Strain-Dependent Effect of Macroautophagy on Abnormally Folded Prion Protein Degradation in Infected Neuronal Cells

Prion diseases are neurodegenerative disorders caused by the accumulation of abnormal prion protein (PrPSc) in the central nervous system. With the aim of elucidating the mechanism underlying the accumulation and degradation of PrPSc, we investigated the role of autophagy in its degradation, using cultured cells stably infected with distinct prion strains. The effects of pharmacological compounds that inhibit or stimulate the cellular signal transduction pathways that mediate autophagy during PrPSc degradation were evaluated. The accumulation of PrPSc in cells persistently infected with the prion strain Fukuoka-1 (FK), derived from a patient with Gerstmann–Sträussler–Scheinker syndrome, was significantly increased in cultures treated with the macroautophagy inhibitor 3-methyladenine (3MA) but substantially reduced in those treated with the macroautophagy inducer rapamycin. The decrease in FK-derived PrPSc levels was mediated, at least in part, by the phosphatidylinositol 3-kinase/MEK signalling pathway. By contrast, neither rapamycin nor 3MA had any apparently effect on PrPSc from either the 22L or the Chandler strain, indicating that the degradation of PrPSc in host cells might be strain-dependent.


Introduction
Transmissible spongiform encephalopathies, so-called prion diseases, are fatal neurodegenerative disorders that include Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy in cattle and scrapie in sheep. They are transmitted by prions, unconventional infectious agents that mainly consist of proteinase-resistant and β-sheet-rich amyloidogenic isoforms (PrP Sc ) of the normal host protein PrP (referred to as the conformational isoform, PrP C ) [1,2].
The degradation of cellular organelles and cytoplasmic proteins is carried out by a process referred to as autophagy, of which there are three types: macroautophagy, microautophagy and chaperone-mediated autophagy (CMA). In macroautophagy, cytoplasmic proteins or previously described [23][24][25][26]. Briefly, a 10% brain homogenate (BH) was prepared from priondisease-onset ddY mice and used to infect N2a-58 cells. All animal experiments were approved by the Committees on Animal Care and Use of Nagasaki University and were performed according to their recommendations (Permit No.: 1102170900). The cells were cultured in DMEM (Sigma) containing 10% heat-inactivated foetal bovine serum and 1% penicillin-streptomycin (Life Technologies, Japan) and split every 3 days at a 5 to 10-fold dilution. All cultured cells were maintained at 37°C in 5% CO 2 in the biohazard prevention area of the author's institution. In drug treatment studies, cells (2 × 10 4 cells/well) were grown in 12-well plate for 24 h prior to the addition of each drug. In mouse Atg5 knockdown study, psiRNA-mATG5 plasmid (InvivoGen, USA) was transduced by Fugene 6 (Roche Diagnostics K.K.) as transfectant into N2a-FK cells and the cells were harvested after 48 h. In knockdown study, psiRNA-Luc GL3 plasmid (InvivoGen) as control was used. Starvation conditions were obtained by replacing the growth medium with Hank's balanced salt solution (HBSS; Wako, Japan) and culturing the cells for 24 h. Fluorescence imaging of autophagic induction was achieved by Lipofectamine LTX (Life Technologies)-mediated transfection of the cells with plasmid pEGFP-LC3, prepared by inserting a mouse LC3 cDNA between the BglII and EcoRI sites in the multiple cloning site (MCS) of pEGFP-C1 (Clontech Laboratories, CA, USA). The plasmid-transfected cells were treated with the various drugs after 24 h and continuously cultured for 24 h. In autophagic level evaluation study using immunoblotting and microscope imaging, to inhibit protein degradation in lysosomes, the cells were pre-treated with 10 mM NH 4 Cl (Nacalai Tesque). After 24 h from NH 4 Cl treatment, the cells were appropriately added drugs to use for each study and cultured for a further 24 h.

