Four related peptides rescue MBNL-dependent missplicing events in DM1 myotubes
Immortalized human DM1 muscle cell lines display disease-associated molecular features such as nuclear RNA aggregates and alternative splicing defects23. DM1-related phenotypes can be used as readouts to screen candidate therapeutics in vitro for effects on RNA toxicity associated with the DM1 mutation. MBNL protein depletion explains most aberrant splicing patterns observed in DM124,25,26. Thus, we established three splicing events typically altered in DM1 as screening criteria for the 15 d-amino-acid hexapeptides previously identified (Supplementary Table 1)22: the inclusion of exon 5 of cardiac troponin T gene (cTNT; Entrez ID: 7139) and the exclusion of exon 78 of the dystrophin gene (DMD; Entrez ID: 1756), both MBNL1-dependent and the exclusion of exon 23 of spectrin alpha non-erythrocytic gene (SPTAN1; Entrez ID: 6709), which is MBNL2-dependent27. Immortalized control and DM1 fibroblasts were transdifferentiated into myotubes for 48 h. After that, they were incubated two more days with 10 µM of each peptide dissolved in myotube differentiation medium (MDM). Semiquantitative RT-PCR evaluated the activity of the peptides on the missplicing events. Despite most peptides being able to improve inclusion of at least one of the alternative exons, only peptides cpyaqe (79), cpyawe (80), cpytqw (81), and cpytwe (82) rescued all of them in a statistically significant manner (Fig. 1a–d and Supplementary Fig. 1). Notably, the four peptides shared 4 out of 6 amino acids, and only the fourth and the fifth positions changed, generating the consensus sequence cpy(a/t)(q/w)e, which strongly suggests a structure–function relationship. Treatment with these peptides did not change the inclusion of exon 8 of the CAPZB gene, regulated by CELF128, nor exon 19 of the DLG1 gene, which remains unchanged in DM1 patients29, suggesting a specific effect on the regulatory factors MBNL1 and 2 in the disease (Fig. 1e–g). Finally, we tested whether such activity was (CUG)exp-specific or not; thus, we treated control cells with 10 µM of peptide 80 and quantified their activity on the alternative exons of cTNT and SPTAN. The peptide produced no significant change, suggesting that its activity depended on the presence of the mutation that causes DM1 (Fig. 1h–j). Peptides 79, 80, 81, and 82 were selected for further evaluation. We treated control myotubes (Fig. 1k) and fibroblasts (Supplementary Fig. 2) with peptides 79, 80, 81, and 82 at concentrations ranging from 0.1 to 100 µM and assayed their toxicity profile. We did not observe any toxic effect even at the highest evaluated concentration. Thus, we performed following experiments at 10 µM, a concentration at which compounds were not toxic for cells.
Peptides 79, 80, 81, and 82 rescued MBNL-dependent mis-splicing events in DM1 myotubes. Control (CNT, with no DMSO) and DM1 myotubes (96 h of differentiation, 3 biological replicates of each condition) were treated with 10 μM of the indicated peptides or DMSO (0.1%) for 48 h and the percentage of inclusion of cTNT exon 5 (a), DMD exon 78 (b), and SPTAN1 exon 23 (c) was determined. (d) Representative 2% agarose gels showing semiquantitative RT-PCR amplicons with or without the indicated exons and GAPDH internal control. None of the tested peptides induced changes in the splicing of the CAPZB gene, altered in DM1 and regulated by the CELF1 protein (e, g) or in the splicing of the DLG1 gene, which remains unchanged in DM1 (f, g). The inclusion of exon 5 of the cTNT gene, regulated by MBNL1, (h, j) and the inclusion of the exon 23 of the gene SPTAN1, regulated by MBNL2 (i, j), did not respond to peptide 80 in CNT myotubes, thus supporting the specificity of its activity. Cell growth inhibition assay by MTS method. Human CNT myotubes were transfected with increasing concentrations of the indicated peptides (4 biological replicates per condition) (k). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns p > 0.05 according to Student’s t-test.
