Stimulation of Autophagy Prevents Amyloid-Ð¯ Peptide ... - IOS Press

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Journal of Alzheimer’s Disease 40 (2014) 929–939 DOI 10.3233/JAD-132270 IOS Press

Stimulation of Autophagy Prevents Amyloid-␤ Peptide-Induced Neuritic Degeneration in PC12 Cells Yi Yanga,b,c,∗ , Sicong Chenc , Jiafeng Zhanga , Chentan Lia , Yonghong Sunc , Lihui Zhanga and Xiaoxiang Zhengb,c,∗ a Department of Pharmacology, Hangzhou Key Laboratory of Medical Neurobiology, School of Medicine, Hangzhou

Normal University, Hangzhou, P.R. China b Qiushi Academy of Advanced Studies, Zhejiang University, Hangzhou, P.R. China c Department of Biomedical Engineering, Zhejiang Provincial Key Laboratory of Cardio-Cerebral Vascular Detection Technology and Medicinal Effectiveness Appraisal, Zhejiang University, Hangzhou, P.R. China Handling Associate Editor: Chengxin Gong

Accepted 27 December 2013

Abstract. Autophagy is a lysosomal degradative process essential for neuronal homeostasis, whereas autophagic failure has been linked to accumulating neurodegenerative disorders. However, the precise role of autophagy in axonal and dendritic degeneration in Alzheimer’s disease (AD) remains unclear. In this study, we aim to investigate the precise effect of autophagy in amyloid-␤ peptide (A␤)25-35 -mediated neurite degeneration. A␤35-25 , the non-neurotoxic reverse sequence analogue of A␤25-35 , was used as a negative control. Our results showed that A␤25-35 dose-dependently suppressed PC12 proliferation and induced autophagy induction in neurites (axons and dendrites). A high proportion of autophagic structures in PC12 neurites were autolysosomes after 24 h of A␤25-35 treatment. Autophagy inhibition by 3-methyladenine (3MA), LY294002, and chloroquine (CQ) could not relieve the A␤25-35 -induced neurite degeneration, while administration of autophagy stimulator rapamycin or AR-12 efficiently suppressed neurite degeneration. Autophagosomes colocalized with fragmented mitochondria after A␤25-35 treatment. Similar results were obtained using in vitro cultured superior cervical ganglion neurons. These findings demonstrate that autophagy stimulation may prevent neuritic degeneration following A␤25-35 treatment. Upregulation of autophagic activity may provide a valuable approach for the treatment of axonal and dendritic dystrophy in AD patients. Keywords: Alzheimer’s disease, amyloid-␤ peptide, autophagy, neuritic degeneration

INTRODUCTION Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the presence of senile plaques composed primarily of amyloid-␤ peptide ∗ Correspondence

to: Dr. Yi Yang, Room 401, Building 8, Hangzhou Normal University, No. 16 Xuelin Street, Xiasha Higher Education Zone, Hangzhou 310036, Zhejiang, P.R. China. Tel.: +86 571 28865673; E-mail: [email protected]; Dr. Xiaoxiang Zheng, Room 505, Zhou Yiqing Building, Zhejiang University, No. 38 Zheda Road, Hangzhou 310027, Zhejiang, P.R. China. Tel./Fax: +86 571 87951091; E-mail: [email protected].

(A␤), intracellular neurofibrillary tangles (NFT) with hyperphosphorylated tau, dystrophic axons and dendrites (neurites), degenerating neurons, and activated astrocytes and microglia, especially around the senile plaques [1]. According to the classical amyloid cascade hypothesis, among all events, the extracellular deposition of A␤ in the brain is an initiator of disease pathogenesis in early-onset AD [2], while emerging lines of evidences indicate that this peptide can also accumulate intraneuronally and may contribute to disease progression [3]. A␤25-35 , which is a synthetic peptide of 11 amino acids that corresponds to a

