Article pubs.acs.org/Biomac
Urea and Thiourea Modified Polypropyleneimine Dendrimers Clear Intracellular α‑Synuclein Aggregates in a Human Cell Line Kristoffer Laumann,†,‡ Ulrik Boas,† Hjalte M. Larsen,†,‡ Peter M. H. Heegaard,† and Ann-Louise Bergström*,‡ †
Innate Immunology Group, National Veterinary Institute, Technical University of Denmark, 1870 Frederiksberg C, Denmark Department of Neurodegeneration, H. Lundbeck A/S, 2500 Valby, Denmark
‡
ABSTRACT: Synucleinopathies are neurodegenerative pathologies in which disease progression is closely correlated to brain accumulation of insoluble α-synuclein, a small protein abundantly expressed in neural tissue. Here, two types of modified polypropyleneimine (PPI) dendrimers having either urea or methylthiourea (MTU) surface functional groups were investigated in a cellular model of synucleinopathy. Dendrimers are synthetic macromolecules that may be produced in a range of well-defined molecular sizes. Using cellomics array scan high-content screening, we show that both types of dendrimers are able to significantly reduce intracellular levels of α-synuclein aggregates dependent on the concentration, the type and molecular size of the dendrimer with the bigger size MTU-dendrimers having the highest potency. The intracellular clearance of α-synuclein aggregates by dendrimers was achieved at noncytotoxic concentrations.
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symptoms and LB formation.13,14 Also, grafting is invasive and associated with significant risks for the patient. An alternative disease modifying strategy would be to directly target the α-synuclein aggregates by using protein solubilizing drugs such as dendrimers. Dendrimers are synthetic, monodisperse, highly branched, globular polymers in which the number of branching points from the core to the periphery defines the generation number. Dendrimers are characterized by a high number of functional groups at their surfaces, for example, primary amino groups in the case of polypropyleneimine (PPI) dendrimers. These can easily be further modified into functional groups such as urea15 or thiourea (Figure 1).16 The nature of such dendrimer surface groups has a major effect on the physicochemical and ligand binding properties of the dendrimer. Despite their relatively large size, studies have shown that dendrimers have the potential to cross the blood− brain barrier (BBB) in healthy tissue when they are conjugated to targeting ligands toward receptors commonly expressed on the BBB endothelial cells.17 Also, a recent paper showed that PPI glycodendrimers were able to cross the BBB and reach neuronal cells when administered intranasally in an animal model for AD.18 Guanidino-modified dendrimers have been shown to inhibit the fibrillation of the prion peptide PrP106-126, a synthetic peptide frequently used to model the pathogenic PrPSc protein associated with transmissible spongiform encephalopathies.19 In
INTRODUCTION
Parkinson’s disease (PD) is a progressive neurodegenerative disorder with accelerated loss of dopaminergic neurons in the Substantia Nigra pars compacta. An important hallmark of PD is the formation of intracellular Lewy Bodies (LB) mainly consisting of an fibrillized and insoluble form of the protein αsynuclein; both presence and intensity of LBs is closely correlated to disease progression.1 Brain accumulation of αsynuclein fibrils is also a biochemical hallmark of additional synucleinopathies including Dementia with Lewy Bodies (DLB)2 and Multiple System Atrophy (MSA).3 At present, only symptomatic treatment of PD is available, as no disease-modifying strategies have proven useful so far.4 Levodopa, a pro-drug that is converted to dopamine after its entry into the brain, is considered the gold standard for this kind of treatment. Levodopa treatment is however associated with several adverse effects, and with prolonged use, symptoms generally become unresponsive to the treatment.5,6 Also, it is speculated that the excess extracellular dopamine levels resulting from Levodopa treatment might actually promote αsynuclein aggregation and thus speed up the disease progression.7 Surgical perturbation of affected tissues as well as subthalamic nucleus stimulation have shown positive effects, however, are also symptomatic interventions that do not stop the PD progression.8−10 Neuronal grafts from fetal mesencephalic tissues11 and, more recently, from stem cells12 have shown promising results; however, host-to-graft propagation of aggregated α-synuclein species eventually results in neuronal death in the grafted tissues and reemergence of clinical © XXXX American Chemical Society
Received: August 22, 2014 Revised: October 24, 2014
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dx.doi.org/10.1021/bm501244m | Biomacromolecules XXXX, XXX, XXX−XXX
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Two types of modified dendrimers were investigated; one having a urea modified surface and another with a methylthiourea (MTU) modified surface. The N-methyl thiourea group was introduced at the dendrimer surface to give increased hydrophobicity compared to the dendrimers with urea surface, which lack the N-methyl group. The thiourea functionality was introduced to dendrimer as this has stronger hydrogen bonding properties compared to the corresponding urea functionality in the interaction with small peptides.16,28 In the present study we found that both urea- and MTU-modified dendrimers were able to significantly reduce the intracellular levels of α-synuclein fibrils in a dose- and generation-dependent manner with MTU-modified dendrimers showing the highest potency. Although the dendrimers were associated with a certain dose- and generation-dependent cytotoxicity potentiated by coincubation with α-synuclein fibrils, the cleareance effect was nevertheless sustained at noncytotoxic concentrations.
