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. 2015 Apr 10;10:20.
doi: 10.1186/s13024-015-0014-y.

Calcium-responsive Transactivator (CREST) Protein Shares a Set of Structural and Functional Traits With Other Proteins Associated With Amyotrophic Lateral Sclerosis

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Free PMC article

Calcium-responsive Transactivator (CREST) Protein Shares a Set of Structural and Functional Traits With Other Proteins Associated With Amyotrophic Lateral Sclerosis

Michail S Kukharsky et al. Mol Neurodegener. .
Free PMC article

Abstract

Background: Mutations in calcium-responsive transactivator (CREST) encoding gene have been recently linked to ALS. Similar to several proteins implicated in ALS, CREST contains a prion-like domain and was reported to be a component of paraspeckles.

Results: We demonstrate that CREST is prone to aggregation and co-aggregates with FUS but not with other two ALS-linked proteins, TDP-43 and TAF15, in cultured cells. Aggregation of CREST affects paraspeckle integrity, probably by trapping other paraspeckle proteins within aggregates. Like several other ALS-associated proteins, CREST is recruited to induced stress granules. Neither of the CREST mutations described in ALS alters its subcellular localization, stress granule recruitment or detergent solubility; however Q388stop mutation results in elevated steady-state levels and more frequent nuclear aggregation of the protein. Both wild-type protein and its mutants negatively affect neurite network complexity of unstimulated cultured neurons when overexpressed, with Q388stop mutation being the most deleterious. When overexpressed in the fly eye, wild-type CREST or its mutants lead to severe retinal degeneration without obvious differences between the variants.

Conclusions: Our data indicate that CREST and certain other ALS-linked proteins share several features implicated in ALS pathogenesis, namely the ability to aggregate, be recruited to stress granules and alter paraspeckle integrity. A change in CREST levels in neurons which might occur under pathological conditions would have a profound negative effect on neuronal homeostasis.