Candidate peptides enhance MBNL expression and its normal distribution in the cell
To understand what could trigger the rescue of the analyzed splicing events, we used cDNA from treated cells to perform quantitative PCR (qPCR) and detect any modification in the mRNA levels of MBNL1 and MBNL2. We found that peptides 79, 80, 81, and 82 doubled MBNL1 expression while peptides 79 and 81 slightly increased MBNL2 mRNA amounts (Fig. 2a, b). This finding was encouraging as the increase in MBNL1 and 2 gene expression has been proposed as a valid strategy to improve the clinical outcome of DM15,6,16,18,19,30. However, at the protein level, only cells treated with the peptides 80 and 81 showed a statistically significant increase in MBNL1 levels by western blot compared to untreated DM1 cells (Fig. 2c, Supplementary Fig. 3). Finally, since the subcellular localization of MBNL1 is altered in DM119,30, we evaluated this phenotype using an anti-MBNL1 antibody. Immunofluorescence images confirmed that the MBNL1 signal was increased in the cytoplasm of the treated cells compared to the untreated and approached normal intensity and subcellular distribution (Fig. 2d–i). Taken together, these results indicate that the candidate peptides were able to target the upregulation of MBNL1. Therefore, we studied additional molecular phenotypes related to MBNL1 in the pathogenesis pathway.
Candidate peptides enhanced the expression of MBNL proteins in a DM1 cell model. Quantification by RT-qPCR of relative expression of MBNL1 and MBNL2 transcripts in DM1 cells differentiated for four days and treated with the four candidate peptides (three biological replicates and three technical replicates per biological sample were performed). Levels were referenced to GAPDH as endogenous control (a, b). Western blot quantification relative to beta-Actin confirmed a significant increase in the levels of MBNL1 protein in the cells treated with the 80 and 81 peptides (3 biological replicates per condition). Black lines indicate those lanes cropped from different blots (c). The molecular weight marker band at 37 kDa is indicated to the right of the blot. Confocal microscopy micrographs of control (CNT, d; without DMSO) and DM1 myotubes (e–i) treated with vehicle (DMSO 0.1%; e) or the indicated peptides (f–i) stained for MBNL1 (green channel) and DAPI. All images were taken at the same settings. Note the general increase in the intensity of MBNL1 fluorescence in (f–i) panels. *p < 0.05, **p < 0.01, ***p < 0.001 according to Student’s t-test. Scale bar corresponds to 100 microns.
A 48-h treatment is sufficient to reduce the number of foci with no effect on the DMPK transcripts.
A prime mechanism to enhance functional MBNL1 levels in cells is to prevent its sequestering into ribonuclear foci by either blocking its binding to CUG repeats or changing the secondary structure of the toxic RNA so it is less prone to bind to the protein15,22,31. We quantified the number of foci per nucleus to shed light on this problem, using fluorescence in situ hybridization with an RNA probe (Cy3-(CAG)7-Cy3) and an IN Cell Analyzer High-Content Cellular Analysis System to acquire images (Fig. 3a–h). The treatment with each of the peptides produced a significant, although mild, change in the number of cells without foci, which increased, and in the percentage of foci per cell, which was significantly reduced (Fig. 3i, j). These changes occurred without altering the relative expression levels of DMPK transcripts (Fig. 3k), half of which carry the expanded CUG triplets, suggesting the possibility of a direct interaction of the peptides with the RNA, with or without a subsequent influence on its secondary structure.
Candidate peptides reduced ribonuclear foci in DM1 cells. Representative micrographs of foci in control (CNT, a; without DMSO), DM1 (DMSO 0.1%, b), and DM1 cells treated with negative control (peptide 89 or scrambled peptide (SC); c, d) or candidate peptides (e–h) obtained with an IN Cell Analyzer high-content imaging system (three biological replicates and three technical replicates per biological sample were performed in each condition. Four different fields were analyzed in each sample). Accumulation of mutant transcripts was detected using fluorescent in situ hybridization (FISH) with a Cy3-labeled RNA probe (red dots). Nuclei were counterstained with Hoechst 33258 (blue). Quantification of the images revealed that peptides 79, 80, and 81 significantly increased the percentage of cells without foci (i) and reduced the number of foci per cell, in this case including peptide 82 (j). The observed reduction in mutant DMPK accumulation was not due to the repression of the DMPK gene expression itself, which was quantified by real-time PCR using primers against a non-repetitive sequence and was found not significantly different from DM1 controls (three biological replicates and three technical replicates per biological sample were performed; k). *p < 0.05, **p < 0.01, ***p < 0.001 according to Student’s t-test. Scale bar (a–h) measures 20 microns.