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EXPERIMENTAL SECTION
Materials and Methods. Chemical Synthesis. General Remarks. PPI-dendrimers were purchased from SyMO-Chem BV, all other chemicals were purchased from Sigma-Aldrich and used without further purification. Solvents were purchased from Fischer Scientific and were of HPLC grade. Reversed-phase HPLC was performed on a Shimadzu LCMS 2010, using a Phenomenex Jupiter C5 column (5 μm, 300 Å) and 1 mL/min gradient from 3 to 95% buffer B over 18 min (buffer A: 0.025% TFA in 10% aqueous acetonitrile, buffer B: 0.025% TFA in 90% aqueous acetonitrile). Mass spectrometry was performed in electrospray (ESI+) mode on the Shimadzu LCMS 2010 and by MALDI-TOF on a Bruker Autoflex Speed. 2,4,6-Trihydroxyacetophenone (THAP) and fucose was applied as matrix for MALDITOF as follows: Stock solution: TFA (25 μL), Milli-Q water (475 μL), and acetonitrile (500 μL). Matrix solution: THAP (25 mg) dissolved in stock solution (1 mL). Sample solution: Sample (50 mg) dissolved in stock solution (1 mL). Sample solution (1 μL) was applied to the MALDI-TOF steel target plate and allowed to air-dry for 3 min. Matrix solution (1 μL) was added onto the dried sample droplet and the resulting droplet was allowed to air-dry for 3 min before mass analysis was carried out. In case of the G4-PPI-MTU dendrimer, 1 μL of fucose solution (25 mg/mL in Milli-Q water) was added to the dried matrix/ sample droplet and the resulting sample/matrix/fucose droplet was allowed to dry for 3 min before mass analysis. Infrared spectroscopy (IR) was performed directly on the crystalline products by a Shimadzu IRaffinity spectrometer using attenuated technique of reflectance (ATR). Nuclear magnetic resonance spectroscopy NMR (1H and HSQC) was performed in deuterium oxide on a 300 MHz Bruker Avance 300 equipped with a BBO probe and autosampler operating at 300 (1H) and 75 MHz (13C). PPI Dendrimers Modified with Urea Surface Groups (PPI-Urea). PPI-dendrimers modified with urea groups were prepared according to previously published procedures.20 PPI Dendrimers Modified with Methylthiourea Surface Groups (PPI-MTU). Commercially available PPI dendrimer, generation 2, 3, 4 (0.3 g, end group concentration appr 3 mmol), was dissolved in dichloromethane (3 mL). The reaction mixture was cooled on an ice bath, methylisothiocyanate (3.7 mmol) was added in the cold, and the mixture was stirred 48 h at rt. The solvent was removed by evaporation in vacuo, and the residue was suspended in diethyl ether (10 mL) and stirred on an icebath for 1 h, allowing the product to precipitate as a sticky solid. This was repeated 3−6 times using 15 min incubations. This afforded the alkaline methylthiourea (MTU)-modified PPI dendrimer as a white crystalline in 60−80% yields. The G2- and G3-PPI-MTU dendrimer derivatives were converted to water-soluble hydrochloride salts by suspending 1 equiv dendrimer in 3 equiv 1 M aqueous HCl. For the G4-PPI-MTU, 10 equiv of HCl were needed. The suspension was sonicated for 10 min and 5 volumes of Milli-Q water was added giving a clear solution. The aqueous dendrimer
Figure 1. Structures of a second, third, and fourth generation PPI dendrimer (G2-PPI, G3-PPI, and G4-PPI) carrying methyl thiourea (R: -(CS)-NHCH3, MTUs) or urea (R: -(CO)-NH2) groups at their surfaces.