Figures

Figure 1
Figure 1
CREST protein is aggregation-prone in vivo . (A) CREST aggregates upon its accumulation in the nucleus. GFP-tagged, Flag-tagged or untagged CREST are largely confined to the nucleus where they are diffusely distributed or can form dot-like aggregates. In high-expressing cells CREST aggregates are visibly larger and the protein undergoes a shift to the cytoplasm where it also aggregates (‘large + cyt’ panel). SH-SY5Y cells expressing tagged or untagged CREST were analysed 24 hours post-transfection. (B) CREST-GFP is almost entirely confined to the nucleus where it is excluded from the nucleolus visualised by ethidium bromide (EtBr) staining. It is not enriched in PML bodies (anti-PML staining), Gems (anti-SMN staining) or Cajal bodies (anti-coilin p80 staining), but is concentrated around speckles (FISH with MALAT1 probe) and highly enriched in paraspeckles (FISH with NEAT1 probe) in low-expressing cells. (C) Nuclear aggregates formed by CREST-GFP overexpressed in SH-SY5Y cells do not overlap with PML bodies, Gems, Cajal bodies or paraspeckles but surround speckles. Arrowheads denote nuclear bodies in CREST-expressing cells. Scale bars, 10 μm.
Figure 2
Figure 2
Characterisation of the aggregation capacity of CREST. (A) In cells with diffuse distribution of CREST, it re-localizes to nucleolar caps (arrowheads) in response to transcriptional inhibition. (B) The pool of CREST in nuclear dot-like aggregates fails to redistribute to nucleolar caps upon inhibition of transcription, and the aggregates persist under these conditions, although weakly CREST-positive nuclear caps can be observed (arrowheads). In A and B SH-SY5Y cells were exposed to actinomycin D for 1 hour. (C) CREST is recovered in detergent-insoluble fractions. HEK293 cells expressing untagged CREST were subjected to sequential protein extraction as described in Materials and methods. For total lysate (L) and high-salt (HS) fraction 10% of the amount relative to other fractions was loaded. Bar chart shows relative protein amounts (±s.e.m.) in each fraction quantified by densitometry. (D) Untagged CREST, Flag-CREST and GFP-CREST do not form SDS-resistant oligomeric forms. Cleared lysates of CREST-expressing SH-SY5Y cells were run in SDS-containing agarose gel; all variants were visualized using anti-CREST antibody. Mutant tau protein from spinal cord lysate of a transgenic P301S mouse (detected by phospho-tau-specific antibody) was used to demonstrate typical behavior of amyloid species in this assay. (E) Schematic representation of CREST deletion constructs used in the study. All variants were expressed as GFP-fusion proteins. (F, G) Distribution of CREST deletion mutants in SH-SY5Y cells. CR_dNT and CR_dNT-Met were shifted to the cytoplasm and formed nuclear dot-like aggregates less frequently than full-length protein (G). Bar chart in G shows the fraction of cells (mean ± s.e.m.) with nuclear aggregates for each variant (*** - p < 0.001; at least 150 cells counted per variant in each of the three independent experiments). CREST was expressed for 24 hours prior to actinomycin D exposure, cell lysis or fixation. Scale bars, 10 μm.
Figure 3
Figure 3
CREST is targeted to stress granules by various stresses. (A) CREST-Flag (top panel) and CREST-GFP (three bottom panels) are detected in stress granules induced by oxidative stress (sodium arsenite, SA), ER stress (thapsigargin, thaps) or inhibition of eIF4E (15d-PGJ2) and visualized with stress granule markers TIAR, FMRP and G3BP1. SA and 15d-PGJ2 were applied to SH-SY5Y cells for 1 hour and thapsigargin – for 4 hours. (B) In SH-SY5Y cells subjected to oxidative stress (SA for 1 hour) CREST-GFP undergoes significant shift to the cytoplasm. (C, D) CREST deletion mutant lacking autoregulatory domain (CR_dNT) is readily recruited to stress granules (C) and shows higher enrichment in these structures compared to full-length protein (D, left graph). This phenomenon is related to higher cytoplasmic levels of CR_dNT since the fluorescence intensity ratio stress granules/cytoplasm is similar for full-length and truncated protein (D). (E) Cytoplasmic aggregates of CREST-GFP do not overlap with SA-induced stress granules but are found in their immediate vicinity. (F) Cytoplasmic aggregates of CREST-GFP do not overlap with P-bodies (visualized by anti-Dcp1a staining, arrowheads in the enlarged panel). (G) Aggresomes formed by GFP-tagged CREST lacking MFD domain are negative for a SG marker G3BP1. In B and D, fluorescence was measured in stress granules and/or cytoplasm of GFP-positive cells as described in Materials and methods, and cytoplasmic intensity for non-stressed cells (B) or full-length CREST-GFP (D) (mean ± s.e.m.) was taken as equal 1 (***p < 0.001). Scale bars, 10 μm.
Figure 4
Figure 4
CREST aggregation disrupts paraspeckles. (A) CREST aggregates might originate from the sites of paraspeckle formation. Paraspeckles (anti-NONO/p54nrb staining, arrowheads) and CREST nuclear aggregates exist as distinct structures in COS7 cells (top panel). In a fraction of cells CREST aggregates are found in close apposition to/partially overlapping with paraspeckles (bottom panel). (B) In response to transcriptional inhibition CREST redistributes to the same nucleolar caps as a typical paraspeckle protein FUS but not to the caps formed by coilin p80. (C) CREST lacking autoregulatory domain is efficiently recruited in paraspeckles (top panel) and redistributes to nucleolar caps (bottom panel). (D) Endogenous FUS is not essential for nuclear aggregation of CREST. Cells were co-transfected with FUS siRNA and a plasmid to express CREST-Flag and were analysed 48 hours post-transfection. (E-G) CREST efficiently sequesters endogenous FUS into dot-like nuclear aggregates in COS7 cells. In contrast, two other paraspeckle components, p54nrb and PSPC1, are not recruited to small and medium-sized CREST aggregates (F and G, top panels), and are detected in aggregates only in nuclei with extensive CREST aggregation (F and G, bottom panels). (H, I) Presence of CREST aggregates in the nucleus negatively affects paraspeckles. The fraction of cells with paraspeckles among COS7 cells expressing CREST-Flag (anti-p54nrb staining) or CREST-GFP (FISH with NEAT1 probe) was quantified separately for cells with diffuse CREST distribution and with nuclear CREST aggregates (mean ± s.e.m, *p < 0.05, **p < 0.01; 150-250 cells counted from each of the four or three independent experiments). (J) NEAT1 levels are decreased in CREST-expressing cells. Untagged CREST or GFP (vector) were expressed in SH-SY5Y cells for 24 hours; NEAT1 levels were measured by qPCR (**p < 0.01; results from four independent experiments run in duplicates). Scale bars, A – 5 μm; B-G – 10 μm.