Western blotting
The drug treated cells were harvested with lysis buffer (50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate and 2 mM EDTA). After 2 min of centrifugation at 10,000 × g, the supernatant was collected and its total protein concentration was measured using the BCA protein assay kit (Nacalai Tesque). To detect PrP Sc , the protein concentration was adjusted to 5 mg/ml and the samples were digested with 20 μg proteinase K (PK; Sigma)/ml at 37°C for 30 min, followed by boiling for 10 min with sample buffer (50 mM Tris-HCl, pH 6.8, containing 5% glycerol, 1.6% SDS, 100 mM dithiothreitol and a moderate amount of bromophenol blue). After SDS-polyacrylamide (15%) gel electrophoresis, the proteins were transferred onto a PVDF membrane (Immobilon-P; Merck Millipore) which was blocked with 5% skim milk in TBST (10 mM Tris-HCl, pH 7.8, 100 mM NaCl, 0.1% Tween 20) for 1 h at room temperature. The membrane was then reacted with diluted primary (1:1000) and HRP-conjugated secondary antibodies (1:5000). Immunoreactive bands were visualized by ECL prime (GE Healthcare). To quantify the signals, the intensity of each band was measured using the NIH image J software. A detailed description of the methods was previously provided [22].

Lysosomal purification
Lysosomes were purified from mammalian cells as described previously, with several modifications [27]. All steps were carried out at 4°C unless otherwise noted. Cells at an initial concentration of 1 × 10 9 , corresponding to the number of cells cultured under starvation conditions in four 75-cm 2 flasks, were grown to 90% confluency and harvested by trypsinization. All subsequent lysosomal isolation steps were performed according to the protocol included in the lysosome isolation kit (Sigma). In brief, the cells were centrifuged at 600 × g for 5 min and resuspended as a 1mL packed cell volume (PCV) in PBS. After the dilution of 2.7 × PCV mL with 1 × extraction buffer, the cells were finally subjected to five freeze-thaw cycles. The degree of breakage was checked until 80-85% of the cells were broken. Cellular debris such as nuclei was removed by centrifugation at 2,000 × g for 10 min and the supernatant further was centrifuged at 20,000 × g for 1 h. The resulting pellet, containing the crude lysosomal fraction (CLF), was resuspended in 1 mL of extraction buffer, as the minimal volume. To enrich the lysosomes, the suspension was further purified by density gradient ultracentrifugation at 150,000 × g for 4 h followed by filling of Diluted Optiprep Fraction (DOF) along with the a part of CLF by 27 to 8% Optiprep Density Gradient Medium solutions, included in the kit, according to the manufacturer's instructions. After centrifugation, several fractions of the appropriate volume were collected from the top of the gradient. Each fraction was assayed for the amount of Lamp-1, as a standard of lysosomal protein, and for β-actin and PrP Sc , which was treated with final 40 μg of proteinase K /ml at 37°C for 30 min.

Immunocytochemistry
The cells were treated with 10 mM 3MA and 1 μM rapamycin, either separately or together, for 24 h or by incubation in HBSS for 8 h, and then incubated with 0.1 mM MDC in PBS for 30 min at 37°C. The cells were then washed twice with PBS and observed using an Axio Observer Z1 microscope (Carl Zeiss, Deutsch). MDC-positive granules were counted in all treated cells as previously described [21]. For immunofluorescence analysis, the drug-treated cells were fixed for 20 min at room temperature in 4% paraformaldehyde buffer and permeabilized with 0.5% Triton X-100 for 5 min at room temperature. For PrP Sc staining, the cells were treated with 3M guanidine thiocyanate for 5 min, blocked for 1 h at room temperature in TBST containing 5% skim milk and incubated overnight at 4°C with SAF61 antibody (1:100). The cells were washed in PBS and then incubated with Alexa Fluor-488-conjugated secondary antibody (Life Technologies) (1:200) for 60 min at 37°C. In this PrP Sc detection, prion-uninfected N2a-58 cells as a negative control were used. For lysosomal staining, the cells were incubated and pre-stained with 1 μM Lysotracker dye (Life Technologies) for 30 min in a CO 2 incubator and then fixed as described above. For nuclear staining, the cells were labelled in mounting medium containing the DNA counterstain DAPI. All images were obtained using a confocal laser-scanning microscope 700 (Carl Zeiss). The immunofluorescence staining protocols used in this study were previously described in detail [22].

Statistical analysis
Student's t-test and the Mann-Whitney U-test were used in comparisons of two groups, and the one-way ANOVA followed by the Tukey-Kramer test in multiple comparisons. The logrank test was used to analyse the mortality of prion-infected mice. Statistical analysis of all data was performed using Statcel 2 of the Excel and GraphPad Prism software.

