Pre- and postsynaptic upregulation of FasII synergistically underlies neuropathological and behavioral phenotypes in a Drosophila model of myotonic dystrophy

Domain: neuroscience
Published: 2025-12-18
Editorial outcome: major_revision

Abstract
Myotonic dystrophy type 1 is a multisystemic disorder that has been extensively studied for decades, yet our understanding of its neuropathological aspect remains rudimentary. Building on an established Drosophila model, we study the neuropathological features of the disease by expressing untranslated expanded CUG repeats at the Drosophila larval neuromuscular junction. In this model, we show that both pre- and postsynaptic expressions of CUG repeats participate in inducing phenotypes in synaptic boutons, arbors, transmission and larval locomotor activity. Furthermore, expression of CUG repeats in either motor neurons or body wall muscles induces upregulation of the cell adhesion molecule FasII (NCAM1 in mammals), and the knockdown of fasII is sufficient to rescue the phenotypes. Overexpression of FasII-C, a FasII isoform with no cytoplasmic domain, mimics the phenotypes of expanded CUG expression at the neuromuscular junction. In contrary, overexpression of FasII-A-PEST+ rescues the synaptic and behavioral defects. Our study provides insights into the fundamental mechanisms underlying synapse dysregulation in myotonic dystrophy type 1.

Introduction
Myotonic dystrophy type 1 (DM1), also known as Steinert disease, is the most common form of adult-onset muscular dystrophy, affecting as many as one in every 2100 individuals1. This multisystemic disorder is characterized not only by progressive myotonia and muscle degeneration but also by cataracts, heart dysfunction, and neuropathology2. Congenital DM1 can also occur in infants and children, who present with severe muscle weakness and hypotonia rather than myotonia, as well as cognitive impairment3. DM1 is caused by a CTG trinucleotide repeat expansion in the 3′ UTR of the dystrophia myotonica protein kinase (DMPK) gene4–6. Normal individuals may have fewer than 37 CTG repeats, whereas DM1 patients may have hundreds or even thousands of repeats7. The gain-of-function from mRNA transcripts harboring these expansions of untranslated CUG repeats was found to contribute to some of the major pathological features of DM1 independently of the DMPK locus8. In fact, the severity of DM1 was found to correlate with the number of repeats and age of onset9. CUG repeat-containing transcripts were shown to be retained in the nucleus and recruited into ribonuclear foci10, leading to the sequestration of RNA-binding proteins such as muscleblind-like 1 (MBNL1) and subsequently compromising the RNA-splicing machinery11,12.

RNA toxicity is well-known to be associated with the neurodegenerative features of numerous repeat expansion diseases, including polyglutamine diseases, spinocerebellar ataxias, and C9ORF72-associated amyotrophic lateral sclerosis/frontotemporal dementia13. DM1 is no exception. Despite its original designation as a muscular dystrophy, DM1 is known to manifest many neuropathological features14–16. DM1 patients commonly exhibit degeneration of the pigmentary retina and loss of photoreceptor neurons2. They also may exhibit cognitive impairment, speech and language difficulties, attention deficit, autism spectrum disorder, autistic features, sleep disorder, social anxiety, and peripheral neuropathy17–23. Nevertheless, the neuropathological aspect of DM1 is much less explored than the muscle pathology. In neurons, mutant mRNAs containing CUG repeats were found to accumulate in ribonuclear foci within the nuclei and to colocalize with MBNL1, similar to the process observed in muscles12. However, in a transgenic mouse model of DM1 (DM300), an expansion of 300 CUG repeats did not induce detectable neuropathology24. In contrast, an expansion of 1300 repeats in DMSXL mice was found to result in motor neuropathy, suggesting that a large CUG expansion can indeed result in neuropathology25. The end-plates at the neuromuscular junctions (NMJs) of DMSXL mice exhibit decreased size and complexity, and 23% are disconnected from the axonal branches; furthermore, the motor neurons exhibit defects in conducting action potentials25. Synaptic proteins such as RAB3A and synapsin I are respectively upregulated and hyperphosphorylated in DMSXL mice, in association with synaptic transmission and behavioral deficits15. Neuronal progeny cells derived from human embryonic stem cells carrying the DM1 mutation exhibit defects in neurite outgrowth and synapse formation16. Despite these findings, the mechanism underlying the influence of expanded CUG repeats on neurons and synapses remains unclear.

Drosophila melanogaster has emerged as a valuable model for studying the molecular pathology of DM1 in vivo. Transgenic flies expressing expanded CUG repeats in muscles recapitulate hallmark features of both the mouse DM1 model and human disease, including the formation of RNA foci, misregulation of alternative splicing, hypercontraction of muscles, splitting of fibers, progressive degeneration of muscle, and genome-wide changes in expression that are both splice-dependent and -independent26–28. These models provide a genetically tractable platform for dissecting pathogenic mechanisms and identifying potential therapeutic targets.

Here, we used the highly stereotypical and accessible Drosophila larval NMJ system to study the effects of untranslated CUG repeats on synaptic functions and behavior. We found that the simultaneous expression of expanded CUG in the presynaptic motor neurons (MNs) and postsynaptic body wall muscles (BWMs) resulted in synergistic functional and structural impairment of the NMJ. We characterized this DM1 model of neuropathology and identified the upregulation of Fasciclin II (FasII, a.k.a. Fas2), the Drosophila orthologue of mammalian neural cell adhesion molecule 1 (NCAM1), as a major contributor to the observed NMJ phenotypes. We observed dysregulation of NCAM1 in both the DMSXL mouse model and patients with DM1, supporting the relevance of our findings in Drosophila. Overexpression of FasII-C, an isoform of FasII with no cytoplasmic domain, mimicked the phenotypes of expanded CUG expression at the NMJ. We further rescued our DM1 model by either knocking down the upregulated fasII or overexpressing the major neuronal isoform of FasII at the NMJ, suggesting that synaptic loss might be alleviated by restoring the proper ratios of cell adhesion molecule isoforms. These findings provide important insights into the mechanisms underlying motor defects associated with DM1 neuropathology.

