Nanoscale Analysis Reveals the Maturation of Neurodegeneration-Associated Protein Aggregates: Grown in mRNA Granules then Released by Stress Granule Proteins Sanae Abrakhi,† Dmitry A. Kretov,†,‡,§ Bénédicte Desforges,† Ioana Dobra,† Ahmed Bouhss,† David Pastré,† and Loic Hamon*,† †
SABNP, Univ Evry, INSERM U1204, Université Paris-Saclay, 91025 Evry, France Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia
‡
S Supporting Information *
ABSTRACT: TDP-43 and FUS are two mRNA-binding proteins associated with neurodegenerative diseases that form cytoplasmic inclusions with prion-like properties in affected neurons. Documenting the early stages of the formation of TDP-43 or FUS protein aggregates and the role of mRNA stress granules that are considered as critical intermediates for protein aggregation is therefore of interest to understand disease propagation. Here, we developed a single molecule approach via atomic force microscopy (AFM), which provides structural information out of reach by fluorescence microscopy. In addition, the aggregation process can be probed in the test tube without separating the interacting partners, which would affect the thermodynamic equilibrium. The results demonstrate that isolated mRNA molecules serve as crucibles to promote TDP-43 and FUS multimerization. Their subsequent merging results in the formation of mRNA granules containing TDP-43 and FUS aggregates. Interestingly, TDP-43 or FUS protein aggregates can be released from mRNA granules by either YB-1 or G3BP1, two stress granule proteins that compete for the binding to mRNA with TDP-43 and FUS. Altogether, the results indicate that age-related successive assembly/disassembly of stress granules in neurons, regulated by mRNA-binding proteins such as YB-1 and G3BP1, could be a source of protein aggregation. KEYWORDS: atomic force microscopy, RNP nanostructures, RNA−protein interactions, protein aggregation, stress granules and FUS form insoluble cytoplasmic aggregates1,11−16 that can further spread the disease to other areas of the brain.17,18 The prion-like behavior of TDP-43 and FUS has been attributed to their long self-attracting domains of low complexity (LCD)19−23 that enable weak and multivalent homotypic interactions to mediate the formation of liquid droplets.24 In line with the role of LCD in ALS and FTLD, many mutations identified as genetic factors driving these
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AR DNA-binding protein 43 (TDP-43) and fused in sarcoma (FUS), two RNA-binding proteins (RBPs), have recently been the subject of increased attention due to their role in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD).1−5 In nonpathological conditions, these proteins are mostly found in the nucleus where they participate in mRNA biogenesis6 and alternative splicing.7,8 However, TDP-43 and FUS are also present in the cytoplasm since they shuttle from one compartment to another. Cytoplasmic TDP-43 and FUS notably participate to mRNA transport and localized translation,9,10 which are of critical importance for neuron physiology. In neurons of ALS and FTLD patients, TDP-43 © 2017 American Chemical Society
Received: May 3, 2017 Accepted: June 28, 2017 Published: June 28, 2017 7189
DOI: 10.1021/acsnano.7b03071 ACS Nano 2017, 11, 7189−7200
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Figure 1. Recombinant TDP-43 and FUS form protein aggregates. (A) Purified recombinant (YB-1, GST-TDP-43, and GST-FUS) and commercial (G3BP1) proteins used in this study as observed after their migration in a denaturating gel. (B) AFM images of TDP-43 and FUS proteins (25 nM) at different incubation times in deposition buffer. After 30 min incubation, TDP-43 and FUS form particles with a granular appearance. Size: 1 × 1 μm. Inset: Zooms on specific structures. (C) Box plot representing the heights of TDP-43 and FUS particles measured in AFM images after 1, 15, and 30 min incubation in deposition buffer. Box charts are shown as a box (25−75 percentiles); vertical bars (5−95 percentiles) and data points (circles, n = 50 particles) overlap with the mean value (empty square) and median value (horizontal line in the box). **P < 0.01,*P < 0.05, two-tailed t test.
