of
aqueous NH4OH was added to 39 mL of a 0.05 (M) aqueous solution of europium(III)
nitrate at a molar ratio of [OH/Eu] ¼ 4 in a 100 mL round bottomed flask. A colloidal
material, without any special morphology, was obtained upon the addition of
NH4OH to the Eu(III) nitrate solution. The colloidal mixture was heated at 150?C for
60 min to obtain the as-synthesized europium hydroxide nanorods. After the
completion of the reaction, the resulting products were collected, centrifuged at
6000 rpm, washed 3 times with millipore water followed by ethanol and millipore
water again and then dried overnight in a hot air oven.
The structure and phase purity of the as-synthesized europium hydroxide
nanorods sample were determined by X-ray diffraction (XRD) analysis using a
Bruker AXS D8 Advance Powder X-ray diffractometer (using CuKa l ¼ 1.5406 Å
radiation). The morphology and shape of nanomaterials were examined on an FEI
Tecnai F12 (Philips Electron Optics, Holland) instrument operated at 100 kV.
Selected area electron diffraction (SAED) patterns were also taken using this in-
strument. Fourier transformed infrared (FTIR) spectral analysis is an important
requisite tool for the identification of functional groups present in a chemical
compound on the basis of their vibrational energy. The FTIR spectrum of the euro-
pium hydroxide nanorods sample was recorded using thermo Nicolet Nexus 670
spectrometer in the diffuse reflectance mode at a resolution of 4 cm?1in KBr pellets.
2.3. Cell culture
HeLa and GFPeLC3/HeLa cell lines have been described previously , PC12
and Neuro 2a cells stably expressing EGFP-Htt(Q74) were constructed previously
and rescreened using G418. All cells have grown continuously as a monolayer at
37?C and 5% CO2in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented
with 10% FBS.
2.4. Observation of GFPeLC3 dots and GFP-tagged aggregates
GFPeLC3 dot formation in GFPeLC3/HeLa cells and GFP-tagged huntingtin ag-
gregation in Neuro 2a and PC12 cells were observed under fluorescence microscopy
(Olympus IX71). Pictures were captured randomly. The incubation times and con-
centrations of EuIII(OH)3nanorods are stated in figure legends and/or figures.
2.5. Autophagic marker dye staining
HeLa-LC3 cells, after europium hydroxide nanorods treatment, were stained for
10 min with 10 mM monodansylcadaverine (MDC) or 75 nM Lyso Tracker Red. After
washing twice with PBS, cells were observed under fluorescence microscopy
(Olympus IX71). The incubation times and concentrations of EuIII(OH)3nanorods are
stated in figure legends and/or figures.
2.6. Cell death assay
Cells were firstly washed with PBS twice, and total cell nuclei were stained with
10 mg/mL Hoechst 33342 for 5 min. And then, dead cells were stained with 10 mg/mL
propidium iodide (PI) for 5 min. Images were obtained using fluorescence micro-
scopy (Olympus IX71). The incubation times and concentrations of EuIII(OH)3
nanorods are stated in figure legends and/or figures.
2.7. Cell viability assay
Neuro 2A or PC12 cells were seeded in 96-well plates at a density of w6000 cells
every well. After incubation for 20 h at 37?C and 5% CO2in Dulbecco’s Modified
Eagle’s Medium (DMEM) supplemented with 10% FBS, the cells were treated with
EuIII(OH)3nanorods and TE buffer for 48 h or 72 h at indicated concentrations. Then
10 mL MTT (5 mg/mL) was added to each well followed by incubation for 4 h at 37?C.
P.-F. Wei et al. / Biomaterials 35 (2014) 899e907
Then the media was removed out and formazan crystals were completely dissolved
into 100 mL of dimethyl sulfoxide (DMSO). Finally the absorbance of the plate was
measured at 570 nm using microplate reader (ELx 808, Bio-Tek, USA). All the
experimental absorbance values of the treatments were recorded in triplicates and
the untreated cells were regarded as the controls.
2.8. Western blot analysis
Harvested cells were resuspended by the lysis buffer (0.5% Nonidet? P-40/
10 mM TriseHCl, pH 7.5/100 mM NaCl) on ice. One-quarter volumeof 5? SDS sample-
loading buffer (100 mM TriseHCl, pH 6.8, 2% b-mercaptoethanol, 4% SDS, 20% glyc-
erol, and 0.02% bromphenol blue) was added, followed by boiling for 10e15 min.