Results
Simultaneous pre- and postsynaptic expression of expanded CUG repeats causes neuromuscular phenotypes
We used the Drosophila larval NMJ system to examine how CUG repeat-mediated toxicity affected neuronal growth and development. The larval NMJ is characterized by a high level of plasticity during development and large, easily visualized synapses that produce easily recordable data29. Importantly, this system also allows easy manipulation of the presynaptic MNs and postsynaptic BWMs separately. C380-Gal4 (C380) and C57-Gal4 (C57) are, respectively presynaptic MN and postsynaptic BWM driver lines. Using these Gal4 drivers, we overexpressed either a control construct (UAS-CTG60) containing fewer untranslated CUG repeats than the disease threshold or an untranslated expanded CUG repeat construct (UAS-CTG480), which was previously characterized in an adult Drosophila DM1 model27,30. Fluorescence in situ hybridization showed that CUG480 expression in MNs and BWMs of 3rd instar larvae resulted in RNA foci containing CUG repeats in the nuclei of these cells (Supplementary Fig. 1). Compared with the CUG60 control, the expression of CUG480 in either MNs or BWMs alone did not result in any change in synaptic bouton numbers in late 3rd instar larvae. However, a robust bouton reduction phenotype was observed when both the C57 and C380 drivers were used to drive CUG480 expression in the pre- and postsynaptic compartments simultaneously (Fig. 1a, b). This phenotype was not observed when we overexpressed an untranslated CAG repeat expansion construct (UAS-CAG250)31 using both drivers, suggesting that the bouton phenotype is specific to expanded CUG repeats (Fig. 1b).

This phenotypic reduction in boutons could have been caused by reduced synaptic growth during development, the disassembly/retraction of mature boutons after they were formed or a combination of both. If the phenotype is present in early larval stages, then it is likely to be due to reduced synaptic growth during development. Thus, we dissected 1st and 2nd instar larvae expressing CUG480 via C380 and C57 drivers to investigate whether they had reduced bouton numbers. Interestingly, we found that both the 1st and 2nd instar larvae had no detectable reductions in bouton numbers, whereas 3rd instar larvae exhibited a robust decrease (Fig. 1c). These results suggest that the NMJs of CUG480-expressing animals were morphologically normal during the early larval stages, with the bouton reduction phenotype appearing only around the 3rd instar stage.

Checking for the presence of mature postsynaptic markers after synaptic disassembly/retraction would be a more direct way to determine whether this process is present in CUG480-expressing larvae. Discs large (DLG) is the Drosophila counterpart of the mammalian postsynaptic MAGUK scaffolding proteins, which include SAP-97, SAP-70, and PSD-9532,33, and it is only present in the postsynaptic density of a mature bouton. Through a detailed analysis of the NMJs, we identified some disconnected, disassembling boutons from seemingly degenerating arbors in larvae expressing CUG480 using both the C380 and C57 drivers (Fig. 1d, e). We observed faint expression of DLG on the BWMs near these disassembling boutons, indicating that these were once mature boutons with postsynaptic densities (Fig. 1d). Thus, these disassembling boutons differ from ghost boutons (i.e., premature boutons that lack DLG on postsynaptic BWMs34). Although we cannot rule out the possibility that the bouton reduction phenotype in these animals is due to reduced synaptic growth during development, we are certain that the disassembly of mature boutons and arbors contributed to this phenotype, at least in part. Interestingly, although the expression of CUG480 using the presynaptic MN driver (C380) alone did not induce a detectable bouton reduction phenotype (Fig. 1b), it resulted in a small but significant increase in arbor disassembly (Fig. 1e). However, the expression of CUG480 using both the presynaptic (C380) and postsynaptic (C57) drivers resulted in a significantly higher amount of arbor disassembly than using the C380 driver alone (Fig. 1e). These results suggest that although the presynaptic expression of CUG480 alone does not yield a bouton number phenotype, it does cause morphological defects. Our results also suggest that the presence of CUG RNA toxicity in both the presynaptic MNs and postsynaptic BWMs has a synergistic effect on arbor disassembly.

Structural changes at the NMJ are usually accompanied by functional changes. Thus, after analyzing the morphology of the NMJs, we explored whether the expression of CUG480 would also affect larval crawling behavior. A previous study demonstrated that the expression of expanded CUG repeats in postsynaptic BWMs alone was sufficient to induce locomotor defects in Drosophila larvae28. As the C380-driven expression of CUG480 in MNs alone was sufficient to cause arbor disassembly (Fig. 1e), we hypothesized that it may also be sufficient to cause locomotor defects in larvae. Indeed, we found that C380-driven expression of CUG480 alone induced a small but significant locomotor defect in 3rd instar larvae, while expression of CUG480 using the postsynaptic driver (C57) alone was sufficient to produce a stronger locomotor defect; the expression of CUG480 using both the C380 and C57 drivers produced the strongest phenotype (Fig. 1f). These results suggest that CUG RNA toxicity in both presynaptic MNs and postsynaptic BWMs contributes to a robust behavioral defect in larva.

To better understand how expanded CUG RNA affects NMJ functions, we examined synaptic transmission in CUG-expressing animals through electrophysiological analyses. The control CUG60 or expanded CUG480 was expressed presynaptically (C380), postsynaptically (C57), or pre+postsynaptically (C380+C57), and the miniature excitatory postsynaptic potential (mEPSP) and excitatory postsynaptic potential (EPSP) were measured. Expression of CUG480 in presynaptic MNs led to increases in mEPSP and EPSP amplitudes (Fig. 2a, b, and e) but no detectable changes in quantal content (QC) or mEPSP frequency (Fig. 2c–e). Expression of CUG480 in postsynaptic BWMs led to decreases in mEPSP and EPSP amplitudes (Fig. 2f, g, and j) but no detectable changes in QC or mEPSP frequency (Fig. 2h–j). Expression of CUG480 pre+postsynaptically resulted in no detectable changes in mEPSP amplitude (Fig. 2k, o), possibly because the increasing effect of presynaptic expression (Fig. 2a) and decreasing effect of postsynaptic expression (Fig. 2f) cancelled each other. However, pre+postsynaptic expression of CUG480 actually caused a significant increase in EPSP amplitude (Fig. 2l, o), despite the slight decreasing effect due to postsynaptic expression (Fig. 2g). Interestingly, although no change in QC was detected when CUG480 was expressed either pre- or postsynaptically (Fig. 2c, h), simultaneous pre+postsynaptic expression of CUG480 resulted in a significant increase in QC, indicating a synergistic effect (Fig. 2m). This increase in QC may indicate an increased release of vesicles per evoked response. In contrast, no detectable change in mEPSP frequency was observed when CUG480 was expressed pre+postsynaptically (Fig. 2n, o).