diseases4,19,25−30 are found within the LCD of both TDP-43 and FUS. Consequently, preventing the aggregation of free TDP-43 and FUS has been considered as a putative therapeutic route for disease treatments but has been so far disappointing as many weak interactions in the LCD are involved. Exploring the role of mRNA in the formation of TDP-43 and FUS inclusions may provide an interesting angle to tackle this issue.22,31,32 Indeed, mRNA favors phase separation of RBPs with LCD.33 In addition, the toxicity of TDP-43 and FUS requires their intact mRNA binding capacity.34,35 Furthermore, stress granules (SGs) which are large mRNA-rich cytoplasmic granules formed during cellular stress36 also sequester FUS and TDP-43.37−39 Thereby, stress granules are considered as putative crucibles for the genesis of TDP-43 and FUS inclusions,40−42 although this hypothesis remains to be further supported by experimental evidence. To better document the role of mRNA and stress granules in TDP-43 and FUS aggregation, we propose a single-molecule approach using atomic force microscopy (AFM). Previous in vitro investigations have been focused on phase-separation observations by fluorescence microscopy.30,33 Although very useful, resolution limits preclude the structural analysis of large and heterogeneous structures that would indicate the molecular mechanism underlying liquid-phase formation and possibly the transition toward insoluble aggregates. Regarding classical biochemical methods such as high-speed centrifugation, chromatographies, and gel-shift assays, these methods are not appropriate for exploring dynamic structures such as mRNA granules, which result from the permanent exchange of mRNA and proteins with the surrounding.43 The thermodynamic equilibrium is indeed disrupted whenever mRNA granules are isolated as required by classical biochemical methods. In line
with this, SG integrity is disrupted upon cell permeabilization with detergents, which renders their isolation a challenging issue.44 In addition, while centrifugation has been used successfully to identify proteins enriched in mRNA granules,23 the structural composition of the pellet could be heterogeneous as mRNA granules and protein aggregates can coexist. Here, we explored by AFM the interaction of mRNA and RBPs in order to detect the resulting structural heterogeneities without any separation step. We considered the contribution of two RBPs, Y-box-binding protein 1 (YB-1 or YBX1), and RasGAP SH3-domain binding protein 1 (G3BP1) to TDP-43 and FUS aggregation. YB-1 and G3BP1 are abundant nonaggregating RBPs that lead to the formation of isolated messenger ribonucleoproteins (mRNPs) in vitro. As components of SGs,45−48 both YB-1 and G3BP1 may interfere with the maturation of protein aggregates inside SGs.42 The nanometric resolution of AFM enables to distinguish the structural signatures of protein aggregates, granules containing mRNA (mRNA granules) and isolated mRNPs such as YB-1− mRNA complexes that form typical “beads-on-a-string” structures.49−52 Based on the results obtained, we propose a mechanistic model for the appearance of protein aggregates which emphasizes the roles played by mRNA as a scaffold for TDP-43 and FUS aggregation and reveals stress granule proteins such as G3BP1 and YB-1 as key factors for the release of TDP-43 and FUS aggregates from mRNA granules.
RESULTS AFM Analysis of the Structure and Kinetic Properties of TDP-43 and FUS Aggregates. TDP-43 and FUS proteins are intrinsically prone to aggregation and form protein 7190
DOI: 10.1021/acsnano.7b03071 ACS Nano 2017, 11, 7189−7200
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ACS Nano
Figure 2. mRNA serves as a scaffold for TDP-43 and FUS aggregation. (A) Gel mobility shift assay showing the binding of TDP-43 and FUS to 2Luc−mRNA (0.2 pmol/lane). (B) AFM images of TDP-43− and FUS−mRNA granules (1 protein per 30 nt) produced along 30 min incubation in deposition buffer. Size: 450 × 450 nm. (C) Comparison of height of TDP-43−, FUS−, or TIA1−mRNA granules measured in AFM images after 1, 15, and 30 min incubation in deposition buffer. Box charts are shown as a box (25−75 percentiles), vertical bars (5−95 percentiles), and data point (circles, n = 50 particles) overlap with the mean value (empty square) and median value (horizontal line in the box). *P < 0.05, **P < 0.01, ***P < 0.005, two-tailed t test. (D) Heights of TDP-43−mRNA granules measured in AFM images after 30 min incubation in deposition buffer using full length protein (TDP-43−FL) or its truncated form (TDP-43−RRM) that lack the low complexity domain. Number of molecules analyzed for each average value: 50. Results are mean ± SD of three independent samples. *P < 0.05, **P < 0.01, ***P < 0.005, two-tailed t test. On top are represented typical structures observed on AFM images. Bottom, schematic representation of recombinant proteins.
