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Characterization of membrane penetration and cytotoxicity of C9orf72-encoding arginine-rich dipeptides

Characterization of membrane penetration and cytotoxicity of C9orf72-encoding arginine-rich... www.nature.com/scientificreports OPEN Characterization of membrane penetration and cytotoxicity of C9orf72-encoding arginine-rich Received: 19 March 2018 dipeptides Accepted: 2 August 2018 Published: xx xx xxxx 1 1 2 3 4 Kohsuke Kanekura , Yuichiro Harada , Mao Fujimoto , Takuya Yagi , Yuhei Hayamizu , 5 1 Kentaro Nagaoka & Masahiko Kuroda Cell-penetrating peptides (CPPs) including arginine-rich peptides are attracting a lot of attention due to their potential as a novel intracellular drug delivery tool without substantial toxicity. On the other hand, disease-associated arginine-rich CPPs, such as poly-PR and poly-GR translated from C9orf72 gene, also efficiently enter neuronal cells and then kill them. Although both non-harmful CPPs and harmful poly-PR/GR penetrate the plasma membrane using same arginine residues, little is known about the factors which determine the toxicity of the pathogenic CPPs. Here, we show that poly-PR and poly-GR, but not other Arg-rich CPPs, specifically distributed to nucleolus via interaction with RNA. Importantly, C9orf72-dipeptides, but not other Arg-rich CPPs, caused inhibition of protein translation and cell death. Raising extracellular pH enhanced the cell penetration of poly-PR. The repeat number of (PR) affected the secondary structure and determined the intracellular delivery rate and neurotoxicity, and enforced intracellular delivery of non-penetrating short poly-PR peptide caused cell death, suggesting that modulation of extracellular environment to inhibit the uptake of Arg-rich dipeptides might be a drug target against poly-PR/GR-mediated neurotoxicity. Dipeptide-repeat proteins (DRPs), translated through repeat-associated non-ATG (RAN) translation from path- ogenic G4C2 expansions in C9orf72 gene, are supposed to play a pivotal role in development of C9orf72-related 1–4 neurological disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) . Among 5 species of DRPs (poly-GA, GP, PR, GR and PA) translated from sense and antisense of G4C2 expan- sions, especially arginine-rich DRPs (poly-PR and poly-GR) are of interest because they caused neuronal loss 3,4 in vitro and in vivo . When these arginine-rich DRPs are applied to the culture medium of neuronal cells, they penetrate the plasma membrane and directly affect intracellular machineries, thus they are classified as 4,5 cell-penetrating peptides (CPPs) . The first CPP discovered was trans-activating transcription activator (TAT) peptide from human immuno- deficiency virus-1 (HIV-1) whose amino acid sequence is shown in Fig.  1A . Since the discovery, a variety of CPPs have been artificially designed (e.g. R8, R12) or identified from natural organisms such as Flock house virus (FHV) . Because of their efficient translocation through biological membranes without significant cellu- lar damage, many of them have been extensively investigated as tools for intracellular delivery of compounds 8–10 including nucleic acids, proteins and drugs . Although several hypotheses such as the endocytotic pathway, energy-independent uptake and cell surface molecules-mediated uptake have been proposed, precise mecha- nisms how these CPPs penetrate the plasma membrane still remain to be investigated . 4,5 Similar to these non-harmful CPPs, ALS-causing poly-PR/GR efficiently enter cells . Once they penetrate the plasma membrane, intracellular poly-PR/GR localize to membrane-less organelles and inhibit their func- tions, resulting in disturbance of protein translation, abnormal formation and malfunction of stress granules, 3–5,12,13 mis-splicing of mRNAs, and ultimately neuronal death in vitro and in vivo . Despite their importance in 1 2 Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan. School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan. Department of Neurology, KEIO University, Tokyo, Japan. 4 5 Department of Organic and Polymeric Materials, Tokyo Institute of Technology, Tokyo, Japan. Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Tokyo, Japan. Correspondence and requests for materials should be addressed to K.K. (email: [email protected]) SCIENTIfIC Repo R Ts | (2018) 8:12740 | DOI:10.1038/s41598-018-31096-z 1 www.nature.com/scientificreports/ AC z-stack TAT GRKKRRQRRRPPQ R12 RRRRRRRRRRRR FHV RRRRNRTRRNRRRVR (PR)20 PRPRPR........PRPRPR (GR)20 GRGRGR........GRGRGR Merge DAPI FITCZoom Merge (PR)20B23 TAT R12 E MergeGFP-(PR)50 (PR)20 F MergeGFP-(GR)50 (GR)20 FHV Figure 1. Poly-PR localized to nucleoli. (A) Amino acid sequences of cell penetrating peptides (CPPs) used in this study. (B) Confocal imaging of live HEK293 cells treated with 10 μM of indicated FITC-labeled CPPs. Nucleus was visualized by Nucblue live dye. (C) Z-stack imaging of live HEK293 cells treated with FITC- (PR)20. (D) Immunocytochemistry of HEK293 cells treated with FITC-(PR)20, immunostained with anti-B23/ nucleophosmin antibody. Nucleus was visualized by DAPI. (E) Hela cells were transfected with GFP-(PR)50 repeats. (F) Hela cells were transfected with GFP-(GR)50 repeats. White arrows show the cells with both nucleolar and cytosolic staining and white arrowheads indicate the cells with cytosolic staining. Scale bar shows 20 μm. neuroscience and drug delivery research fields, the mechanism underlying the membrane penetration as well as the correlation between membrane penetration and neurotoxicity of C9orf72-DRPs remains undetermined. Here we identified key factors which ae ff ct membrane penetration and neurotoxicity by poly-PR/GR peptides. First, we examined a series of arginine-rich CPPs and found that poly-PR and poly-GR, but not other CPPs, dis- tributed to nucleolus. Next, in vitro translation assay and lactate dehydrogenase (LDH) release assay showed that C9orf72-DRPs, but no other CPPs, inhibited protein translation and caused cell death by aberrant interaction with RNA. The cell penetration rate of poly-PR was determined by length of repeats and extracellular pH, and enforced delivery of non-penetrating short poly-PR peptide caused cell death, suggesting that inhibition of intra- cellular delivery of DRPs might be a novel drug target for C9orf72-mediated ALS. Results Cell penetrating peptides (CPP) which can cross the plasma membrane bilayer are subcategorized by their bio- 14,15 chemical characteristics such as hydrophilic CPPs, hydrophobic CPPs and amphipathic CPPs . C9orf72-related Arg-rich dipeptides are classified as the hydrophilic cationic CPP because 50% of their content is polar argi - nine. The most established cationic CPP is TAT peptide (GRKKRRQRRRPPQ: 13 a. a.) identified from HIV-1. Since the discovery of TAT, many cationic CPPs including R12 (RRRRRRRRRRRR: 12 a. a.) and FHV peptide (RRRRNRTRRNRRRVR:15 a. a.) were identified and investigated as possible tools for the drug delivery system . e ad Th vantage of these CPPs as a drug delivery carrier is their high ability to deliver molecules including proteins and nucleotides across biological membranes without cytotoxicity. Although C9orf72-encoding Arg-rich dipep- tides should have similar biochemical characteristics with these CPPs, C9orf72-related DRPs have been shown to cause cell death once they penetrate the plasma membrane . SCIENTIfIC Repo R Ts | (2018) 8:12740 | DOI:10.1038/s41598-018-31096-z 2 (PR)20 www.nature.com/scientificreports/ Figure 2. Poly-PR interacts with RNA. (A) Confocal imaging of HEK293 cells fixed/permeabilized and stained with FITC-(PR)20 in the absence of pretreatment of RNase A. (B) Colocalization analysis of signals from DAPI staining and FITC-(PR)20 in the HEK293 cells in the absence of pretreatment of RNase A. (C) Confocal imaging of HEK293 cells fixed/permeabilized and stained with FITC-(PR)20 in the presence of pretreatment of RNase A. (D) Colocalization analysis of signals from DAPI staining and FITC-(PR)20 in the HEK293 cells in the presence of pretreatment of RNase A. (E) Liquid-liquid phase separation of tetramethylrhodamine (TAMRA)-labeled random 15-mer RNA by FITC-(PR)20. (F) Agarose gel electrophoresis of human ribosomal RNA mixed with (PR)20 peptide at indicated concentration (μM). (G) Structure of RNA (base: adenine). (H) Crosslinking analysis of HA-(PR)20 peptide mixed with adenine, adenosine, ribose or ribose-phosphate in the presence or absence of RNA (poly-A). Arrowheads indicate crosslinked oligomer of HA-(PR)20 peptides. *polymerized HA-(PR)20. To clarify the difference between the C9orf72-related DRPs and non-harmful CPPs, we synthesized well-known cationic CPPs including TAT peptide, R12 peptide and FHV peptide and C9orf72-derived DRPs, (PR)20 peptide (40 a. a.) and (GR)20 peptide (40 a. a.) (Fig. 1A), and examined their penetration to human embryonic kidney (HEK) 293 cells. All of the CPPs examined were uptaken by HEK293 cells as expected (Fig. 1B). The confocal imaging revealed the subcellular localization of FITC-labeled CPPs. TAT made cytosolic large dots, R12 and FHV diffusely localized to both nuclear and cytosol with some punctate structures in cyto- sol, (PR)20 mainly localized to the nucleus and especially accumulated to round-shaped nucleoli and (GR)20 localized to both nucleoli and cytosol (Fig. 1B). The nucleolar localization of (PR)20 was also confirmed with Z-stack imaging and colocalization with a nucleolar marker B23/nucleophosmin (Fig. 1C,D) as reported pre- 4,12 viously . To deny the possibility that nucleolar distribution of poly-PR and poly-GR was an artifact because these peptides were added extracellularly, we overexpressed green fluorescent protein (GFP)-tagged (PR)50 repeat protein and GFP-tagged (GR)50 repeat protein in HEK293 cells and confirmed that both GFP-(PR)50 and GFP-(GR)50 distributed to nucleoli (Fig. 1E,F). Consistent with the subcellular localization of FITC-peptides, GFP-(PR)50 showed almost exclusive distribution to nucleoli and GFP-(GR)50 distributed to both nucleoli and cytosol (Fig. 1E,F). e Th nucleolar distribution of the FITC-labeled DRPs and GFP-fused DRPs implies existence of molecules with high affinity to C9orf72-DRPs in the nucleolus. The nucleolus is a subnuclear membrane-less compartment which is the site for transcription of ribosomal RNA and assembly of ribosome . Because the nucleolus contains a large amount of ribosomal RNA accounting for 90% of total RNA and a number of RNA-binding proteins, the concen- tration of intranucleolar DNA is low, since DNA staining by DAPI excludes the nucleolus . In our previous study, we demonstrated liquid chromatography mass spectrometry to comprehensively identify interactors of poly-PR and found that poly-PR interacted with RNA-binding proteins. Immunoprecipitation assays in the presence of RNAse diminished some of the interactions, suggesting that poly-PR directly binds to RNA . In order to examine whether (PR)20 accumulated to the nucleoli through interaction with RNA or with RNA-binding proteins, we fixed the HEK293 cells with 4% paraformaldehyde (PFA) and incubated the cells with FITC-(PR)20 with or with- out RNAse treatment (Fig. 2A–D). Fixation of HEK293 cells slightly increased cytosolic staining by FITC-(PR)20 compared with the staining of live HEK293 cells (Fig. 1B) but most of (PR)20 localized to nucleoli (Fig. 2A). The RNAse treatment diminished the specific distribution of (PR)20 to nucleoli and increased diffuse nuclear staining SCIENTIfIC Repo R Ts | (2018) 8:12740 | DOI:10.1038/s41598-018-31096-z 3 www.nature.com/scientificreports/ Figure 3. Poly-PR caused cell death through inhibition of protein translation. (A) Scheme of real-time monitoring of protein translation using in vitro translation (IVT) system. tGFP: turboGFP. (B) Real time monitoring of tGFP signal in the IVT assay. Signals from tGFP were measured every 1 min. (C) Fluorescence intensity of translated tGFP in the IVT assay treated with 100 μM of each peptide. N = 3. (D) Cytotoxicity of each CPPs were monitored by release of LDH. NSC34 cells were treated with each CPP at indicated concentration for 24 hr. N = 6. (E) Cytotoxicity of each CPPs were monitored by Celltox glo luminescence. NSC34 cells were treated with each CPP at indicated concentration for 24 hr. N = 6. Each experiment was independently repeated at least 3 times. Asterisks indicate a significant difference analyzed by one-way ANOVA followed by Dunnett’s test (**p < 0.01, *p < 0.05). (Fig. 2C). Colocalization analysis showed that the green fluorescence from FITC-(PR)20 and blue fluorescence from DAPI did not correlate before the RNAse treatment (slope m = −0.11), and the RNAse treatment drastically change the correlation manner (slope m = 0.85), implying that (PR)20 distributed to nucleoli through interaction with RNA, presumably ribosomal RNA, abundant in the nucleolus (Fig. 2B,D). We next investigated how (PR)20 interacts with RNA. It has been reported that poly-PR forms phase-separated 12,13 liquid droplets with anionic molecules in vitro . Therefore, we first tested if (PR)20 induces phase-separated liquid droplets with RNA. For this purpose, we synthesized tetramethylrhodamine (TAMRA)-labeled random 15-mer RNA and mixed the RNA with FITC-labeled (PR)20. As expected, (PR)20 induced the round-shaped droplet formation (Fig. 2E), indicating that (PR)20 induced phase separation of RNA. We also confirmed that (PR)20 induced separation of dense liquid phase fusing each other, and the separated droplets showed high cir- cularity and roundness, suggesting minimization of surface tension (Figs S1A and 1B). To examine whether (PR)20-RNA complex maintains the liquid-like aspect in live cells, we demonstrated the fluorescence recovery after photobleaching (FRAP) assay and confirmed that the nucleolar (PR)20 peptide maintained fluidity even aer 24 ft hr incubation (Fig. S1C,D). Next, we tested if (PR)20 interacts with ribosomal RNA. The ribosomal RNA is negatively charged and runs to anode when applied to Tris-acetate-EDTA (TAE)-buffered agarose gel elec- trophoresis. (PR)20 is positively charged and thus possibly neutralizes the negative charge of ribosomal RNA, inhibiting the running during the electrophoresis. To test if this hypothesis is the case, we mixed the human ribosomal RNA extracted from HEK293 cells and (PR)20 peptide then applied the mixture to the agarose gel elec- trophoresis. As expected, (PR)20 inhibited the running of rRNA to the anode and the rRNA resided to the well applied (Fig. 2F). To support this, we next tested which component of RNA is the binding target for poly-(PR) peptide. For this end, we used a crosslinking assay because RNA enhances polymerization of poly-(PR) . RNA is composed of nucleotides containing bases, sugars and phosphates (Fig. 2G), and each component was mixed with (PR)20 in the presence or absence of poly-A RNA as an enhancer for polymerization. Ribose-3′ phosphate, but not ribose alone, enhanced oligomerization of (PR)20, suggested that the phosphate attracted (PR)20 peptide presumably due to the negative charge (Fig. 2H). As we previously reported, Arg-rich C9orf72 dipeptides inhibit protein translation and cause neuronal cell death . Indeed, ectopic expression of GFP-(PR)50 and GFP-(GR)50 inhibited protein translation in Hela cells (Fig. S2A). To investigate if other cationic CPPs ae ff ct protein translation and exert neurotoxicity as well, we tested the effects of the cationic CPPs on protein translation. For this end, we adopted a cell-free in vitro translation (IVT) system to avoid possible secondary effects of cytotoxicity by the CPPs (Fig.  3A). The efficiency of protein SCIENTIfIC Repo R Ts | (2018) 8:12740 | DOI:10.1038/s41598-018-31096-z 4 www.nature.com/scientificreports/ Figure 4. Extracellular pH ae ff cted penetration of poly-PR. (A) Peptides absorption assay using FITC-R12 or FITC-(PR)20. The pictures show the actual images of the absorbed peptides. The white arrowheads indicate octanol phase and white arrows show aqueous buffer phase. The bar graph shows the fluorescence of FITC- labeled peptides absorbed in the octanol fractions measured by a plate reader. (B) Hela cells were treated with PBS with different pH containing 2 μM FITC-R12 or FITC-(PR)20. Nucleus was visualized by Nucblue live dye. (C) Cell penetration rate of FITC-(PR)20 incubated at 37°C or 4°C or pH9 in HEK293 cells, measured by a plate reader. Asterisks indicate a significant difference analyzed by one-way ANOVA followed by Dunnett’s test (**p < 0.01, *p < 0.05). n.s.; not significant. translation was real-timely monitored by fluorescence emitted from translated turboGFP which does not have significant delay for maturation like jellyfish-derived GFP . As expected, (PR)20 (Arg content: 50%), but not TAT (Arg content: 46%), R12 (Arg content: 100%) or FHV (Arg content: 73%), efficiently inhibited protein trans- lation in vitro (Fig. 3B,C). Next, motor neuronal NSC34 cells were treated with these CPPs and cellular damages were measured by LDH release assay and Cytotox glo assay (Fig. 3D,E). Consistent with the results of IVT assay, (PR)20, but not other CPPs, killed the cells, indicating that not only the ratio of cationic amino acid content but structural factors underlie the cell death by poly-PR. Next, we investigated the molecular mechanism how poly-PR penetrates the plasma membrane. It has been shown that cationic CPPs cross the plasma membrane via both the energy-independent translocation pathway and energy-dependent endocytotic pathway. To confirm if this is the case with poly-PR, we first tested the effect of fatty acids and pH gradient at the cell membrane on the translocation of the (PR)20 peptide, because these two components were reported to ae ff ct the uptake of Arg-rich peptides by live cells through the energy-independent translocation . To see the effects of fatty acids and pH gradient on the translocation of the peptides, we adopted a simple cell free model to show the distribution of fluorescent CPPs between an aqueous buffer and hydro- phobic octanol containing oleic acid, mimicking lipid bilayer environment . When pH was increased higher than 7.5, R12 started to be absorbed into the octanol phase and most of R12 peptide was absorbed into octanol phase at pH 9.0 (Fig. 4A, upper panels). The pH gradient also enhanced the absorption of (PR)20 peptide into the octanol phase and higher pH increased the absorption of the peptide (Fig. 4A, lower panels), indicating that extracellular pH condition may influence the uptake of Arg-rich dipeptides in hydrophobic environments such as the lipid bilayer. e Th change of pH did not enhance the absorption of non-permeable human insulin peptide into the octanol phase (Fig. S2B), indicating that the effect of pH was specific to the cationic CPPs. Next, we chal- lenged Hela cells with buffers of different pH and treated with FITC-R12 or FITC-(PR)20 (Fig.  4B). Consistent with the results from the cell-free model (Fig. 4A) and previous study , higher pH increased the distribution of SCIENTIfIC Repo R Ts | (2018) 8:12740 | DOI:10.1038/s41598-018-31096-z 5 www.nature.com/scientificreports/ Figure 5. Repeat lengths ae ff cted the cytotoxicity of poly-PR peptides. (A,B) Fluorescence intensity of translated tGFP in the IVT assay treated with 100 μM of each peptide. N = 3. (C) Cytotoxicity of poly-PR peptides with different repeat lengths were monitored by release of LDH. NSC34 cells were treated with each peptide at 5 μM or 10 μM for 24 hr. N = 6. (D) Confocal imaging of HEK293 cells stained with 10 μM of FITC- (PR)12 or FITC-(PR)20 before or aer t ft he fixation/permeabilization step. (E) CD spectra of R12, (PR)12 and (PR)20 in Tris-buffer (pH7.4). (F) CD spectra of R12, (PR)12 and (PR)20 in 1 v/w% SDS-Tris buffer (pH7.4). Asterisks indicate a significant difference analyzed by one-way ANOVA followed by Dunnett’s test (**p < 0.01, *p < 0.05). FITC-labeled CPPs into the cells. We also investigated whether cellular uptake of (PR)20 is ae ff cted by inhibition of energy-dependent endocytosis. It has been established that the endocytotic pathway is temperature-sensitive , thus we tested if lowering temperature affects the uptake of the peptide. When the cells were incubated with FITC-(PR)20 at 4 °C, the fluorescent signal was comparable with the signal from the cells incubated at 37 °C (Fig. 4C). es Th e results suggest that (PR)20 penetrated the plasma membrane via the direct translocation rather than the energy-dependent endocytosis. Next, we investigated the effect of repeat length of PR on the cell toxicity. IVT assay indicated that even (PR)8 blocked the protein synthesis and longer repeat tended to have higher inhibition rate (Fig. 5A,B). We also exam- ined the effect of repeat length on the poly-GR toxicity. We performed the IVT assay with poly-GR peptides with 12 repeats and 20 repeats. In this assay, amyloid β (40 a.a.) was used as a negative control peptide with length of 40 a. a., same as (GR)20 (Fig. S3A,B). (GR)20 modestly inhibited protein translation and we did not see inhibitory effect of (GR)12 or amyloid β on protein translation, indicating that (1) poly-GR also inhibited protein translation in a length-dependent manner, (2) the inhibitory ee ff ct of (GR)20 is specific because amyloid β did not inhibit protein translation, (3) poly-PR is more potent in the inhibition of translation. Because there was a significant dif- ference in the degree of translation inhibition between (PR)20 and (GR)20, we next compared their cytotoxicity. SCIENTIfIC Repo R Ts | (2018) 8:12740 | DOI:10.1038/s41598-018-31096-z 6 www.nature.com/scientificreports/ Figure 6. Enforced delivery of poly-(PR)12 rescued the cytotoxicity. (A) Scheme of GE11 peptide-mediated endocytotic delivery system. (B) Cytotoxicity of GE11 peptide, (PR)12 peptide or GE11-(PR)12 peptides were monitored by release of LDH. Human fibroblasts were treated with each peptide at 10 μM, 20 μM or 50 μM for 48 hr. N = 6. (C) Microscopic image of human fibroblasts treated with 50 μM of each peptide for 48 hr. Each experiment was independently repeated at least 3 times. Asterisks indicate a significant difference analyzed by one-way ANOVA followed by Dunnett’s test (**p < 0.01, *p < 0.05). First, we treated NSC34 cells with FITC-conjugated DRPs and compare their penetration rate. As Fig. S3C shows, (PR)20 entered NSC34 cells more efficiently, but (GR)20 also entered the cells as well. Next, we treated NSC34 cells with (PR)20 or (GR)20 and measured their cytotoxicity. Consistent with the rate of translation inhibition as well as the efficiency of penetration, (PR)20 exerted stronger cytotoxicity (Fig. S3D). Based on the results from IVT using poly-PR peptides with different length, even (PR)8 repeat might have cytotoxicity. However, when we challenged NSC34 cells with these poly-PR peptides with various lengths, minimal requirement of peptide repeat length for cytotoxicity was 16 and poly-PR peptides shorter than 16 had little tox- icity even with high concentration (Fig. 5C). To explain the discrepancy of translation inhibition and cytotoxicity, we investigated the rate of cell penetration of these poly-PR variants because the cationic