Results
PrP Sc degradation is strongly reduced by lysosomal but not by proteasomal inhibitors in N2a-FK cells It is known that protein degradation system can be largely classified into two groups as ubiquitin-proteasome and lysosome system. Thus, to confirm PrP Sc degradation system in mouse neuroblastoma cells persistently infected with a mouse-adapted prion strain derived from a patient with Gerstmann-Sträussler-Scheinker syndrome, a genetic form of human prion disease (N2a-FK), we investigated about the degradation pathway using proteasomal and lysosomal inhibitors as a familiar pharmacological approach. Treatment of N2a-FK cells with 0.1 to 10 nM epoxomicin or 0.01 to 1 μM MG132, both of which inhibit proteasome activation, had no effect on PrP Sc degradation after 48 h ( Fig 1A). However, when the cells were treated with 0.1 to 10 mM NH 4 Cl, PrP Sc degradation was inhibited dose-dependently and significantly (Fig 1A and 1B). These results demonstrate the PrP Sc clearance in N2a-FK cells may relate to the autophagy-lysosomal pathway because it is known that NH 4 Cl will block the late stage of the autophagy-lysosomal pathway in lysosomal system.

PrP Sc is markedly influenced by inducers and inhibitors of autophagy in N2a-FK persistently prion-infected cells
To elucidate the detailed degradation mechanism of PrP Sc in autophagy-lysosomal pathway, we analyzed the effects of rapamycin, a widely used macroautophagy activator that inhibits The results in the graph are the mean ± SD of at least three independent experiments. *p < 0.05 and **p < 0.01 (one-way ANOVA followed by Tukey's test).
doi:10.1371/journal.pone.0137958.g001 mTOR, and 3-methyladenine (3MA), a selective inhibitor of macroautophagy that blocks the early stage of the autophagy-lysosomal pathway, by inhibiting type-III phosphatidylinositol 3-kinase (PI3K), on PrP Sc levels in N2a-FK cells. The cells were treated with 1 to 10 mM 3MA and 0.2 to 1 μM rapamycin for 48 h. A dose-dependent increment of PrP Sc was observed in N2a-FK cells treated with 3MA and a dose-dependent reduction in those treated with rapamycin (Fig 2 upper). By contrast, neither drug had any effect on PrP Sc in mouse neuroblastoma cells persistently infected with the scrapie-derived 22L or Chandler strain of prions (N2a-22L and-Ch cells) (Fig 2 middle and bottom).
To determine the level of autophagic activity in N2a-FK cells, we analyzed the expression of the autophagy-related molecules, LC3-II, Beclin-1 and the Atg12-Atg5 complex. The LC3-II / LC3-I ratio was 10-to 40-fold higher in rapamycin-treated cells, but was only slightly increased in 3MA-treated cells. Beclin-1 and Atg12-Atg5 increased in response to rapamycin and decreased in response to 3MA, both in a dose-dependent manner (S1 Fig). Moreover, Fig 2. PrP Sc in N2a-FK cells is markedly degraded by the autophagy pathway. Persistently prion-infected cells were treated with 1 to 10 mM of 3-methyladenine (3MA) and 0.2 to 1 μM rapamycin (Rap) for 48 h. Proteinase K (PK)-treated N2a-FK, -22L and-Ch cells, which vary in the prion strains, were loaded at concentrations of 100, 60 and 35 μg protein per lane onto a 15% polyacrylamide gels and subjected to SDS-PAGE. PrP Sc was detected by western blotting using an anti-PrP antibody. For densitometric analysis, the images were scanned and the intensity of each band on the western blotting was quantified with respect to PrP Sc expression levels in drug-treated prion-infected cells, respectively. The results are representative of at least three independent experiments, with each experiment performed in triplicate. *p < 0.05 and **p < 0.01 (one-way ANOVA followed by Tukey's test).
doi:10.1371/journal.pone.0137958.g002 rapamycin treatment also caused dose-dependent decreases in phosphorylated S6 ribosomal protein and phosphorylated eIF4B protein, two markers of the mTOR pathway (S1 Fig). Furthermore, we also investigated about PrP Sc degradation using Atg5 shRNA and observed the increment of PrP Sc in N2a-FK cells, indicating that the result of other method was also similar to that of chemical compound (S2A Fig). Accordingly, we examined autolysosome, which is the final structure in the autophagy system, levels by first altering the autophagic vesicles such that they expressed an EGFP-fused LC3 transgene and then staining the autolysosomes with the autofluorescent marker monodansylcadaverine (MDC), which specifically detects autolysosomes [28]. LC3-positive vesicles were increased in N2a-FK cells treated with rapamycin for 24 h, but the increment was suppressed by 3MA (Fig 3A). In previous studies in other mammalian cells, the number of MDCpositive cells in cultures treated with either oridonin or starvation via amino acid deprivation, both of which induce autophagy, was reduced by co-treatment with 3MA [28,29]. As controls in our study, MDC-labelled vesicles were abundant in N2a-FK cells incubated for 8 h in Hank's balanced salt solution (HBSS) to induce starvation, whereas the number of vesicles was significantly reduced in starved cells co-treated with 3MA ( S3 Fig). Similarly, vesicle induction in rapamycin-treated N2a-FK cells was strikingly reduced when the cells were co-treated with 3MA for 24 h (Fig 3B). Thus, in our system both rapamycin and starvation strongly induced autophagy in persistently prion-infected cells.
