Biomaterials 35 (2014) 899e907

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Accelerating the clearance of mutant huntingtin protein aggregates through autophagy induction by europium hydroxide nanorods Peng-Fei Wei a,1, Li Zhang a, c,1, Susheel Kumar Nethi b, Ayan Kumar Barui b, Jun Lin a, Wei Zhou a, Yi Shen a, Na Man a, Yun-Jiao Zhang a, Jing Xu a, Chitta Ranjan Patra b, **, Long-Ping Wen a, * a Hefei National Laboratory for Physical Sciences at The Microscale, School of Life Sciences, University of Science and Technology of China, 230027 Hefei, PR China b Biomaterials Group, CSIR e Indian Institute of Chemical Technology, 500007 Hyderabad, India c Department 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

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Article history: Received 13 September 2013 Accepted 5 October 2013 Available online 26 October 2013

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 degradation 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 inhibitors (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. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Europium hydroxide [EuIII(OH)3] nanorods Autophagy Huntingtin aggregation Chloroquine (CQ) Nanomedicine

1. Introduction 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 [1]. Though these diseases vary in their origin and evolution, they have a common feature of deposition of protein aggregates like huntingtin, a-synuclein, tau etc. [2,3]. These misfolded aggregated proteins usually promote neuronal death when processed to the form of toxic multimeric complexes [4]. The ubiquitineproteasome system (UPS) and autophagyelysosomal pathway are the two main mechanistic * Corresponding author. Tel./fax: þ86 551 63600246. ** Corresponding author. Tel.: þ91 402 7191480; fax: þ91 402 7160387. E-mail addresses: [email protected], [email protected] (C.R. Patra), [email protected] (L.-P. Wen). 1 These authors equally contributed to the work. 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.10.024

pathways responsible for eukaryotic intracellular proteolysis and protein modifications [5]. The short-lived nuclear and cytosolic proteins are selectively removed by ubiquitineproteasome system (UPS), while misfolded proteins, protein aggregates, bulk intracellular organelles and long half-lived cytosolic proteins are degraded by virtue of highly regulated autophagyelysosomal pathway [6]. 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 subsequently result in the toxic disruption of normal cellular processes [8]. To counterattack this, autophagy is one such protective mechanism which helps in clearance of these toxic aggregated misfolded proteins from the cells and maintaining homeostasis [9]. Autophagy, also termed as cellular self-digestion, is a lysosomedependent, evolutionarily conserved and dynamic degradation process in eukaryotic organisms for clearance of misfolded proteins and damaged organelles, which is associated with survival,

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differentiation, development and homeostasis [10e14]. It helps in safeguarding the human system against various invading microbial pathogens and strengthening the innate defense system [15]. Also a few essential autophagy-related genes are reported to help in the longevity phenotype and life span extension and thus prevent agerelated diseases [16]. 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 [19], A53T and A30P through autophagic participation in vitro [20]. Autophagyrelated 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 rapamycin 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 of mutant huntingtin and a-synuclein in vitro [24]. Some other small molecules, such as lithium [23], N10-substituted phenoxazine [25], rilmenidine [26], methylene blue [27] etc. can also induce autophagy at proper concentrations. Those previous studies suggest that all of these agents may be useful for their applications in the therapy of neurodegenerative diseases. But these chemical autophagy inducers 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 [28]. An imidazoline receptor 1 agonist called rilmenidine is reported to enhance the mutant huntingtin clearance in a mouse model of Huntington’s disease [26]. As rilmenidine belongs to the class of centrally-acting antihypertensive drugs, its usage may also lead to various unwanted 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 [33], fullerene and its derivatives [34,35], CNTs [36], MnO nanocrystals [37], iron core-gold shell nanoparticles [38], iron oxide nanoparticles [39], graphene oxide [40] and graphene quantum dots [41] 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 especially 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 nanorods by hydrothermal method and investigated their autophagy-inducing activities and mechanisms involved. Subsequently, 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 clearance ability of EuIII(OH)3 nanorods. Finally, we intended to explore the association between the clearance of mutant huntingtin with the autophagy induction by EuIII(OH)3 nanorods. 2. Materials and methods 2.1. Materials 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)3 nanorods. 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, MO). 3-(4,5)-Dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT, 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 purchased 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. HRPconjugated anti-rabbit antibodies (W4011) were purchased from Promega (Wisconsin, USA). Enhanced chemiluminescence (ECL) kits were purchased from Biological 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 at 20  C. 2.2. Preparation and characterization of EuIII(OH)3 nanorods EuIII(OH)3 nanorods 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 instrument. 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 europium hydroxide nanorods sample was recorded using thermo Nicolet Nexus 670 spectrometer in the diffuse reflectance mode at a resolution of 4 cm1 in KBr pellets. 2.3. Cell culture HeLa and GFPeLC3/HeLa cell lines have been described previously [34], 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% CO2 in 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 aggregation in Neuro 2a and PC12 cells were observed under fluorescence microscopy (Olympus IX71). Pictures were captured randomly. The incubation times and concentrations of EuIII(OH)3 nanorods 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)3 nanorods 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 microscopy (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% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS, the cells were treated with EuIII(OH)3 nanorods 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% NonidetÔ P-40/ 10 mM TriseHCl, pH 7.5/100 mM NaCl) on ice. One-quarter volume of 5 SDS sampleloading buffer (100 mM TriseHCl, pH 6.8, 2% b-mercaptoethanol, 4% SDS, 20% glycerol, 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 aggregation would be separated by separation gels. After blockage with Trisbuffered 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% OsO4 at 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