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Y. Yang et al. / Autophagy Stimulation Prevents Neuritic Degeneration

fragment of A␤1-40 and A␤1-42 , represents the biologically active region of A␤ and is thus widely used for the establishment of in vitro cell model of AD [4]. Autophagy (specifically macroautophagy) is a lysosomal pathway for the turnover of organelles and long-lived proteins, and is essential for neuronal homeostasis. Despite insufficient or excessive autophagy both links to various neurodegenerative disorders [5], our recent retrospective study reveals that autophagy failure, characterized by extensive defective autophagic-lysosomal proteolysis especially in axons and dendrites, is a major problem in AD brain [6]. Increased number of autophagosomes and autolysosomes has been observed in dystrophic axons and dendrites of the AD brain [7], whereas the precise role of autophagy in neurite degeneration is not yet fully addressed. Lee et al. reported that selectively disrupting the axonal transport of autophagy-related compartments by lysosomal inhibitors could cause an AD-like axonal dystrophy [8]. Therefore, it is possible that dysfunctional autophagy may lead to neurite degeneration in the progression of AD. In the present study, we investigated the induction of autophagy in neurites of PC12 cells and primary cultured mouse superior cervival ganglion (SCG) neurons following A␤25-35 exposure. The role of autophagy on neurite degeneration was determined using autophagy stimulators and inhibitors. Our findings may provide valuable insights into understanding the pathogenesis of neuritic dystrophy in AD and may offer novel target for the treatment of neurite degeneration in neurodegenerative disorders.

Radio Immunoprecipitation assay (RIPA) lysis buffer was purchased from Beyotime Institute of Biotechnology (Jiangsu, China). All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) except those specifically mentioned. Cell culture Highly differentiated PC12 pheochromocytoma cells were obtained from the Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China), and were maintained in DMEM supplemented with 10% heat-inactivated horse serum, 5% fetal bovine serum (FBS), 50 U/mL penicillin, and 50 ␮g/mL streptomycin at 37◦ C in a humidified atmosphere of 5% CO2 / 95% air. Explant culture of SCG cells were prepared from postnatal 1-day-old C57BL/6 mice as described previously [9]. Procedures involving experimentation on animal subjects were done in accord with the guide of the Animal Care and Use Committee in Hangzhou Normal University. In brief, ganglia were plated on collagen-coated 12-well plates after dissection. Cultures were maintained in AM50 medium: MEM supplemented with 5% FBS, 50 ng/mL nerve growth factor (NGF), 10 ␮M fluorodeoxyuridine, 10 ␮M uridine, 50 U/mL penicillin, and 50 ␮g/mL streptomycin and were grown in a humidified atmosphere of 5% CO2 / 95% air at 37◦ C. Cultures were treated with 5 ␮M aphidicolin at DIV (day in vitro) 1 for two days to eliminate non-neuronal cells. All experiments were started at DIV 5. Preparation of Aβ solution

MATERIALS AND METHODS Reagents Dulbecco’s modified Eagle’s medium (DMEM), Eagle’s minimum essential medium (MEM), and heat-inactivated horse serum were obtained from GIBCO-BRL, New York, USA. A␤35-25 was synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. Cell-Light™ EdU Apollo® 488 In Vitro Imaging Kit was purchased from Guangzhou RiboBio Co., Ltd (Guangdong, China). Rabbit anti-A␤31-35 polyclonal antibody was bought from Beijing Biosynthesis Biotechnology Co., Ltd (Beijing, China). Rapamycin was bought from Alexis Biochemicals (San Diego, CA). AR-12 was provided by Dr. Chen (Ohio State University). DsRed-Mito plasmid was purchased from Clontech Laboratories, Inc.