The localization of PrP Sc after autophagy induction was determined by immunofluorescence staining of PrP Sc in N2a-FK cells treated with the protein denaturant guanidine [30,31]. In this method, PrP C in N2a-58 prion-uninfected cells were not detected. After 24 h, the accumulation of PrP Sc in the cytoplasm was reduced compared to 0 h ( Fig 3C). Meanwhile, in rapamycin-treated N2a-22L cells high levels of PrP Sc accumulated that strongly localized in the vicinity of nuclei after 24 h (S4 Fig). In HBSS-starved N2a-FK cells, the reduction of PrP Sc was similar to that of rapamycin-treated cells after 24 h (S5 Fig), indicating that PrP Sc might be selectively degraded by the autophagolysosome system in N2a-FK cells.
The intracellular signalling cascade in the autophagic pathway contributes to PrP Sc clearance in N2a-FK cells Next, to investigate whether the intracellular signalling cascade of the autophagic pathway had an effect on the degradation of PrP Sc , we tested the effects of PI3K inhibitor, LY294002. The pharmacological properties of LY294002 include an ability to inhibit the macroautophagic degradation of proteins in mammalian cells at autophagic sequestration step [32,33] and to block PI3K-dependent Akt phosphorylation and kinase activity [34,35]. The effect of LY294002 as a PI3K inhibitor was confirmed by the dose-dependent decrease in phosphorylated Akt levels in N2a-FK cells after 48 h (Fig 4A). In similarly treated cells, PrP Sc levels significantly and dosedependently increased (Fig 4B), confirming the role of PI3K in PrP Sc degradation. Next, to investigate the relationship between PrP Sc degradation and the intracellular signalling cascade downstream of PI3K, we assessed the effects of PD98059, an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK: MEK), on PrP Sc degradation. MEK signalling regulates autophagy by regulating Beclin-1, through the mTOR pathway [36,37]. PD98059 inhibits MEK signalling such that the phosphorylation of downstream ERK 1/2 (ERK1/2: P44/P42) is prevented. A role for MEK signalling was verified by the decrease in phosphorylated MAPK levels in N2a-FK cells (Fig 4A). When these cells were treated with 50 μM of the inhibitor for 48 h, PrP Sc levels were significantly increased (Fig 4B). Subsequently, we asked whether PrP Sc degradation by rapamycin-induced autophagy was blocked by these inhibitors of autophagy-related signalling cascades (Fig 5). Indeed, in N2a-FK cells the rapamycin-induced decrease in PrP Sc was significantly overcome by co-treatment with either LY294002 (10 μM) or PD98059 (50 μM) for 48 h (Fig 5A). Moreover, the reduction in PrP Sc was significantly recovered in cells co-incubated for 48 h with rapamycin and the lysosomal inhibitor NH 4 Cl (Fig 5B). These results provide evidence of the involvement of PI3K and MEK signalling in autophagy-mediated degradation of PrP Sc in N2a-FK cells.

Nearly all of the PrP Sc in N2a-FK cells is degraded in lysosomes
Autophagolysosomes are the product of autophagosome and lysosome fusion and their formation is a necessary step in protein clearance by the autophagy system. In HBSS-starved N2a-FK cells, the accumulation of PrP Sc was co-localized in lysosomes in the vicinity of nuclei after 8 h, and the PrP Sc was reduced after 24 h like rapamycin treatment (Fig 3C and S5 Fig). To confirm the localization of PrP Sc within the lysosomal fraction after autophagic induction, we followed the subcellular localization of PrP Sc and the lysosomal marker Lamp1 in N2a-FK cells by isolating a pure lysosomal fraction. Under control conditions, PrP Sc localized to the same lysosomerich fractions as Lamp1 (Fig 6A, left, lanes 1 to 3). However, in N2a-FK cells in which autophagy was induced by starvation conditions (8 h of HBSS treatment), although total PrP, Lamp1 and β-actin band intensities were similar, there was little PrP Sc in the Lamp1 fractions (Fig 6A,  right, lanes 1 to 3). Quantification of the ratio of PrP Sc in each fraction (F1 to F4), based on band intensity, showed significantly less PrP Sc in the lysosome-rich fractions (F1 and F2) of HBSS-starved cells treatment than in those of control cells (Fig 6B), suggesting that under normal conditions PrP Sc might be almost completely degraded in the lysosomal fraction, via the autophagic system.