CPP poly-arginine has 16,20 optimal length between 7–15 for efficient cellular uptake and thus short (PR) peptides might not satisfy the requirement for the cell penetration. To test whether the length of (PR) repeats influences the translocation of the peptides, we performed cell penetration assay using FITC-labeled (PR)12 and FITC-labeled (PR)20 with live or fixed/permeabilized HEK293 cells. When added to the culture medium of live HEK293 cells, FITC-(PR)20, but not FITC-(PR)12, penetrated the plasma membrane and accumulated to nucleoli (Fig. 5D). Aer ft the fixation/ permeabilization, both of the peptides entered the cells and accumulated to nucleoli, indicating that the difference of cell toxicity between (PR)12 and (PR)20 was determined by the degree of cell penetration rather than the degree of inhibition of translation, because the level of translation inhibition by (PR)12 was comparable to the level by (PR)20 (Fig. 5B). It has been reported that HA-tagged (GR)20 peptide has a shorter half-life (less than 1 hr) in the culture medium and that may contribute to the lessor toxicity of poly-GR . To deny the possibility that the lack of cell toxicity of shorter poly-PR peptides is due to their instability, we carried out a functional assay of (PR)12 aer ft incubation in the culture medium. Aer in ft cubation in the culture medium for 2 hr, (PR)12 still could accumulate to nucleoli of fixed/permeabilized HEK293 cells, showing that (PR)12 is stable in the culture medium (Fig. S4). To investigate if the secondary structure of the poly-PR peptides might ae ff ct their efficiency of translocation, we monitored circular dichroism (CD) spectra of R12, (PR)12 and (PR)20 because artificially designed CPP containing (Arg-Arg-Pro) repeats preferably formed α-helix-like structure in cellular environment and thus had enhanced penetration rate . Figure 5E,F showed CD spectra of R12, (PR)12 and (PR)20 in 20 mM Tris bue ff r (pH7.4) solution (Fig. 5E) and in 1.0 w/v% sodium dodecyl sulfate (SDS)-Tris buffer (pH7.4) solution (Fig.  5F), which mimicked amphipathic environments such as the plasma membrane. The spectra of all these peptides exhibited negative maxima at around 200 nm and weak negative maxima at 235 nm, suggesting these peptides basically formed random secondary structure under physiological condition. Mean residue molar ellipticity of (PR)12 and (PR20) were almost identical, indicating that both peptides had similar secondary structure. On the other hand, addition of SDS markedly influenced the secondary structure of poly-PR, resulting in the CD spectra with negative maxima at 210 nm and 230 nm, implying that poly-PR peptide formed a α-helix-like structure in amphipathic condition . The effect of SDS on the structural change was more obvious in (PR)20 compared with (PR)12 (Fig. 5F). Based on these findings, we hypothesized that (PR)12 may cause cell death if it is successfully delivered into cells by a molecular cargo because it still possessed the inhibitory effect on protein translation (Fig.  5A). To exam- ine this idea, we synthesized a (PR)12 peptide fused to GE11 peptide. GE11 sequence (YHWYGYTPQNVI: 12 a. a.) was found using a phage display screen to identify peptide sequences which have high affinity to epidermal growth factor receptor (EGFR) . Despite its high affinity to EGFR (Kd = 22 nM), its mitogenic activity is much lower than EGF. When GE11 binds to EGFR, GE11-EGFR complex becomes internalized thus GE11 is a potential drug delivery carrier into the cells (Fig. 6A). To examine whether internalized (PR)12 by fused GE11 sequence exerts cytotoxicity, we treated human fibroblasts with GE11, (PR)12 or GE11-(PR)12 peptide and incubated the cells for 48 hr. (PR)12 fused to GE11, but not GE11 or (PR)12 alone, caused cell death, showing that although it lacks the ability to enter cells, (PR)12 still possesses the cytotoxic property (Fig. 6B,C). SCIENTIfIC Repo R Ts | (2018) 8:12740 | DOI:10.1038/s41598-018-31096-z 7 www.nature.com/scientificreports/ Discussion Discovery of C9orf72 has opened a new horizon in the research field of ALS and FTD because it dominates the familial cases of these miserable disorders. From the standpoint of biochemistry and molecular biology, find- ings of RAN translation and associated arginine-containing dipeptides are of interests due to their intriguing characteristics including the unique translation system as well as cell penetration and cytotoxicity. Despite of the importance, little was known about the mechanism how Arg-rich dipeptides from C9orf72 penetrate the plasma membrane and cause cell death. In this study, we characterized the poly-PR and poly-GR peptides by comparing with other arginine-rich CPPs and found that only poly-PR and poly-GR, but not other Arg-rich CPPs inhibit protein translation, resulting in cell death. Extracellular pH ae ff cted the penetration rate of poly-PR and enforced delivery of non-penetrating short poly-PR peptide induced cytotoxicity, proposing a possible therapeutic target for C9orf72-mediated neurotoxicity. CPPs have been extensively investigated because of their potential as a drug delivery tool. Especially cationic CPPs are attracting attentions because of their high efficiency of translocation and lower toxicity, and intracellular 23–25 delivery of drugs, nucleotides and proteins by cationic CPPs have been achieved without significant cytotoxicity . e k Th ey component of the cationic CPPs is arginine residue which has a strong positive charge. When compared with CPPs containing another positive amino acid lysine, CPPs containing Arg have higher efficiency to enter cells so that CPPs with high Arg contents attract more attentions . The Arg content of (PR)20 (50%) and (GR)20 (50%) was comparable to those of other cationic CPPs, TAT (46%) and FHV (73%), and all of these peptides penetrated the plasma membrane and entered cells efficiently (Fig.  1B). However, (PR)12 which also has 50% of Arg content did not penetrate the plasma membrane, suggesting that not only the proportion of cationic amino acids but also other factors including the structure of peptide also regulate the efficiency. One such possible factor is formation of α-helix-like structure because artificially enhanced α -helix formation of cationic CPP results in better uptake rate . Because the repeat length of PR regulated the penetration efficiency, we speculated (PR)20 might form more α-helix-like structure than (PR)12 did, and CD spectra under amphipathic condition showing that (PR)20 tended to form α-helix-like structure more than (PR)12 supported the idea (Fig. 5F). Although cationic CPPs TAT, FHV and poly-PR/GR peptides, have been similarly reported to interact with 5,16,26 anionic molecules including RNA , their intracellular distributions were highly varied. R12 and FHV were diffusely localized to both cytosol and nucleus, but (PR)20 almost exclusively localized to the nucleus, especially nucleoli and (GR)20 localized to both nucleoli and cytosol. The difference of subcellular localization of poly-(PR) and poly-(GR) were also confirmed by ectopic expression of GFP-fused proteins. Because their Arg content was identical (50%), the difference was dependent on the nature of Pro and Gly inserted between Arg residues. e s Th pecific distribution of poly-(PR) to the nucleolus was diminished by RNAse treatment aer P ft FA fixation, indicating that RNA, but not RNA-binding proteins, was major binding partner of poly-(PR). Consistent with the aberrantly high an ffi ity of poly-(PR) to RNA, cell toxicity assays and IVT assays showed that poly-PR had cytotox - icity through translation inhibition. Based on the findings that (PR)12, but not (GR)12 or R12, inhibited protein translation (Figs 3D, 5B and S3), not only the number of Arg residues, but also proline residue might play a role in determining the toxicity level. However, which aspect of the proline confers the toxic property to the poly-PR peptide still remains under investigation. Pure Arg oligomer is a good model to investigate molecular mechanisms how these cationic CPPs penetrate plasma membrane. Several hypotheses such as the energy-dependent endocytotic model, energy-independent direct translocation model and cell surface receptor molecules-mediated internalization model, have been proposed . As we demonstrated here, penetration rates of poly-PR as well as poly-Arg were controlled by the extracellular pH, and lowering temperature had little effect on the uptake, indicating that poly-PR entered cells independently from the energy-requiring endocytotic pathway. How the extracellular pH ae ff cts the penetrating rate remains elusive, but deprotonated fatty acids in the plasma membrane are supposed to attract the cationic polypeptide . It has been established that the length of Arg oligomer (n = between 7–15 is optimal) determines the efficiency of translocation and thus it is speculated that not only the positive charge which Arg residue harbors, but also structural factors limit the efficiency . Consistent with this observation, the repeat number of poly-PR also con- trolled the penetrating rate and thus determined cellular toxicity. The (PR)12 peptide, which had the inhibitory effect on protein translation in a cell free system, did not show cytotoxicity because it could not enter cells. When fused to GE11 cargo, however, it was internalized by the endocytotic pathway and exerted its cytotoxicity, indicat- ing that inhibition of uptake of the PR peptides might block neurotoxicity and suppress progression of diseases. Although the physiological significance remains to be determined, DRPs from C9orf72 actually transmit from 27,28 cells to cells in neighborhood via the exosome-dependent and exosome-independent pathways , and even are secreted to cerebrospinal ui fl d of ALS patients , suggesting that intercellular communication via DRPs may con- tribute to the development of diseases and inhibition of uptake of DRPs could be a novel therapeutic target. One possible approach against extracellular DRPs is the antibody-mediated immunotherapy and it has been shown that the anti-GA antibody prevents the transmission of poly-GA and blocks its seeding activity, suggesting that anti-PR antibody may also prevent the penetration of poly-PR . As we reported previously, we proposed the neurotoxicity model of DRPs via inhibition of protein translation, and recently Zhang et al. reported that poly-GR impairs protein translation in vivo and causes neuronal loss in a mouse model of C9orf72-ALS, supporting our hypothesis . In addition to our theory, a number of hypotheses such as aberrant ER stress, deficit in nucleocytoplasmic transport and inhibition of membrane-less organelles have been proposed. It is also reported that poly-(GR) exerts its toxicity through mitochondrial oxidative stress and DNA damages . As we demonstrated here, structural factors, such as the length of dipeptides and existence of glycine residues or proline residues, determined the degree of translation inhibition, and thus it is also possible that these factors may be the determinant of the toxicity through other mechanisms. SCIENTIfIC Repo R Ts | (2018) 8:12740 | DOI:10.1038/s41598-018-31096-z 8 www.nature.com/scientificreports/ Taken together, our study clarified the essential components for cell penetration and cytotoxicity by poly-PR/ GR peptides, and modulation of extracellular environment which can mitigate the uptake and thus toxicity of the peptide might be a novel option to prevent motor neuronal death in ALS and FTD. Materials and Methods Antibodies and reagents. Anti-B23/nucleophosmin antibody was purchased from Cell signaling technol- ogy (Danvers, MA). RNAse A was obtained from Qiagen (Hilden, Germany). 