Four related peptides rescue MBNL-dependent missplicing events in DM1 myoblasts.
Immortalized human DM1 muscle cell lines display disease-associated molecular features such as nuclear RNA aggregates and alternative splicing defects 23. 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 DM1 24 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-dependent 27. Immortalized control and DM1 fibroblasts were transdifferentiated into myoblasts for 48 h. After that, they were incubated two more days with 10 µM of each peptide dissolved in myoblast 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 (Fig. 1a-d). 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 CELF1 28, nor exon 19 of the DLG1 gene, which remains unchanged in DM1 patients 29, 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, including toxicity assays in control myoblasts (Fig. 1k) and fibroblasts (Supplementary Fig. 1). Peptides were not toxic even at 100 µM, which is ten times higher than the concentration at which they rescued missplicing.
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. 2ab). 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 DM1 5 6 30 16 18 19. 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. 2). Finally, since the subcellular localization of MBNL1 is altered in DM1 19,30, 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.
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 protein 15,22,31. 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 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.
Treated DM1 myoblasts reduce the differentiation delay.
Symptoms of myotonia, muscle weakness, and muscular atrophy are the main features of DM1 1, and the molecular contributions to these symptoms are numerous 10. 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 myoblasts transdifferentiation 32 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. 3).
Another molecular mechanism contributing to muscle atrophy in DM1 is the activation of autophagy 12,13,34. To check the autophagy status in DM1 cells after peptide treatments, we used the lysotracker reagent, which stains acidic lysosomes 35. First, we confirmed that the level of autophagy of the diseased cells was considerably higher than that of healthy cells (Fig. 4j,k). Furthermore, we verified that the treatment with the peptide 80 caused a robust 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 vitro 12,13,34.
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 humans 13,36,37. After feeding the DM1 flies with 10 µM of each peptide mixed with the food, 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 increase in the speed of the flies treated with the four peptides compared with DM1 flies fed with DMSO supplemented food or scrambled peptide controls (Fig. 5j). 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.
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 unfolding 38. 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 abp1 22. 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 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 factors 39,40. 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 affinity 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 myoblasts, mainly in the perinuclear area, but no significant overlap was found with the MBNL1 protein signal (Supplementary Fig. 4). 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 constant affinity. 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.