Discussion
In this study, PrP Sc in N2a-FK cells was degraded by an autophagic process enhanced by the inducer rapamycin. By contrast, rapamycin had no effect on PrP C in N2a-58 cells (S2B Fig), indicating that autophagy might be one of the degradation system involved in PrP Sc but not in PrP C degradation. Recently, Xu Y, et al. reported that scrapie-derived PrP Sc is degraded by mTOR-related autophagic system via FBXW7 protein [38]. It is possible that GSS-derived PrP Sc may also relate to the degradation system mediating FBXW7, but the elucidation of its option will need to be further investigated.
The site-specific proteolysis of LC3-I (18 kDa) to LC3-II (16 kDa) is indicative of autophagic activity, as are Beclin-1 and the Atg12-Atg5 complex. The latter are required for nucleation of the phagophore and maturation of the autolysosome in mediating autophagosome formation. In our study, LC3 activation in rapamycin-treated N2a-FK cells was approximately 40-fold stronger than in non-treated control cells (S1 Fig). Rapamycin also increased the levels of Beclin-1 and the Atg12-Atg5 complex, both of which were reduced by 3MA treatment, and plasmid pcDNA 3.1) and the morphological changes in LC3-positive granular vesicles were followed. (B) Autophagolysosomes in cells treated with 1 μM rapamycin for 24 h were visualized using 0.1 mM of monodansylcadaverine (MDC) for 30 min (left, three panels per group). To inhibit rapamycin-induced autophagy, N2a-FK cells were co-treated with 10 mM 3MA. Scale-bars represent 10 μm. To quantify the average number of MDC-labeled autolysosomes in a single cell, the vesicles in the treated cells were shown as a graph represented by the mean ± SD of three independent experiments (right). **p < 0.01 (one-way ANOVA followed by Tukey's test). (C) N2a-FK cells treated with 1 μM rapamycin for 24 h were pre-treated with 3 M guanidine thiocyanate prior to the antibody reaction. PrP Sc in cells was detected using the SAF61 antibody (green) and N2a-58 cells were stained as a negative control. Cell nuclei were counterstained with DAPI (blue). The cells were visualized by confocal laser scanning microscopy. Differential interference contrast (DIC) images were obtained to confirm the consistency of the experimental condition. Scale-bars represent 10 μm.
decreased markers of the mTOR signalling as phosphorylated S6 ribosomal protein and phosphorylated eIF4B protein (S1 Fig). It already have been confirmed that expression of Beclin1 PK-treated or-untreated samples were applied at concentrations of 100 and 50 μg protein per lane onto a 15% polyacrylamide gel and subjected to SDS-PAGE. The proteins were analyzed by western blotting using anti-PrP, anti-Akt, anti-phosphorylated Akt (to determine the Akt activation level), anti-p44/p42 MAPK, anti-phosphorylated p44/p42 MAPK (to determine the p44/p42 MAPK activation level) and anti-β-actin antibodies. (B) The effect of these drugs on PrP Sc was determined by quantifying the PrP Sc band intensities as a percentage of those of the negative controls. The results in the graph are the mean ± SD of at least three independent experiments. *p < 0.05 and **p < 0.01 (one-way ANOVA followed by Tukey's test).
doi:10.1371/journal.pone.0137958.g004 and Atg5 were increased by starvation-induced activation of cardiac autophagy in mice and that of neonatal mice to supply the amino acids for energy homeostasis [39,40]. In N2a-FK cells, some autophagy-related proteins also may be produced by the same mechanism after autophagic activation. However, the detailed mechanism remains to be determined. These results indicate that PrP Sc in N2a-FK cells might be degraded by the canonical autophagic system, which is induced by rapamycin.