1-Step Human Coupled IVT kit, oleic acid, poly-A RNA, Prolong Gold with DAPI and Nucblue live reagent were purchased from ThermoFisher Scientific (Waltham, MA). Cytotoxicity LDH Assay Kit-WST and octanol were obtained from Wako pure chem- icals (Japan). Cell Titer-Glo and Cell-tox glo kits were purchased from Promega (Madison, WI). TAMRA- labeled random 15mer RNA was synthesized by Fasmac (Japan). Anti-puromycin antibody was purchased from Millipore. Peptides. FITC-labeled recombinant human insulin (51 a.a.) was purchased from Sigma. Amyloid beta-40 (40 a.a.) was obtained from Peptide Institute (Osaka, Japan). CPPs and C9orf72-Arg-rich dipeptides were synthe- sized by Genscript (Piscataway, NJ). The purity of the peptides was at least 85%. Ae ft r synthesis, triu fl oroacetate was replaced by acetate. LDH assay. LDH assay was performed with Cytotoxicity LDH Assay Kit-WST from Wako (Japan) following the manufacture’s protocol. Briefly, mouse neuronal NSC34 cells or human fibroblasts were plated on a 96-well plate at 2 × 10 cells/well and then treated with the indicated concentration of peptide for 24 hr (NSC34 cells) or 48 hr (fibroblasts) before measurement of LDH release. The absorbance was measured by iMark microplate absorbance reader (Biorad). Buffers. All buffers used for the peptide absorption assay were prepared with 140 mM NaCl, 2.5 mM KCl, 5 mM HEPES, 5 mM glycine, and the final pH was adjusted with NaOH or HCl. Peptides absorption assay. The assay to show the effect of pH in peptides absorption into a hydropho- bic phase was performed as previously reported . FITC-labeled R12 or (PR)20 peptides were diluted to a final concentration of 10 μM in each buffer. Fifty microliter of the buffer-peptide mix and 50 μl of octanol containing 1% of oleic acid (OA) (v/v) were placed into a microtubes and vortexed for 5 min, followed by centrifugation at 2200 g briefly to separate the octanol phase and buffer phase. Aer s ft eparation, octanol phase containing OA and absorbed peptides were collected and the fluorescence was measured by a plate reader TECAN infinite F200 (Mannedorf, Switzerland). Peptide penetration assay. For imaging, Hela cells were washed twice with phosphate buffered saline (PBS) (pH7.4) and then incubated with PBS of different pH (pH6.0, pH7.4 and pH8.0) containing 2 μM of FITC- (PR)20 or FITC-R12 for 1 hr, followed by washing with PBS twice. For quantification, HEK293 cells were incu- bated with 10 μM FITC-(PR)20 in 10% FBS-DMEM for 30 min at 4 °C or 37 °C. e ce Th lls were washed twice with PBS and then re-suspended in PBS. The fluorescent signal was measured by a plate reader TECAN infinite F200. Crosslinking assay. HA-tagged (PR)20 was mixed with 10 mM of adenine, adenosine, ribose or ribose-3′ phosphate in the presence or absence of 10 μM poly-A and incubated for 5 min at room temperature. Crosslink was performed with 5 mM DSP (Pierce) for 30 min. The crosslinked peptides were then applied to SDS-PAGE followed by Coomassie blue staining. In vitro translation (IVT) assay. IVT assay was performed with 1-step Human-Coupled IVT kit (ThermoFisher) following manufacturer’s protocol. Briefly, turboGFP cDNA and Hela cell lysates were mixed in a 96 white-well PCR plate in the presence or absence of 100 μM of each peptide and incubated at 37 °C. The fluo- rescence from translated turboGFP was measured by LightCycler (Roche lifescience) every 1 min. Imaging of live HEK293 cells treated with fluorescent peptides. HEK293 cells were treated with 10 μM of FITC-labeled CPPs for 1 hr in combination with Nucblue live dye and then washed with PBS twice. The images were obtained by FV10 confocal microscopy (Olympus, Japan) or LSM710 confocal microscopy (Zeiss, Germany). Imaging of fixed HEK293 cells treated with fluorescent peptides in combination with RNAse A treatment. For RNAse A treatment, the HEK293 cells, fixed and permeabilized, were incubated with 10 μg/ml of recom- binant RNAse A for 1 hr at 37 °C, followed by treatment with 10 μM of FITC-labeled peptides for 30 min. The cells were then washed with PBS twice and imaged with a confocal microscopy. Immunostaining of fixed HEK293 treated with fluorescent peptides. HEK293 cells were pretreated with 10 μM of FITC-labeled peptides for 2 hr and fixed with 4% PFA. Aer fixa ft tion, the cells were permeabilized with 0.1% Triton-X100 followed by incubation with 0.1% bovine serum albumin-PBS for blocking. The cells were immunostained with anti-B23 primary antibody and visualized by Cy3 labeled anti-rabbit secondary antibody. Colocalization analysis. Colocalization of signals from FITC-(PR)20 and DAPI were analyzed by ImageJ and Coloc2 plugin. The slope reflects the rate of association. SCIENTIfIC Repo R Ts | (2018) 8:12740 | DOI:10.1038/s41598-018-31096-z 9 www.nature.com/scientificreports/ CD spectra. CD spectra of peptides were measured by J-820 CD spectrometer (JASCO, Japan) following manufacturer’s protocol. The cell length was 1 mm and 100 μM of peptide in buffer was measured at room tem- perature. The spectra shown were the average of eight measurements for each sample and signals from buffer were subtracted. Data availability statement. All materials, data and associated protocols are promptly available to readers without undue qualifications in material transfer agreements. References 1. DeJesus-Hernandez, M. et al. Expanded GGGGCC Hexanucleotide Repeat in Noncoding Region of C9ORF72 Causes Chromosome 9p-Linked FTD and ALS. Neuron 72, 245–256 (2011). 2. Mori, K. et al. The C9orf72 GGGGCC Repeat Is Translated into Aggregating Dipeptide-Repeat Proteins in FTLD/ALS. Science (80- ). 339, 1335–1338 (2013). 3. Mizielinska, S. et al. C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins. Science (80- ). 345, 1192–1194 (2014). 4. Kwon, I. et al. Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells. Science (80-). 345, 1139–1145 (2014). 5. Kanekura, K. et al. Poly-dipeptides encoded by the C9ORF72 repeats block global protein translation. Hum. Mol. Genet. 25, 1803–1813 (2016). 6. Frankel, A. D. & Pabo, C. O. Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55, 1189–1193 (1988). 7. Milletti, F. Cell-penetrating peptides: Classes, origin, and current landscape. Drug Discovery Today 17, 850–860 (2012). 8. Kristensen, M., Birch, D. & Nielsen, H. M. Applications and challenges for use of cell-penetrating peptides as delivery vectors for peptide and protein cargos. International Journal of Molecular Sciences 17, (2016). 9. Trabulo, S. et al. Cell-penetrating peptide-based systems for nucleic acid delivery: A biological and biophysical approach. Methods Enzymol. 509, 277–300 (2012). 10. Vivès, E., Schmidt, J. & Pèlegrin, A. Cell-penetrating and cell-targeting peptides in drug delivery. Biochimica et Biophysica Acta - Reviews on Cancer 1786, 126–138 (2008). 11. Bechara, C. & Sagan, S. Cell-penetrating peptides: 20 years later, where do we stand? FEBS Letters 587, 1693–1702 (2013). 12. Lee, K. H. et al. C9orf72 Dipeptide Repeats Impair the Assembly, Dynamics, and Function of Membrane-Less Organelles. Cell 167, 774–788.e17 (2016). 13. Lin, Y. et al. Toxic PR Poly-Dipeptides Encoded by the C9orf72 Repeat Expansion Target LC Domain Polymers. Cell 167, 789–802. e12 (2016). 14. Hawiger, J. Noninvasive intracellular delivery of functional peptides and proteins. Current Opinion in Chemical Biology 3, 89–94 (1999). 15. Wang, F. et al. Recent progress of cell-penetrating peptides as new carriers for intracellular cargo delivery. Journal of Controlled Release 174, 126–136 (2014). 16. Futaki, S. et al. Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. J. Biol. Chem. 276, 5836–5840 (2001). 17. Sirri, V., Urcuqui-Inchima, S., Roussel, P. & Hernandez-Verdun, D. Nucleolus: The fascinating nuclear body. Histochemistry and Cell Biology 129, 13–31 (2008). 18. Evdokimov, A. G. et al. Structural basis for the fast maturation of Arthropoda green fluorescent protein. EMBO Rep. 7, 1006–1012 (2006). 19. Herce, H. D., Garcia, A. E. & Cardoso, M. C. Fundamental molecular mechanism for the cellular uptake of guanidinium-rich molecules. J. Am. Chem. Soc. 136, 17459–17467 (2014). 20. Mitchell, D. J., Steinman, L., Kim, D. T., Fathman, C. G. & Rothbard, J. B. Polyarginine enters cells more efficiently than other polycationic homopolymers. J. Pept. Res. 56, 318–325 (2000). 21. Yamashita, H. et al. Development of a Cell-penetrating Peptide that Exhibits Responsive Changes in its Secondary Structure in the Cellular Environment. Sci. Rep. 6, (2016). 22. Li, Z. et al. Identification and characterization of a novel peptide ligand of epidermal growth factor receptor for targeted delivery of therapeutics. FASEB J. 19, 1978–85 (2005). 23. Yukawa, H. et al. Quantum dots labeling using octa-arginine peptides for imaging of adipose tissue-derived stem cells. Biomaterials 31, 4094–4103 (2010). 24. Nakamura, Y., Kogure, K., Futaki, S. & Harashima, H. Octaarginine-modified multifunctional envelope-type nano device for siRNA. J. Control. Release 119, 360–367 (2007). 25. Looi, C. Y. et al. Octa-Arginine mediated delivery of wild-type Lnk protein inhibits TPO-induced M-MOK Megakaryoblastic leukemic cell growth by promoting apoptosis. PLoS One 6, (2011). 26. Hamy, F. et al. An inhibitor of the Tat/TAR RNA interaction that effectively suppresses HIV-1 replication. Proc. Natl. Acad. Sci. USA 94, 3548–53 (1997). 27. Westergard, T. et al. Cell-to-Cell Transmission of Dipeptide Repeat Proteins Linked to C9orf72-ALS/FTD. Cell Rep. 17, 645–652 (2016). 28. Zhou, Q. et al. Antibodies inhibit transmission and aggregation of C9orf72 poly‐GA dipeptide repeat proteins. EMBO Mol. Med. 9, 687–702 (2017). 29. Gendron, T. F. et al. Poly(GP) proteins are a useful pharmacodynamic marker for C9ORF72-associated amyotrophic lateral sclerosis. Sci. Transl. Med. 9, (2017). 30. Zhang, Y. J. et al. Poly(GR) impairs protein translation and stress granule dynamics in C9orf72-associated frontotemporal dementia and amyotrophic lateral sclerosis. Nat. Med. in press (2018). 31. Lopez-Gonzalez, R. et al. Poly(GR) in C9ORF72-Related ALS/FTD Compromises Mitochondrial Function and Increases Oxidative Stress and DNA Damage in iPSC-Derived Motor Neurons. Neuron 92, 383–391 (2016). Acknowledgements We thank Prof. Atsushi Maruyama and Dr. Naohiko Shimada at Tokyo Institute of technology for helping us obtaining CD spectra data. i Th s work was partly supported by the grants from the JSPS KAKENHI Grant number (16H06247 and 17H03923 to K. Kanekura., 17H03923 to K. Nagaoka, 17K15671 to Y. Harada, 16H05973 to Y. Hayamizu, 17H04067 to M. Kuroda). This work was also supported in part by Japan Agency for Medical Research and Development (AMED) (16ek0109180h0001 and 17ae0101016s0904), Strategic Research Foundation Grant- aided Project for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Takeda Science Foundation (K. K.), Japan Intractable Diseases (Nanbyo) Research Foundation (K. K.), Tokyo Biochemistry Research Foundation (K.K.) and The Ichiro Kanehara foundation (K.K.). SCIENTIfIC Repo R Ts | (2018) 8:12740 | DOI:10.1038/s41598-018-31096-z 10 www.nature.com/scientificreports/ Author Contributions K. Kanekura and M. Kuroda contributed to conception and design of the study. K. Kanekura, Y. Harada, T. Yagi, M. Fujimoto, Y. Hayamizu, and K. Nagaoka contributed to data collection and analysis. K. Kanekura and M. Kuroda wrote and edited the manuscript. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-31096-z. 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Characterization of membrane penetration and cytotoxicity of C9orf72-encoding arginine-rich dipeptides

