Defined α-synuclein prion-like molecular assemblies spreading in cell culture
- Suzana Aulić†1,
- Tran Thanh Nhat Le†1,
- Fabio Moda2,
- Saïda Abounit3,
- Stefania Corvaglia4,
- Loredana Casalis4,
- Stefano Gustincich5,
- Chiara Zurzolo3,
- Fabrizio Tagliavini2 and
- Giuseppe Legname1, 4Email author
© Aulić et al.; licensee BioMed Central Ltd. 2014
Received: 3 April 2014
Accepted: 22 May 2014
Published: 4 June 2014
α-Synuclein (α-syn) plays a central role in the pathogenesis of synucleinopathies, a group of neurodegenerative disorders that includes Parkinson disease, dementia with Lewy bodies and multiple system atrophy. Several findings from cell culture and mouse experiments suggest intercellular α-syn transfer.
Through a methodology used to obtain synthetic mammalian prions, we tested whether recombinant human α-syn amyloids can promote prion-like accumulation in neuronal cell lines in vitro. A single exposure to amyloid fibrils of human α-syn was sufficient to induce aggregation of endogenous α-syn in human neuroblastoma SH-SY5Y cells. Remarkably, endogenous wild-type α-syn was sufficient for the formation of these aggregates, and overexpression of the protein was not required.
Our results provide compelling evidence that endogenous α-syn can accumulate in cell culture after a single exposure to exogenous α-syn short amyloid fibrils. Importantly, using α-syn short amyloid fibrils as seed, endogenous α-syn aggregates and accumulates over several passages in cell culture, providing an excellent tool for potential therapeutic screening of pathogenic α-syn aggregates.
Keywordsα-Synuclein Protein aggregation Seeding Prion
α-Synuclein (α-syn) is a key player in the pathogenesis of a group of neurodegenerative diseases defined as synucleinopathies, including Parkinson disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). The discovery that aggregated α-syn is the major component of Lewy bodies (LBs)  indicates a role of α-syn in PD. Neurons containing LBs are the hallmark of PD and DLB, whereas in MSA α-syn is deposited in oligodendrocytes referred to as glial cytoplasmic inclusions (GCIs) [2–4]. In its aggregated form, α-syn is enriched in β-sheet structure, orderly organized into oligomers or amyloid fibrils . As such, many amyloid proteins are characteristic of specific neurodegenerative disorders, for instance Alzheimer disease (AD) and prion diseases. For several decades it has been speculated that the key to understanding age-related neurodegenerative disorders may be found in the unusual biology of prion diseases. This hypothesis is being increasingly supported by experimental evidence. The self-propagation of amyloid-β (Aβ) aggregates is also known from in vitro studies  and inoculation experiments . However, only recent experiments using genetically modified rodents have established that Aβ can be induced to deposit in brain by a prion-like mechanism. Intracerebral injection of Aβ-rich brain extracts from AD patients or from aged APP-transgenic mice stimulated the premature formation of plaques in these models [8, 9]. Aβ lesions in APP transgenic mice are also inducible by injections of pure, synthetic human Aβ fibrils, although synthetic seeds are less powerful than aggregates formed within the living brain. Like prions, Aβ seeds vary in size from small, soluble, protease-sensitive aggregates to large, insoluble, protease-resistant fibrils .
Accumulating experimental data indicate that the seeding principle also applies to other pathogenic proteins that form amyloid-like inclusions within the cells. This is also the case of α-syn which, in its misfolded state, forms assemblies known as LBs . In PD, α-syn aggregates arise first in the brainstem and then spread to telencephalic structures —a dynamics indicative of prion-like spread of protein aggregation. Numerous studies have shown cell-to-cell transmission of soluble or aggregated α-syn, both in cultured cells and in mouse brains, resulting in α-syn aggregation and neuronal dysfunction in the recipient cells [6, 13–15]. Importantly, injections of synthetic (human or mouse) α-syn fibrils also induce the recruitment of endogenous soluble α-syn protein to form LB-like pathology and neuronal degeneration in primary cell culture  and in non-transgenic (wild type) host mice [17, 18]. Lastly, Melki et al. have shown that two different α-syn polymorphs (fibrils and ribbons) exhibit marked differences in their propensity to penetrate the cells, as well as in their toxicity and seeding aggregation in cells, suggesting the existence of different α-syn strains .
On the basis of these findings, we considered the possibility that recombinant human α-syn could acquire prion-like properties, once it is converted into a β-sheet-rich structure, and determine the fate of endogenous α-syn in human immortalized cell lines.
Aggregation properties and structural characterization of recombinant α-syn assemblies
Evidence in literature shows that, under different experimental conditions, the formation of LB-like inclusions can be induced in cultured cells overexpressing human α-syn . To test whether the same process may be induced in the non-transfected human SH-SY5Y cell line we applied different α-syn fibril assemblies to the cell culture.