Treated DM1 myotubes reduce the differentiation delay
Symptoms of myotonia, muscle weakness, and muscular atrophy are the main features of DM11, and the molecular contributions to these symptoms are numerous10. One of them is a delay in the process of muscle differentiation, which can be quantified as a reduction in the fusion index after the induction of the fibroblasts to myotubes transdifferentiation32,33. After incubating with MDM both the control and DM1 fibroblasts for four and seven days and treatment with the peptides for 48 h, we carried out immunofluorescence with an anti-Desmin antibody and quantified Desmin-positive (differentiated) cells. While the percentage of terminally differentiated cells remained unchanged after four days in MDM medium, after seven days the percentage of Desmin-positive DM1 cells increased significantly upon treatment with peptides 79 and 81 and remained unchanged in the presence of a scrambled control peptide (Fig. 4a–i). The fusion index, however, did not significantly increase (Supplementary Fig. 4).
Differentiation delay and autophagy hyperactivation were rescued by peptides. Representative micrographs of control (CNT, a; without DMSO), DM1 (DMSO 0.1%; b) and DM1 cells treated with negative control (c, d) or candidate peptides (e–h) stained for Desmin (green signal) as a marker of myogenic differentiation. Cells differentiated in DMEM for seven days and were treated with 10 µM of the indicated peptides. Quantification of the number of Desmin-positive cells relative to the total number of cells (i) revealed a significant increase in myogenic cells when treated with peptides 79 and 81 (counting over 250 nuclei from randomly chosen Desmin-positive cells from 5–7 micrographs). Human myotubes stained with LysoTracker (red fluorescence; j–o). Autolysosomal labeling is observed in DM1 myotubes (DM1) but not in controls (CNT), denoting increased autophagy in DM1 cells. Cells treated with peptide 80 showed a general reduction of the signal from auto lysosomal vesicles while for cells treated with peptides 81 and 82, an increase in the number of cells without autophagic vesicles around the nucleus was observed. (a–h, j–o) Nuclei were counterstained with Hoechst 33258 (blue). Three independent experiments were carried out. *p < 0.05, **p < 0.01, ***p < 0.001 according to Student’s t-test. Scale bar corresponds to 100 (a–h) and 40 microns (j–o).
Another molecular mechanism contributing to muscle atrophy in DM1 is the activation of autophagy12,13,34. To check the autophagy status in DM1 cells after peptide treatments, we used the lysotracker reagent, which stains acidic lysosomes35. First, we confirmed that the level of autophagy of the diseased cells was considerably higher than that of healthy cells (Fig. 4j, k). Furthermore, a qualitative analysis of the autophagic pathway by LysoTracker staining suggested that the treatment with the peptide 80 caused reduction in the signal associated with the lysosomal vesicles, indicating a recovery of the normal autophagy levels. Along the same lines, peptides 81 and 82 increased the number of cells devoid of autophagic vesicles around the nucleus (Fig. 4j–o). Thus, these observations indicate a general reduction in the autophagy pathway, which has previously been shown to contribute to muscle atrophy in Drosophila and human cells in vitro12,13,34.
Candidate peptides rescue muscle atrophy of a Drosophila model of the disease
Rescue of two molecular phenotypes related to muscle atrophy in the cell model, namely delayed differentiation and hyperactivated autophagy, prompted us to verify if these peptides were also active in vivo in a muscle phenotype in Drosophila. In this model, the expression of toxic CUG repeats is controlled by the myosin heavy chain promoter and reproduces muscle phenotypes observed in humans13,36,37. After feeding the DM1 flies with standard food supplemented with the indicated peptides at a final concentration of 10 µM or DMSO as a control, we embedded the thorax of the flies to obtain cross-sections of indirect flight muscles. The quantification of the muscle area from these images showed a marked improvement in the atrophic phenotype in peptide-treated flies, bringing the muscle area to values very close to those observed in control flies (Fig. 5a–i). Concomitant to muscle atrophy, model flies have reduced locomotor abilities, which in flies can be assessed through climbing, taking advantage of Drosophila’s negative geotropism, and flight assays. First, we used 30 male flies for the climbing experiment to measure the height climbed by the flies in a given time. The results revealed a significant effect of the scrambled peptide, suggesting an unspecific effect of the compounds. However, comparing values obtained for flies fed with food supplemented with peptide 79, 80, 81 or 82 with the scrambled version we observed a significant increase in the speed of the flies treated with peptides 80, 81, and 82 (Fig. 5j). It is worthy of mentioning that climbing ability achieved by flies treated with the peptides was significantly higher than values obtained for control flies (p < 0.0008 in all cases). No significant differences were observed when comparing flies treated with control peptides and control flies. In flight tests, while there were no significant increases in the height of the landing distance (indicative of better flight capabilities), we found an increase in the percentage of flies capable of flying, especially in the case of peptide 82 treatment, where it reached statistical significance and almost doubled the value observed in control-treated DM1 flies (p = 0.0044, Fisher’s exact test; Fig. 5k). In conclusion, the increase in the number of flies showing the ability to fly indicates a partial rescue of the Drosophila muscle function consistent with the increase in the IFM muscle area.