of TDP-43 and FUS globular structures also indicates the association of small unmerging protein particles leading to the formation of large granular aggregates in AFM images. To test whether FUS has formed irreversible aggregates, these structures were exposed to SDS.29,53 Their dissociation indicated the reversibility of such aggregates at this stage (Figure S1B). Free mRNA Serves As a Scaffold for the Growth of TDP-43 and FUS Aggregates. To decipher the role of mRNA on protein aggregation, we first analyzed whether TDP43 and FUS retain their ability to bind to mRNA (Figure 2A). Gel mobility shift assays were performed in the presence of 2Luc mRNA considered here as a nonspecific mRNA of about 3000 nt. The results revealed the binding of TDP-43 and FUS to 2Luc mRNA, albeit with a lower affinity for the former. We then analyzed by AFM the structures resulting from the interaction of TDP-43 or FUS with free mRNA at a
inclusions when overexpressed in bacteria. To keep a significant fraction of these proteins in a soluble state, GST-tag was fused to the N-terminus of these proteins (Figure 1A). After purification, recombinant proteins were then stored at high ionic strength.4,29 To analyze the aggregation kinetics of these proteins, they were diluted in a low ionic strength buffer that is compatible with the analysis of protein−mRNA interactions undertaken in the present study. After 1 min, TDP-43 and FUS appeared as isolated molecules on the mica surface, which indicates that the incubation time was not sufficient to observe a significant protein aggregation (Figure 1B). However, at longer incubation times (15 and 30 min), TDP-43 and FUS formed large globular structures (Figure 1B,C). In the case of TDP-43, small pore-shaped oligomers that have been previously evidenced53 were scarcely detected. Rod- or globular-shaped particles were rather preferentially formed under our experimental conditions (Figure S1A). The analysis 7191
DOI: 10.1021/acsnano.7b03071 ACS Nano 2017, 11, 7189−7200
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Figure 3. YB-1 and G3BP-1 dissociate TDP-43 aggregates from RNA granules. (A) Right panel: AFM images of YB-1 and G3BP1 mRNPs formed in the presence of 0.25 nM mRNA and 20 nM proteins after 5 min incubation in deposition buffer. Left panel: Measurement of the heights and surfaces of the mRNPs. Number of molecules analyzed for each average value: 50. Results are mean ± SD of three independent samples. *P < 0.05, **P < 0.01, ***P < 0.005, two-tailed t test. (B) Incubation of 25 nM TDP-43 with mRNA in deposition buffer during 15 min leads to the formation TDP-43−mRNA granules. Addition of 20 nM of YB-1 or G3BP1 for 5 min is sufficient to dissociate these granules into YB-1 or G3BP1 mRNPs and TDP-43-rich aggregates with granular appearance similar to those observed in Figure 1B. (C) Maximum height (left) and average surface (right) of G3BP1 and YB-1 mRNPs obtained after the interaction of G3BP1 or YB-1 with free mRNA or TDP-43−mRNA granules. The initial presence of TDP-43 interacting with mRNA did not change the structures of the G3BP1 or YB-1 mRNPs. Number of molecules analyzed for each average value: 50. *P < 0.05, **P < 0.01, ***P < 0.005, n.s., not significant, two-tailed t test.
incorporation of additional proteins (Figure 2B).51 To further document that mRNA acts as a scaffold for protein aggregation, we measured the size of TDP-43 and FUS aggregates versus time (Figure 2C), and the growth rate appeared higher in the presence of mRNA than in its absence. To determine whether the ability of 2Luc mRNA to promote TDP-43 and FUS aggregation is mostly due to the capture of
concentration ratio of 1 protein per 30 nucleotides, which is below the saturation value.54 At short incubation times (