In summary, simultaneous pre- and postsynaptic expression of expanded CUG repeats synergistically induced morphological and functional phenotypes at the larval NMJ and locomotor behavioral defects. These results have helped to establish the Drosophila larval NMJ as a model for studying neuropathology in DM1. As expanded CUG repeats must be expressed in both pre- and postsynaptic components to induce certain phenotypes, the ability to flexibly manipulate pre- and postsynaptic cells in the NMJ provided us with a prime system for dissecting the neuropathology underlying DM1.

FasII/NCAM1 is upregulated in both DM1 models and patients with DM1
We next sought to determine the cause of the phenotypes in our DM1 model. Although many molecules can alter bouton numbers at the NMJ, few are present in both presynaptic MNs and postsynaptic BWMs and regulate bouton numbers synergistically. A potential candidate is the cell adhesion molecule FasII, the Drosophila orthologue of mammalian NCAM1. FasII-mediated regulation of bouton numbers at the NMJ is complex. First, the overexpression of total FasII presynaptically, postsynaptically, or both can lead to different bouton number phenotypes35. Second, fasII null mutant-induced lethality can only be rescued by expressing FasII in both the central nervous system (CNS) and BWMs36,37, indicating that it plays both key pre- and postsynaptic roles. Most importantly, fasII hypomorphic mutants exhibit a synaptic retraction phenotype36,37, which is similar to the arbor disassembly phenotype we observed in our DM1 NMJ model (Fig. 1d, e). Therefore, we hypothesized that expanded CUG RNA might dysregulate FasII.

To test this hypothesis, we expressed CUG480 in the CNS and muscles and collected the respective tissues for mRNA or protein analysis. The MN driver C380 was not suitable for this purpose, as its expression was limited to only a few neurons in the CNS. Instead, we used elav GeneSwitch-Gal4 (elavGS), an inducible pan-neuronal Gal4 driver expressed only in the presence of RU48638. This GeneSwitch driver was used to avoid expressing CUG480 throughout the CNS at a too-early stage of embryonic development, as its expression in the CNS was shown to affect viability27. First, we verified that the elavGS+C57 model was also sufficient to cause NMJ bouton phenotypes, while elavGS alone was insufficient (Supplementary Fig. 2). Then, we simultaneously expressed CUG480 in the CNS and muscles of larva using elavGS and C57, reared the animals in standard Drosophila medium containing 50 µM RU486 since hatching, dissected the CNS and BWMs at the 3rd instar larval stage, and performed semi-quantitative RT-PCR to determine the total fasII levels in these tissues. Our results demonstrated the upregulation of fasII transcripts in both types of tissues (Fig. 3a–d).

To show that this upregulation was not limited to the larval stage, we allowed these animals to grow to the adult stage, collected the fly heads and muscles, and performed slot blot to detect total FasII proteins. We observed significant upregulation of FasII in both the CNS and muscles of CUG480-expressing adult flies (Fig. 3e–h). This result suggests that both fasII transcript and overall FasII protein expression are upregulated by the expanded CUG repeats, and this upregulation persists from the larval stages to adulthood.

To investigate the presence of a similar upregulation of cell adhesion molecules in a mammalian model of DM1, we analyzed neural and skeletal muscle samples from DMSXL mice, a well-characterized model of DM115,39,40. Using the slot blot technique, we found that NCAM1 protein was indeed upregulated in the spinal cord and tibialis anterior of DMSXL mice (Fig. 3i–l), confirming that our findings in Drosophila are relevant to mammals.

To further verify that the upregulation of FasII in Drosophila and of NCAM1 in mice is relevant to humans with DM1, we detected human NCAM1 by performing slot blots using the frontal cortex of either control individuals or patients with DM1 and quantitative dot blots using transdifferentiated myotubes derived from the myoblasts of controls or patients with DM1. We again observed the upregulation of NCAM1 protein in these human tissues (Fig. 3m, n), confirming that our findings are truly relevant to patients with DM1.

Knockdown of fasII rescues NMJs in the Drosophila DM1 model
As DM1 is known to be associated with splicing machinery defects11,12, we wondered whether particular fasII isoforms were upregulated in our Drosophila model. At least four known isoforms of FasII are expressed in Drosophila: the two transmembrane isoforms, FasII-A-PEST+ and FasII-A-PEST−, are the major isoforms in neurons41. FasII-C has no transmembrane or cytoplasmic domain and is tethered to the cell membrane via a GPI anchor42. FasII-B is a less-characterized isoform lacking a clear transmembrane domain and a GPI anchor43. To investigate the transcript expression of fasII isoforms, we overexpressed CUG480 using either elavGS or C57 and reared the animals in either medium containing 50 µM RU486 or plain medium, then dissected either the CNS or BWMs at the late 3rd instar larval stage. Semi-quantitative RT-PCR was performed to analyze the transcript levels of fasII-A-PEST+, fasII-A-PEST−, and fasII-C isoforms. All three fasII isoforms were upregulated in both the CNS and BWMs (Fig. 4a–d).

