Aggresomes do not represent a general cellular response to protein misfolding in mammalian cells
© Beaudoin et al; licensee BioMed Central Ltd. 2008
Received: 11 March 2008
Accepted: 20 October 2008
Published: 20 October 2008
Aggresomes are juxtanuclear inclusion bodies that have been proposed to represent a general cellular response to misfolded proteins in mammalian cells. Yet, why aggresomes are not a pathological characteristic of protein misfolding diseases is unclear. Here, we investigate if a misfolded protein inevitably forms aggresomes in mammalian cells.
We show that a cytoplasmic form of the prion protein may form aggresomes or dispersed aggregates in different cell lines. In contrast to aggresomes, the formation of dispersed aggregates is insensitive to histone deacetylase 6 inhibitors and does not result in cytoskeleton rearrangements. Modulation of expression levels or proteasome inhibitors does not alter the formation of dispersed aggregates.
Our results establish that aggresomes are not obligatory products of protein misfolding in vivo.
The deposition of protein aggregates is a pathological feature of a large number of diseases targeting the nervous system and/or peripheral organs. Neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and related polyglutamine disorders, amyotrophic lateral sclerosis (ALS), and prion diseases, [1–3]. Besides the brain, other organs affected in aggregation disorders include the liver and/or the lung in alpha1-antitrypsin deficiency and cystic fibrosis, and the heart in familial amyloid cardiomyopathy .
In order to elucidate the relationship between protein aggregation and cell dysfunction, protein aggregation has been recapitulated in cultured cells by overexpressing wild-type or mutant proteins. These proteins are alpha-synuclein and parkin in PD [5, 6], huntingtin in HD , presenilin1 and presenilin-binding proteins in AD , polyglutamine-containing proteins in polyglutamine diseases , superoxide dismutase in ALS , the prion protein (PrP) in prion diseases [11, 12], cystic fibrosis transmembrane conductance regulator in cystic fibrosis . These studies have defined several features including the coalescence of protein deposit at the centrosome, and the collapse of intermediate filament vimentin protein forming a cage around the deposits. Such juxtanuclear protein deposits were termed aggresomes, and it was originally proposed that aggresome formation is a general cellular response to the accumulation of misfolded proteins .
There is recent evidence that protein aggregates in animal models of human neurodegenerative diseases resemble aggresomes. Mutant superoxide dismutase molecules form aggresome-like particles in a mouse model of ALS . Prion-infected mice also produce perinuclear aggresomal-like particles . Moreover, it was proposed that Lewy-bodies formation in PD patients is similar to the formation of aggresomes in cultured cells [15, 16]. However, aggresomes are not a key pathological feature of all neurodegenerative diseases in humans, which suggests that they may not represent a general response to protein misfolding in vivo.
We have previously reported that a cytoplasmic form of PrP termed CyPrP forms aggresomes in murine N2a and human embryonic kidney 293 cells . In the present study, we have characterized the cellular and molecular response to CyPrP expression in various cells. Our data indicate that although CyPrP misfolds and produces insoluble particles in all cell lines tested, aggregates display two types of molecular morphology. We confirmed that CyPrP spontaneously forms aggresomes in N2a cells. By contrast, other cells including Hela, Cos-7, Huh-7 cells exclusively produce dispersed aggregates which are not juxtanuclear and are not associated with a cage-like structure of vimentin. These findings lead us to propose that cellular management of protein misfolding is complex, and that aggresomes are not obligatory end-products of protein misfolding in cells.
Cell culture, transfections and treatment
Human cervical cancer HeLa, embryonic kidney 293, mammary adenocarcinoma MCF-7, mouse neuroblastoma N2a, fibroblasts NIH3T3, and monkey fibroblasts COS-7 were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS. Transfections were conducted with exponentially growing cells using Lipofectamine 2000 as described by the manufacturer (Invitrogen).