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lead citrate and examined under a transmission electron microscope (JEOL-1230, Japan). 2.10. Statistical analysis All data were 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)3 nanorods 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 reflections [42]. The TEM image (Fig. 1b) clearly indicates that the assynthesized 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)3 nanorods using different physico-chemical techniques. (a) XRD pattern of EuIII(OH)3 nanorods after hydrothermal heating at 150  C for 60 min. (b) TEM image of as-synthesized EuIII(OH)3 nanoparticle 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)3 nanorods.

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(Fig. 1d) that represents the typical FTIR spectra of europium hydroxide nanorods. The appearance of characteristic peaks [46] at w3609 cm1 and w700 cm1 attributes that the as-synthesized nanomaterial is europium hydroxide nanorods. 3.2. Enhanced clearance of GFP-Htt(Q74) by EuIII(OH)3 nanorods These as-synthesized and thoroughly characterized nanorods have been used for the autophagy induction and clearance of huntingtin proteins study. In order to investigate the clearance of this protein, we have incubated Neuro 2a cells stably expressing GFPHtt(Q74) with nanorods and trehalose (positive autophagy inducer) and analyzed the expression of the GFP-Htt(Q74) using fluorescence microscopy and western blot techniques. Here, insoluble protein aggregation would be detained in the sample-loading well, while soluble protein aggregation would be separated by separation gels. Interestingly, the expression of green (in web version) fluorescence due to GFP-Htt(Q74) has been reduced in Neuro 2a cells treated with EuIII(OH)3 nanorods 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 corresponding densitometry analyses have been shown in Fig. 2ced. These figures clearly show the clearance of huntingtin protein aggregation in nanorods treated Neuro 2a cells compared to vehicle control experiment. Time-dependent and dose-dependent removals 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 MTT assay did not significantly alter when treated with EuIII(OH)3 nanorods 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)3 nanorods and vehicle-treated groups, which corroborates with the MTT results (Fig. S2). 3.3. Autophagy induction by EuIII(OH)3 nanorods Atg8/LC3, one of the autophagy-related proteins, is the most widely used autophagic marker protein for the evaluation of autophagy intensity [47]. 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 phosphatidylethanolamine (PE), which subsequently connects to the outer and inner membranes of autophagosome [48]. 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 different cell 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)3 nanorods for 48 h. Fluorescence microscopy study (a) and Western blot analysis (b) clearly show that EuIII(OH)3 nanorods 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)3 nanorods (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)3 nanorods; Tre, trehalose, a positive control).

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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)3 nanorods for 72 h. Fluorescence microscopy study (a) and western blot analysis (b) clearly show that EuIII(OH)3 nanorods 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)3 nanorods. (Mean  s.e.m., n ¼ 3,*p < 0.05, **p < 0.01, compared to control of each group; TE, vehicle control; Eu, EuIII(OH)3 nanorods).

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)3 nanorods (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)3 nanorods for 24 h, the autophagosomes were indicated with the red arrows. (d) Fluorescent microscopy images of HeLa-LC3 cells stained with 10 mM monodansylcadaverine (MDC) or 75 nM Lyso Tracker Red when treated with EuIII(OH)3 nanorods (50 mg/mL) for 24 h. (TE, vehicle control; Eu, EuIII(OH)3 nanorods; 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.).