If upregulated FasII expression in MNs and BWMs caused the NMJ morphological defects and larval crawling behavioral defects observed in our DM1 model, then we should be able to rescue these phenotypes by reducing FasII expression in these tissues using RNAi. We thus knocked down fasII using a previously characterized UAS-Total-FasII-RNAi line44,45. First, we tested the effect of expressing UAS-Total-FasII-RNAi using the presynaptic C380 and postsynaptic C57 drivers on a control background and observed a small but significant decrease in synaptic bouton numbers (Fig. 4e, f). Second, we co-expressed UAS-Total-FasII-RNAi and CUG60 (UAS-CTG60) and found that, intriguingly, CUG60 nullified the bouton reduction effect caused by UAS-Total-FasII-RNAi (Fig. 4f). Third, we sought to determine whether UAS-Total-FasII-RNAi could rescue the bouton reduction phenotype caused by CUG480. However, one caveat of this experiment was that the extra UAS expressed by the RNAi construct might reduce the Gal4 available for CUG480 expression, resulting in a false rescue. Thus, we introduced UAS-CD8::GFP to control for the extra UAS in the rescue genotype. We observed no significant difference in bouton numbers between C380+C57>CUG480 and C380+C57>CUG480, CD8::GFP, indicating that the extra UAS transgene expression did not significantly affect the bouton reduction phenotype caused by CUG480 (Fig. 4f). We then co-expressed UAS-Total-FasII-RNAi and CUG480 (UAS-CTG480) and found that fasII knockdown rescued the bouton numbers (Fig. 4e, f).

To further examine whether the knockdown of particular fasII isoforms was responsible for the observed rescue, we used two fly lines designed to knock down specific fasII isoforms. The previously characterized UAS-FasII-A-RNAi can knock down both fasII-A-PEST+ and fasII-A-PEST−44. UAS-FasII-C-RNAi was designed to target exon 8 of fasII, which encodes the GPI anchor. Thus, it was expected to knock down only fasII-C. However, RT-PCR analysis of Drosophila BWMs expressing this construct revealed that it actually knocked down both the fasII-A-PEST+ and fasII-A-PEST− isoforms, in addition to fasII-C (Supplementary Figs. 3 and 4). Therefore, we renamed this line UAS-FasII-A&C-RNAi. When either UAS-FasII-A-RNAi or UAS-FasII-A&C-RNAi was expressed at the NMJ on the wild-type background, we observed reductions in bouton numbers and locomotor activity (Supplementary Fig. 5). When we knocked down fasII-A and fasII-C simultaneously using UAS-FasII-A&C-RNAi in CUG480-expressing animals, we observed a rescue of bouton numbers, similar to that observed with UAS-Total-FasII-RNAi (Fig. 4e, f). In contrast, fasII-A knockdown alone could not rescue the bouton numbers in CUG480-expressing animals (Fig. 4e, f). Our data suggest that the rescue in our DM1 model was not due to the knockdown of fasII-A isoforms.

As fasII knockdown rescued the bouton numbers at the NMJ, we further explored whether this manipulation could also rescue larval crawling behavior. Consistent with our findings regarding bouton numbers, we found that the expression of UAS-Total-FasII-RNAi and UAS-FasII-A&C-RNAi under the C380+C57 drivers could rescue crawling behavior in DM1 model animals, whereas FasII-A-RNAi was incapable of rescue (Fig. 4g). Intriguingly, the expression of UAS-Total-FasII-RNAi and UAS-FasII-A&C-RNAi on the DM1 background led to an over-rescue phenotype in locomotor activity.

In Fig. 1, we show that the expression of CUG480 in either presynaptic MNs or postsynaptic BWMs alone was not sufficient to reduce bouton numbers. Hypothetically, rescuing either the presynaptic MNs or postsynaptic BWMs via fasII knockdown should be sufficient to rescue bouton numbers at the NMJ in our DM1 model. To test this hypothesis, we needed to perform tissue-specific fasII knockdown independent of the Gal4/UAS system, as CUG480 was overexpressed by C380 and C57 simultaneously in our model. We thus used the LexA/LexAop binary expression system in conjunction with the Gal4/UAS system46. nSyb-LexA (nSyb) is a pan-neuronal driver line expressed in all neurons, while CassM-LexA (CassM) is a muscle driver line expressed in BWMs (Supplementary Fig. 6). We generated LexAop-FasII-A&C-RNAi using the same construct used in UAS-FasII-A&C-RNAi. Similar to our findings with UAS-FasII-A&C-RNAi, we found that LexAop-FasII-A&C-RNAi also knocked down both the fasII-A and fasII-C isoforms (Supplementary Fig. 7). We then expressed LexAop-FasII-A&C-RNAi to knock down fasII using either nSyb or CassM on the C380+C57>CUG480 DM1 model background. As expected, fasII-A and fasII-C knockdown using either nSyb or CassM was sufficient to rescue the bouton numbers in our DM1 model (Fig. 4h, i). In fact, knockdown of fasII-A and fasII-C using CassM resulted in an over-rescue phenotype. These results suggest that the upregulation of FasII by CUG480 in this DM1 model must be present in both the presynaptic MNs and postsynaptic BWMs to cause a decrease in bouton number at the NMJ.

Overexpression of FasII-C results in NMJ morphological defects and behavioral defects that strongly resemble the DM1 model
At this point, we had accumulated several observations. First, a previous study suggested that FasII-C is usually not the major isoform expressed in neurons41. Thus, its upregulation in CUG480-expressing MNs and BWMs may negatively impact NMJ functions. Second, UAS-Total-FasII-RNAi and UAS-FasII-A&C-RNAi, but not UAS-FasII-A-RNAi, could rescue bouton numbers and behavior (Fig. 4e–g), suggesting that FasII-C upregulation may have caused the NMJ defects observed in our DM1 model. To test this possibility, we overexpressed UAS-FasII-A-PEST+, UAS-FasII-A-PEST−, or UAS-FasII-C both pre- and postsynaptically by using the C380 and C57 drivers simultaneously (on the w1118 control background). We found that although overexpression of FasII-A-PEST+ or FasII-A-PEST− did not affect bouton numbers, overexpression of FasII-C reduced bouton numbers, similar to the DM1 model (Fig. 5a, b). Overexpression of FasII-C also led to an increase in arbor disassembly (Fig. 5c, d), another phenotype observed when we overexpressed CUG480 at the NMJ (Fig. 1d, e). This phenotype was not observed with FasII-A-PEST+ or FasII-A-PEST− overexpression (Fig. 1d).

Regarding larval crawling behavior, pre- and postsynaptic overexpression of FasII-A-PEST+ resulted in a significant increase in locomotor activity, while overexpression of FasII-A-PEST− had no impact and overexpression of FasII-C resulted in a decrease in locomotor activity (Fig. 5e). Again, overexpression of FasII-C mimicked the phenotype of the DM1 model.