cell models, both cationic phosphorus dendrimers and unmodified PAMAM dendrimers have been shown to be able to clear protease-resistant aggregates of PrPSc efficiently.20−22 The same type of dendrimers have been shown to inhibit the aggregation process, leading to formation of fibrillar α-synuclein polymers in cell-free systems.23−26 No studies so far have reported on effects of dendrimers on intracellular α-synuclein aggregates in living cells. Here, we have investigated the effect of surface-modified PPI dendrimers in a cellular model of synucleinopathy in which SKMEL-5 cells were incubated with fluorescently labeled preformed α-synuclein fibrils. SK-MEL-5 is a human melanoma cell line with a high endogenous expression of α-synuclein.27 B
dx.doi.org/10.1021/bm501244m | Biomacromolecules XXXX, XXX, XXX−XXX
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Figure 2. Examples of images from Cellomics: (A) Composite (blue and green) image; (B) Nuclei (in gray) defining “Circ” area (blue line) and green line defining “Ring” region; Orange line defines rejected nuclei; (C) Algorithm defined areas as in B, but with Ring spots in yellow and Circ spots in red. SK-MEL-5 cells were fixed with 8% paraformaldehyde (PFA) to the medium-containing wells in 1:1 volume ratio (yielding a total of 4% PFA), and the fixed cells were washed twice with phosphate buffered saline (PBS). Fixed SK-MEL-5 cell nuclei were stained with Hoechstdye and washed repeatedly with PBS. The plates were then analyzed with Cellomics ArrayScan technology. From every well, 20 images (Fields) were taken; in channel 1, the Hoechst-staining (blue) was used to identify the nuclei, and in channel 2, the green fluorescence associated with α-synuclein fibril was recorded. In Figure 2A, the composite image of a selected field is shown. In Figure 2B, the applied algorithm is shown; the blue line surrounding the nuclei defines an area denoted “Circ”, and the area surrounding this (restricted by the green line) is a user-defined area denoted “Ring”, which roughly corresponds to the cellular outline. All of the green fluorescence located in the “Ring” area (yellow in Figure 2C) is by the algorithm interpreted as intracellular (nonnuclear, which is red in Figure 2C) aggregates. The average area of these “Ring” spots was in the following experiments used as a measure of the intracellular load of fibrils. The average number of valid nuclei per field in a well is in the following being used as a measure of the cell density and viability, as dead cells will either not be fixed or will appear too small to be accepted by the algorithm. MTT Viability Assay. Initial cytotoxicity assays: SK-MEL-5 cells were added to Nunclon 96-well plates at densities of 6000 cells per well (for 24 h α-synuclein fibril exposure) or 3000 cells per well (for the 48 h α-synuclein fibril exposures). After ended incubation with fibrils or dendrimers, as indicated in figure legends, MTT was added directly to the cells and the viability assay was carried out according to the method of Mosmann.29 The viability of cells was determined relative to SK-MEL-5 cells not exposed to fibrils. Fibrillation Procedure. Recombinantly expressed (E. coli) monomeric α-synuclein was purchased from rPeptide (Cat. No. S-1001) as a lyophilized powder and dissolved in water giving a solution of 1 mg/ mL in 10 mM Tris/HCl and 200 mM NaCl at pH = 7.4. Monomeric α-synuclein was then labeled with Atto488 from Sigma following the instructions from the supplier. Fibrillation was initiated by incubating a mixture of 30% Atto488-labeled and 70% unlabeled α-synuclein at 37 °C with agitation of 200 rpm in a Vortemp 56 agitator for 12 days. Presence of fibrils was analyzed with Thioflavin T assay and Western Blot (not shown). Immunocytochemistry. In 96-well plates, SK-MEL-5 cells and additives were incubated as described above using unlabeled αsynuclein fibrils. After fixation, the SK-MEL-5 cells were permeabilized with 0.25% Tween-20 in PBS for 10 min and blocked in 3% BSA in PBS for 1 h. The mouse monoclonal antibody LB509 (ab27766 from Abcam) was diluted 1:2000 in blocking buffer then added and incubated for 1 h at room temperature. After washing with PBS, a secondary goat-antimouse antibody conjugated with Alexa-Flour 568 nm (A11031, Sigma, 1:400) was added together with Hoechst dye (1:800) for nuclear staining. The plate was read in the Cellomics using the wavelengths optimized for the Alexa-568 flourescent label.