Proteins were separated on an SDS/PAGE gel and transferred to PVDF membrane
(Millipore) or nitrocellulose transfer membrane (GE Healthcare). Here, insoluble
protein aggregation would be detained in the sample-loading well, while soluble
protein aggregationwould be separated by separation gels. After blockage with Tris-
buffered saline containing 0.1% Tween-20 and 5% nonfat dry milk, the membrane
was incubated overnight at 4?C or for 2 h at the room temperature with a primary
antibody at an appropriate dilution (1:2000e1:1000 dilution), then washed four
times for 8 min each with TBST (TBS containing 0.1% Tween-20), incubated with
horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution) for 1 h
at RT, extensively washed, and finally visualized with enhanced chemiluminescence
(ECL) kit. The incubation times and concentrations of agents are stated in figure
legends and/or figures.
2.9. Bio-electron microscopy for the observation of autophagosome
HeLa cells were grown in the 24-well plates and either untreated or treated with
[EuIII(OH)3] nanorods for 24 h. After harvesting, cell pellet was fixed in 0.1 M Nae
phosphate buffer (pH 7.4) containing 2% glutaraldehyde for 1 h. After post-fixing in
1% OsO4at room temperature for 60 min, cells were dehydrated with a graded series
of ethanol and embedded in epoxy resin. Areas containing cells were block mounted
and cut into ultrathin sections. The sections were stained with uranyl acetate and
lead citrate and examined under a transmission electron microscope (JEOL-1230,
2.10. Statistical analysis
All datawere calculated and exhibited as mean ? s.e.m. *p < 0.05, **p < 0.01 and
***p < 0.001 were considered statistically significant. Statistical comparisons of
densitometry results were analyzed by two-tailed student’s t-tests.
3. Results and discussion
3.1. Preparation and characterization of EuIII(OH)3nanorods
The crystallinity, morphology and functional group analyses of
the materials have been investigated by several physico-chemical
techniques and the results are presented in Fig. 1aed. The XRD
pattern of the as-synthesized europium hydroxide nanorods
(Fig.1a) indicates crystalline nature of the materials. All reflections
can be distinctly indexed to a pure hexagonal phase of EuIII(OH)3
nanorods. The diffraction peaks of nanorods are consistent with the
standard data files (the JCPDS card No. 01-083-2305) for all re-
flections . The TEM image (Fig.1b) clearly indicates that the as-
synthesized materials entirely consist of nanorods with an
approximate length 80e160 nm and a diameter 25e40 nm. The
corresponding selected area electron diffraction (SAED) pattern
(Fig. 1c) indicates the crystallinity of the as-synthesized materials,
which corroborates with the XRD pattern. The as-synthesized
nanomaterials have further been confirmed by FTIR spectroscopy
Fig.1. Characterization of EuIII(OH)3nanorods using different physico-chemical techniques. (a) XRD pattern of EuIII(OH)3nanorods after hydrothermal heating at 150?C for 60 min.
(b) TEM image of as-synthesized EuIII(OH)3nanoparticle clearly showing its rod shaped structure. (c) SAED pattern of as-synthesized nanorods indicating its crystalline nature. (d)
FTIR spectrum of the as-synthesized EuIII(OH)3nanorods.
P.-F. Wei et al. / Biomaterials 35 (2014) 899e907
(Fig. 1d) that represents the typical FTIR spectra of europium hy-
droxide nanorods. The appearance of characteristic peaks  at
w3609 cm?1and w700 cm?1attributes that the as-synthesized
nanomaterial is europium hydroxide nanorods.
3.2. Enhanced clearance of GFP-Htt(Q74) by EuIII(OH)3nanorods
These as-synthesized and thoroughly characterized nanorods
have been used for the autophagy induction and clearance of hun-
tingtin proteins study. In order to investigate the clearance of this
protein, we have incubated Neuro 2a cells stably expressing GFP-
Htt(Q74)with nanorods and
inducer) and analyzed the expression of the GFP-Htt(Q74) using
fluorescence microscopy and western blot techniques. Here, insol-
uble protein aggregation would be detained in the sample-loading
well, while soluble protein aggregation would be separated by sep-
aration gels. Interestingly, the expression of green (in web version)
treated with EuIII(OH)3nanorods compared to vehicle control [Trise
EDTA (TE) buffer] treatment indicating that the nanorods effectively
augment the clearance of huntingtin proteins (Fig. 2a).