In Fig. 1, we show that simultaneous pre- and postsynaptic overexpression of CUG480 was required for a reduction in bouton numbers at the NMJ (Fig. 1b). We explored whether there was a similar requirement underlying the effects of FasII-C overexpression on the NMJ. Indeed, overexpression of FasII-C using a single driver (either C380 or C57) was insufficient to induce significant changes in bouton numbers at the NMJ (Fig. 5f, g), indicating a synergistic effect between pre- and postsynaptic expression of FasII-C on NMJ morphology. Overexpression of FasII-C in BWMs alone (using C57) was also insufficient to induce arbor disassembly, while overexpression of FasII-C in MNs alone (using C380) slightly but significantly increased arbor disassembly (Fig. 5h), similar to that observed with CUG480 overexpression (Fig. 1e). Presynaptic overexpression of FasII-C resulted in a slight but significant decrease in locomotor activity, while postsynaptic overexpression caused a further decrease; pre+postsynaptic overexpression caused the most severe decrease in mobility (Fig. 5i). These phenotypes also strongly resemble those of CUG480 overexpression (Fig. 1f).

Simultaneous pre- and postsynaptic overexpression of FasII-C synergistically induces transmission phenotypes at the NMJ
To further evaluate the effects of FasII-C overexpression on NMJ functions, we performed electrophysiological analyses to examine synaptic transmission. We overexpressed UAS-FasII-C using either C380, C57 or both drivers simultaneously and measured the mEPSPs and EPSPs. We found that presynaptic or postsynaptic overexpression of FasII-C alone had no impact on mEPSP amplitude (Fig. 6a, b, m, n). In contrast, simultaneous pre- and postsynaptic overexpression of FasII-C resulted in a significant decrease in mEPSP amplitude, indicating a decrease in either vesicle size or glutamate receptor expression at the NMJ (Fig. 6c, o). Presynaptic or postsynaptic overexpression of FasII-C resulted in a small but significant decrease in EPSPs, while simultaneous pre- and postsynaptic overexpression resulted in a further decrease (Fig. 6d–f, m–o), indicating an overall decrease in the evoked response. Presynaptic overexpression had no impact on QC, while postsynaptic overexpression resulted in a small but significant decrease and simultaneous pre- and postsynaptic overexpression resulted in a further decrease (Fig. 6g–i), indicating a decrease in the number of vesicles released per evoked response. Lastly, in terms of mEPSP frequency, presynaptic overexpression resulted in a small but significant decrease, postsynaptic overexpression had no effect and simultaneous pre- and postsynaptic overexpression caused a further decrease (Fig. 6j–o), indicating a decrease in the number of spontaneously released vesicles. All these data strongly suggest that simultaneous presynaptic and postsynaptic overexpression of FasII-C synergistically impairs NMJ functions.

Overexpression of FasII-C exacerbates and FasII-A rescues NMJ phenotypes of the Drosophila DM1 model
As our data suggest that FasII-C upregulation is the major cause of the NMJ phenotypes of our Drosophila DM1 model, we wondered whether the overexpression of FasII-C on our disease model background would worsen the phenotypes and whether overexpression of FasII-A isoforms would ameliorate the phenotypes. To test whether FasII-C expression would exacerbate the pathological phenotypes of our disease model, we co-expressed CUG480 and UAS-FasII-C using C380 and C57 and compared the resulting animals with those co-expressing CUG480 and UAS-CD8::GFP. Animals co-expressing CUG480 and UAS-CD8::GFP displayed a bouton reduction phenotype when comparing with CUG60-expressing control animals, while those co-expressing CUG480 and UAS-FasII-C exhibited an even stronger bouton reduction phenotype (Fig. 7a, b). Similarly, animals co-expressing CUG480 and UAS-FasII-C exhibited more strongly impaired larval locomotor activity than those co-expressing CUG480 and UAS-CD8::GFP (Fig. 7c). These results suggest that the upregulation of FasII-C in the DM1 model may not have reached the saturation point, such that a further increase in FasII-C expression resulted in more severe phenotypes.

To test whether overexpression of FasII-A isoforms could ameliorate the pathological phenotypes of our disease model, we overexpressed either UAS-FasII-A-PEST+ or UAS-FasII-A-PEST− in conjunction with CUG480 (UAS-CTG480) using C380 and C57. Intriguingly, overexpression of either of the two FasII-A isoforms was sufficient to rescue the bouton numbers in our DM1 model (Fig. 7d–f). Moreover, overexpression of UAS-FasII-A-PEST+ also rescued locomotor impairment in our DM1 model (Fig. 7g), even resulting in over-rescue. However, overexpression of UAS-FasII-A-PEST− did not lead to an observable improvement of locomotor activity (Fig. 7g). To determine why UAS-FasII-A-PEST− could not rescue locomotor activity, we analyzed the bouton morphology in UAS-FasII-A-PEST− animals using high-magnification confocal microscopy, and found the boutons displaying abnormalities (Fig. 7h, i). These morphological abnormalities might have contributed to the lack of behavioral rescue in the UAS-FasII-A-PEST− animals.

Methods
Mouse tissue samples and ethics compliance
The DMSXL mice with an expansion of over 1300 CTG repeats and control mice used in the study were provided by Mário Gomes-Pereira and Genevieve Gourdon. For each genotype, 2 female and 3 male mice aged between 16 and 21 days were analyzed. All experiments involving the use of DMSXL research mice have complied with all relevant ethical regulations according to the Ministere de L’Enseignement Superieur, de la Recherche et de L’Innovation of France. Animal ethics was approved by Ministry of Higher Education, Research and Innovation (Paris). Authorization for animal experimentation number #23473. Animal facility approval number B751320. The study protocol was approved by Prefecture de Police (Paris) and the French Veterinary Department. Authorization for animal experimentation number 75003. Animal facility approval number B91228107. The Mbnl1−/−; Mbnl2c/c; Nestin-Cre+ conditional double-knockout mice (Mbnl1/Mbnl2 DKO) and wild type (WT) P30 mouse brain samples were provided by Professor Maurice Swanson (Department of Molecular Genetics and Microbiology, Center for NeuroGenetics and the Genetics Institute, University of Florida, College of Medicine, USA). All Mbnl1/Mbnl2 DKO and WT animal procedures were approved by the Institutional Animal Care & Use Committee (IACUC) of the University of Florida (Approval number: IACUC202300000652).