solutions were freeze-dried to afford the hydrochloride salts as crystalline compounds. G2-PPI-MTU analytical data: HPLC, one peak, tR: 12.2 min. Mass spectrometry (C56H120N22S8): ESI+ (MH22+) Calcd, 680.2; found, 680. MH33+Calcd, 453.8; found, 453. MH44+ Calcd, 340.6; found, 341. MALDI-TOF: MH+ Calcd, 1359.3; found, 1359.4. IR (ATR): ν in cm−1 1257.6 (CS, thiourea), 1340.5 (C−H, methyl), 2945.3 (C−H satd), 3248.1 (N−H stretch, thiourea). NMR (1H and HSQC, 300 MHz, D2O): δ in ppm 1.79 (br s, 4H, CH2(core)), 1.95 (br qn, 16H, CH2), 2.16 (br. s., 8H, CH2), 2.77 (s, 24H, CH3-N), 3.24 (m, 36H, CH2-N), 3.49 (br s, 16H, CH2-N). 13C (75 MHz): δ in ppm 18.7, 20.7, 23.5, 29.9, 36.5, 49.3, 49.9, 50.3, 52.24, 65.9, 179.5. G3-PPI-MTU analytical data: HPLC, one peak, tR: 12.7 min. Mass spectrometry (C120H256N46S16): ESI+ (MH44+) Calcd, 715.0; found, 715. MH55+ Calcd, 572.2; found, 572. MH66+ Calcd, 477.0; found, 474. MALDI-TOF: (MH+) Calcd, 2858.2; found, 2861.9. IR (ATR): ν in cm−1 1257.6 (CS, thiourea), 1346.3 (C−H, methyl), 2945.3 (C−H satd), 3234.6 (N−H stretch, thiourea). NMR (1H and HSQC, 300 MHz, D2O): δ in ppm 1.99 (m, 4H+32H, CH2), 2.21 (br s, 24H, CH2), 2.79 (s, 48H, CH3-N), 3.22−3.31 (m, 84H, CH2-N), 3.51 (br s, 32H, CH2-N). 13C (75 MHz): δ in ppm 18.7, 19.1, 20.8, 23.5, 29.9, 40.9, 49.3, 50.1, 50.3, 52.7, 65.9, 179.6. G4-PPI-MTU analytical data: HPLC, one broad peak, tR: 13.4 min. Mass spectrometry (C248H528N94S32): MALDI-TOF (MH+) Calcd, 5855.7; found, 5856.2. IR (ATR): ν in cm−1 1257.6 (CS, thiourea), 1348.2 (C−H, methyl), 2949.2 (C−H satd), 3248.1 (N−H stretch, thiourea). NMR (1H and HSQC, 300 MHz, D2O): δ in ppm 1.99 (br s, 4H+64H,CH2), 2.22 (br s, 56H, CH2), 2.81 (br s, 96H, CH3-N), 3.29 (br s, 180H, CH2-N), 3.56 (m, 64H, CH2−N). 13C (75 MHz): δ in ppm 18.9, 19.4, 21.8, 23.7, 29.3, 31.2, 41.3, 49.4, 50.4, 66.2, 179.2. Cell Cultures. SK-MEL-5 cells (human melanoma, ATCC, HTB70) were cultured in Dulbecco’s Modified Eagle Medium with 4.5 g/L glucose and pyruvate (1 mM pyruvate; Gibco). Fetal Calf Serum (FCS, Gibco) was added as 10% of total volume upon opening, as well as penicillin and streptomycin at concentrations of 10.000 U/mL and 10.000 μg/mL, respectively, added as 1% of the total volume. Cells were cultured in culturing flasks containing 10 mL of medium, which was changed every 48 h. Culturing was done in incubators at 37 °C, 100% relative humidity, and 5% CO2. Passage of cells were done when confluencies reached >90% of the surface of the flasks. Cell cultures were used in passages 2−10. Cellomics ArrayScan Technology. SK-MEL-5 cells were added to 96-well Falcon plates at densities 2000−3000 cells per well of 100 μL and allowed to attach for 24 h (incubation conditions as above). Atto488-labeled fibrils of α-synuclein (see below) were added to the media to a total concentration of 0.01 mg/mL and incubated for 24 h, whereafter the cells were washed twice with media to remove extracellular fibrils. Dendrimers of various generations and concentrations or in-house antibodies against α-synuclein epitopes (comparative control) were then added to the cells and the plates were incubated for a further 48 h as above. C
dx.doi.org/10.1021/bm501244m | Biomacromolecules XXXX, XXX, XXX−XXX
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Transmission Electron Microscopy. Samples of either untreated αsynuclein fibrils or fibrils treated with MTU-G3 dendrimer for 48 h were mounted on carbon-coated mesh grids, stained 2% with uranyl acetate and analyzed immediately using a Philips CM 100 TEM, operated at an accelerating voltage of 80 kV and equipped with an OSIS Veleta digital slow scan 2k × 2k CCD camera. Digital images were recorded with the ITEM software package.