Candidate peptides rescued muscle atrophy in a Drosophila DM1 model. Quantification of indirect flight muscle (IFM) cross-sectional area (a) of control (CNT), DM1 flies taking vehicle (DMSO) and DM1 flies taking negative controls (89 or sc) or the indicated candidate peptides. All four candidate peptides increased the mean muscle area relative to DMSO-treated flies (6 flies and 6 micrographs per condition were analyzed). (b–i) Representative bright-field microscope images of transversal sections of resin-embedded adult IFM of Mhc-Gal4 UAS-i(CTG)480 (Rec2) heterozygous flies treated with DMSO (0.01%) or with peptides 79–82 (10 μM) that were used to generate the data shown in (a). Muscle recovery at the histological level leads to functional improvements. The climbing assay (n = 30; j) showed a significant increase in the speed of the treated flies compared to the untreated ones, calculated as the distance traveled in 10 s. In the flight assay (n = 100; k), the average value of the landing height did not significantly improve, but there was a clear increase in the percentage of flies that were able to fly (colored sectors in pie charts underneath the graph), especially for flies treated with peptide 82. *p < 0.05, **p < 0.01, ***p < 0.001 according to Student’s t-test. Scale bar for (b)–(i) panels measures 100 microns.
The secondary structure of (CUG)23 RNA remains unchanged after candidate peptides binding
We used a Differential Scanning Fluorimetry (DSF) assay to monitor CUG RNA thermodynamics in the presence of increasing concentrations of candidate peptides. DSF is technique used to study the effect of compounds on RNA stability as RNA undertakes structural conversions upon thermal unfolding38. When RNA changes its structure the single stranded form increases the available binding sites for RiboGreen dye. We represented the first derivatives of normalized fluorescence of RiboGreen with an RNA probe containing 23 repeats of CUG versus temperature in the presence of concentrations of each hexapeptide ranging from 1 to 100 µM (Fig. 6a, d, g, j). The titration with increasing concentrations of peptides 79, 80, and 81 only changed the height of the peak indicating interference in the intrinsic folding properties of the RNA probe rather than stabilization or destabilization of the RNA hairpins. Peptide 82, however, did slightly shift the curve peak towards lower temperatures, which meant that the interaction between the hexapeptide and the probe does not stabilize the single strain RNA conformation, which is in striking contrast with the proposed destabilization of CUG RNA by abp122. Taken together, the DSF experiments strongly support that the candidate peptides, at least 79, 80, and 81, do not significantly modify the secondary structure of the CUG RNA.
Candidate peptides interact with CUG RNA without affecting thermodynamic stability. The graphs show the first derivatives of RiboGreen fluorescence (DSF assay) versus temperature (a, d, g, j; 4 technical replicates per condition). and Thiazole orange fluorescence titration experiments (b, e, h, k; FID assay) for peptides 79 (a–c), 80 (d–f), 81 (g–i) and 82 (j–l). Experiment was performed per duplicate. DMSO 0.1% was used as a negative control. In Thiazole orange titrations, the intensity of the fluorescence progressively decreased with increasingly higher peptide concentrations (concentrations ranged from 0.03 up to 4 µM). The association constants (Ka) were calculated from FID assay data, which indicate that peptide 79 interacts with the (CUG)23 RNA probe with the highest affinity, although differences in binding affinity were low among all four peptides. DSF data also supports the binding of peptides to the RNA probe but did not detect significant changes in its secondary structure.