Stock solution of A␤25-35 was prepared by dissolution in distilled water to a 0.5 mM concentration. Prior to use, A␤25-35 was diluted in DMEM at a concentration of 50 ␮M and was maintained at 37◦ C for 7 days to pre-age the peptide. Just before use, the aged A␤ solution was adjusted to indicated concentrations with DMEM and 2% of heat-inactivated horse serum and 1% of FBS was applied. Reverse sequence peptide A␤35-25 prepared in the same way was used as control. Thioﬂavin T (ThT)-based ﬂuorimetric assay A␤ peptide aggregation was evaluated by ThT-based fluorimetric assay, in which fluorescence intensity reflected the degree of aggregation [10]. Different concentrations of pre-aged A␤ solutions were added


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into 50 mM pH 8.5 glycine-NaOH buffer, containing 1.5 mM ThT. Fluorescence intensity was determined at 446 nm (excitation) and 490 nm (emission) by microplate reader (Flex Station II, Molecular Devices, Sunnyvale, CA, USA).

tured under a 60 × 1.45 NA oil-immersion objective (PlanApoN, Olympus), using standard filter sets and an Hg lamp. The colocalization pattern was analyzed by ImageJ 1.45 software and the Pearson’s Coefficient was recorded.

Determination of cell proliferation

Immunocytochemistry

Cell proliferation capability was determined using EdU (5-ethynyl-2 -deoxyuridine) incorporation assay with a Cell-Light™ EdU Apollo® 488 In Vitro Imaging Kit. After exposed to different concentrations of A␤, cells were treated with 50 ␮M of EdU for 2 h at 37◦ C. Then, cells were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.5% Triton X-100 and exposed to 1×Apollo® reaction solution for 30 min. The cell nuclei were counterstained with 5 mg/mL Hoechst 33342. After phosphate buffered saline (PBS) washing, samples were examined under fluorescence microscope (IX-81, Olympus). Every experimental condition was repeated at least in sextuplicate wells and data were calculated from three independent experiments.

After treatment with 10 ␮M A␤25-35 for 24 h, GFPLC3-labeled PC12 cells were fixed with 4% PFA, permeabilized with 0.3% Triton X-100, and blocked with 1% bovine serum albumin (BSA). Then, cells were incubated with anti-A␤31-35 antibody (1 : 300 dilution) in PBS containing 1% BSA and 0.1% Triton X-100 overnight at 4◦ C. Cells were subsequently stained with Alexa Fluor 568-conjugated anti-rabbit IgG secondary antibody (1 : 100 dilution, Molecular Probes, Inc.) at room temperature for another 1 h. After three times washing with PBS, nuclei were further stained with 5 mg/mL Hoechst 33342 for 15 min. Images were visualized under fluorescence microscope (IX-81, Olympus). Western blotting

Transmission electron microscopy (TEM) analysis After fixation with 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate-buffered (pH 7.4) solution for 2 h, PC12 cells were then post-fixed with 1% OsO4 in the same buffer for 1 h. After dehydration in an ethanol gradient, samples were incubated with propylenoxid (2 × 10 min), impregnated with a mixture of propylenoid/LX-112 (1 : 1; Ladd Research Industries, Williston, VT), and embedded in LX-112. Ultrathin sections were stained with uranyl acetate and lead citrate. Sections were visualized under a Jeol 100CX-II TEM operating at 80 kV. Transfection Transient transfection with GFP-LC3, mRFPGFP-LC3 plasmid (provided by Dr. Yoshimori) or cotransfection with GFP-LC3 and DsRed-Mito plasmids was carried out one day after PC12 subculture by using Lipofectamine 2000 (Invitrogen Life Technologies) transfection reagent according to the manufacturer’s instructions. Two days after transfection, cells were washed three times with fresh culture medium and incubated with A␤ for 24 h at 37◦ C to induce autophagy activation. Cells were visualized under an inverted microscope (IX-81, Olympus) equipped with a live cell station. Images were cap-