Additionally, these nanorods significantly reduce the level of
insoluble and soluble huntingtin protein aggregation in Neuro 2a
cells observed by the Western blot analysis (Fig. 2b). The corre-
sponding densitometry analyses have been shown in Fig. 2ced.
These figures clearly show the clearance of huntingtin protein ag-
gregation in nanorods treated Neuro 2a cells compared to vehicle
control experiment. Time-dependent and dose-dependent re-
movals of protein aggregation from this cell line using nanorods are
also shown in Fig. S1aeb. Furthermore, these nanorods did not
affect the Neuro 2a cell viability which is clearly revealed by the
MTT assay (Fig. 2e).
To gain more concrete data, similar experiments with prolonged
treatment duration have been carried out to assess the clearance of
the aggregated huntingtin protein in PC12 cells stably expressing
GFP-Htt(Q74). It has been observed that after 72 h treatment of
PC12 cells with nanorods, the expression of GFP-Htt(Q74) has been
decreased compared to vehicle-treated cells (Fig. 3a). Western
blotting results have also confirmed that these nanorods reduced
both soluble and insoluble huntingtin protein aggregation (Fig. 3b).
The cell viability measured by using MTTassay did not significantly
alter when treated with EuIII(OH)3nanorods compared with vehicle
control group in this PC12 cell line (Fig. 3c). Notably, fluorescence
microscopy study reveals that after the treatment with these
nanorods for 72 h, a very few and almost equal amount of dead cells
(propidium iodide positive) can be found in EuIII(OH)3nanorods and
vehicle-treated groups, which corroborates with the MTT results
3.3. Autophagy induction by EuIII(OH)3nanorods
Atg8/LC3, one of the autophagy-related proteins, is the most
widely used autophagic marker protein for the evaluation of
autophagy intensity . LC3 is constitutively expressed in
mammalian cells, which is immediately processed to be cytosolic
LC3-I. During induction of autophagy, membrane-binding LC3-II is
generated via fusion between soluble LC3-I and phosphatidyleth-
anolamine (PE), which subsequently connects to the outer and in-
ner membranes of autophagosome . Hence, conversion from
LC3-I to LC3-II in different cell lines such as Neuro 2a, PC12 and
HeLa treated with nanorods has been analyzed by western blot
analysis (Fig. 4a, Fig. S3a and b). As shown in Fig. 4a, Fig. S3a and b,
these nanorods trigger the conversion from LC3-I to LC3-II and
increase the relative LC3-II/GAPDH ratio in differentcell lines, while
TE buffer alone does not show any significant increase of LC3-II
expression. The dose-dependent conversion of LC3 in Neuro 2a
cells has also been illustrated in Fig. S1b. Trehalose, a natural
inducer of autophagy, served as a positive control here. Moreover,
Fig. 2. Fluorescence microscopy study, Western blot analysis and cell viability in GFP-Htt(Q74) expressing Neuro 2a cells treated with 50 mg/mL EuIII(OH)3nanorods for 48 h.
Fluorescence microscopy study (a) and Western blot analysis (b) clearly show that EuIII(OH)3nanorods enhance the clearance of soluble and insoluble GFP-Htt(Q74). Here, 100 mM
trehalose was used as a positive control experiment. Densitometry analyses of insoluble (c) and soluble (d) GFP-Htt(Q74) relative to GAPDH in Neuro 2a cells were performed with at
least three independent experiments. (e) Cell viability was analyzed in Neuro 2a cells by MTT assay after the cells were incubated with EuIII(OH)3nanorods (50 mg/mL) for 48 h.
(Mean ? s.e.m., n ? 3,*p < 0.05, **p < 0.01, compared to control of each group; TE, vehicle control; Eu, EuIII(OH)3nanorods; Tre, trehalose, a positive control).