For tetracycline-regulated expression, cells were transfected with pRevTet-On (Invitrogen) and selected with 200 μg/ml G418 (Sigma) to obtain individual clones. Selected clones were propagated and transfected with pRevTRE-CyPrPEGFP. Cells were then tested for their tetracycline-regulated CyPrPEGFP expression by western blotting. For inhibition of histone deacetylase, cells were transfected with CyPrPEGFP and incubated for 24 h in the presence of 5 μM scriptaid or its inactive structural analog nullscript (BioMol), or in the presence of 5 μM tubacin or its inactive structural analog (kindly provided by Dr Ralph Mazitschek, Broad Institute, Cambridge, MA, USA).
Plasmid constructs and antibodies
Cloning of CyPrPEGFP, a form of PrP without N-terminal secretion and C-terminal glycosylphosphatidylinositol anchor signal peptides was described previously . CyPrPDsRed2 was obtained from PrPDsRed2 , by deleting the N- and C-terminal signal peptides. DsRed2, like EGFP, was introduced in the natural SmaI restriction site of human gene encoding PrP, Prnp, at bp113 (amino-acid 38). To construct CyPrPmOrange, mOrange was amplified as a Sma1 fragment from pREST-BmOrange kindly provided by Dr Roger Tsien (University of California San Diego, CA, USA). The PCR product was introduced in the natural SmaI restriction site of Prnp. A clone containing mOrange in the right orientation was selected. CyPrP124stopmOrange was obtained by inserting the PCR product in the SmaI restriction site of CyPrP124stop . For tetracycline-regulated expression of CyPrPEGFP, CyPrPEGFP was cloned between the BamH1 and HindIII restriction sites of pRevTRE (Invitrogen). All clones were sequenced and their expression verified by western blot analysis.
The construct encoding GFP-250 was kindly provided by Dr Elisabeth Sztul (University of Alabama at Birmingham, AL USA).
Monoclonal anti-PrP (clone 3F4), anti-tubulin-alpha (clone 236–10501), and anti-vimentin (clone V9) antibodies were purchased from Chemicon, Molecular Probes, and Sigma, respectively.
To fractionate protein aggregates on sucrose gradient, cells from a 10 cm-petri dish were harvested, washed twice with ice-cold PBS, and resuspended in 2 ml hypotonic fractionation buffer (KCl 10 mM; β-mercaptoethanol 1 mM; MgCl2 1.5 mM; Hepes 10 mM, pH7.9). Cells were mechanically broken with 20 strokes in a Dounce homogenizer. Nuclei and unbroken cells were removed by centrifugation 5 min at 1,500 × g. The supernatant was centrifuged 5 min at 10,000 × g to pellet the aggregates. The pellet was resuspended with 0.5 ml fractionation buffer and layered on top of a continuous 1.2 M-2 M sucrose gradient in fractionation buffer. After a 2 h centrifugation at 150,000 g in a Beckman SW60 Ti rotor at 4°C, 1 ml fractions were collected and proteins were precipitated with 12% trichloroacetic acid. The resulting pellets were dissolved in sample buffer, separated by SDS-PAGE and analyzed by immunoblotting.
To assay the detergent insolubility of PrP, cells were solubilized in lysis buffer (0.5% sodium deoxycholate, 0.5% Triton X-100, 150 mM NaCl, and 50 mM Tris-HCl, pH 7.5) supplemented with protease inhibitors(mini-tablets from Roche). The lysate was clarified by centrifugation at 16,000 × g for 3 min, followed by ultracentrifugation at 186,000 × g for 40 min. The pellet from the second centrifugation, containing detergent-insoluble PrP, was resuspended in SDS-PAGE sample buffer. Detergent-soluble PrP was precipitated from the supernatant by addition of 0.05 volumes of 10% SDS and 4 volumes of methanol at -20°C. PrP was visualized by SDS-PAGE and Western blotting using antibody 3F4 and ECL detection .