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www.nature.com/scientificreports OPEN Characterization of membrane penetration and cytotoxicity of C9orf72-encoding arginine-rich Received: 19 March 2018 dipeptides Accepted: 2 August 2018 Published: xx xx xxxx 1 1 2 3 4 Kohsuke Kanekura , Yuichiro Harada , Mao Fujimoto , Takuya Yagi , Yuhei Hayamizu , 5 1 Kentaro Nagaoka & Masahiko Kuroda Cell-penetrating peptides (CPPs) including arginine-rich peptides are attracting a lot of attention due to their potential as a novel intracellular drug delivery tool without substantial toxicity. On the other hand, disease-associated arginine-rich CPPs, such as poly-PR and poly-GR translated from C9orf72 gene, also efficiently enter neuronal cells and then kill them. Although both non-harmful CPPs and harmful poly-PR/GR penetrate the plasma membrane using same arginine residues, little is known about the factors which determine the toxicity of the pathogenic CPPs. Here, we show that poly-PR and poly-GR, but not other Arg-rich CPPs, specifically distributed to nucleolus via interaction with RNA. Importantly, C9orf72-dipeptides, but not other Arg-rich CPPs, caused inhibition of protein translation and cell death. Raising extracellular pH enhanced the cell penetration of poly-PR. The repeat number of (PR) affected the secondary structure and determined the intracellular delivery rate and neurotoxicity, and enforced intracellular delivery of non-penetrating short poly-PR peptide caused cell death, suggesting that modulation of extracellular environment to inhibit the uptake of Arg-rich dipeptides might be a drug target against poly-PR/GR-mediated neurotoxicity. Dipeptide-repeat proteins (DRPs), translated through repeat-associated non-ATG (RAN) translation from path- ogenic G4C2 expansions in C9orf72 gene, are supposed to play a pivotal role in development of C9orf72-related 1–4 neurological disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) . Among 5 species of DRPs (poly-GA, GP, PR, GR and PA) translated from sense and antisense of G4C2 expan- sions, especially arginine-rich DRPs (poly-PR and poly-GR) are of interest because they caused neuronal loss 3,4 in vitro and in vivo . When these arginine-rich DRPs are applied to the culture medium of neuronal cells, they penetrate the plasma membrane and directly affect intracellular machineries, thus they are classified as 4,5 cell-penetrating peptides (CPPs) . The first CPP discovered was trans-activating transcription activator (TAT) peptide from human immuno- deficiency virus-1 (HIV-1) whose amino acid sequence is shown in Fig.  1A . Since the discovery, a variety of CPPs have been artificially designed (e.g. R8, R12) or identified from natural organisms such as Flock house virus (FHV) . Because of their efficient translocation through biological membranes without significant cellu- lar damage, many of them have been extensively investigated as tools for intracellular delivery of compounds 8–10 including nucleic acids, proteins and drugs . Although several hypotheses such as the endocytotic pathway, energy-independent uptake and cell surface molecules-mediated uptake have been proposed, precise mecha- nisms how these CPPs penetrate the plasma membrane still remain to be investigated . 4,5 Similar to these non-harmful CPPs, ALS-causing poly-PR/GR efficiently enter cells . Once they penetrate the plasma membrane, intracellular poly-PR/GR localize to membrane-less organelles and inhibit their func- tions, resulting in disturbance of protein translation, abnormal formation and malfunction of stress granules, 3–5,12,13 mis-splicing of mRNAs, and ultimately neuronal death in vitro and in vivo . Despite their importance in 1 2 Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan. School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan. Department of Neurology, KEIO University, Tokyo, Japan. 4 5 Department of Organic and Polymeric Materials, Tokyo Institute of Technology, Tokyo, Japan. Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Tokyo, Japan. Correspondence and requests for materials should be addressed to K.K. (email: [email protected]) SCIENTIfIC Repo R Ts | (2018) 8:12740 | DOI:10.1038/s41598-018-31096-z 1 www.nature.com/scientificreports/ AC z-stack TAT GRKKRRQRRRPPQ R12 RRRRRRRRRRRR FHV RRRRNRTRRNRRRVR (PR)20 PRPRPR........PRPRPR (GR)20 GRGRGR........GRGRGR Merge DAPI FITCZoom Merge (PR)20B23 TAT R12 E MergeGFP-(PR)50 (PR)20 F MergeGFP-(GR)50 (GR)20 FHV Figure 1. Poly-PR localized to nucleoli. (A) Amino acid sequences of cell penetrating peptides (CPPs) used in this study. (B) Confocal imaging of live HEK293 cells treated with 10 μM of indicated FITC-labeled CPPs. Nucleus was visualized by Nucblue live dye. (C) Z-stack imaging of live HEK293 cells treated with FITC- (PR)20. (D) Immunocytochemistry of HEK293 cells treated with FITC-(PR)20, immunostained with anti-B23/ nucleophosmin antibody. Nucleus was visualized by DAPI. (E) Hela cells were transfected with GFP-(PR)50 repeats. (F) Hela cells were transfected with GFP-(GR)50 repeats. White arrows show the cells with both nucleolar and cytosolic staining and white arrowheads indicate the cells with cytosolic staining. Scale bar shows 20 μm. neuroscience and drug delivery research fields, the mechanism underlying the membrane penetration as well as the correlation between membrane penetration and neurotoxicity of C9orf72-DRPs remains undetermined. Here we identified key factors which ae ff ct membrane penetration and neurotoxicity by poly-PR/GR peptides. First, we examined a series of arginine-rich CPPs and found that poly-PR and poly-GR, but not other CPPs, dis- tributed to nucleolus. Next, in vitro translation assay and lactate dehydrogenase (LDH) release assay showed that C9orf72-DRPs, but no other CPPs, inhibited protein translation and caused cell death by aberrant interaction with RNA. The cell penetration rate of poly-PR was determined by length of repeats and extracellular pH, and enforced delivery of non-penetrating short poly-PR peptide caused cell death, suggesting that inhibition of intra- cellular delivery of DRPs might be a novel drug target for C9orf72-mediated ALS. Results Cell penetrating peptides (CPP) which can cross the plasma membrane bilayer are subcategorized by their bio- 14,15 chemical characteristics such as hydrophilic CPPs, hydrophobic CPPs and amphipathic CPPs . C9orf72-related Arg-rich dipeptides are classified as the hydrophilic cationic CPP because 50% of their content is polar argi - nine. The most established cationic CPP is TAT peptide (GRKKRRQRRRPPQ: 13 a. a.) identified from HIV-1. Since the discovery of TAT, many cationic CPPs including R12 (RRRRRRRRRRRR: 12 a. a.) and FHV peptide (RRRRNRTRRNRRRVR:15 a. a.) were identified and investigated as possible tools for the drug delivery system . e ad Th vantage of these CPPs as a drug delivery carrier is their high ability to deliver molecules including proteins and nucleotides across biological membranes without cytotoxicity. Although C9orf72-encoding Arg-rich dipep- tides should have similar biochemical characteristics with these CPPs, C9orf72-related DRPs have been shown to cause cell death once they penetrate the plasma membrane . SCIENTIfIC Repo R Ts | (2018) 8:12740 | DOI:10.1038/s41598-018-31096-z 2 (PR)20 www.nature.com/scientificreports/ Figure 2. Poly-PR interacts with RNA. (A) Confocal imaging of HEK293 cells fixed/permeabilized and stained with FITC-(PR)20 in the absence of pretreatment of RNase A. (B) Colocalization analysis of signals from DAPI staining and FITC-(PR)20 in the HEK293 cells in the absence of pretreatment of RNase A. (C) Confocal imaging of HEK293 cells fixed/permeabilized and stained with FITC-(PR)20 in the presence of pretreatment of RNase A. (D) Colocalization analysis of signals from DAPI staining and FITC-(PR)20 in the HEK293 cells in the presence of pretreatment of RNase A. (E) Liquid-liquid phase separation of tetramethylrhodamine (TAMRA)-labeled random 15-mer RNA by FITC-(PR)20. (F) Agarose gel electrophoresis of human ribosomal RNA mixed with (PR)20 peptide at indicated concentration (μM). (G) Structure of RNA (base: adenine). (H) Crosslinking analysis of HA-(PR)20 peptide mixed with adenine, adenosine, ribose or ribose-phosphate in the presence or absence of RNA (poly-A). Arrowheads indicate crosslinked oligomer of HA-(PR)20 peptides. *polymerized HA-(PR)20. To clarify the difference between the C9orf72-related DRPs and non-harmful CPPs, we synthesized well-known cationic CPPs including TAT peptide, R12 peptide and FHV peptide and C9orf72-derived DRPs, (PR)20 peptide (40 a. a.) and (GR)20 peptide (40 a. a.) (Fig. 1A), and examined their penetration to human embryonic kidney (HEK) 293 cells. All of the CPPs examined were uptaken by HEK293 cells as expected (Fig. 1B). The confocal imaging revealed the subcellular localization of FITC-labeled CPPs. TAT made cytosolic large dots, R12 and FHV diffusely localized to both nuclear and cytosol with some punctate structures in cyto- sol, (PR)20 mainly localized to the nucleus and especially accumulated to round-shaped nucleoli and (GR)20 localized to both nucleoli and cytosol (Fig. 1B). The nucleolar localization of (PR)20 was also confirmed with Z-stack imaging and colocalization with a nucleolar marker B23/nucleophosmin (Fig. 1C,D) as reported pre- 4,12 viously . To deny the possibility that nucleolar distribution of poly-PR and poly-GR was an artifact because these peptides were added extracellularly, we overexpressed green fluorescent protein (GFP)-tagged (PR)50 repeat protein and GFP-tagged (GR)50 repeat protein in HEK293 cells and confirmed that both GFP-(PR)50 and GFP-(GR)50 distributed to nucleoli (Fig. 1E,F). Consistent with the subcellular localization of FITC-peptides, GFP-(PR)50 showed almost exclusive distribution to nucleoli and GFP-(GR)50 distributed to both nucleoli and cytosol (Fig. 1E,F). e Th nucleolar distribution of the FITC-labeled DRPs and GFP-fused DRPs implies existence of molecules with high affinity to C9orf72-DRPs in the nucleolus. The nucleolus is a subnuclear membrane-less compartment which is the site for transcription of ribosomal RNA and assembly of ribosome . Because the nucleolus contains a large amount of ribosomal RNA accounting for 90% of total RNA and a number of RNA-binding proteins, the concen- tration of intranucleolar DNA is low, since DNA staining by DAPI excludes the nucleolus . In our previous study, we demonstrated liquid chromatography mass spectrometry to comprehensively identify interactors of poly-PR and found that poly-PR interacted with RNA-binding proteins. Immunoprecipitation assays in the presence of RNAse diminished some of the interactions, suggesting that poly-PR directly binds to RNA . In order to examine whether (PR)20 accumulated to the nucleoli through interaction with RNA or with RNA-binding proteins, we fixed the HEK293 cells with 4% paraformaldehyde (PFA) and incubated the cells with FITC-(PR)20 with or with- out RNAse treatment (Fig. 2A–D). Fixation of HEK293 cells slightly increased cytosolic staining by FITC-(PR)20 compared with the staining of live HEK293 cells (Fig. 1B) but most of (PR)20 localized to nucleoli (Fig. 2A). The RNAse treatment diminished the specific distribution of (PR)20 to nucleoli and increased diffuse nuclear staining SCIENTIfIC Repo R Ts | (2018) 8:12740 | DOI:10.1038/s41598-018-31096-z 3 www.nature.com/scientificreports/ Figure 3. Poly-PR caused cell death through inhibition of protein translation. (A) Scheme of real-time