P.-F. Wei et al. / Biomaterials 35 (2014) 899e907
Fig. 3. Fluorescence microscopy study, Western blot analysis and cell viability in GFP-Htt(Q74) expressing PC12 cells treated with 50 mg/mL EuIII(OH)3nanorods for 72 h. Fluo-
rescence microscopy study (a) and western blot analysis (b) clearly show that EuIII(OH)3nanorods enhance the clearance of soluble and insoluble GFP-Htt(Q74). Densitometry
analysis (b) of soluble and insoluble GFP-Htt(Q74) relative to GAPDH in PC12 cells was also performed with three independent experiments. (c) Cell viability was analyzed in PC12
cells by MTT assay after incubation with 50 mg/mL EuIII(OH)3nanorods. (Mean ? s.e.m., n ¼ 3,*p < 0.05, **p < 0.01, compared to control of each group; TE, vehicle control; Eu,
Fig. 4. LC3 conversion and puncta formation induced by europium hydroxide nanorods. (a) The conversion of LC3-I to LC3-II in PC12, Neuro 2a and HeLa cells was analyzed by
Western blot technique when treated with EuIII(OH)3nanorods (50 mg/mL) and trehalose (100 mM) for 24 h. (b) GFPeLC3 puncta formation in GFPeLC3 expressing HeLa cells was
randomly captured under fluorescence microscopy, the puncta were indicated with the red arrows. (c) Bio-TEM images of GFPeLC3 HeLa cells treated with vehicle control and
EuIII(OH)3nanorods for 24 h, the autophagosomes were indicated with the red arrows. (d) Fluorescent microscopy images of HeLa-LC3 cells stained with 10 mM mono-
dansylcadaverine (MDC) or 75 nM LysoTracker Red when treated with EuIII(OH)3nanorods (50 mg/mL) for 24 h. (TE, vehicle control; Eu, EuIII(OH)3nanorods; Tre, trehalose, a positive
control). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).
P.-F. Wei et al. / Biomaterials 35 (2014) 899e907
the europium hydroxide nanorods elicited a dose-dependent
autophagic induction in PC12 cells and Neuro 2a cells through the
conversion of LC3 (Fig. S3c and Fig. S1b respectively). To obtain
more convincing evidence, we have used GFPeLC3/HeLa , a
human epithelial carcinoma cell line (HeLa) stably expressing
exogenous fusion protein (GFPeLC3) [green fluorescent protein
(GFP) and microtubule-associated light chain 3 (LC3) protein] to
assess the autophagy-inducing ability of europium hydroxide
nanorods. It has been observed that along with positive control
trehalose, these nanorods can up-regulate the formation of GFP-
puncta (green (in web version) fluorescence dot) in GFPeLC3/
HeLa whereas no such puncta formation has found in vehicle TE
buffer treated cells (Fig. 4b). Fig. S3d also reveals that there was no
such puncta formation in untreated or TE-treated control cells.
Electron microscopy is a useful tool for observing the formation of
autophagosome to analyze autophagy . Therefore, the forma-
tion of double-membrane autophagosomes in the cytoplasm of
HeLa cells (as shown by the TEM images) has illustrated that
autophagy was induced by EuIII(OH)3nanorods (Fig. 4c). To lend
more concrete proof that EuIII(OH)3 nanorods induced genuine
autophagy, we have presented several additional lines of evidence.
As we all know, autophagy was characterized by autophagosomes.
Consistent with their presumed autophagosome/autolysosome
identity, the majority of GFPeLC3 dots observed after EuIII(OH)3
nanorods treatment were stained by monodansylcadaverine
(MDC), a dye that stains acidic vesicles (Fig. 4d). Besides, extensive
co-localizations were observed between the GFPeLC3 dots and
Lyso-Tracker Red (LT), a selective dye of the lysosome, which
strongly suggested that these GFPeLC3 dots were probably auto-
lysosomes (Fig. 4d). Besides, these results also hinted that the
autophagic flux induced by EuIII(OH)3nanorods was complete .