Human tissue samples and ethics compliance
All experiments using human samples were approved by the Ethics Committees of the host institutions. Written informed consent of specimen use for research was obtained from all patients. No payments of any form have been given to the subjects or their families. Human frontal cortex samples were collected from two different laboratories: Dr. Yasuhiro Suzuki (Asahikawa Medical Center, Japan) and Dr. Tohru Matsuura (Okayama University, Japan). Information relative to patients was previously described15,62,63. For the transdifferentiated myoblasts, samples were obtained from the Institute of Myology, Paris. In brief, skin biopsies were obtained from MyoBank-AFMbank of tissues for research, a partner in the EU network Euro-BioBank, in accordance with European recommendations and French legislation. Under these regulations, informed consent was obtained from donors prior to biopsy collection. The samples originated from an 11-year-old female donor with DM1 (1300 CTG repeats) and a healthy 25-year-old male donor. The cells were immortalized using hTERT expression, which was previously described. Fibroblasts were transduced to express MyoD with the supplement of doxycycline, which drove the transdifferentiation of fibroblasts into myotubes64. For experiments involving deltoid muscles, muscle biopsies were taken after informed consent by patients and approval by the Experimentation Ethics Committee of the University Hospital La Fe (Valencia, Spain; authorization number: 2014/0799), according to the Helsinki Declaration.

Fly Stocks
The following stocks were used: w1118, elavGS-GAL4 (43642), UAS-CD8::GFP (5137), UAS-Gbb-GFP (63057), and UAS-Tkv.CA (36537) were acquired from Bloomington Drosophila Stock Center. UAS-CTG60 and UAS-CTG48027 were obtained from Ruben Artero. Transgene expression levels of these constructs can be found in (Supplementary Fig. 19). UAS-CAG25031 was obtained from Nancy Bonini. C380-GAL4 (a.k.a. BG380)65 and C57-GAL4 (a.k.a. BG57)65 were obtained from Vivian Budnik. Expression patterns of these drivers can be found in (Supplementary Fig. 20). UAS-FasII-A-PEST+36,37, UAS-FasII-A-PEST−66, UAS-FasII-C66, UAS-Total-FasII-RNAi (a.k.a. UAS-Total-FasII-dsRNA or P[KK100888]VIE-260B45, Vienna Drosophila RNAi Centre, #v103807), and UAS-FasII-A-RNAi44 and UAS-FasII-A&C-RNAi (unpublished) were obtained from Brian McCabe. UAS-FasII-A&C-RNAi was originally named UAS-FasII-C-RNAi (#38) since it was designed against Exon 8 of fasII, which encodes for the GPI anchor of FasII-C. However, the construct was found to knock down FasII-A-PEST+ and FasII-A-PEST− as well (Supplementary Figs. 3 and 4). Hence, it was renamed as UAS-FasII-A&C-RNAi in this study. LexAop-FasII-A&C-RNAi was generated by subcloning the same RNAi construct (Amplicon sequence: GCTAATAACA ATCTCGGCAC GTTGCTCTAT TCGGCCGGAT TTAATTCCGG TGTCGGTGCG CTACACAAAC GACTGTTCAC AACAACAACA ACAA- CAACAGCCACATCAACAACAACAATCACATCGATAACAACAGCAAC AACAACAATC ATTACGCTGG CCAC) into the pJFRC19-13XLexAop2-IVS-myr::GFP vector (Addgene plasmid #26224), and inserted onto the 2nd chromosome using the Phi3C1 system (The flylines generation process was done by BestGene Inc., U.S.A.). nSyb-LexA (a.k.a. nSyb-LexA-GAD)67 was obtained from Ching-Po Yang and Tzumin Lee. CassM-LexA (unpublished) was obtained from Vivian Budnik. Flies were reared in standard Drosophila medium at 25°C. For genotypes involving elavGS-GAL4, RU486 was dissolved in 100% ethanol before added to the medium to achieve a final concentration of 50 µM.

Semi-quantitative RT-PCR
mRNA was extracted from either dissected larval CNSs or BWMs using standard TRIzol extraction method, homogenizing the tissues in TRIzol, adding chloroform to conduct phase separation, following with isopropanol RNA precipitation and resuspension of RNA using DEPC-treated water at the end68. Synthesis of cDNA was performed using the ImProm-II™ Reverse Transcription System (Promega). Primers used for amplification of fasII were as follow: Total-FasII Forward: GCAAC CAGGTGGGATTAGG (on Exon 6), Total-FasII Reverse: TAACGCCCG GACAGTATTTG (partially on Exon 6 and partially on Exon 7), FasII-A Forward: CTGTCCGGGCGTTAAGATC (partially on Exon 6 and partially on Exon 7), FasII-A Reverse: ACGTCAATTCCTCGTGTCG (partially on Exon 7 and partially on TM domain), FasII-A-PEST+ Forward: ACAC GAGGAATTGACGTCATC (partially on Exon 7 and partially on TM domain), FasII-A-PEST+ Reverse: GTGGCTCCTTTACCAGCTG (on the PEST domain), FasII-C Forward: GCGTTAAGATCAGCGGCAC (on Exon 7), FasII-C Reverse: GAATCGGACTCACCTCGTG (partially on Exon 7 and partially on GPI anchor). The designs of these primers had been previously published44. Other primers used in this study were as follow: Beag Forward: GCATCAGAGGAGCCGATAATATC, Beag Reverse: GGCCTTCTCGTTCTTCTCC. Dsmu1 Forward: TGCAGTTCTCGCGAGA TAAC, Dsmu1 Reverse: TCACTGTAGCTGGCCAGTAG. Elav Forward: CAAGTCGCAGGTCTACATCG, Elav Reverse: CTCCTTTCGTCTGCGTA TCG. Mbl Forward: CAACGTGGAGGTCCAGAAC, Mbl Reverse: CCGGTCAGATAGGGGTTTG. Actin Forward: ATGTGCAAGGCCGG TTTCGC, Actin Reverse: CGACACGCAGCTCATTGTAG. SV40 Forward: GGAAAGTCCTTGGGGTCTTC, SV40 Reverse: GGAACTGATGAATGG GAGCA. Hs promoter Forward: TCCTCCGAGCGGAGACTC, Hs promoter Reverse: TGGCAGATTTCAGTAGTTGCAG. All the primers were ordered and synthesized from the Thermo Fisher Scientific Inc. (Hong Kong). Quantification of gel bands was performed using ImageJ. The housekeeping gene, Actin, was used as a reference standard in each group to observe the expression level of the interested genes. Each of the interested gene band intensity was first normalized to its Actin band intensity. The fold change of each of the experiment group was then calculated by using the normalized intensity of each group over the normalized intensity of the control group.

