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. 2018 Jun 12;131(11):jcs209122.
doi: 10.1242/jcs.209122.

SOD1A4V aggregation alters ubiquitin homeostasis in a cell model of ALS

Natalie E Farrawell  1   2 , Isabella Lambert-Smith  1   2 , Kristen Mitchell  1   2 , Jessie McKenna  3 , Luke McAlary  1   2   4 , Prajwal Ciryam  5   6   7 , Kara L Vine  1   2 , Darren N Saunders  3 , Justin J Yerbury  8   2
Affiliations

SOD1A4V aggregation alters ubiquitin homeostasis in a cell model of ALS

Natalie E Farrawell et al. J Cell Sci. .

Abstract

A hallmark of amyotrophic lateral sclerosis (ALS) pathology is the accumulation of ubiquitylated protein inclusions within motor neurons. Recent studies suggest the sequestration of ubiquitin (Ub) into inclusions reduces the availability of free Ub, which is essential for cellular function and survival. However, the dynamics of the Ub landscape in ALS have not yet been described. Here, we show that Ub homeostasis is altered in a cell model of ALS induced by expressing mutant SOD1 (SOD1A4V). By monitoring the distribution of Ub in cells expressing SOD1A4V, we show that Ub is present at the earliest stages of SOD1A4V aggregation, and that cells containing SOD1A4V aggregates have greater ubiquitin-proteasome system (UPS) dysfunction. Furthermore, SOD1A4V aggregation is associated with the redistribution of Ub and depletion of the free Ub pool. Ubiquitomics analysis indicates that expression of SOD1A4V is associated with a shift of Ub to a pool of supersaturated proteins, including those associated with oxidative phosphorylation and metabolism, corresponding with altered mitochondrial morphology and function. Taken together, these results suggest that misfolded SOD1 contributes to UPS dysfunction and that Ub homeostasis is an important target for monitoring pathological changes in ALS.This article has an associated First Person interview with the first author of the paper.

Keywords: ALS; Degron; Neurodegeneration; Proteasome; Protein aggregation; Proteostasis; SOD1; Supersaturation; Ubiquitin; Ubiquitomics.