Immunofluorescence and fluorescence in situ hybridization
Cells grown on coverslips were fixed and processed for immunofluorescence using anti-PrP monoclonal antibodies 3F4 as previously described . For in situ staining, permeabilized cells were incubated 10 min with 2 × SSC, and hybridized with 1 nM of an end-labeled biotinylated oligo-dT (50 nucleotide, IDT) overnight at 40°C. After washing twice with 2 × SSC and once with 0.5 × SSC, cells were equilibrated in 1 × PBS containing 1 mg/ml BSA. Cells were incubated with 2 μg/ml Alexa Fluor 633-labeled strepavidin (Molecular Probes) in 1 × PBS containing 1 mg/ml BSA. After a 1 h incubation, cells were washed and mounted as previously described . For fluorescence analysis, cells were examined with an Eclipse TE2000-E visible/epifluorescence inverted microscope (Nikon Corporation) equipped with band pass filters for fluorescence of Hoechst (Ex. D340/40: Em. D420), GFP (Ex. D450/40: Em. D500/50) and tetramethylrhodamine isothiocyanate (TRITC) (Ex. D528/25: Em. D590/60) (Nikon Corporation). Photomicrographs of 1344 × 1024 pixels were captured using a 100 × oil immersion objective and Orca cooled color digital camera (Hamamatsu Photonics). Images were processed using NIS Elements AR software (Nikon Corporation). Within the same figure, all pictures were taken with identical exposure time except in figure 3.
106 cells were transfected either with the empty vector, EGFP or CyPrPEGFP and incubated 24 hours at 37°C. Cells were washed twice with PBS and incubated 20 minutes in starvation media (DMEM without methionine and cysteine, Gibco). Cells were then pulsed with 25 μCi/ml 35S labelling mix (Easy Tag Express protein labelling mix, NEG772, Perkin-Elmer) for 1 h at 37°C. In control experiments, the translation inhibitor cycloheximide was added at 30 μg/ml prior to starvation. After labelling, cells were washed twice with PBS, scraped, collected in 1 ml PBS and centrifuged 5 minutes at 5000 rpm at 4°C. Pellets were lysed with 100 μl of buffer B [10 mM Tris pH 8.0, 100 mM NaCl, 0,5% Nonidet-P40, 0,5% Sodium deoxycholate and 1 mini EDTA-free protease inhibitor tablet (Roche) per 10 ml]. Proteins were dosed with BCA protein assay (Pierce) and 50 μg of total proteins were loaded on 10% polyacrylamide denaturing gels. Gels were then exposed 24 hrs to a Phosphor screen (GE Healthcare) and scanned on a Storm 860 Imager (Molecular Dynamics).
Results and discussion
It was of interest to test the generality of these findings with another aggresome-forming protein. To address this point, we expressed a cytosolic chimera termed GFP-250. This chimeric polypeptide composed of the entire soluble protein GFP fused at its COOH terminus to a 250-amino acid fragment of the cytosolic protein, p115, has been used to determine the dynamics of aggresomes formation . Similar to CyPrPEGFP, GFP-250 formed aggresomes in N2a cells and dispersed aggregates in Hela, COS-7, and Huh-7 cells (Fig. 1A). The solubility of GFP-250 was independent of the formation of aggresomes or dispersed aggregates, and the protein was largely insoluble in N2a or Hela cells (Fig. 1B). Like CyPrPEGFP, GFP-250 aggresomes and dispersed aggregates also displayed differences of density in a sucrose gradient (Fig. 1C).
We determined the proportion of aggresomes and dispersed aggregates 24 h after transfection (Fig. 1D). N2a and 293 cells produced a majority of aggresomes, while the proportion of dispersed aggregates was largely dominant in Hela and Huh-7 cells. One possibility to explain that Hela and Huh-7 cells did not produce aggresomes after 24 h of expression could be that the kinetics of coalescence of individual aggregates at the centrosome are slower in these cells. To address this question, we determined the proportion of cells producing aggresomes and dispersed aggregates different times post-transfection. After 12 h of transfection, cells produced mostly dispersed aggregates (Fig. 1E). At 24 h, N2a cells mainly produced aggresomes, indicating that dispersed aggregates had coalesced at the centrosome (Fig. 1E). In contrast, over 80% of Hela cells displayed dispersed aggregates even 96 h post-transfection (Fig. 1E). Inhibiting proteasomes accelerate the formation of perinuclear aggresomes . Yet, two proteasome inhibitors, epoxomycin and MG132, did not induce the coalescence of dispersed aggregates in a perinuclear aggresome (not shown). From these data, we conclude that end-products of misfolded proteins are aggresomes in N2a cells, and dispersed aggregates in Hela cells. Since similar results were obtained using CyPrPEGFP and GFP-250, the following experiments were carried out with CyPrPEGFP.