We reasoned that these diverse β-sheet-rich assemblies might differ in quaternary structure. As predicted, atomic force microscopy measurements showed diverse assemblies: oligomers, and short and long amyloid fibrils (Figure 1C). From the statistical analysis of the three different preparations (Figure 1D) we identified three different assemblies enriched in: (i) oligomeric structure, indicating spherical, ring-like characteristics; (ii) short fibrils, the protofibril-like structure, and (iii) long fibrils (Additional file 2: Table S1 lists all height and length values observed).
Toxicity of α-syn aggregates in cells
Effects of exposure of human neuroblastoma cell lines to different α-syn assemblies
Short amyloid fibrils of α-syn induce accumulation earlier in transfected SH-SY5Y cells
Endogenous α-syn aggregates display LB-like properties
Together, these observations indicate that the endogenous α-syn aggregates in this cell culture model exhibit LB-like defining features.
Immortalized cell lines and primary-cultured cells are commonly used in the research of synucleinopathies. Volpicelli-Daley et al. have reported a seeded inclusion formation in primary neuronal cultures that did not overexpress α-syn, and Sacino et al. have investigated the aggregation process in primary mixed neuronal-glial cultures using recombinant wild-type α-syn and PD-linked mutations (A53T and E46K). Nevertheless, the drawback of employing primary cell models is that they have a limited use for transmission experiments. Moreover, the cellular populations might be altered during repeated sub-passaging. This is one reason why we focused on immortalized cell lines, as they yield more homogenous cell preparations, cells can be passaged for long periods, and they harbor a sufficient quantity of samples for biochemical analyses. Indeed, here we show that one defined molecular assembly of extracellular α-syn is able to induce endogenous α-syn to aggregate, thus supporting the hypothesis that α-syn pathology can spread via a prion-like self-templating mechanism. This finding is a shared feature with other cell culture models that are permissive to prion replication, such as neuroblastoma N2a cells . This infected cell culture model has provided valuable insights into the biogenesis of the scrapie prion protein (PrPSc) in terms of subcellular localization, conversion, and physiopathological consequences. Therefore, using the immortalized SH-SY5Y cell line, which replicates α-syn aggregates, is important to better understand the mechanism involved in α-syn aggregation. We focused our attention on non-transfected SH-SY5Y cells, and these findings were also confirmed in another cell line that does not overexpress α-syn protein. The GT1 cell line  was treated with short amyloid fibrils of recombinant human α-syn, and we observed the same results, indicating that this aggregation phenomenon extends to multiple cell types, even to those that do not overexpress α-syn (Additional file 5: Figure S4). These cell cultures will allow us to study the mechanism of cellular uptake of fibrils (p0), the conversion mechanism of endogenous α-syn protein into aggregates (from p4), and their sub-cellular localization.
In this study we characterized three different fibril assemblies of recombinant α-syn protein (oligomers, short fibrils, and long fibrils) and evaluated their effect on immortalized cell lines. Our findings show that recombinant human α-syn can adopt a conformation (i.e., short amyloid fibrils) able to recruit cellular α-syn into aggregates that can replicate and accumulate in vitro in non-transfected cells. Although we used synthetic recombinant assemblies, and not the aggregates from brain homogenates either from PD patients or transgenic mice, we observed prolonged and sustained aggregation and accumulation of endogenous α-syn in non-transfected and transfected SH-SY5Y cell lines. Our findings point to α-syn short amyloid fibrils as the pathological species of α-syn aggregates primarily involved in the transmission of α-syn pathology. Our data support the hypothesis that the sequence of events leading to LB formation can be recapitulated in cultured cells. In summary, we developed a new approach for the study of α-syn aggregates in cell culture. The advantage of our system is that it can be employed for prolonged experimental periods since the accumulation of α-syn is sustained over time. This approach systematically minimizes the possibility of clonal artifacts and represents a valid tool for potential therapeutic screening that targets pathologic α-syn aggregates.
The GT1-1 cells were obtained from the laboratory of Professor Krister Kristensson, Department of Neuroscience, Karolinska Institutet Stockholm, Sweden, following an MTA agreement with Dr. Pamela Mellon of the University of California, San Diego, USA. This line is often used in prion biology and it is established from gonadotropin hormone-releasing neurons immortalized by genetically targeted tumorigenesis in transgenic mice . The SH-SY5Y cell line is a thrice-cloned sub-line of SK-N-SH cells, which were originally established from a bone marrow biopsy of a neuroblastoma patient with sympathetic adrenergic ganglial origin . GT1 cells were seeded in 10-cm plates containing 10 mL of Dulbecco’s modified Eagle’s medium (DMEM) culture media, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. SH-SY5Y normal cells were cultivated in 10-cm plates, containing 10 mL of minimal EMEM: Ham F12 (1:1) culture media, supplemented with 15% FBS, 1% non-essential amino acids, 0.5% L-glutamax, 1% penicillin-streptomycin and 1% G-418 (only for SH-SY5Y wild-type cells transfected for overexpressing α-syn). The cells were grown at 37°C in 5% CO2 to 95% confluence for 1 week before splitting at 1:10 for further cultivation.