Proteins were extracted from PC12 cells using RIPA lysis buffer. Protein extracts (25 ␮g) were separated on 12% SDS-polyacrylamide gel, transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Burlington, MA), blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween (TBS-T), and then probed with rabbit anti-LC3A/B (1 : 2000, Cell Signaling Technology) at 4◦ C for overnight. Secondary horseradish peroxidase (HRP)conjugated anti-rabbit IgG was used at a dilution of 1 : 500 for 1.5 h at room temperature. Visualization of immunoreactive bands was done with an enhanced chemoluminescence detection kit (ECL; Amersham Biosciences, Piscataway, NJ). Anti-GAPDH (1 : 2000, Cell Signaling Technology) was used for normalization. Western blot analysis was performed in triplicate. Quantification of the intensities of the immunoreactive bands was carried out using the Photoshop imaging system. Labeling with Calcein-AM and MitoTracker Orange Cells were labeled with 500 nM Calcein-AM and 500 nM MitoTracker Orange chloromethyl tetramethylrosamine (CMTMRos; Molecular Probes, Inc., Eugene, OR, USA) in MEM for 1 h at 37◦ C in

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dark. The loading solution was then replaced by fresh pre-warmed medium, and the cells were observed using the fluorescence microscope (IX-81, Olympus). Determination of cell apoptosis PFA-fixed and Triton X-100-permeabilized cells were washed with PBS, and exposed to 5 mg/mL Hoechst 33342 at room temperature in the dark for 10 min. After PBS washing, samples were examined under fluorescence microscope (IX-81, Olympus). MTT assay Cell viability was analyzed by the 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) assay. Each data point represented results from 3 independent experiments and each treatment was performed in sextuplicate. After 24 h of drug treatment, 15% volume of MTT (5 mg/mL) was added into each well. After another 4 h of incubation at 37◦ C, the medium was removed and 100 ␮L dimethyl sulfoxide was added to each well to resuspend the MTT metabolic product. The absorbance of the dissolved formazan was measured at 570 nm (A570 ) with microplate reader (Versa Max, Molecular Devices). The percentage of cell viability was calculated by the formula: Viability (%) = 100-[(A570,Control A570,A␤ )/A570,Control ]∗ 100. Evaluation of neurite degeneration Neurite degeneration was determined as previously described [9]. Briefly, photomicrographs were captured and 15 fields were randomly selected from each treatment group. At least ten neurites were evaluated in each field and the number of beadings per 100 ␮m neurites was counted. Data were calculated from three independent experiments. To analyze the extent of fragmentation, the number of fragmented neurites was counted per field and was expressed as a percentage of the total number of neurites in the same photomicrograph. Statistical analysis Data were plotted as mean ± SEM from three independent experiments. Statistical significant differences were carried out by one-way ANOVA, followed by Tukey’s post-hoc tests. p value 0.05). Thus the dose of 10 ␮M was used for the following experiments. Additionally, A␤35-25 (1–50 ␮M) appeared to have no influence on cell proliferation (p > 0.05). Induction of autophagy by Aβ25-35 exposure Twenty-four hours after 10 ␮M A␤25-35 or A␤35-25 treatment, the subcellular morphology of PC12 cells was examined under TEM. Nuclear membrane was intact in appearance after A␤35-25 application, and rough endoplasmic reticulum with ribosomes attached to its outer surface was clearly visualized (Fig. 2A, arrowheads). Mitochondria were rod-like or elongated in shape (Fig. 2A, asterisks). Most of the principal cellular organelles were not distinctly from those of control cells at 24 h after A␤25-35 treatment, whereas abundant single-membrane-bound vacuoles with characteristics of autolysosomes were found (Fig. 2A, arrows), demonstrating the activation of autophagic processes. GFP-LC3 has been widely used to label autophagosomes in live cells. LC3 distributed in a diffuse pattern throughout the cytoplasm upon A␤35-25 treatment (Fig. 2B). However,


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Fig. 1. A␤25-35 dose-dependently suppressed PC12 proliferation. A) ThT fluorescence of pre-aged A␤35-25 and pre-aged A␤25-35 . The peptide aggregation of different concentrations of 7-day pre-aged A␤ was evaluated by ThT-based fluorimetric assay. The data were presented as percentage of the control (0 ␮M) and were plotted as mean ± SEM from three independent experiments. B) Cell proliferation and viability was determined by EdU incorporation assay after PC12 cells were incubated with different concentrations of A␤35-25 or A␤25-35 for 24 h. The percentage of EdU-positive cells was counted on five randomly selected fields for each well. Data were plotted as mean ± SEM from three independent experiments. ∗ p < 0.05 compared with control. Representative data of varied dose of A␤25-35 treatment were presented in (C). Cell nuclei were counterstained with Hoechst 33342. Scale bar, 50 ␮m.