In autophagic signalling, LY294002 specifically blocks PI3K-dependent Akt phosphorylation and kinase activity, resulting in a drastic inhibition of autophagy [32][33][34][35]. In another  study, the activation of autophagy by HBSS was more strongly prevented by LY294002 than by wortmannin, another inhibitor of PI3K [37]. Here we showed that PrP Sc levels in N2a-FK cells are regulated by an autophagic system involving PI3K and MEK signalling (Figs 4 and 5). In Beclin-1-dependent autophagic activation, an important pathway is that in which Bcl2 is phosphorylated by phosphorylated ERK. In HBSS-starved cells, phosphorylated ERK is suppressed by PD98059 via the inhibition of P44/P42 MAPK, and autophagic sequestration is prevented by increment of Beclin-1 and unphosphorylated Bcl2 complex [37]. In autophagic sequestration inhibitory system by nitric oxide (NO), disruption of hVps34/Beclin-1 complex formation is leaded by increment of Beclin-1/Bcl2 interaction after phosphorylated Bcl2 reduction due to inhibition of JNK1 by NO [41]. It is furthermore likely that PD98059 inhibits autophagy via MAPK-regulated mTOR signalling [42]. Thus, the mechanism underlying the autophagic degradation of PrP Sc in N2a-FK seems to involve a pathway that includes mTOR, PI3K and MEK signalling.
In this study, we were not able to find the evidence of the autophagic degradation of PrP Sc in N2a-Ch and N2a-22L cells, whereas it has reported that PrP Sc in RML-inefcted N2a cells significantly reduced after rapamycin treatment [17,19]. Likewise, we previously reported that PrP Sc in N2a-FK cells was degraded by autophagy in a process activated by FK506 (tacrolimus) [21], while Karapetyan et al. showed that tacrolimus reduces PrP Sc in RML-infected cells in the absence of autophagic activation [43]. According to Kawasaki et al., compound-B strongly reduces PrP Sc of the RML strain but had only marginally effects on prion strains 22L and FK-1 [44]. These results raise the question, why does the degradation of PrP Sc differ for each prion strain? A difference in the biochemical properties of PrP Sc from different strains such that they differ in their sensitivity to proteolytic enzymes is unlikely because there is no difference in the conformational stabilities of PrP Sc in N2a-22L and-FK cells following their denaturation by guanidine hydrochloride [45]. It was previously reported that low-dose and long-term treatment of 22L-and RML-prion infected GT-1 cells with rapamycin reduces PrP Sc levels, by suppressing protein translation [46]. However, similar experiments were beyond the scope of this study. Evidence of alternative pathways of PrP Sc degradation comes from a study showing that both tamoxifen and 4-hydroxytamoxifen decrease PrP Sc in an autophagy-independent manner, by bringing PrP to lysosomes [47], indicating that the degradation mechanism of PrP Sc might have various pathway. Thus, these verification need to be further examined using various prion strains.
In summary, our results demonstrated that PrP Sc in Fukuoka-1 prion strain-derived cells might be efficiently degraded in canonical autophagic system-dependently compared with those in other prion strains-derived cells. This finding indicates that we might have to alter the therapeutic strategies by patients with Creutzfeldt-Jakob disease, and suggests the need for new therapeutic strategies, such as the use of autophagy-inducing compounds, in patients with Gerstmann-Sträussler-Scheinker syndrome. Further studies of the autophagy-mediated degradation of PrP Sc will provide additional therapeutic insights and the necessary advances to one day allow the complete cure of prion diseases. 1 μM rapamycin (Rap) for 48 h. PK-untreated samples containing 10 to 30 μg of protein were used to investigate the expression of total PrP, LC3, Beclin-1 and the Atg12-Atg5 complex as indicators of autophagic flux. To confirm the levels of autophagy, LC3-II/-I ratio was assessed by LC3 blotting. β-actin was used as the internal standard for each sample. Phosphorylated S6 ribosomal protein (Ser235 and Ser236) (p-S6) and phosphorylated eIF4B protein (Ser422) (p-eIF4B) levels, as indicators of a rapamycin effect, were performed with western blotting. The rapamycin-treated the N2a-22L cells were pre-treated with 3M guanidine thiocyanate, prior to antibody reaction. PrP Sc was detected by SAF61 antibody (green). Cell nuclei were counterstained with DAPI (blue). All images were visualized by CLSM700. The differential interference contrast (DIC) images were demonstrated to confirm the consistency in all experimental condition. Scale-bars represent 10 μm.