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Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
SOD1A4V co-aggregates with Ub. (A) NSC-34 cells co-transfected with SOD1A4V–GFP and mRFP–Ub were imaged every 15 min for 17 h. (B) Ub incorporation into SOD1A4V inclusions was quantified using ImageJ. (C) NSC-34 cells overexpressing SOD1A4V–GFP were fixed, permeabilised and stained for K48-linked and K63-linked polyubiquitin chains at 48 h post transfection. (D) The spinal cord from the SOD1G93A mouse was fixed, permeabilised and stained for neuron-specific βIII tubulin, SOD1 and K48-linked Ub. Colocalization of SOD1 and Ub is indicated with arrows. Scale bars: 10 µm.
Fig. 2.
Fig. 2.
Mutant SOD1A4V alters UPS activity. (A) Dose-dependent response of UPS activity (tdTomatoCL1 fluorescence) in NSC-34 cells co-transfected with SOD1WT–GFP or SODA4V–GFP after overnight treatment with the indicated concentration of the proteasome inhibitor MG132. Data represent mean±s.e.m. tdTomatoCL1 fluorescence (n=3). (B) Frequency distribution analysis of SOD1A4V–GFP fluorescence was performed on cells expressing soluble (diffuse) SOD1A4V and aggregated SOD1A4V. (C) Cells containing SOD1A4V aggregates (Agg) exhibited significantly higher fluorescence than cells that did not contain aggregates. Data shown are mean±s.e.m. (n≥100, combined from four independent experiments). (D) 80% of cells expressing the highest SOD1A4V–GFP signal (top 10% of GFP-expressing cells) contained aggregates. Data shown are mean±s.e.m. from four independent experiments where a minimum of 100 cells were counted. (E) Cells expressing the highest GFP signal (top 10% of GFP-expressing cells) typically contained aggregates and revealed greater significant differences in tdTomatoCL1 fluorescence between cells expressing SOD1WT and SOD1A4V. **P<0.01; ***P<0.001; ****P<0.0001 (one-way ANOVA with a Tukey's multiple comparison post test). Scale bars: 10 µm.
Fig. 3.
Fig. 3.
Ub distribution is not significantly altered in cells containing SOD1A4V aggregates. (A) NSC-34 cells co-transfected with SOD1WT–GFP or SOD1A4V–GFP (cells classified according to whether SOD1A4V was either soluble or insoluble) and mCherry–Ub were photobleached in either the nucleus or cytoplasm, and recovery of Ub fluorescence was monitored for 120 s. Data shown are means±s.e.m. (n=3) and are representative of three independent experiments. (B) Representative confocal images of pre-bleach, post-bleach and the recovery endpoint (final read) are shown, with the ROI marked in yellow. Scale bars: 10 µm. (C) Diffusion rates (T1/2) of mCherry–Ub measured in both the nucleus and cytoplasm of co-transfected NSC-34 cells. Data shown are means±s.e.m. (n≥6, combined from three independent experiments). (D) Quantification of the proportion of mobile Ub in the nucleus and cytoplasm of cells expressing either SOD1WT, soluble SOD1A4V or insoluble SOD1A4V. Data shown are means±s.e.m. combined from three independent experiments (n≥7). One-way ANOVA with a Tukey's multiple comparison post-test was used to compare differences, which were not significant.
Fig. 4.
Fig. 4.
Reduced levels of free monomeric Ub in NSC-34 cells containing SOD1A4V aggregates. (A) Western blot analysis of cell lysates of NSC-34 cells transiently transfected with SOD1WT–GFP or SOD1A4V–GFP, mRFP–Ub or Turbo (t)GFP control. UT, not transfected. Samples were separated under reducing conditions and probed with anti-Ub antibody. (B) The entire nucleus of co-transfected NSC-34 cells was photobleached and the recovery of nuclear Ub was monitored as a proportion of cytoplasmic fluorescence for 120 s. Data shown are means±s.e.m. (n≥17) combined from three independent experiments. Cells expressing SOD1A4V were classified according to whether SOD1A4V was either soluble (s) or insoluble (i). (C) Representative confocal images of pre-bleach, post-bleach and final read. (D) The percentage of mobile Ub in the nucleus at the final read was quantified as a proportion of cytoplasmic fluorescence. Data represent mean±s.e.m. (n≥17, combined from three independent experiments). *P<0.05; **P<0.01; ***P<0.001 (one-way ANOVA with a Tukey's comparison post test). Scale bars: 10 µm.
Fig. 5.
Fig. 5.
The ubiquitylated proteome of transfected NSC-34 cells. (A) NSC-34 cells expressing GFP fusions of SOD1WT or SOD1A4V were subjected to high-affinity purification to isolate ubiquitylated proteins, which were subsequently identified by LC-MS/MS. (B) Venn analysis comparing the number of ubiquitylated proteins in NSC-34 cells transfected with either SOD1A4V (n=2) or SOD1WT (n=3). (C) STRING analysis of the protein–protein interaction network of ubiquitylated proteins unique to cells expressing SOD1A4V or SOD1WT. Proteins were identified as present in each condition if they were present in at least two replicates for that condition. Additionally proteins were only considered ‘unique’ to either SOD1WT or SOD1A4V if they were not identified in any of the replicates for any of the other conditions. (D) The median supersaturation score calculated for the unfolded (σu) and native (σf) states of proteins unique to the SOD1A4V ubiquitome (Ubiq). Fold Δ refers to the increase in supersaturation score from the known mouse proteome (Prt). The box represents the 25–75th percentiles, and the median is indicated. The whiskers range from the lowest to highest value data points within 150% of the interquartile ranges. Statistical significance was assessed by the one-sided Wilcoxon/Mann–Whitney U-test with Holm–Bonferroni-corrected P values (****P<0.0001).
Fig. 6.
Fig. 6.
Cells containing SOD1A4V aggregates display altered mitochondrial morphology and dysfunction. (A) NSC-34 cells transiently transfected with SOD1WT–GFP or SOD1A4V–GFP (or tGFP as a control) were stained for mitochondria with Mitotracker Deep Red at 48 h post transfection. Scale bars: 10 µm. The number of mitochondria (B) and their circularity (C) was determined using a mitochondrial morphology macro in ImageJ. Data represent mean±s.e.m. (n≥23). (D) Mitotracker fluorescence was also quantified by flow cytometry in cells expressing the highest levels of SOD1WT–GFP or SOD1A4V–GFP. (E) Mitochondrial membrane potential was examined through the accumulation of Mitotracker Red CMXRos. Data represent mean±s.e.m. (n=3). *P<0.05; **P<0.01; ***P<0.001 (one-way ANOVA with a Tukey's multiple comparison post test).
Fig. 7.
Fig. 7.
Disrupted Ub homeostasis in ALS. Mutations in SOD1-associated ALS disrupt Ub homeostasis, either directly or through sequestration of Ub into protein aggregates. These changes result in altered Ub distribution and subsequent depletion of free Ub, eventually reaching a threshold below which vital cellular functions are severely compromised, and ultimately result in cell death.
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