GFP-LC3-labeled dot like structures, representing autophagosomes, were formed in the juxtanuclear regions and along the neurites after 24 h of A␤25-35 incubation. In addition, immunocytochemical analysis with an anti-A␤31-35 antibody further revealed the enhanced peptide immunoreactivity during A␤25-35 treatment. These results confirm the hypothesis that A␤25-35 can initiate the autophagy activation. A high proportion of LC3-labeled autophagic compartments are actually autolysosomes During autophagic process, the double-membrane autophagosomes finally fuse with lysosomes to form single-membrane autolysosomes where incorporated materials are degraded. A novel reporter protein mRFP-GFP-LC3, constructed by Yoshimori’s research

group, has been used for assessing the fusion step of autophagosomes with lysosomes. After fusion with lysosomes, GFP signal is attenuated, while mRFP signal could still be detected [11]. Twenty-four hours after 10 ␮M A␤35-25 treatment, mRFP-labeled autolysosomes were found concentrated at the juxtanuclear region, whereas GFP signals dispersed throughout the cytoplasm (Fig. 3A), indicating administration of A␤35-25 would not induce the induction of autophagy in both cell body and neurites. In contrast, both mRFP and GFP signals could be detected in A␤25-35 -treated PC12 cells, and increased number of mRFP-labeled autolysosomes was observed as compared with GFPlabeled autophagosomes (Fig. 3A). The number of autophagosomes/autolysosomes in neurites was quantified as shown by Fig. 3B. It could be clearly seen that the neuritic accumulation of autolysosomes occurred

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Fig. 2. A␤25-35 -induced induction of autophagy in PC12 neurites. A) TEM analysis showed the ultrastucture of 10 ␮M A␤35-25 - or A␤25-35 treated PC12 cells. Note that the single membrane autolysosomes, containing dense, amorphous materials, were presented after 24 h of A␤25-35 treatment (arrows). N, nucleus; asterisks, mitochondria; arrowheads, endoplasmic reticulum. Scale bars, 0.5 ␮m. B) PC12 cells were transfected with GFP-LC3 plasmid. After treated with 10 ␮M A␤25-35 for 24 h, GFP-LC3-labeled cells were stained with anti-A␤31-35 antibody and Hoechst 33342. Representative images were presented. Scale bars, 20 ␮m.

since 6 h after A␤25-35 addition, and peaked at 24 h. A similar trend was detected in the autophagosome appearance, while the number of autophagosomes was obviously lower than that of autolysosomes at 18 and 24 h after treatment (p < 0.05). These observations demonstrated that 24 h after A␤25-35 treatment, a high proportion of LC3-labeled autophagic compartments were actually autolysosomes. MitoTracker staining further revealed that the mitochondria membrane potential was gradually decreased in neurites after 18 h of A␤25-35 incubation (Fig. 3C). Effects of autophagy inhibitors and stimulators on PC12 cells exposed to Aβ25-35 We next investigated the role of autophagy in A␤25-35 -induced PC12 cell death. For this purpose, the widely used autophagy inhibitors such as 3-methyladenine (3MA), LY294002, and chloroquine (CQ) and autophagy stimulator rapamycin were applied. As shown by Fig. 4A, autophagy inhibition could not relieve the A␤25-35 -induced neurite degeneration. The administration of 3MA and CQ even aggravated neuritic death. Importantly, application of autophagy stimulator rapamycin efficiently suppressed neurite degeneration. Moreover, the novel