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RESULTS Synthesis of Dendrimers. Methylthiourea- (MTU-) and urea-modified dendrimers were synthesized from commercially available PPI dendrimers having primary amines as surface functional groups. Urea-derivatized dendrimers were synthesized as described previously20 and MTU dendrimers (G2− G4) were synthesized by reaction of PPI dendrimers with methylisothiocyanate in dichloromethane at ambient temperature to afford PPI dendrimers completely derivatized with surface MTU groups in good yields. In their alkaline form, these MTU dendrimers are not soluble in water and, therefore, the dendrimer tertiary amines were converted to water-soluble hydrochloride salts by ultrasonication with dilute hydrochloric acid. This procedure afforded the MTU dendrimers as highly water-soluble crystalline compounds with high purity according to reversed-phase HPLC (measured at 220 nm; Figure 3). Furthermore, mass spectrometry analysis (electrospray and MALDI-TOF) gave molecular masses corresponding to the masses expected for the products (Figure 3). In MALDI-TOF analysis, 2,4,6-trihydroxyacetophenone (THAP) was preferred as matrix in comparison to α-hydroxy-cyano-cinnamic acid (HCCA) which gave poorer spectra due to fragmentation. Infrared spectroscopy on all the dendrimer conjugates showed strong CS and N−H stretch bands (approximately 1255 and 3230 cm−1, respectively), indicating the formation of thiourea surfaced dendrimers. Furthermore, the presence of methyl groups were indicated by a characteristic weak band at approximately 1350 cm−1 in accordance with the presence of methyl groups in PPI-MTU. Urea and MTU Dendrimers Show Low Cytotoxicity up to 0.1 mg/mL. Dendrimers have previously been found to be cytotoxic.20 The cytotoxicity of urea- and MTU-modified PPI dendrimers was therefore analyzed in order to define nontoxic concentration ranges in the human melanoma cell line, SKMEL-5 expressing high, endogenous levels of α-synuclein and using the MTT assay for assessing cell viability. A guanidinium-modified dendrimer was used as a positive control for dendrimer cytotoxicity as initial experiments established that guanidinium-modified dendrimers show high cytotoxicity, confirming previous findings (Cordes et al. 2007). Urea-modified PPI dendrimers of generation 2, 3, and 5 (G2PPI-urea, G3-PPI-urea, G5-PPI-urea) and MTU-modified PPI dendrimers of generation 2, 3, and 4 (G2-PPI-MTU, etc.) were tested. Within the concentration range tested (0.002−0.1 mg/ mL), urea-modified dendrimers showed no or very low cytotoxicity, with the exception of generation 5, which caused a slight reduction in cell viability at 0.1 mg/mL (Figure 4A). For the MTU-modified dendrimers, the same pattern was observed, with only generation 4 showing significant cytotoxicity at the highest concentration used (0.1 mg/mL, Figure 4C). Cell density was also analyzed using the Cellomics technology and was found to correlate well with cell viability, as assessed by the MTT assay. Both types of dendrimers reduced the cell density significantly only for the highest
Figure 3. RP-HPLC (top three) and MALDI-TOF mass analysis (bottom three) of the synthesized PPI-MTU dendrimers: G2-PPIMTU (top), G3PPI-MTU (middle), G4-PPI-MTU (bottom).