3.4. Enhanced autophagic flux by EuIII(OH)3nanorods
None of the above-mentioned methods adequately evaluate the
autophagic flux that involves the complete flux from autophago-
somes formation to their fusion with the lysosomes. Autophago-
some accumulation might also result from the impairment of
autophagosomeelysosome fusion, leading to a fake appearance of
autophagy. For instance, gold nanoparticles have been confirmed to
elicit autophagosome accumulation through lysosome alkaliniza-
tion . Besides this, both autophagosome formation and impair-
ment of autophagosomeelysosome fusion have been found after
calcium phosphate treatment . Therefore, it is essential to
evaluate the autophagic flux induced by EuIII(OH)3nanorods and
check whether this flux outcomes through the autophagosome
formation without the blockage of autophagosomeelysosome
fusion. Chloroquine (CQ), a classical lysosomotropic compound, is
one of the widely-used agents to measure autophagy flux via
neutralizing the lysosomal pH . Earlier report has been
demonstrated that after the treatment of cells with CQ at the
impaired . In this study, it is observed that in the absence or
presence of CQ, europium hydroxide nanorods at 50 mg/mL can
induce more accumulation of LC3-II compared to vehicle control in
PC12 cells, Neuro 2a cells and HeLa cells, indicating that these
formation, rather than the blockage of autophagosomeelysosome
fusion (Fig. 5aec).
To further verify the induction of autophagy flux by EuIII(OH)3
nanorods, we have examined the expression levels of another
autophagy specific marker protein p62/SQSTM1 in different cell
lines. p62/SQSTM1 is a multidomain protein which participates in
The p62 protein is generally accumulated when autophagy is
inhibited and their levels are observed to be decreased during
autophagy induction [31,55,56]. p62 is one of the proteins which
mediates autophagosomal removal of p62-associated huntingtin
protein aggregates and it mechanistically functions by guiding the
the blockade of the autophagyelysosome pathway results in the
accumulation of p62 which eventually leads to various neurode-
generative diseases by activating the cellular stress responses .
In the present work, it is clearly shown that EuIII(OH)3nanorods
significantly reduce the amount of p62 protein in different cell lines
such as HeLa, Neuro 2a and PC12 (Fig. 5cee), which suggests the
genuine induction of autophagy by these nanorods. In addition, the
p62 degradation in HeLa cells has been found to be retarded by
using autophagy inhibitors like CQ (Fig. 5c). This supporting evi-
dence obtained by utilizing these autophagy inhibitors, firmly
confirmed that EuIII(OH)3nanorods induced complete autophagy
flux. We can therefore infer that the removal of huntingtin protein
aggregates may be dependent on p62-mediated specific autophagy
3.5. The role of autophagy in EuIII(OH)3nanorods-mediated
clearance of GFP-Htt(Q74)
It is well known that huntingtin protein aggregates are mainly
removed by non-selective macroautophagy . A few reports
state that autophagy induction by trehalose also plays a vital role in
enhancing the clearance of the mutant protein because reduction of
mutant huntingtin was abrogated after autophagy inhibition
[4,23,24]. After studying the autophagic flux and protein aggrega-
tion degradation effects by EuIII(OH)3nanorods, it was required to
check whether the nanorods-induced clearance of protein aggre-
gation was mediated by autophagy. Wortmannin (WM) is an
established autophagic inhibitor, which suppresses the initiation of
autophagy via selectively inhibiting phosphatidylinositol-3-kinase
[60,61]. Wortmannin has also been reported to enhance the ag-
gregation of mutant huntingtin protein inclusions by the inhibition
of autophagy . In the present study, to analyze the role of
autophagy in the clearance of huntingtin protein, we have checked
the expression of soluble GFP-Htt(Q74) in the presence or absence
of wortmannin by western blot analysis in Neuro 2a cell line. The
results are represented in the Fig. 6awhich shows that wortmannin
is able tosimultaneously inhibit autophagy induction and clearance
of protein aggregation elicited by EuIII(OH)3 nanorods. Further-
more, we have applied another autophagy inhibitor CQ which can
suppress the autophagosomeelysosome fusion to gain a more
concrete proof of our view points. However, it is well known that
CQ has obvious cytotoxicityespecially at high concentration and for
prolonged treatment time. In addition, cells stably expressing
protein aggregation were more sensitive to extracellular stress.