Figure 5
Figure 5
CREST co-aggregates with FUS but not with TDP-43 or TAF15. (A) Endogenous FUS co-immunoprecipitates with CREST-GFP. Pull-down of GFP-tagged CREST from transfected cells was performed with GFP-Trap beads as described in Material and methods, endogenous FUS was detected in the immunoprecipitate (IP) by Western blotting (WB). (B) CREST-Flag recruits GFP-tagged FUS (top panel) and its cytoplasmically mislocalised variant bearing R522 substitution (FUS R522G, middle panel) into nuclear aggregates upon co-expression in SH-SY5Y cells. In a small fraction of co-expressing cells the latter variant also co-aggregates with CREST in the cytoplasm (middle, arrowheads, border of the nucleus is indicated by a dashed line in the inset) but in the majority of such cells it is trapped in the nucleus leading to its lowered cytoplasmic levels and significant decrease in the proportion of cells bearing cytoplasmic FUS aggregates (FAs, bottom panel). The number of cells with aggregates was quantified for cells expressing GFP-tagged FUS R522G only (FUS-GFP) and those co-expressing GFP-tagged FUS R522G and CREST-Flag (FUS + CREST). The bar chart shows the fraction of cells (mean ± s.e.m.) bearing FAs (***p < 0.001; at least 100 cells counted per variant from three independent experiments). (C) CREST-GFP and FUS-Flag with R522G substitution co-aggregate. (D) N-terminally truncated protein (FUS-GFP CT, aa. 360-526) cannot be recruited to CREST-Flag aggregates (top panel), while C-terminally truncated FUS (FUS-GFP NT, aa.1-359) retains the ability to co-aggregate with CREST (bottom panel). (E) CREST does not sequester wild-type TDP-43 into nuclear aggregates and is not recruited to cytoplasmic (arrow) or nuclear (arrowheads) aggregates formed by mislocalised TDP-43 (TDP-43-GFP dNLS) or C-terminal TDP-43 fragment (TDP-43-GFP CT, aa.193-414). NLS of TDP-43 was deleted to achieve cytoplasmic re-distribution and aggregation of the protein. (F) Nuclear aggregates of Flag-tagged CREST and GFP-tagged TAF15 do not overlap. Scale bars, 10 μm.
Figure 6
Figure 6
ALS-associated CREST mutation Q388stop increases steady-state levels of the protein and its ability to aggregate. (A) A map of CREST with the positions of single amino acid substitutions or a deletion found in ALS indicated. (B) All CREST variants are localized predominantly to the nucleus in SH-SY5Y cells. (C) Q388stop mutant aggregates in the nucleus more often compared to the normal protein and other mutants. Among cells expressing each of the untagged CREST variants those with aggregates in the nucleus were quantified on the entire coverslip 24 hours post-transfection and obtained values were normalized against the value for wild-type CREST. The bar chart shows means ± s.e.m. of these normalized values from four independent experiments (*p < 0.05). (D) Steady-state protein levels for Q388stop mutant variant are increased compared to the wild-type protein without significant difference at the transcript level. The bar charts show means ± s.e.m. of seven independent experiments (*p < 0.05, Mann-Whitney U-test). (E) All CREST variants are stable proteins with half-lives of approximately 36 hours. Cycloheximide was used to block protein synthesis; levels of a short-lived protein, cyclin A, were measured to confirm successful block of translation. (F) Solubility of mutant CREST variants upon sequential protein extraction was comparable to that of wild-type protein. Fractionation was performed as described in Materials and methods. (G) CREST mutants do not affect the ability of the protein to be recruited to stress granules. The fraction of cells with CREST-positive stress granules after sodium arsenite exposure was quantified in the population of CREST-GFP expressing cells for each variant (at least 100 cells counted per variant in each of the two independent experiments). Scale bar, 10 μm.
Figure 7
Figure 7
Overexpression of normal or mutant CREST affects complexity of dendritic tree in primary hippocampal neurons. (A) CREST-GFP is largely confined in the nucleus when expressed in primary mouse neurons but is also found in multiple dot-like aggregates in the processes in some cells. Untagged CREST displays nuclear distribution in neurons. CREST-GFP or untagged CREST was co-transfected into primary hippocampal neurons together with dsRed2 to visualise neuronal morphology and allowed to express for 48 hours prior to analysis. (B) Total dendritic length in micrometers measured in mouse hippocampal neurons expressing CREST variants. Mouse primary hippocampal neurons were co-transfected with vectors to express GFP (to visualize neuronal morphology) and each of the untagged CREST variants and allowed to express the proteins for 48 hours prior to analysis. Control neurons were transfected with GFP-expressing vector only. The bar charts show means ± s.e.m. for at least 100 neurons per variant from four independent experiments (*p < 0.05, ***p < 0.001). (C) Sholl analysis of the same neurons as in B. Number of dendrite intersections of 15-μm spaced shells as a function of the radial distance from the soma was plotted. **p < 0.01, ***p < 0.001. Scale bar, 50 μm.
Figure 8
Figure 8
Overexpression of normal or mutant CREST induces retinal degeneration in Drosophila melanogaster. (A) Protein expression levels of CREST variants in the heads of transgenic flies of each line as determined by Western blotting and subsequent quantification of band intensities (mean ± s.d). (B) Images of external head surface of 5-day-old flies overexpressing wild-type CREST, CREST I123M, CREST Q388stop or lacZ in the retina. Overexpression of wild-type CREST or its variants in photoreceptor neurons results in rough and depigmented eyes. (C) H&E staining of retinal sections of 5-day-old flies overexpressing wild-type CREST or its variants reveals disruption of regularly ordered arrays of photoreceptor neurons. Data for representative transgenic fly lines with similar (intermediate) level of CREST protein expression are shown (#7, #7 and #2 for WT, I123M and Q388stop variants, respectively, – see Additional file 5: Figure S4). Scale bar, 50 μm.

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