small-molecule agent, AR-12, which has been demonstrated to induce autophagy in cancer cells [12], also delayed neurite degeneration, suggesting the activation of autophagy can delay or relieve the neurite degeneration caused by AD. It should be noted that blockage of lysosomal degradation by CQ reversed the neuritic protection of rapamycin (Fig. 4A). Hoechst staining indicated that A␤25-35 induced cell soma apoptosis, which could be prevented by the co-administration of rapamycin or AR-12 (Fig. 4A). In addition, stimulation of autophagy by either rapamycin or AR-12 greatly suppressed neuritic degeneration, with the appearance of decreased beading formation (Fig. 4B) and fragmentation (Fig. 4C), and reserved cell viability (Fig. 4D). As the upregulation of LC3-II protein level is one of the biomarker for autophagy induction, western blotting with an anti-LC3 antibody was performed. As expected, 24 h of A␤25-35 treatment promoted the protein expression of LC3-II (Fig. 4E, F). Co-administration of either rapamycin or AR12 significantly upregulated the protein expression of LC3-II as compared with control cells, while 3MA or LY294002 greatly downregulated LC3-II levels to normal value. Note that combined administration of CQ also caused a substantial increase of LC3-II level possibly due to accumulation of autophagosomes


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Fig. 3. A high proportion of autophagic structures in PC12 neurites were autolysosomes after 24 h of A␤25-35 treatment. A) mRFP-GFP-LC3 labeled PC12 cells were incubated with 10 ␮M A␤35-25 or A␤25-35 . Twenty-four hours after A␤ treatment, both mRFP and GFP signals were detected in A␤25-35 -treated PC12 neurites, and increased number of mRFP-labeled autolysosomes (arrows) were observed as compared with GFP-labeled autophagosomes (arrowheads). Higher magnifications of the boxed regions were presented. B) The number of autophagosomes/autolysosomes per 150 ␮m neurites was counted. ∗ p < 0.05 compared with 0 h; #p < 0.05 compared with autophagosomes. C) After indicated period of A␤ incubation, cells were stained with MitoTracker Orange. The mitochondria membrane potential was gradually decreased since 18 h of A␤25-35 treatment. Scale bars, 20 ␮m.

upon autophagy inhibition. Furthermore, autophagy inhibitors also dramatically reduced the numbers of autophagosomes and autolysosomes in neurites, whereas autophagy stimulator obviously elevated the neuritic deposition of these vesicles (Fig. 4G).

eliminating damaged cellular organelles such as mitochondria.

Colocalization of autophagosomes and fragmented mitochondria

Explant culture of ganglion neurons is a widely used cell model for the analysis of neuritic outgrowth and degeneration. We thus investigated the potential role of autophagy in A␤-induced neurite degeneration of primary cultured SCG neurons using autophagy inhibitors and stimulators. In accordance with results obtained from PC12 neurites, rapamycin significantly suppressed A␤25-35 -induced neurite degeneration in in vitro cultured SCG neurons (Fig. 6A, B). The neuritic protective effects of rapamycin might be achieved by elevation of neurite viability and mitochondria membrane potential in neurites, as significantly increased fluorescence intensity of Calcein-AM and MitoTracker Orange was detected (Fig. 6C, D). On the contrary, blockage of autophagy activity by 3MA, LY294002,

Using TEM analysis, some of the mitochondria were found to be encapsulated in autolysosomes, implying the process of specific removal of mitochondria by autophagy. To prove this, PC12 cells were cotransfected with GFP-LC3 and DsRed-Mito plasmids. Enhanced colocalization of autophagosomes and fragmented mitochondria were detected in PC12 neurites after 24 h of A␤25-35 incubation as compared with A␤35-25 treatment (Fig. 5) (Pearson’s coefficient: A␤35-25 , r = 0.489; A␤25-35 , r = 0.786). These observations indicated that autophagy may play a self-protective role in neurites through

Similar patterns were noted in the neurites of primary cultured sympathetic neurons