Thus, we have performed both western blot and fluorescence mi-
croscopy analysis using 10 mM concentration of CQ which actually
inhibited the clearance of protein aggregation mediated by the
EuIII(OH)3nanorods, as clearly illustrated by the Fig. 6bec. These
results obtained from the EuIII(OH)3nanorods treatment in pres-
ence of specific autophagy inhibitors such as wortmannin and CQ
confirm that the clearance of huntingtin protein in the Neuro 2a
cells is mediated through autophagy induction by the nanorods.
In this study, we have demonstrated that europium hydroxide
EuIII(OH)3nanorods synthesized by hydrothermal method initiate
authentic and cell type-independent autophagy via inducing the
autophagosomes formation and reduce the protein levels of the
P.-F. Wei et al. / Biomaterials 35 (2014) 899e907
autophagy specific substrate p62. In addition to autophagy induc-
tion, these nanorods enhance the clearance of GFP-tagged hun-
tingtin protein without affecting the cell viability and without
causing any notable cell toxicity, as observed from the MTT assay
and Hoechst 33342/PI double-staining assay, respectively. More-
over, autophagy inhibition obliterated the clearance of GFP-Htt
(Q74) protein, indicating that autophagy is primarily involved in
the EuIII(OH)3nanorods-mediated clearance of the protein aggre-
gation (Fig. 7). In summary, we have reported that europium hy-
droxide nanorods can be regarded as a kind of autophagy inducers
to accelerate the degradation of protein aggregation, which may
boost the applications of these nanomaterials in the therapy of
many neurodegenerative diseases.
The authors acknowledge financial support from the National
Basic Research Program of China (2013CB933900), the National
Natural Science Foundation of China (Grants 30721002, 31071211,
30830036, 31170966, 31101020, 81170698), the Innovation Pro-
gram of the Chinese Academy of Sciences (Grant KSCX2-YW-R-
139), the Fundamental Research Funds for the Central Universities
Fig. 5. EuIII(OH)3nanorods induced complete autophagy. (a) LC3 conversion and densitometry analyses were carried out in stably-expressing GFP-Htt(Q74) Neuro 2a cells treated
with EuIII(OH)3nanorods with or without CQ for 24 h. (b) Determination of LC3 conversion was carried out by Western blot and densitometry analyses in stably-expressing GFP-
Htt(Q74) PC12 cells treated with EuIII(OH)3nanorods with or without CQ for 24 h. (c) Determination of LC3 conversion was carried out by Western blot and densitometry analyses in
HeLa cells treated with EuIII(OH)3nanorods with or without CQ for 24 h, and p62 protein level was also measured in those HeLa cells. (d) p62 protein level determination in stably-
expressing GFP-Htt(Q74) Neuro 2a cells treated with EuIII(OH)3nanorods by Western blot and densitometry analyses. (e) p62 protein level determination in stable-expressing GFP-
Htt(Q74) PC12 cells treated with EuIII(OH)3nanorods and trehalose (100 mM, Tre) by Western blot and densitometry analyses. (Mean ? s.e.m., n ¼ 3,*p < 0.05, **p < 0.01,
***p < 0.001, NS, non-significant, compared to control of each group; TE, vehicle control; Eu, EuIII(OH)3nanorods; Tre, trehalose, a positive control).
P.-F. Wei et al. / Biomaterials 35 (2014) 899e907
(Grant WK2070000008) and the Clinical Key Subjects Program of
the Ministry of Public Health. CRP acknowledges DST, Government
of India, New Delhi for the award of Ramanujan Fellowship &
financial support (SR/S2/RJN-04/2010; GAP0305) and CSIR, New
Delhi for ‘CSIReMayo Clinic Collaboration for Innovation and
Translational Research’ (CMPP 09; MLP0020). SKN and AKB are
thankful to DST and UGC, New Delhi, respectively for their
research fellowships. We also thank Dr. Noboru Mizushima (Tokyo
Medical and Dental University, Japan) and Dr. Tamotsu Yoshimori
(Osaka University, Japan) for providing the LC3 plasmid. Heartfelt
thanks to Fang Zheng, Jiqian Zhang, Shuai Zhao, Yang Lu, Liang
Dong and An Xu at the University of Science and Technology of
China, and Dr. Wen Hu at Anhui Provincial Hospital for their
technical support in the work.
Appendix A. Supplementary data
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