dendrimer generation (G5 for urea and G4 for MTU) at the highest concentration tested (Figure 4B,D). α-Synuclein Fibrils Increase the Cytotoxicity of MTUDendrimers but Not of Urea-Dendrimers. Next, cytotoxicity of dendrimers in the presence of α-synuclein fibrils was investigated. For this purpose, SK-MEL-5 cells were incubated with α-synuclein fibrils at 0.01 mg/mL for 24 h, then dendrimers were added and the cells were incubated for another 48 h. Toxicity was assessed by quantifying cell density using the Cellomics ArrayScan. α-Synuclein fibrils at 0.01 mg/ mL were not by themselves cytotoxic toward then SK-MEL-5 cells (Figure 5B,D). For the urea-modified dendrimers, only concentrations where the dendrimers were not cytotoxic per se were analyzed (Figure 5A) and coincubation with α-synuclein D
dx.doi.org/10.1021/bm501244m | Biomacromolecules XXXX, XXX, XXX−XXX
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Figure 4. Cytotoxicity of PPI-urea and PPI-MTU dendrimers in SK-MEL-5 cells, as assessed by MTT assay (A, C) and quantification of living cells density measurement by the Cellomics assay (B, D). Plates (96-well) containing samples for the two methods were processed in parallel and treated identically until analysis. The graphs in A and C are MTT-values normalized to untreated control. Error bars refer to SEM (standard error of mean) and asterisks indicate significant differences toward untreated control cells in an unpaired students t test. N = 3 for each concentration and dendrimer type.
Figure 5. Cytotoxicity (48 h) of PPI-Urea and PPI-MTU Alone (A, C) and in Combination with α-Synuclein Fibrils (B, D). The cytotoxicity was assessed as relative cell density (average number of cells per valid field per well) normalized to untreated control cells. Error bars refer to SEM (standard error of mean) and asterisks indicate significant differences toward untreated control cells in an unpaired students t test. N = 3 for each concentration and dendrimer type.
fibrils did not result in cytotoxicity (Figure 5B). However, for the MTU-modified dendrimers, the presence of α-synuclein fibrils increased cytotoxicity, for example, G3-PPI-MTU alone
was only weakly cytotoxic (Figure 5C), whereas when coincubated with α-synuclein fibrils, it was significantly toxic toward SK-MEL-5 cells at the highest concentration (0.1 mg/ E
dx.doi.org/10.1021/bm501244m | Biomacromolecules XXXX, XXX, XXX−XXX
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Figure 6. (A, B) Cellomics quantification of intracellular α-synuclein fibril load (Ring Spot average area; thus, the average area per spot per cell). Cells were incubated with α-synuclein fibrils for 24 h, then washed in media, and dendrimers (urea or MTU modified in the indicated generations and concentrations) were added in fresh cell culture media and incubated for a further 48 h. The error bars refer to SEM (standard error of mean) and asterisks indicate significant differences toward fibrils alone in a student’s unpaired t test. N = 3 for each concentration and dendrimer type.
Figure 7. Cellomics quantification of SK-MEL-5 cell viability (average number of living cells per field) and intracellular fibril load (RingSpot average area) after treatment with unlabeled α-synuclein fibrils (24 h) and subsequently dendrimers (as indicated) for 48 h. Light gray bars = urea-PPI and dark gray bars = MTU-PPI. After fixation, the cells were stained for α-synuclein with LB509. Error bars indicate SEM (standard error of mean) and asterisks indicate significant differences (to fibrils only) in student’s nonpaired t test. N = 3 for each dendrimer type and concentration.