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Fig. 4. Effects of autophagy inhibitors and stimulators on PC12 cells exposed to A␤25-35 . A) PC12 cells were treated with 10 ␮M A␤25-35 in combination with 3-methyladenine (3MA, 10 mM), LY294002 (LY, 10 ␮M), chloroquine (CQ, 10 ␮M), rapamycin (Rapa, 10 nM), rapamycin (10 nM) plus CQ (10 ␮M) (Rapa + CQ), or AR-12 (1 ␮M), respectively. Twenty-four hours after A␤25-35 treatment, the cellular morphology was examined by phase-contrast microscope. A␤35-25 treatment was used as negative control. Some of the cell samples were stained with Hoechst 33342. Apoptotic cells with the appearance of condensed nucleus were indicated by arrows. Scale bars, 50 ␮m. The number of beadings per 100 ␮m neurites and the percentage of neurite fragmentation were quantified in (B) and (C). D) Cell viability was determined by MTT assay at 24 h following drug treatment. E) Immunoblotting analysis of LC3 expression in cells after 24 h of drug incubation. F) The amount of LC3-II was quantified relative to the level of GAPDH. Results are representative of three independent experiments. The number of autophagosomes/autolysosomes per 150 ␮m neurites was counted and presented in (G). Wort, wortmannin (100 nM) (autophagy inhibitor). ∗ p < 0.05 compared with control; #p < 0.05 compared with A␤ 25-35 treatment.


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Fig. 5. Autophagosomes colocalized with fragmented mitochondria after A␤25-35 treatment. PC12 cells were co-transfected with GFP-LC3 and dsRed-Mito plasmids. Two days after transfection, cells were incubated with 10 ␮M A␤35-25 or A␤25-35 for another 24 h. GFP-labeled autophagosomes and dsRed-labeled mitochondria were visualized under fluorescent microscope. Higher magnifications of the boxed regions were presented in the lower panels. The colocalization of autophagosomes and fragmented mitochondria was indicated by arrows. Scale bars, 20 ␮m.

and CQ could not delay or relieve neurite degeneration induced by A␤25-35 deposition. DISCUSSION Using electron microscopy analysis, increased autophagic activation has been detected in the brain biopsies of AD patients [13]. Abundant numbers of autophagosomes, multivesicular bodies, multilamellar bodies, and cathepsin-containing autolysosomes were accumulated in AD brains particularly, within the dystrophic neurites [13]. Similar results are seen in dystrophic neurites from mice carrying mutant amyloid-␤ protein precursor (A␤PP) and mutant presenilins (PS1) (PS/A␤PP) [14]. Yu et al. showed that autophagosomes may constitute a unique site of production/accumulation of the pathogenic A␤ [15]. Nevertheless, the underlying mechanism of autophagy-mediated neurite degeneration is not yet fully understood. Here, A␤25-35 was used to induce Alzheimer-like neuritic dystrophy. Consistent with our previous findings [16, 17], prominent neurotoxicity was observed when PC12 cells were exposed to 10–50 ␮M A␤25-35 . Extensive autophagosome and autolysosome accumulation was found extremely in cytoplasm as well as neurites, which is in accordance with previous findings using either AD animal model [14] or biopsies obtained from AD patients [13]. It is speculated that autophagosomes and autolysosomes accumulate in dystrophic neurons of AD brain, presumably as a result of impairment in autolysosomal maturation [15]. Recent study indicated that primary lysosomal dysfunction caused cargo-specific deficits of axonal transport leading to Alzheimer-like neuritic dystrophy [18]. Therefore, it is

possible that the deposition of A␤ protein may lead to the dysfunction of lysosomal proteolysis, accounting for the accumulation of autophagic structures. In order to understand the actual role of autophagy during neurite degeneration, several autophagy inhibitors and stimulators were applied. We found that autophagy inhibition had no obvious effect in preventing A␤25-35 -induced neuritic dystrophy, and 3MA and CQ even aggravated neuritic death. In contrast, application of autophagy stimulator rapamycin or a novel small-molecule agent AR-12 efficiently suppressed neurite degeneration. Explant culture of primary sympathetic ganglion neurons is widely used for the study of axonal and dendritic degeneration. Here, similar results were obtained in the primary cultured SCG neurons treated with A␤25-35 . Autophagy is recognized as a double-edged sword in the regulation of health and disease [19]. Accumulating evidences show that over-activated autophagy is harmful for the maintenance of axonal and dendritic integrity. Upon insults, including mechanical injury [9, 20, 21], pharmacological toxin exposure [22, 23], nutrition withdrawal [9], lysosomal inhibition [14, 23], blockage of autophagic activity could prevent neurite degeneration (for review, see [6]). Interestingly, in the case of AD, autophagy is proven to be beneficial [8, 15, 24, 25]. It has been demonstrated that the autophagic-lysosomal degradative system clears the disease-related protein aggregates or inclusions, such as A␤PP in AD [8]. Hence, it is possible that upregulation of autophagy may protect against the neurite degeneration induced by A␤ exposure. According to western blotting analysis, A␤25-35 treatment induced approximately 2-fold in LC3-II expression as compared with control group. Co-admini-