monitoring of protein translation using in vitro translation (IVT) system. tGFP: turboGFP. (B) Real time monitoring of tGFP signal in the IVT assay. Signals from tGFP were measured every 1 min. (C) Fluorescence intensity of translated tGFP in the IVT assay treated with 100 μM of each peptide. N = 3. (D) Cytotoxicity of each CPPs were monitored by release of LDH. NSC34 cells were treated with each CPP at indicated concentration for 24 hr. N = 6. (E) Cytotoxicity of each CPPs were monitored by Celltox glo luminescence. NSC34 cells were treated with each CPP at indicated concentration for 24 hr. N = 6. Each experiment was independently repeated at least 3 times. Asterisks indicate a significant difference analyzed by one-way ANOVA followed by Dunnett’s test (**p < 0.01, *p < 0.05). (Fig. 2C). Colocalization analysis showed that the green fluorescence from FITC-(PR)20 and blue fluorescence from DAPI did not correlate before the RNAse treatment (slope m = −0.11), and the RNAse treatment drastically change the correlation manner (slope m = 0.85), implying that (PR)20 distributed to nucleoli through interaction with RNA, presumably ribosomal RNA, abundant in the nucleolus (Fig. 2B,D). We next investigated how (PR)20 interacts with RNA. It has been reported that poly-PR forms phase-separated 12,13 liquid droplets with anionic molecules in vitro . Therefore, we first tested if (PR)20 induces phase-separated liquid droplets with RNA. For this purpose, we synthesized tetramethylrhodamine (TAMRA)-labeled random 15-mer RNA and mixed the RNA with FITC-labeled (PR)20. As expected, (PR)20 induced the round-shaped droplet formation (Fig. 2E), indicating that (PR)20 induced phase separation of RNA. We also confirmed that (PR)20 induced separation of dense liquid phase fusing each other, and the separated droplets showed high cir- cularity and roundness, suggesting minimization of surface tension (Figs S1A and 1B). To examine whether (PR)20-RNA complex maintains the liquid-like aspect in live cells, we demonstrated the fluorescence recovery after photobleaching (FRAP) assay and confirmed that the nucleolar (PR)20 peptide maintained fluidity even aer 24 ft hr incubation (Fig. S1C,D). Next, we tested if (PR)20 interacts with ribosomal RNA. The ribosomal RNA is negatively charged and runs to anode when applied to Tris-acetate-EDTA (TAE)-buffered agarose gel elec- trophoresis. (PR)20 is positively charged and thus possibly neutralizes the negative charge of ribosomal RNA, inhibiting the running during the electrophoresis. To test if this hypothesis is the case, we mixed the human ribosomal RNA extracted from HEK293 cells and (PR)20 peptide then applied the mixture to the agarose gel elec- trophoresis. As expected, (PR)20 inhibited the running of rRNA to the anode and the rRNA resided to the well applied (Fig. 2F). To support this, we next tested which component of RNA is the binding target for poly-(PR) peptide. For this end, we used a crosslinking assay because RNA enhances polymerization of poly-(PR) . RNA is composed of nucleotides containing bases, sugars and phosphates (Fig. 2G), and each component was mixed with (PR)20 in the presence or absence of poly-A RNA as an enhancer for polymerization. Ribose-3′ phosphate, but not ribose alone, enhanced oligomerization of (PR)20, suggested that the phosphate attracted (PR)20 peptide presumably due to the negative charge (Fig. 2H). As we previously reported, Arg-rich C9orf72 dipeptides inhibit protein translation and cause neuronal cell death . Indeed, ectopic expression of GFP-(PR)50 and GFP-(GR)50 inhibited protein translation in Hela cells (Fig. S2A). To investigate if other cationic CPPs ae ff ct protein translation and exert neurotoxicity as well, we tested the effects of the cationic CPPs on protein translation. For this end, we adopted a cell-free in vitro translation (IVT) system to avoid possible secondary effects of cytotoxicity by the CPPs (Fig.  3A). The efficiency of protein SCIENTIfIC Repo R Ts | (2018) 8:12740 | DOI:10.1038/s41598-018-31096-z 4 www.nature.com/scientificreports/ Figure 4. Extracellular pH ae ff cted penetration of poly-PR. (A) Peptides absorption assay using FITC-R12 or FITC-(PR)20. The pictures show the actual images of the absorbed peptides. The white arrowheads indicate octanol phase and white arrows show aqueous buffer phase. The bar graph shows the fluorescence of FITC- labeled peptides absorbed in the octanol fractions measured by a plate reader. (B) Hela cells were treated with PBS with different pH containing 2 μM FITC-R12 or FITC-(PR)20. Nucleus was visualized by Nucblue live dye. (C) Cell penetration rate of FITC-(PR)20 incubated at 37°C or 4°C or pH9 in HEK293 cells, measured by a plate reader. Asterisks indicate a significant difference analyzed by one-way ANOVA followed by Dunnett’s test (**p < 0.01, *p < 0.05). n.s.; not significant. translation was real-timely monitored by fluorescence emitted from translated turboGFP which does not have significant delay for maturation like jellyfish-derived GFP . As expected, (PR)20 (Arg content: 50%), but not TAT (Arg content: 46%), R12 (Arg content: 100%) or FHV (Arg content: 73%), efficiently inhibited protein trans- lation in vitro (Fig. 3B,C). Next, motor neuronal NSC34 cells were treated with these CPPs and cellular damages were measured by LDH release assay and Cytotox glo assay (Fig. 3D,E). Consistent with the results of IVT assay, (PR)20, but not other CPPs, killed the cells, indicating that not only the ratio of cationic amino acid content but structural factors underlie the cell death by poly-PR. Next, we investigated the molecular mechanism how poly-PR penetrates the plasma membrane. It has been shown that cationic CPPs cross the plasma membrane via both the energy-independent translocation pathway and energy-dependent endocytotic pathway. To confirm if this is the case with poly-PR, we first tested the effect of fatty acids and pH gradient at the cell membrane on the translocation of the (PR)20 peptide, because these two components were reported to ae ff ct the uptake of Arg-rich peptides by live cells through the energy-independent translocation . To see the effects of fatty acids and pH gradient on the translocation of the peptides, we adopted a simple cell free model to show the distribution of fluorescent CPPs between an aqueous buffer and hydro- phobic octanol containing oleic acid, mimicking lipid bilayer environment . When pH was increased higher than 7.5, R12 started to be absorbed into the octanol phase and most of R12 peptide was absorbed into octanol phase at pH 9.0 (Fig. 4A, upper panels). The pH gradient also enhanced the absorption of (PR)20 peptide into the octanol phase and higher pH increased the absorption of the peptide (Fig. 4A, lower panels), indicating that extracellular pH condition may influence the uptake of Arg-rich dipeptides in hydrophobic environments such as the lipid bilayer. e Th change of pH did not enhance the absorption of non-permeable human insulin peptide into the octanol phase (Fig. S2B), indicating that the effect of pH was specific to the cationic CPPs. Next, we chal- lenged Hela cells with buffers of different pH and treated with FITC-R12 or FITC-(PR)20 (Fig.  4B). Consistent with the results from the cell-free model (Fig. 4A) and previous study , higher pH increased the distribution of SCIENTIfIC Repo R Ts | (2018) 8:12740 | DOI:10.1038/s41598-018-31096-z 5 www.nature.com/scientificreports/ Figure 5. Repeat lengths ae ff cted the cytotoxicity of poly-PR peptides. (A,B) Fluorescence intensity of translated tGFP in the IVT assay treated with 100 μM of each peptide. N = 3. (C) Cytotoxicity of poly-PR peptides with different repeat lengths were monitored by release of LDH. NSC34 cells were treated with each peptide at 5 μM or 10 μM for 24 hr. N = 6. (D) Confocal imaging of HEK293 cells stained with 10 μM of FITC- (PR)12 or FITC-(PR)20 before or aer t ft he fixation/permeabilization step. (E) CD spectra of R12, (PR)12 and (PR)20 in Tris-buffer (pH7.4). (F) CD spectra of R12, (PR)12 and (PR)20 in 1 v/w% SDS-Tris buffer (pH7.4). Asterisks indicate a significant difference analyzed by one-way ANOVA followed by Dunnett’s test (**p < 0.01, *p < 0.05). FITC-labeled CPPs into the cells. We also investigated whether cellular uptake of (PR)20 is ae ff cted by inhibition of energy-dependent endocytosis. It has been established that the endocytotic pathway is temperature-sensitive , thus we tested if lowering temperature affects the uptake of the peptide. When the cells were incubated with FITC-(PR)20 at 4 °C, the fluorescent signal was comparable with the signal from the cells incubated at 37 °C (Fig. 4C). es Th e results suggest that (PR)20 penetrated the plasma membrane via the direct translocation rather than the energy-dependent endocytosis. Next, we investigated the effect of repeat length of PR on the cell toxicity. IVT assay indicated that even (PR)8 blocked the protein synthesis and longer repeat tended to have higher inhibition rate (Fig. 5A,B). We also exam- ined the effect of repeat length on the poly-GR toxicity. We performed the IVT assay with poly-GR peptides with 12 repeats and 20 repeats. In this assay, amyloid β (40 a.a.) was used as a negative control peptide with length of 40 a. a., same as (GR)20 (Fig. S3A,B). (GR)20 modestly inhibited protein translation and we did not see inhibitory effect of (GR)12 or amyloid β on protein translation, indicating that (1) poly-GR also inhibited protein translation in a length-dependent manner, (2) the inhibitory ee ff ct of (GR)20 is specific because amyloid β did not inhibit protein translation, (3) poly-PR is more potent in the inhibition of translation. Because there was a significant dif- ference in the degree of translation inhibition between (PR)20 and (GR)20, we next compared their cytotoxicity. SCIENTIfIC Repo R Ts | (2018) 8:12740 | DOI:10.1038/s41598-018-31096-z 6 www.nature.com/scientificreports/ Figure 6. Enforced delivery of poly-(PR)12 rescued the cytotoxicity. (A) Scheme of GE11 peptide-mediated endocytotic delivery system. (B) Cytotoxicity of GE11 peptide, (PR)12 peptide or GE11-(PR)12 peptides were monitored by release of LDH. Human fibroblasts were treated with each peptide at 10 μM, 20 μM or 50 μM for 48 hr. N = 6. (C) Microscopic image of human fibroblasts treated with 50 μM of each peptide for 48 hr. Each experiment was independently repeated at least 3 times. Asterisks indicate a significant difference analyzed by one-way ANOVA followed by Dunnett’s test (**p < 0.01, *p < 0.05). First, we treated NSC34 cells with FITC-conjugated DRPs and compare their penetration rate. As Fig. S3C shows, (PR)20 entered NSC34 cells more efficiently, but (GR)20 also entered the cells as well. Next, we treated NSC34 cells with (PR)20 or (GR)20 and measured their cytotoxicity. Consistent with the rate of translation inhibition as well as the efficiency of penetration, (PR)20 exerted stronger cytotoxicity (Fig. S3D). Based on the results from IVT using poly-PR peptides with different length, even (PR)8 repeat might have cytotoxicity. However, when we challenged NSC34 cells with these poly-PR peptides with various lengths, minimal requirement of peptide repeat length for cytotoxicity was 16 and poly-PR peptides shorter than 16 had little tox- icity even with high concentration (Fig. 5C). To explain the discrepancy of translation inhibition and cytotoxicity, we investigated the rate of cell penetration of these poly-PR variants because the cationic CPP poly-arginine has 16,20 optimal length between 7–15 for efficient cellular uptake and thus short (PR) peptides might not satisfy the requirement for the cell penetration. To test whether the length of (PR) repeats influences the translocation of the peptides, we performed cell penetration assay using FITC-labeled (PR)12 and FITC-labeled (PR)20 with live or fixed/permeabilized HEK293 cells. When added to the culture medium of live HEK293 cells, FITC-(PR)20, but not FITC-(PR)12, penetrated the plasma membrane and accumulated to nucleoli (Fig. 5D). Aer ft the fixation/ permeabilization, both of the peptides entered the cells and accumulated to nucleoli, indicating that the difference of cell toxicity between (PR)12 and (PR)20 was determined by the degree of cell penetration rather than the degree of inhibition of translation, because the level of translation inhibition by (PR)12 was comparable to the level by (PR)20 (Fig. 5B). It has been reported that HA-tagged (GR)20 peptide has a shorter half-life (less than 1 hr) in the culture medium and that may contribute to the lessor toxicity of poly-GR . To deny the possibility that the lack of cell toxicity of shorter poly-PR peptides is due to their instability, we carried out a functional assay of (PR)12 aer ft incubation in the culture medium. Aer in ft cubation in the culture medium for 2 hr, (PR)12 still could accumulate to nucleoli of fixed/permeabilized HEK293 cells, showing that (PR)12 is stable in the culture medium (Fig. S4). To investigate if the secondary structure of the poly-PR peptides might ae ff ct their efficiency of translocation, we monitored circular dichroism (CD) spectra of R12, (PR)12 and (PR)20 because artificially designed CPP containing (Arg-Arg-Pro) repeats preferably formed α-helix-like structure in cellular environment and thus had enhanced penetration rate . Figure 5E,F showed CD spectra of R12, (PR)12 and (PR)20 in 20 mM Tris bue ff r (pH7.4) solution (Fig. 5E) and in 1.0 w/v% sodium dodecyl sulfate (SDS)-Tris buffer (pH7.4) solution (Fig.  5F), which mimicked amphipathic environments such as the plasma membrane. The spectra of all these peptides exhibited negative maxima at around 200 nm and weak negative maxima at 235 nm, suggesting these peptides basically formed random secondary structure under physiological condition. Mean residue molar ellipticity of (PR)12 and (PR20) were almost identical, indicating that both peptides had similar secondary structure. On the other hand, addition of SDS markedly influenced the secondary structure of poly-PR, resulting in the CD spectra with negative maxima at 210 nm and 230 nm, implying that poly-PR peptide formed a α-helix-like structure in amphipathic condition . The effect of SDS on the structural change was more obvious in (PR)20 compared with (PR)12 (Fig. 5F). Based on these findings, we hypothesized that (PR)12 may cause cell death if it is successfully delivered into cells by a molecular cargo because it still possessed the inhibitory effect on protein translation (Fig.  5A). To exam- ine this idea, we synthesized a (PR)12 peptide fused to GE11 peptide. GE11 sequence (YHWYGYTPQNVI: 12 a. a.) was found using a phage display screen to identify peptide sequences which have high affinity to epidermal growth factor receptor (EGFR) . Despite its high affinity to EGFR (Kd = 22 nM), its mitogenic activity is much lower than EGF. When GE11 binds to EGFR, GE11-EGFR complex becomes internalized thus GE11 is a potential drug delivery carrier into the cells (Fig. 6A). To examine whether internalized (PR)12 by fused GE11 sequence exerts cytotoxicity, we treated human fibroblasts with GE11, (PR)12 or GE11-(PR)12 peptide and incubated the cells for 48 hr. (PR)12 fused to GE11, but not GE11 or (PR)12 alone, caused cell death, showing that although it lacks the ability to enter cells, (PR)12 still possesses the cytotoxic property (Fig. 6B,C). SCIENTIfIC Repo R Ts | (2018) 8:12740 | DOI:10.1038/s41598-018-31096-z 7 www.nature.com/scientificreports/ Discussion Discovery of C9orf72 has opened a new horizon in the research field of ALS and FTD because it dominates the familial cases of these miserable disorders. From the standpoint of biochemistry and molecular biology, find- ings of RAN translation and associated arginine-containing dipeptides are of interests due to their intriguing characteristics including the unique translation system as well as cell penetration and cytotoxicity. Despite of the importance, little was known about the mechanism how Arg-rich dipeptides from C9orf72 penetrate the plasma membrane and cause cell death. In this study, we characterized the poly-PR and poly-GR peptides by comparing with other arginine-rich CPPs and found that only poly-PR and poly-GR, but not other Arg-rich CPPs inhibit protein translation, resulting in cell death. Extracellular pH ae ff cted the penetration rate of poly-PR and enforced delivery of non-penetrating short poly-PR peptide induced cytotoxicity, proposing a possible therapeutic target for C9orf72-mediated neurotoxicity. CPPs have been extensively investigated because of their potential as a drug delivery tool. Especially cationic CPPs are attracting attentions because of their high efficiency of translocation and lower toxicity, and intracellular 23–25 delivery of drugs, nucleotides and proteins by cationic CPPs have been achieved without significant cytotoxicity . e k Th ey component of the cationic CPPs is arginine residue which has a strong positive charge. When compared with CPPs containing another positive amino acid lysine, CPPs containing Arg have higher efficiency to enter cells so that CPPs with high Arg contents attract more attentions . The Arg content of (PR)20 (50%) and (GR)20 (50%) was comparable to those of other cationic CPPs, TAT (46%) and FHV (73%), and all of these peptides penetrated the plasma membrane and entered cells efficiently (Fig.  1B). However, (PR)12 which also has 50% of Arg content did not penetrate the plasma membrane, suggesting that not only the proportion of cationic amino acids but also other factors including the structure of peptide also regulate the efficiency. One such possible factor is formation of α-helix-like structure because artificially enhanced α -helix formation of cationic CPP results in better uptake rate . Because the repeat length of PR regulated the penetration efficiency, we speculated (PR)20 might form more α-helix-like structure than (PR)12 did, and CD spectra under amphipathic condition showing that (PR)20 tended to form α-helix-like structure more than (PR)12 supported the idea (Fig. 5F). Although cationic CPPs TAT, FHV and poly-PR/GR peptides, have been similarly reported to interact with 5,16,26 anionic molecules including RNA , their intracellular distributions were highly varied. R12 and FHV were diffusely localized to both cytosol and nucleus, but (PR)20 almost exclusively localized to the nucleus, especially nucleoli and (GR)20 localized to both nucleoli and cytosol. The difference of subcellular localization of poly-(PR) and poly-(GR) were also confirmed by ectopic expression of GFP-fused proteins. Because their Arg content was identical (50%), the difference was dependent on the nature of Pro and Gly inserted between Arg residues. e s Th pecific distribution of poly-(PR) to the nucleolus was diminished by RNAse treatment aer P ft FA fixation, indicating that RNA, but not RNA-binding proteins, was major binding partner of poly-(PR). Consistent with the aberrantly high an ffi ity of poly-(PR) to RNA, cell toxicity assays and IVT assays showed that poly-PR had cytotox - icity through translation inhibition. Based on the findings that (PR)12, but not (GR)12 or R12, inhibited protein translation (Figs 3D, 5B and S3), not only the number of Arg residues, but also proline residue might play a role in determining the toxicity level. However, which aspect of the proline confers the toxic property to the poly-PR peptide still remains under investigation. Pure Arg oligomer is a good model to investigate molecular mechanisms how these cationic CPPs penetrate plasma membrane. Several hypotheses such as the energy-dependent endocytotic model, energy-independent direct translocation model and cell surface receptor molecules-mediated internalization model, have been proposed . As we demonstrated here, penetration rates of poly-PR as well as poly-Arg were controlled by the extracellular pH, and lowering temperature had little effect on the uptake, indicating that poly-PR entered cells independently from the energy-requiring endocytotic pathway. How the extracellular pH ae ff cts the penetrating rate remains elusive, but deprotonated fatty acids in the plasma membrane are supposed to attract the cationic polypeptide . It has been established that the length of Arg oligomer (n = between 7–15 is optimal) determines the efficiency of translocation and thus it is speculated that not only the positive charge which Arg residue harbors, but also structural factors limit the efficiency . Consistent with this observation, the repeat number of poly-PR also con- trolled the penetrating rate and thus determined cellular toxicity. The (PR)12 peptide, which had the inhibitory effect on protein translation in a cell free system, did not show cytotoxicity because it could not enter cells. When fused to GE11 cargo, however, it was internalized by the endocytotic pathway and exerted its cytotoxicity, indicat- ing that inhibition of uptake of the PR peptides might block neurotoxicity and suppress progression of diseases. Although the physiological significance remains to be determined, DRPs from C9orf72 actually transmit from 27,28 cells to cells in neighborhood via the exosome-dependent and exosome-independent pathways , and even are secreted to cerebrospinal ui fl d of ALS patients , suggesting that intercellular communication via DRPs may con- tribute to the development of diseases and inhibition of uptake of DRPs could be a novel therapeutic target. One possible approach against extracellular DRPs is the antibody-mediated immunotherapy and it has been shown that the anti-GA antibody prevents the transmission of poly-GA and blocks its seeding activity, suggesting that anti-PR antibody may also prevent the penetration of poly-PR . As we reported previously, we proposed the neurotoxicity model of DRPs via inhibition of protein translation, and recently Zhang et al. reported that poly-GR impairs protein translation in vivo and causes neuronal loss in a mouse model of C9orf72-ALS, supporting our hypothesis . In addition to our theory, a number of hypotheses such as aberrant ER stress, deficit in nucleocytoplasmic transport and inhibition of membrane-less organelles have been proposed. It is also reported that poly-(GR) exerts its toxicity through mitochondrial oxidative stress and DNA damages . As we demonstrated here, structural factors, such as the length of dipeptides and existence of glycine residues or proline residues, determined the degree of translation inhibition, and thus it is also possible that these factors may be the determinant of the toxicity through other mechanisms. SCIENTIfIC Repo R Ts | (2018) 8:12740 | DOI:10.1038/s41598-018-31096-z 8 www.nature.com/scientificreports/ Taken together, our study clarified the essential components for cell penetration and cytotoxicity by poly-PR/ GR peptides, and modulation of extracellular environment which can mitigate the uptake and thus toxicity of the peptide might be a novel option to prevent motor neuronal death in ALS and FTD. Materials and Methods Antibodies and reagents. Anti-B23/nucleophosmin antibody was purchased from Cell signaling technol- ogy (Danvers, MA). RNAse A was obtained from Qiagen (Hilden, Germany). 