The cytotoxic effect of α-syn fibrils was assessed by measuring cellular redox activity with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Briefly, 100,000 SH-SY5Y and GT1 cells/well were cultured in a 96-well microtiter plate and treated with α-syn oligomers, short fibrils and long fibrils (suspended by vortexing). Following a 24-h incubation, the cytotoxic effect was assessed by measuring cellular redox activity, following the manufacturer’s (Sigma) instructions.
Expression and purification of recombinant human α-syn
Expression and purification of recombinant human α-syn were performed in accordance with the method previously described . Briefly, the human α-syn gene was cloned and expressed in pET11a vector using BL21 (DE3) E. coli strain. Expression of α-syn was obtained by growing cells in Luria-Bertani broth medium with 100 mg/mL ampicillin at 37°C until an O.D.600 of about 0.6, followed by induction with 0.6 mM isopropyl β-D-thiogalactoside (IPTG) for 5 hours. The protein was purified according to the method of Huang et al..
α-syn amyloid preparations
All solutions were sterilized by filtration through a 0.22-μm filter prior to each assay run. Reactions were prepared in a 96-well black plate (BD Falcon), and each well contained 200 μL of reaction solution [1.5 mg/mL recombinant human α-syn, 100 mM NaCl, 10 μM Thioflavin T (ThT) in 20 mM Tris–HCl pH 7.4]. Each sample analysis was performed in fifteen replicates. Each well contained one 3-mm glass bead (Sigma). The plate was covered with sealing tape (Fisher Scientific), incubated at 37°C under continuous shaking, and read on SpectraMax M5 fluorescence plate reader (Molecular Devices) by top fluorescence reading every 5 min at excitation of 444 nm and emission of 485 nm.
AFM analysis was performed in accordance with the method previously described . Three to five μL of fibril solution was deposited onto a freshly cleaved piece of mica and left to adhere for 30 min. Samples were then washed with distilled water and blow-dried under a flow of nitrogen. Images were collected at a line scan rate of 0.5-2 Hz in ambient conditions. The AFM free oscillation amplitudes ranged from 25 nm to 40 nm, with characteristic set points ranging from 75% to 90% of these free oscillation amplitudes. AFM data were analyzed with Gwyddion (gwyddion.net) and SPIPTM (http://www.imagemet.com) software.
α-syn amyloid solution for cell infection
The α-syn amyloid solution was transferred from the 96-well plate into a 1.5 mL Eppendorf tube and collected under sterile conditions. The solution was ultracentrifuged at 100,000 g for 30 min at 4°C (Beckman Coulter). Pellets were resuspended in 1X PBS and then sonicated (Branson 2510) for 5 min prior to adding to the cultured cell plate.
α-syn fibril infection in cell lines
Three hundred μg of α-syn amyloids was added to GT1 and SH-SY5Y cell plates (10 cm-plate) and exposed in the cell culture media for 7 days before the next splitting and media change. Cells were split and maintained for six additional passages. Cell lysates were collected at each passage for Western blotting and immunofluorescence studies. For immunofluorescence of amyloids internalization, cells were cultured in 12-well plates with coverslips; 30 μg of α-syn amyloids was added to the cell culture media and incubated for 7 days.
Detection of α-syn aggregates in infected cells and analysis by Western blotting
The total protein content of samples was measured by bicinchoninic acid assay (BCA) (Pierce). Fifty μg of cell lysates protein was used and 5X loading buffer was added in a 1:5 ratio. The samples were boiled for 5 min at 100°C, loaded onto a 10% Tris-Glycine SDS-PAGE gel, and transferred overnight onto Immobilon P PVDF membranes (Millipore). Membranes were blocked by 5% nonfat milk, incubated with 0.4 μg/mL rabbit polyclonal anti-α-syn antibody (Santa Cruz) followed by incubation with goat anti-rabbit IgG F(ab)2 fragment conjugated with horseradish peroxidase. Blots were developed with the enhanced chemiluminescent system (ECL, Amersham Biosciences) and visualized on Hyperfilm (Amersham Biosciences). To analyze α-syn aggregation in cell samples, cells were scraped in TBS buffer containing 1% Triton X100, protease cocktail inhibitors and phosphatase inhibitor. After sonication (Sonicrep 150) with 10 amplitude microns for 3 times (30 seconds of sonication and 30 seconds intermediate stop) cells were centrifuged at 2000 RPM for 5 min before the protein was quantified. Fifty μg of sample was centrifuged at 100,000 g for 30 min. Supernatant (in term S-TX) was collected and the pellets were resuspended in 2% SDS. Samples in 2% SDS were centrifuged, supernatant (S) and pellet (P) were collected. All the fractions were added to loading buffer 5X in ratio 1:5 and prepared similarly for Western blotting.