Semi-quantitative RT-PCR for the analysis of alternative exon usage
RNA extraction was performed with the RNeasy Mini kit (QIAGEN; 74104) following the manufacturer’s protocol, including a DNase digestion step (RNase-Free DNase Set; QIAGEN; 79254) after the first wash with RW1 buffer. RNA concentration was assessed using the NanoDrop (Thermo Scientific) and RNA quality was verified by electrophoresis on agarose gel. cDNA synthesis and semi-quantitative reverse-transcriptase PCR analysis of alternative exon usage were performed by using PCR amplification (mostly around 21–26 cycles) to obtain PCR product following with resolved the PCR product through 2.5% (w/v) agarose gels and stained with ethidium bromide62,63. Oligonucleotide primers used for RT-PCR analysis of Ncam1 were the following: Ncam1 E16 (forward): ACGTCATGCTCAAGTCCCTG. Ncam1 E17 (reverse): AGTCACCGCAGAGAAAAGCA. Ncam1 E18 (reverse): ATGAGCAGGCCACACTTGTT.

Locomotor behavioral assay for larvae
Larval crawling assay was previously published study69. In brief, wandering third instar larvae were loaded onto a 2% agarose plate with a grid (0.5 cm2 per square) placed underneath. The total number of gridlines crossed by the animals in one minute was counted, and the actual distance calculated. n represents the number of larvae analyzed.

Immunohistochemistry
Larval BWMs were dissected and fixed for 10 min in 4% paraformaldehyde, and permeabilized with 0.2% Triton-× 100 at room temperature70. After that, samples were incubated with primary antibodies overnight at 4°C. Primary antibodies and their concentrations used: anti-HRP-Alexa Fluor 488 1:500 (Jackson), anti-HRP-Alex Fluor 647 1:500 (Jackson), anti-DLG1 1:750 (4F3, DSHB), anti-FasII 1:50 (affinity purified 1D4, DSHB)71, anti-FasII 1:5 (34B3, DSHB)42. Secondary antibodies conjugated to Alexa Fluor 488/594 (Abcam) were used at a concentration of 1:200. Samples were incubated with secondary antibodies at room temperature for 1 to 2 h. Lastly, samples were mounted in the glass slice using anti-fade fluorescence mounting medium (Abcam, ab104135).

Quantification of boutons
The number of type I boutons was obtained at muscles 6 and 7 of abdominal segment A3 of late 3rd instar larvae unless specified otherwise. n represents the number of analyzed NMJs. At most two NMJs were quantified in each animal, and each animal is defined as a biological replicate70,72. The development of larvae expressing CUG480 using C380, elavGS (and fed with food with RU486) or other combinations involving these drivers were slower than that of controls by approximately 24 h. Thus, crosses involving these genotypes were set up one day in advance so the dissections and immunostaining can be carried out at the same time. No notable abnormalities were found in the bouton numbers of the genetic controls for the major UAS lines used in this study (Supplementary Fig. 21).

Quantification of arbor disassembly
An NMJ with at least one disassembling arbor was defined as an NMJ with arbor disassembly. A disassembling arbor was defined as a stretch of at least two boutons that are severed from the arbor and from one another (immunostained by anti-HRP), in which at least one of these boutons showed DLG staining on the postsynaptic muscle.

Quantification of muscle areas
Lengths and widths of muscle 6 of segment A3 were measured under a 20× objective on a wide field microscope, and the areas were subsequently calculated (Supplementary Fig. 22). n represents the number of analyzed muscles. At most two muscles were quantified in each animal.

Slot blot assay for Drosophila FasII and mice NCAM1
Fly heads, fly thorax muscles, mouse CNS tissues, or mouse muscle tissues were homogenized in the lysis buffer (100 mM Tris/HCl, pH 6.8; 2% Sodium Dodecyl Sulfate; 40% w/v Glycerol). The homogenates were boiled at 99°C for 10 min, then centrifuged at 14,000 × g for 2 min and the supernatant was collected. Before the slot blot assay, samples were diluted with 2% SDS to a final volume of 200 µL and heated at 99°C for 10 min. Protein samples were loaded to a 48-well Bio-Dot® microfiltration apparatus (Bio-Rad Laboratories, Hercules, CA, USA) with BioTrace™ NT nitrocellulose membrane (Pall Life Sciences, Portsmouth, UK; pore size 0.2 µm). The membrane was blocked at room temperature in 5% non-fat milk and incubated in 34B3 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA; Mouse; 1:10 https://dshb.biology.uiowa.edu/)42 at 4°C overnight for the detection of FasII in fly protein samples. For mouse protein samples, the membrane was incubated in ab154566 (Abcam, Cambridge, UK; Rabbit; 1:1000) for NCAM1 detection. The protein chemiluminescence signal was obtained and visualized with the ChemiDoc™ Touch Gel Imaging System (Bio-Rad Laboratories, Hercules, CA, USA). After FasII or NCAM1 detection, the membrane was stripped in stripping buffer (ThermoFisher Scientific, Grand Island, NY, USA) and blocked again in 5% non-fat milk. β-tubulin was used as the internal loading control, where the membrane was re-probed with ab6046 (Abcam, Cambridge, UK; Rabbit; 1:500) for fly protein samples or 2G7D4 (GenScript, Piscataway, NJ, USA; Mouse; 1:2000) for mouse protein samples, respectively. The images were analyzed with ImageLab™ software (Bio-Rad Laboratories, Hercules, CA, USA) and band intensities were quantified using the Image J software (Research Services Branch, National Institute of Mental Health).