Protein aggregation is a major characteristic of most neurodegenerative diseases [2, 3]. In order to elucidate the relationship between protein aggregation and cell dysfunction, in vitro models using recombinant proteins have been developed. The reconstitution of protein aggregation in cultured cells has led to the proposition that aggresomes are a general response to protein misfolding . In this report, we show that CyPrP may either form aggresomes or dispersed aggregates independently of protein levels and of proteasomal activity. This result is significant since it demonstrates that aggresome is not an obligatory final product of protein misfolding, and it may explain in part why aggresome is not a conspicuous pathological feature of all neurodegenerative diseases in humans.
Several lines of evidence indicate that the mechanisms involved in the assembly of cytoplasmic protein aggregates are more complex than a simple accumulation of misfolded proteins. First, the observation that CyPrP does not form aggresomes in some cell lines, including Cos-7, Hela, and Huh-7 may indicate that such cells are not able to assemble aggresomes. Yet, Cos-7 has been used as a cell model to investigate the dynamics of aggresome formation . Similarly, several proteins are able to form aggresomes in Hela and Huh-7 cells [25–27]. Therefore, all cells probably possess the molecular machinery to form aggresomes. However, CyPrPEGFP does not accumulate in an aggresome in all cells. Second, in contrast to monomeric EGFP and mOrange fluorescent proteins, the obligate tetrameric DsRed2 protein does not produce fluorescence when fused to CyPrP. This observation suggests that structural constraints imposed on individual molecules within aggregates are important and that the arrangement of protein molecules may not be random. Third, in contrast to other proteins or other mutant forms of PrP, the formation of CyPrP aggresomes is independent of expression levels and proteasome inhibition [11, 13]. Furthermore, proteasome inhibition does not result in the conversion of dispersed aggregates into aggresomes. Forth, by co-expressing two unrelated misfolded proteins, it was observed that nonspecific co-aggregation between hydrophobic proteins does not occur and that protein aggregation is highly specific .
Our data using HDAC6 inhibitors and nocodazole indicate that dispersed aggregates do not require the microtubules network to assemble. A possible interpretation of these observations is that dispersed aggregates represent intermediate particles in an incomplete aggresomal pathway in Cos-7, Hela, and Huh-7 cells. Why dispersed aggregates are not transported to and accumulated at the centrosome in these cells remains to be determined. Another interpretation is that dispersed aggregates form through a novel pathway independent from the aggresomal pathway.
Consequently, there is accumulating evidence that protein aggregation in vivo cannot be considered to result from the simple coalescence of misfolded proteins, driven by interactions between solvent-exposed hydrophobic surfaces that are normally buried. The elucidation of the mechanisms governing protein aggregation, assembly and cellular localization of aggregates will help understanding aggregation diseases including a majority of neurodegenerative disorders.
cytoplasmic prion protein genetically fused to EGFP
histone deacetylase 6.
We thank Dr Roger Tsien (University of California San Diego, CA, USA) for providing pREST-BmOrange. We also thank Dr Ralph Mazitschek from the Broad Institute (Cambridge, MA, USA) and ICG (Initiative for Chemical Genetics – National Cancer Institute) for providing tubacin and its inactive structural analog niltubacin. We are grateful to Dr Leonid Volkov for his assistance with the microscope. This work was supported by grants from the Canadian Institutes for Health Research to XR. XR is a Junior 2 research scholar from the Fonds de la Recherche en Santé du Québec.
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