After 24 h, the medium was changed to regular DMEM with supplements, and the day after that to DMEM with 150C200 g/ml of hygromycin B (Sigm-Aldrich, 108435550019) and 15 g/ml of blasticidin (Sigma-aldrich, {“type”:”entrez-protein”,”attrs”:{“text”:”SBR00022″,”term_id”:”1075795620″,”term_text”:”SBR00022″}}SBR00022). development of FXTAS. 0.001; ** 0.01; * 0.05. The exact model for FXTAS have demonstrated that inhibition of UPS increases neurodegeneration, while inhibiting autophagy can improve the phenotype (Oh et al., 2015). Moreover, mayor players in the UPS, namely ubiquitin and the proteasome, are present in FXTAS inclusions (Iwahashi et al., 2006; Lin et al., 2013). With this in mind, we asked whether protein components of the UPS and/or the autophagy machinery co-localized with FMRpolyG-aggregates in our system. For this purpose, cells containing FMRpolyG aggregates were stained with antibodies to marker proteins for UPS (20S proteasome and ubiquitin) and autophagy (LC3B and p62), and analyzed by fluorescence confocal microscopy. The majority of aggregates contained both ubiquitin and the 20S proteasome (Figures 8ACC). Interestingly, p62, an autophagy receptor involved in both autophagic and proteasomal degradation of proteins (Pankiv et al., 2007; Geetha et al., 2008), was enriched in ~35C50% of the aggregates (Figures 8A,D). p62 has previously been found in FXTAS-inclusions (De Pablo-Fernandez et al., PKI-402 2015). In contrast, LC3B, a major adaptor and marker in the autophagy pathway, was not found to be present in the aggregates (Figure 8E). Importantly, we find the numbers of p62-, proteasome-, and ubiquitin positive aggregates to be similar in wtHP-99Gly-GFP and mutHP-90Gly-GFP expressing cells. Open in a separate window Figure 8 Proteasomes are recruited to FMRpolyG aggregates. (A) Representative confocal fluorescence microscopy images of HEK293 cells transfected with wtHP-99Gly-GFP (upper panel) or mutHP-90Gly-GFP (lower panel) and immunostained with antibodies to the proteasome, ubiquitin and p62. Fraction of FMRpolyG-GFP aggregates which co-localized with the proteasome (B), ubiquitin (C), p62 (D), or LC3B (E), after transfection of wtHP-99Gly-GFP (black bars) or mutHP-90Gly-GFP (white bars). Cells were stained for the indicated endogenous proteins. Quantifications were performed using the image analyzing software Volocity, and are based on 3C6 experiments. For (B) the PKI-402 total number of aggregates included in the quantification was 65 per construct. The remaining graphs (CCE) are based on analysis of a total of 190 GFP-positive aggregates per construct. (FCH) FMRpolyG is mainly degraded by the Rabbit Polyclonal to PLG proteasome. Except for the negative controls (uninduced cells), HEK-FlpIn cells were treated with tetracycline (1 g/ml) for 48 h to induce accumulation of GFP-p62 (F) or FMRpolyG-GFP (G,H), respectively. Degradation was PKI-402 then measured by flow cytometry of the entire cell population ( 20,000 cells for each condition, per experiment), as a loss in mean GFP intensity after the removal of tetracycline (Tet Off). The experiments were performed as indicated in the absence or presence of Baf-A1 or MG132. All graphs are based on a minimum of three independent experiments. The exact model of FXTAS (Jin et al., 2007), patient material reveal inclusions exclusively in the nucleus (Greco et al., 2002; Hunsaker et al., 2011). We therefore cannot exclude that formation of intranuclear aggregates in patients arise through other pathways than the aggregates observed in this study, and in the model. Nonetheless, our main finding concerning aggregate formation is that presence or absence of the CGG mRNA does not affect aggregate formation, localization or mobility. In addition, we have applied electron microscopy to reveal that the ultrastructure of these aggregates is mainly filamentous, dense and non-membrane bound. Importantly, inclusions in FXTAS patients are reported to have similar morphological features (Greco et al., 2002; Gokden et al., 2009). This is to our knowledge the first study of the ultrastructure of FMRpolyG-induced aggregates. Interestingly, polyGlycineAlanine (poly-GA) aggregates have recently been studied using cryoelectron tomography (Guo et al., 2018). This dipeptide is part of a protein produced by RAN translation across the G4C2 repeats in C9ORF72 ALS/FTD. The authors show that poly-GA PKI-402 aggregates recruit the proteasomes (Guo et al., 2018). Since the FMRpolyG aggregates stain positive for the 20S proteasome, it is possible that the glycine in both poly-GA and FMRpolyG aggregates interacts directly with the proteasome to mediate this sequestration. Finally, our study is the first to assess important features of the FMRpolyG protein such as its mobility in different cellular compartments and the rate.
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