Reverse transcribed–polymerase chain reaction
Total RNA was extracted from cultured cells using the Trizol® Reagent (Life Technologies) extraction method. From 1 μg of total RNA, cDNA was synthesized using 200 units MoMuLV-reverse transcriptase (SuperScrip™ III RT; Life Technologies), oligo (dT) primers and 1 μl 10 mM dNTP Mix in a final volume of 13 μl. Ten microliters of the 25-fold diluted solution was subjected to quantitative PCR analysis. The following primer pairs were used for α-syn: Hu_Syn_FW 5′-ATGGATGTATTCATGAAA-3′, Hu_Syn REV 5′-TTAGGCTTCAGGTTCGTA-3′. PCR amplification was conducted using the Phusion® High-Fidelity DNA Polymerase protocol, under the following conditions: initial denaturation at 98°C for 30s, 10s at 98°C, 30s at 45°C and 30s at 72°C for 25 cycles, followed by 10 min at 72°C. After amplification, 10 μl aliquots were electrophoresed in 1.5% agarose gel, followed by photographic recording of the gel stained with ethidium bromide.
Immunocytochemistry and ThS staining of α-syn fibril-infected cells
Cells on coverslips were washed with PBS and fixed with 4% paraformaldehyde, then washed twice, 15 min/time with PBS and blocked in blocking buffer [5% normal goat serum (NGS) in PBS + 0.3% Triton] for 1 hour. For Thioflavin S (ThS) staining, fixed cells were incubated with 0.025% of the fluorophore (Sigma) for 8 min and washed three times with 80% ethanol for 5 min each time, prior to the antibody incubations. Fluorescence immunocytochemistry was performed using the primary and secondary antibodies listed in Additional file 6: Table S2. Primary antibodies were made up in 1% blocking buffer and PBS. After incubation, the cells were washed 5 times for 5 min/time with PBS; secondary antibodies were incubated in 1% blocking buffer and PBS for 45 min. Finally, cells were washed 5 times, 5 min/time with PBS, and counterstained with DAPI to reveal nuclei, then mounted in Vectashield Mounting Medium. Cell coverslips were stored at 4°C for confocal fluorescence microscopy. The fluorescence was measured in at least 10 randomly chosen observation fields for each experimental condition using Leica SP5 confocal laser-scanning microscope. The increase in fluorescence was measured calculating CORRECTED TOTAL CELL FLUORESCENCE (CTCF) using the formula: CTCF = Integrated density – (Area of selected cell X Mean fluorescence of Background readings) using the ImageJ 1.47 Software (NIH). Quantification experiments were carried out independently at least three times; more than 150 cells were counted for each condition. Individual differences were assessed using individual student’s t-tests in GraphPad Prism software (San Diego, CA). Data are shown as mean ± standard deviation (SD).
Immunofluorescence microscopy analysis of phosphorylated aggregates
All dopaminergic neuroblastoma SH-SY5Y cells were grown on ibidi dishes (Biovalley) for microscopy. After washing in PBS, cells were fixed using 4% (w/v) paraformaldehyde (Sigma) in PBS for 15 min and permeabilized with 0.01% Triton X-100 (Sigma) in PBS for 3 min, washed and blocked in 2% Bovine serum albumin (Sigma) in PBS for 20 minutes. Cells were immunostained using anti-α-syn (Santa Cruz Biotechnology, INC) and anti-phospho S129 α-syn (Abcam) followed by secondary antibody coupled to Alexa 488 (Invitrogen). Cells were mounted in Aqua Poly/Mount (Polysciences) and pictures were acquired using white field Axiovision microscope (Zeiss) with a 63x objective. Quantification experiments were carried out independently at least three times; more than 150 cells were counted for each condition. Individual differences were assessed using individual student’s t-tests in GraphPad Prism software (San Diego, CA). Data are shown as mean ± standard error of the mean (SEM).
Dementia with lewy bodies
Multiple system atrophy.
This work is supported by the European Community's Seventh Framework Programme (FP7 grant agreement n. 222887 - the PRIORITY project) and the Italian Ministry of Health.
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