dependently reduced the intracellular fibril load by ∼35−55%, with all three generations used (G2, G3, and G5, Figure 6A), with no clear generation-dependent effect. In comparison, the anti-α-synuclein antibody reduced fibril load by 60−80% (Figure 6A,B). Incubation with PPI-MTU dendrimers resulted in a dose- and generation-dependent reduction in intracellular fibril load and in this case the effect increased with both concentration and generation (Figure 6B). The maximum effect (50−75% reduction) was obtained with G4-MTU-PPI used at 0.025 mg/mL. There was only very little variation in well-towell cell density, verifying that none of the treatments were cytotoxic (not shown). This set of experiments was run independently three times with similar results. As the observed reduction in average spot area could be due to unspecific reduction of fluorescence, either due to cleavage of the Atto488 tag from the α-synuclein fibrils or due to quenching of fluorescence by the added dendrimers, a control experiment was performed in which SK-MEL-5 cells were incubated with unlabeled α-synuclein fibrils followed by dendrimer-treatment as above and analysis of intracellular fibril load by immunocytochemistry. Although this setup led to more well-to-well variation in cell density (Figure 7A), probably due
mL; Figure 5D.) The G4-PPI-MTU dendrimer, which was cytotoxic alone at 0.1 mg/mL, was rendered more cytotoxic by the presence of α-synuclein fibrils, 0.05 mg/mL being also cytotoxic (Figure 5D). Both Urea- and MTU-Modified Dendrimers Clear Internalized α-Synuclein Fibril in SK-MEL-5 Cells. Having established the noncytotoxic concentration range of dendrimers in the presence of α-synuclein fibrils, it was investigated whether treatment with dendrimers in a noncytotoxic range was able to reduce the intracellular accumulation of α-synuclein fibrils. SK-MEL-5-cells were loaded with fluorescently (Atto488) labeled α-synuclein fibrils by incubation of cells with fibrils for 24 h followed by washing to remove extracellular fibrils. This treatment makes the cells internalize and accumulate the fibrils, quantifiable as intracellular “Ring Spots”, as described in Materials and Methods. As a positive control for fibril clearance, an in-house anti-α-synuclein antibody that was previously shown to be very potent in clearing intracellular α-synuclein accumulation in this model (not published) was used. After loading of cells with fibrils, they were incubated with dendrimers (or anti-α-synuclein antibody for positive control) for 48 h. Incubation with PPI-urea doseF
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structures (Figure 8C) that were not seen in the nontreated fibrils.
to the increased number of washing steps in the immunocytochemistry protocol, a significant decrease in average spot area after incubation with dendrimers could still be seen (Figure 7B). The concentration and generation dependence were not as clear in the immunohistochemistry analysis, as seen by the direct analysis of fluorescent fibrils. Thus, it was confirmed that both types of dendrimers were able to decrease the intracellular α-synuclein fibril load significantly at noncytotoxic concentrations. The reason why the cell density increases significantly in all of the cells receiving dendrimers compared to untreated cells and fibril-only treated cells (Figure 7A) is not certain. It could be an assay artifact due to increased attachment of cells to the polymer surface when dendrimers are present resulting in higher retained cell numbers compared to cells not treated with dendrimers. Electron Microscopy Shows Dendrimer-Induced Fragmentation of α-Synuclein Fibrils. As MTU- and ureamodified dendrimers were able to significantly decrease intracellular α-synuclein fibril load, we next investigated whether dendrimers were able to directly fragment or solubilize α-synuclein fibrils in an acellular assay. Preformed α-synuclein fibrils were incubated with the G3-PPI-MTU dendrimer, established by the previous experiments to have a good, biological effect) for 48 h and subsequently inspected by electron microscopy. For comparison, untreated fibrils were also analyzed. Whereas the untreated fibrils presented themselves as fibrillar structures of up to 900 nm in length (10−12 nm wide) as well as larger aggregates (Figure 8A), the