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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 [49], 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 GFPpuncta (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 [50]. Therefore, the formation 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)3 nanorods (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 autolysosomes (Fig. 4d). Besides, these results also hinted that the autophagic flux induced by EuIII(OH)3 nanorods was complete [43]. 3.4. Enhanced autophagic flux by EuIII(OH)3 nanorods None of the above-mentioned methods adequately evaluate the autophagic flux that involves the complete flux from autophagosomes formation to their fusion with the lysosomes. Autophagosome 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 alkalinization [51]. Besides this, both autophagosome formation and impairment of autophagosomeelysosome fusion have been found after calcium phosphate treatment [52]. Therefore, it is essential to evaluate the autophagic flux induced by EuIII(OH)3 nanorods 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 [53]. Earlier report has been demonstrated that after the treatment of cells with CQ at the saturating dose (50 mM), the degradation of LC3-II will be completely impaired [54]. 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 nanorods induce authentic autophagy flux through autophagosome 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 degradation of ubiquitinated proteins and autophagy regulation. 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 entrapment of such polyubiquitinated protein aggregates (commonly known as cargo) by the autophagy machinery [57]. Thus, the blockade of the autophagyelysosome pathway results in the accumulation of p62 which eventually leads to various neurodegenerative diseases by activating the cellular stress responses [58]. In the present work, it is clearly shown that EuIII(OH)3 nanorods 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 evidence obtained by utilizing these autophagy inhibitors, firmly confirmed that EuIII(OH)3 nanorods induced complete autophagy flux. We can therefore infer that the removal of huntingtin protein aggregates may be dependent on p62-mediated specific autophagy degradation. 3.5. The role of autophagy in EuIII(OH)3 nanorods-mediated clearance of GFP-Htt(Q74) It is well known that huntingtin protein aggregates are mainly removed by non-selective macroautophagy [59]. 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 aggregation degradation effects by EuIII(OH)3 nanorods, it was required to check whether the nanorods-induced clearance of protein aggregation 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 aggregation of mutant huntingtin protein inclusions by the inhibition of autophagy [59]. 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. 6a which shows that wortmannin is able to simultaneously inhibit autophagy induction and clearance of protein aggregation elicited by EuIII(OH)3 nanorods. Furthermore, 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 cytotoxicity especially 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 microscopy analysis using 10 mM concentration of CQ which actually inhibited the clearance of protein aggregation mediated by the EuIII(OH)3 nanorods, as clearly illustrated by the Fig. 6bec. These results obtained from the EuIII(OH)3 nanorods treatment in presence 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. 4. Conclusion In this study, we have demonstrated that europium hydroxide EuIII(OH)3 nanorods synthesized by hydrothermal method initiate authentic and cell type-independent autophagy via inducing the autophagosomes formation and reduce the protein levels of the

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Fig. 5. EuIII(OH)3 nanorods 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)3 nanorods with or without CQ for 24 h. (b) Determination of LC3 conversion was carried out by Western blot and densitometry analyses in stably-expressing GFPHtt(Q74) PC12 cells treated with EuIII(OH)3 nanorods 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)3 nanorods with or without CQ for 24 h, and p62 protein level was also measured in those HeLa cells. (d) p62 protein level determination in stablyexpressing GFP-Htt(Q74) Neuro 2a cells treated with EuIII(OH)3 nanorods by Western blot and densitometry analyses. (e) p62 protein level determination in stable-expressing GFPHtt(Q74) PC12 cells treated with EuIII(OH)3 nanorods 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)3 nanorods; Tre, trehalose, a positive control).

autophagy specific substrate p62. In addition to autophagy induction, these nanorods enhance the clearance of GFP-tagged huntingtin 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. Moreover, autophagy inhibition obliterated the clearance of GFP-Htt (Q74) protein, indicating that autophagy is primarily involved in the EuIII(OH)3 nanorods-mediated clearance of the protein aggregation (Fig. 7). In summary, we have reported that europium hydroxide 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. Acknowledgments 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 Program of the Chinese Academy of Sciences (Grant KSCX2-YW-R139), the Fundamental Research Funds for the Central Universities

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Fig. 6. (a) Western blot analysis clearly shows that the enhanced LC3 conversion and accelerated clearance of soluble GFP-Htt(Q74) induced by EuIII(OH)3 nanorods in Neuro 2a cells are reversed after the autophagic inhibitor wortmannin (100 nM) treatment. (bec) Autophagy inhibition by chloroquine (10 mM) weakens the degradation of GFP-Htt(Q74) in Neuro 2a cells treated with EuIII(OH)3 confirmed from both western blot (b) and fluorescence microscopic (c) studies. But still the clearance of GFP-Htt(Q74) is more compared with the vehicle control group. (Mean  s.e.m., n ¼ 3,*p < 0.05, **p < 0.01, NS, non-significant, compared to control of each group; TE, vehicle control; Eu, EuIII(OH)3 nanorods).

Fig. 7. EuIII(OH)3 nanorods accelerate the clearance of mutant huntingtin through autophagy induction, depression of autophagy via specific inhibitors such as wortmannin (WM), chloroquine (CQ) can abrogate the enhanced clearance of mutant huntingtin protein.

(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.

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