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Fig. 6. Autophagy stimulator suppressed neurite degeneration induced by A␤25-35 in primary cultured sympathetic neurons. DIV 5 explant cultures of mouse SCG neurons were treated with 10 ␮M A␤25-35 in combination with rapamycin (10 nM), 3MA (10 mM), LY294002 (10 ␮M), CQ (10 ␮M), or rapamycin (10 nM) plus CQ (10 ␮M), respectively. A␤35-25 (10 ␮M) was used as control. A) Twenty-four hours after A␤ treatment, the cellular morphology was examined by phase-contrast microscope (upper panels). Neurites were stained Calcein-AM (middle panels) or MitoTracker Orange (lower panels), and fluorescence photomicrographies were recorded under fluorescent microscope. Scale bars, 20 ␮m. The neurite fragmentation was quantified in (B), and the relative fluorescent intensities of Calcein-AM or MitoTracker Orange staining were presented in (C) or (D), respectively. ∗ p < 0.05 compared with A␤35-25 treatment; #p < 0.05 compared with A␤25-35 treatment.

stration of rapamycin or AR-12 also induced 2- or 1.3fold in LC3-II expression as compared with A␤25-35 treatment alone. We speculate that the autophagy induction caused by A␤25-35 exposure may serve as a self-defence mechanism by clearance of peptide deposition intracellularly or damaged cellular organelles such as mitochondria. Such hypothesis was further confirmed by electron microscopy analysis as well as co-transfection of PC12 cells with GFP-LC3 and dsRed-Mito plasmids, both of which showed the co-localization of damaged mitochondria within autophagic structures. Autophagy participates in the clearance of injured intracellular organelles, such as mitochondria, which is known as mitophagy [26]. Mitophagy prevents the release of pro-apoptotic proteins from damaged mitochondria upon certain stimulus, and therefore serves as a quality control of mitochondria and prevents against neurodegen-

erative disorders and aging [27]. Thus, it is likely that insufficient autophagic clearance capability may lead to the accumulation of damaged mitochondria and subsequent neurite degeneration. Moreover, other pathways may also contribute to the protective effects of rapamycin since rapamycin increased mitochondrial activity in degenerating neurites. Future study is needed for understanding the interplay between autophagy and mitochondrial clearance/activity. In summary, our current study showed that autophagy actively participated in the A␤25-35 -induced neurite degeneration. Upregulation of autophagic activity by autophagy stimulators may prevent neuritic dystrophy. These data provide valuable insights into understanding the mechanism of neurite degeneration in AD and may offer a promising target for the treatment of neuritic dystrophy in the early onset of AD.


ACKNOWLEDGMENTS We are grateful to Dr. Yoshimori for GFP-LC3 and mRFP-GFP-LC3 plasmids; Dr. Chen for AR12. This study was supported by Zhejiang Provincial Natural Science Foundation of China (Grant No. LQ13H310004, LY12H31010), Health Bureau of Zhejiang Province (Grant No. 2013KYA147), the Key Laboratory of Hangzhou City Project (Grant No. 20090233T12) and National Basic Research Program of China (973 Program) (Grant No. 2011CB504400). Authors’ disclosures available online (http://www. j-alz.com/disclosures/view.php?id=2084).

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