Candidate peptides interact with CUG RNA with similar affinities
A fluorescent indicator displacement (FID) assay was used to investigate the nature of the interaction between the candidate peptides and the toxic RNA. Thiazole orange (TO) is an asymmetric cyanine intercalator with little fluorescence when free in an aqueous solution but strong emission when forming complexes of different nature with nucleic acids. These characteristics can be exploited to study changes in the interaction between the dye and the nucleic acid of interest in response to external factors39,40. Specifically, the fluorescent reporter interacted with the (CUG)23 RNA probe resulting in fluorescent emission. By adding the peptide, it was possible to displace the TO, causing its fluorescence to decrease. In this way, by analyzing the fluorescence emission at different RNA-peptide ratios, the value of the peptide’s association constant for the CUG sequence could be indirectly calculated. For all peptides, a progressive reduction in fluorescence was observed in response to increasing concentrations of each peptide (Fig. 6b, e, h, k); in the case of peptide 79 (klog = 5.273 ± 0.007), the greatest decrease in fluorescence was observed, followed by peptide 80 (klog = 5.04 ± 0.01) and the smallest change was observed with peptides 82 and 81 (respectively klog = 4.74 ± 0.02 and klog = 4.65 ± 0.02) (Fig. 6c, f, i, l). However, it should be noted that in none of the cases did the level of fluorescence observed reach the characteristic values of a single-stranded RNA. This indicates that, although there was a clear interaction between the peptides and the RNA molecule, peptides did not interfere with the stability of the secondary structure of CUG RNA, which was consistent with the data generated by the DSF assay.
In parallel with the above experiments, we used the peptide showing the highest affinity for CUG repeats to investigate a potential direct interaction with MBNL1 proteins as an alternative mechanism of action since it might similarly prevent sequestration by the repeats. Double immunostaining with biotin-labeled peptide 79 revealed it accumulated in the cytoplasm of DM1 myotubes, mainly in the perinuclear area, but no significant overlap was found with the MBNL1 protein signal (Supplementary Fig. 5). Thus, candidate peptides do not seem to interact physically with MBNL proteins, at least peptide 79.
Study of the interaction mechanism by molecular docking
In molecular modeling, docking is a method that predicts the preferred orientation of one molecule to a second when they bind together to form a stable complex. Multiple docking studies were performed using Autodock VINA and Molecular Operating Environment (MOE) software to assess the preferred binding mechanism between hexapeptides and CUG repeats. Next, the results obtained with blind docking and guided docking techniques were compared; in the latter case, tests were carried out keeping the RNA rigid or admitting certain flexibility. The final docking protocol was validated by correlating the binding affinities predicted by docking (score) and FID results. According to the analysis, peptides 79 and 80 showed the most remarkable tendency to interact in the exposed part of the RNA (Fig. 7a, b). More in detail, peptide 79 was the only d-hexapeptide capable of recognizing two uracils of the two RNA chains by means of the two terminal amino acid residues. This observation could explain that it had the highest RNA binding association constant. Hexapeptide 80 would interact with the two strands of RNA but showing only the recognition of one uracil. According to the results, Trp would not be favorably available for interaction with the RNA backbone. This result is in agreement with the experimental data in which the presence of Trp did not contribute to the increase in the RNA binding constant (cpyaqe > cpyawe, cpytqe ~ cpytwe). Although this conclusion could be due to a limitation of the simulation protocol used, which only contemplates slight flexibility of the RNA, it can be inferred that Trp would not interact by ππ-stacking (attractive and non-covalent interactions between aromatic rings). As for the two peptides with the lowest experimental interaction energy, it was surprising that the binding mechanism obtained, both for peptides 81 and 82, only showed interaction with one strand of RNA (Fig. 7c, d). Of the two, 82 could interact with three consecutive nucleotides, providing a slight additional stabilization.
Modeling of the proposed interaction between the candidate peptides and the CUG RNA. Representation of the results obtained by means of a flexible docking directed to the area of the RNA exposed to the solvent. Two different binding mechanisms are proposed: peptides 79 (a) and 80 (b) might interact with the double-stranded RNA (red lines), recognizing the indicated uracils (red circles); peptides 81 (c) and 82 (d) might interact with one-stranded RNA (red line). Molecular representations included were generated using MOE 2019.01 software (Chemical Computing Group, https://www.chemcomp.com).