1-Step Human Coupled IVT kit, oleic acid, poly-A RNA, Prolong Gold with DAPI and Nucblue live reagent were purchased from ThermoFisher Scientific (Waltham, MA). Cytotoxicity LDH Assay Kit-WST and octanol were obtained from Wako pure chem- icals (Japan). Cell Titer-Glo and Cell-tox glo kits were purchased from Promega (Madison, WI). TAMRA- labeled random 15mer RNA was synthesized by Fasmac (Japan). Anti-puromycin antibody was purchased from Millipore. Peptides. FITC-labeled recombinant human insulin (51 a.a.) was purchased from Sigma. Amyloid beta-40 (40 a.a.) was obtained from Peptide Institute (Osaka, Japan). CPPs and C9orf72-Arg-rich dipeptides were synthe- sized by Genscript (Piscataway, NJ). The purity of the peptides was at least 85%. Ae ft r synthesis, triu fl oroacetate was replaced by acetate. LDH assay. LDH assay was performed with Cytotoxicity LDH Assay Kit-WST from Wako (Japan) following the manufacture’s protocol. Briefly, mouse neuronal NSC34 cells or human fibroblasts were plated on a 96-well plate at 2 × 10 cells/well and then treated with the indicated concentration of peptide for 24 hr (NSC34 cells) or 48 hr (fibroblasts) before measurement of LDH release. The absorbance was measured by iMark microplate absorbance reader (Biorad). Buffers. All buffers used for the peptide absorption assay were prepared with 140 mM NaCl, 2.5 mM KCl, 5 mM HEPES, 5 mM glycine, and the final pH was adjusted with NaOH or HCl. Peptides absorption assay. The assay to show the effect of pH in peptides absorption into a hydropho- bic phase was performed as previously reported . FITC-labeled R12 or (PR)20 peptides were diluted to a final concentration of 10 μM in each buffer. Fifty microliter of the buffer-peptide mix and 50 μl of octanol containing 1% of oleic acid (OA) (v/v) were placed into a microtubes and vortexed for 5 min, followed by centrifugation at 2200 g briefly to separate the octanol phase and buffer phase. Aer s ft eparation, octanol phase containing OA and absorbed peptides were collected and the fluorescence was measured by a plate reader TECAN infinite F200 (Mannedorf, Switzerland). Peptide penetration assay. For imaging, Hela cells were washed twice with phosphate buffered saline (PBS) (pH7.4) and then incubated with PBS of different pH (pH6.0, pH7.4 and pH8.0) containing 2 μM of FITC- (PR)20 or FITC-R12 for 1 hr, followed by washing with PBS twice. For quantification, HEK293 cells were incu- bated with 10 μM FITC-(PR)20 in 10% FBS-DMEM for 30 min at 4 °C or 37 °C. e ce Th lls were washed twice with PBS and then re-suspended in PBS. The fluorescent signal was measured by a plate reader TECAN infinite F200. Crosslinking assay. HA-tagged (PR)20 was mixed with 10 mM of adenine, adenosine, ribose or ribose-3′ phosphate in the presence or absence of 10 μM poly-A and incubated for 5 min at room temperature. Crosslink was performed with 5 mM DSP (Pierce) for 30 min. The crosslinked peptides were then applied to SDS-PAGE followed by Coomassie blue staining. In vitro translation (IVT) assay. IVT assay was performed with 1-step Human-Coupled IVT kit (ThermoFisher) following manufacturer’s protocol. Briefly, turboGFP cDNA and Hela cell lysates were mixed in a 96 white-well PCR plate in the presence or absence of 100 μM of each peptide and incubated at 37 °C. The fluo- rescence from translated turboGFP was measured by LightCycler (Roche lifescience) every 1 min. Imaging of live HEK293 cells treated with fluorescent peptides. HEK293 cells were treated with 10 μM of FITC-labeled CPPs for 1 hr in combination with Nucblue live dye and then washed with PBS twice. The images were obtained by FV10 confocal microscopy (Olympus, Japan) or LSM710 confocal microscopy (Zeiss, Germany). Imaging of fixed HEK293 cells treated with fluorescent peptides in combination with RNAse A treatment. For RNAse A treatment, the HEK293 cells, fixed and permeabilized, were incubated with 10 μg/ml of recom- binant RNAse A for 1 hr at 37 °C, followed by treatment with 10 μM of FITC-labeled peptides for 30 min. The cells were then washed with PBS twice and imaged with a confocal microscopy. Immunostaining of fixed HEK293 treated with fluorescent peptides. HEK293 cells were pretreated with 10 μM of FITC-labeled peptides for 2 hr and fixed with 4% PFA. Aer fixa ft tion, the cells were permeabilized with 0.1% Triton-X100 followed by incubation with 0.1% bovine serum albumin-PBS for blocking. The cells were immunostained with anti-B23 primary antibody and visualized by Cy3 labeled anti-rabbit secondary antibody. Colocalization analysis. Colocalization of signals from FITC-(PR)20 and DAPI were analyzed by ImageJ and Coloc2 plugin. The slope reflects the rate of association. SCIENTIfIC Repo R Ts | (2018) 8:12740 | DOI:10.1038/s41598-018-31096-z 9 www.nature.com/scientificreports/ CD spectra. CD spectra of peptides were measured by J-820 CD spectrometer (JASCO, Japan) following manufacturer’s protocol. The cell length was 1 mm and 100 μM of peptide in buffer was measured at room tem- perature. The spectra shown were the average of eight measurements for each sample and signals from buffer were subtracted. Data availability statement. All materials, data and associated protocols are promptly available to readers without undue qualifications in material transfer agreements. References 1. DeJesus-Hernandez, M. et al. Expanded GGGGCC Hexanucleotide Repeat in Noncoding Region of C9ORF72 Causes Chromosome 9p-Linked FTD and ALS. Neuron 72, 245–256 (2011). 2. Mori, K. et al. The C9orf72 GGGGCC Repeat Is Translated into Aggregating Dipeptide-Repeat Proteins in FTLD/ALS. Science (80- ). 339, 1335–1338 (2013). 3. Mizielinska, S. et al. C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins. Science (80- ). 345, 1192–1194 (2014). 4. Kwon, I. et al. Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells. Science (80-). 345, 1139–1145 (2014). 5. Kanekura, K. et al. Poly-dipeptides encoded by the C9ORF72 repeats block global protein translation. Hum. Mol. Genet. 25, 1803–1813 (2016). 6. Frankel, A. D. & Pabo, C. O. Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55, 1189–1193 (1988). 7. Milletti, F. Cell-penetrating peptides: Classes, origin, and current landscape. Drug Discovery Today 17, 850–860 (2012). 8. Kristensen, M., Birch, D. & Nielsen, H. M. Applications and challenges for use of cell-penetrating peptides as delivery vectors for peptide and protein cargos. International Journal of Molecular Sciences 17, (2016). 9. Trabulo, S. et al. Cell-penetrating peptide-based systems for nucleic acid delivery: A biological and biophysical approach. Methods Enzymol. 509, 277–300 (2012). 10. Vivès, E., Schmidt, J. & Pèlegrin, A. Cell-penetrating and cell-targeting peptides in drug delivery. Biochimica et Biophysica Acta - Reviews on Cancer 1786, 126–138 (2008). 11. Bechara, C. & Sagan, S. Cell-penetrating peptides: 20 years later, where do we stand? FEBS Letters 587, 1693–1702 (2013). 12. Lee, K. H. et al. C9orf72 Dipeptide Repeats Impair the Assembly, Dynamics, and Function of Membrane-Less Organelles. Cell 167, 774–788.e17 (2016). 13. Lin, Y. et al. Toxic PR Poly-Dipeptides Encoded by the C9orf72 Repeat Expansion Target LC Domain Polymers. Cell 167, 789–802. e12 (2016). 14. Hawiger, J. Noninvasive intracellular delivery of functional peptides and proteins. Current Opinion in Chemical Biology 3, 89–94 (1999). 15. Wang, F. et al. Recent progress of cell-penetrating peptides as new carriers for intracellular cargo delivery. Journal of Controlled Release 174, 126–136 (2014). 16. Futaki, S. et al. Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. J. Biol. Chem. 276, 5836–5840 (2001). 17. Sirri, V., Urcuqui-Inchima, S., Roussel, P. & Hernandez-Verdun, D. Nucleolus: The fascinating nuclear body. Histochemistry and Cell Biology 129, 13–31 (2008). 18. Evdokimov, A. G. et al. Structural basis for the fast maturation of Arthropoda green fluorescent protein. EMBO Rep. 7, 1006–1012 (2006). 19. Herce, H. D., Garcia, A. E. & Cardoso, M. C. Fundamental molecular mechanism for the cellular uptake of guanidinium-rich molecules. J. Am. Chem. Soc. 136, 17459–17467 (2014). 20. Mitchell, D. J., Steinman, L., Kim, D. T., Fathman, C. G. & Rothbard, J. B. Polyarginine enters cells more efficiently than other polycationic homopolymers. J. Pept. Res. 56, 318–325 (2000). 21. Yamashita, H. et al. Development of a Cell-penetrating Peptide that Exhibits Responsive Changes in its Secondary Structure in the Cellular Environment. Sci. Rep. 6, (2016). 22. Li, Z. et al. Identification and characterization of a novel peptide ligand of epidermal growth factor receptor for targeted delivery of therapeutics. FASEB J. 19, 1978–85 (2005). 23. Yukawa, H. et al. Quantum dots labeling using octa-arginine peptides for imaging of adipose tissue-derived stem cells. Biomaterials 31, 4094–4103 (2010). 24. Nakamura, Y., Kogure, K., Futaki, S. & Harashima, H. Octaarginine-modified multifunctional envelope-type nano device for siRNA. J. Control. Release 119, 360–367 (2007). 25. Looi, C. Y. et al. Octa-Arginine mediated delivery of wild-type Lnk protein inhibits TPO-induced M-MOK Megakaryoblastic leukemic cell growth by promoting apoptosis. PLoS One 6, (2011). 26. Hamy, F. et al. An inhibitor of the Tat/TAR RNA interaction that effectively suppresses HIV-1 replication. Proc. Natl. Acad. Sci. USA 94, 3548–53 (1997). 27. Westergard, T. et al. Cell-to-Cell Transmission of Dipeptide Repeat Proteins Linked to C9orf72-ALS/FTD. Cell Rep. 17, 645–652 (2016). 28. Zhou, Q. et al. Antibodies inhibit transmission and aggregation of C9orf72 poly‐GA dipeptide repeat proteins. EMBO Mol. Med. 9, 687–702 (2017). 29. Gendron, T. F. et al. Poly(GP) proteins are a useful pharmacodynamic marker for C9ORF72-associated amyotrophic lateral sclerosis. Sci. Transl. Med. 9, (2017). 30. Zhang, Y. J. et al. Poly(GR) impairs protein translation and stress granule dynamics in C9orf72-associated frontotemporal dementia and amyotrophic lateral sclerosis. Nat. Med. in press (2018). 31. Lopez-Gonzalez, R. et al. Poly(GR) in C9ORF72-Related ALS/FTD Compromises Mitochondrial Function and Increases Oxidative Stress and DNA Damage in iPSC-Derived Motor Neurons. Neuron 92, 383–391 (2016). Acknowledgements We thank Prof. Atsushi Maruyama and Dr. Naohiko Shimada at Tokyo Institute of technology for helping us obtaining CD spectra data. i Th s work was partly supported by the grants from the JSPS KAKENHI Grant number (16H06247 and 17H03923 to K. Kanekura., 17H03923 to K. Nagaoka, 17K15671 to Y. Harada, 16H05973 to Y. Hayamizu, 17H04067 to M. Kuroda). This work was also supported in part by Japan Agency for Medical Research and Development (AMED) (16ek0109180h0001 and 17ae0101016s0904), Strategic Research Foundation Grant- aided Project for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Takeda Science Foundation (K. K.), Japan Intractable Diseases (Nanbyo) Research Foundation (K. K.), Tokyo Biochemistry Research Foundation (K.K.) and The Ichiro Kanehara foundation (K.K.). SCIENTIfIC Repo R Ts | (2018) 8:12740 | DOI:10.1038/s41598-018-31096-z 10 www.nature.com/scientificreports/ Author Contributions K. Kanekura and M. Kuroda contributed to conception and design of the study. K. Kanekura, Y. Harada, T. Yagi, M. Fujimoto, Y. Hayamizu, and K. Nagaoka contributed to data collection and analysis. K. Kanekura and M. Kuroda wrote and edited the manuscript. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-31096-z. Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 SCIENTIfIC Repo R Ts | (2018) 8:12740 | DOI:10.1038/s41598-018-31096-z 11

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