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DISCUSSION In this study, we investigated the solubilizing effect on intracellular α-synuclein fibrils of two types of polypropyleneimine (PPI) dendrimers; one type modified with surface urea groups and one modified with methylthiourea (MTU) surface groups. Different generations (G2, G3, and G5 for urea-PPI and G2, G3, and G4 for MTU-PPI) and concentrations were tested. We were able to show that both types of dendrimers could partially clear human melanoma cells from internalized αsynuclein fibril deposits in a generation- and concentrationdependent way. Both MTU- and urea-PPI dendrimers were able to clear the intracellular α-synuclein aggregates in noncytotoxic concentrations, although both types of dendrimers did show cytotoxicity at higher concentrations. The PPI-MTU dendrimers were found to be more potent in clearing the cells from the intracellular α-synuclein aggregates than the PPI-urea dendrimers, with the G4-PPI-MTU dendrimers showing the strongest effect in clearing fibrils from the cells; however, PPI-MTU dendrimers were also more cytotoxic than the urea-PPIs. The MTU-group is more hydrophobic than the urea-group and this may lead to a higher cell membrane penetrating ability of the MTU-PPIs in comparison to the urea-PPIs. Higher cell penetrating abilities may therefore be the explanation for both the higher fibril clearance properties and higher cytotoxicity. However, whether the lower polarity of the methyl thiourea results in an increased uptake/interaction with the cell wall or in a better direct solubilization of the fibrils is not evident from the present results. In contrast to, for example, silicon dendrimers, PPI dendrimers are, to a large extent, governed by the properties of their surface groups, as PPI dendrimers have a more dense surface. The correlation between type of dendrimer-surface group and cytotoxicity and biological effect of modified PPIdendrimers in another cell line, the prion-infected SMB-cell line has previously been shown.20 For both groups of PPIs, fibril clearance properties and cytotoxicity increased with generation number, high generation PPIs being both more cytotoxic and giving better cellular clearance than lower generation PPIs. This also is in agreement with earlier findings20 and can be explained by the increased number of surface groups in the higher generation dendrimers resulting in an increased clearance ability as more active/surface groups may interact with the α-synuclein fibrils. However, the higher number of surface groups combined with higher molecular volume also results in increased disruption of the cellular membrane leading to increased cytotoxicity. Transmission electron microscopy was applied to show the direct fibril-solubilizing effect of the G3-PPI-MTU dendrimer. Here, the coincubation of fibrils with G3-MTU-PPI resulted in dissolution and fragmentation of α-synuclein fibrils into smaller fibrils and less organized aggregates. As fibrils were cleared from fibril-loaded cells, we hypothesize that the dendrimers are able to enter the cells and directly interact with α-synuclein fibrils by partly fragmenting and solubilizing them into smaller aggregates that are more easily degraded by the cell. If dendrimers indeed are able to enter the cells and clear intracellular aggregates, then the observed higher potency of the hydrophobic MTU-PPI dendrimers could be explained by a higher cell penetrance.
Figure 8. Representative transmission electron microscopical images showing α-synuclein fibrils alone (A) or after 48 h of coincubation with MTU-G3-PPI (B−D).
fibrils in the G3-PPI-MTU incubated samples were around 200 nm in length and only few larger aggregates were present (Figure 8B−D). This seems to indicate that coincubation with dendrimer fragmented α-synuclein fibrils into smaller aggregates. At higher magnification, these fractions could be seen more clearly (Figure 8C,D) as well as some “floral-like” fibrillar G
dx.doi.org/10.1021/bm501244m | Biomacromolecules XXXX, XXX, XXX−XXX
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Although fibril-solubilizing effects are hypothesized to be able to rescue cells from the detrimental protein aggregate load, it is possible that the solubilization of larger fibrils into smaller fibrils and oligomeric structures could increase the cytotoxicity of the pathogenic protein species. In this regard, we observed that the MTU-PPI dendrimers became more cytotoxic in the presence of α-synuclein fibrils. This could be due to a direct interaction between fibrils and the dendrimer and possibly to the formation of fibril fragments (oligomers) with increased cytotoxicity.
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CONCLUSION PPI-dendrimers modified with N-methyl-thiourea (MTU) and urea surface groups are able to clear α-synuclein aggregates from human cells and dissolve α-synuclein fibrils into smaller fragments at noncytotoxic concentrations. The potency of the dendrimers was found to be dependent on both the hydrophobicity of the surface groups as well as on dendrimer generation. The cytotoxicity of the MTU modified PPI dendrimers increased in the presence of α-synuclein fibrils which may indicate that the fibril solubilizing effect of the dendrimer results in the formation of cytotoxic fibril oligomers.
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AUTHOR INFORMATION
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Nikki Jane Damsgaard, Louise Buur Vesterager, Karolina Morawska, and Heidi Gertz Andersen are gratefully thanked for their excellent technical assistance during the project.
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REFERENCES
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