Slot blot assay for human frontal cortex
Total protein was extracted from 20 to 30 mg human frontal cortex using RIPA buffer (Pierce™ RIPA Buffer, ThermoScientific, 89901) supplemented with 0.05% CHAPS (Sigma, C3023), 1× complete protease inhibitor (Sigma-Aldrich, 04693124001), 1× PhosSTOP phosphatase inhibitor (Sigma-Aldrich, 04906845001), and 1 mM sodium orthovanadate (Sigma, S6508). Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, 23227). Porablot NCP nitrocellulose membranes (Macherey-Nagel, 741280) and Bio-Dot® SF filter paper (Bio-Rad, 1620161) were soaked and equilibrated in 1× PBS (Gibco, 20012-019) and then placed in the Bio-Dot® SF Apparatus (Bio-Rad, 1706542). Membrane slots were rinsed with 100 µL 1× PBS, and then loaded with 20 µg frontal cortex protein in 100 µL RIPA buffer under vacuum. The membranes were carefully removed from the Bio-Dot® SF Apparatus and stained with ATX Ponceau S red solution (Sigma-Aldrich, 09189-1L-F) for total protein quantification. Membranes were blocked in 5% Blotto (Santa Cruz Biotech, sc-2324) diluted in 1× TBS-T (10 mM Tris-HCl, 0.15 M NaCl, 0.05% Tween 20) for 1 h at room temperature, and incubated overnight at 4°C with primary anti-NCAM1 antibody (GeneTex, GTX111684) diluted 1:10,000 in 5% Blotto. After three washes in 1× TBS-T, membranes were incubated with secondary HRP-conjugated goat anti-rabbit IgG (Life Technologies, 31460) diluted 1:5000 in 5% Blotto, for 1 h at room temperature. Membranes were finally washed another three times in 1× TBS-T and developed with Clarity Max ECL Substrate (BioRad, 1705062). Image acquisition and quantification were conducted with BioRad ChemiDoc™ MP Imaging System and software (Image Lab 6.0.1). Relative protein levels in non-DM controls were normalized to 1 and statistical analysis was performed with Prism (GraphPad Software, Inc.).

Transdifferentiation of myotubes
Control or DM1 patient myoblasts were differentiated for 7 days into transdifferentiated myotubes. Briefly, the cells were first immortalized using hTERT expression. After that, fibroblasts were transduced to express MyoD in the presence of doxycycline. This process induced expression of MyoD facilitates the transdifferentiation of fibroblasts into myotubes64.

Quantitative dot blot assay
1 µg/well of protein samples were denatured (100°C for 5 min) and loaded in quantitative dot blot (QDB) plates (Quanticision Diagnostics Inc, Research Triangle Park, NC, USA). Each cell sample was loaded in quadruplicate in two different plates; one was used to detect NCAM1 (1:1000, Proteintech, Manchester, UK) and the other for GAPDH (1:500, (G-9) Santa Cruz Biotechnology, Dallas, TX, USA), which was used as an endogenous control. ECL substrate (Pierce™ ECL Western blotting substrate) was used to detect and visualize the result and TECAN infinite M200 Pro with an initial acquisition time of 1 s/well in the luminescence was used to acquire data73.

Electrophysiology
Electrophysiology was performed as previously published study74. For detailed methods on how to perform Drosophila NMJ electrophysiology, please see published protocols75,76. Briefly, wandering third instar larvae were collected and filleted for NMJ analysis. Driver control and experimental electrophysiological recordings were performed in parallel using identical conditions. Larval dissections and recordings were performed in a modified, low-magnesium HL3 saline77. (70 mM NaCl, 5 mM KCl, 5 mM HEPES, 10 mM NaHCO3, 115 mM sucrose, 4.2 mM trehalose, 0.5 mM CaCl2 (unless otherwise noted), 10 mM MgCl2, pH 7.2. Neuromuscular junction sharp electrode recordings were performed at muscles 6 and 7 of abdominal segments A2 or A3. For a muscle to be acceptable for recording, it needed to have an input resistance of ≥4 MΩ and a resting potential more hyperpolarized than −58 mV.

Recordings were performed on an Olympus BX51WI microscope and acquired using an Axoclamp 900A amplifier, Digidata 1440A acquisition system, and pClamp10.7 (Molecular Devices) software. Data were analyzed using Mini Analysis (Synaptosoft) and the Clampfit (Molecular Devices) programs. mEPSPs and excitatory postsynaptic potentials (EPSPs at 1 Hz stimulus) were collected74. For each NMJ recorded, four quantifications were made: quantal size (mEPSP size); quantal frequency (mEPSP frequency); evoked potential size (EPSP size); and quantal content (QC). In the case of QC, uncorrected QC was calculated per NMJ by dividing the average EPSP by the average mEPSP.

Fluorescent in situ hybridization (FISH) and immunofluorescence
Primary astrocytes were cultured for 2 weeks, washed 3 times in 1× PBS and then fixed for 15 min in 4% PFA (J61899.AP, VWR) at 4°C. Following 5× 2 min washes in 1× PBS, cells were permeabilized in 2% ice-cold acetone in 1× PBS for 5 min at room temperature. Prehybridization was carried out in 30% formamide/2× SSC for 10 min at room temperature. Hybridization with 1 ng/mL 5′-Cy3-labeled (CAG)5 PNA probe in hybridization buffer (30% formamide, 2× SSC, 0.02% BSA, 66 mg/ml yeast tRNA, 2 mM vanadyl complex) was carried out for 2 h at 37°C in a dark humidified chamber. Next, cells were washed for 30 min in 30% formamide/2× SSC at 50°C, followed by one last wash of an additional 30 min in 1× SSC at room temperature.

Cells were then incubated in blocking and permeabilization solution for 1 h at room temperature (1× PBS; 0.1% Triton ×-100; 10% normal goat serum, G6767, Sigma). Primary antibody was diluted in permeabilization (1× PBS; 0.1% Triton ×-100) and incubated overnight at 4°C (SMU1, Sc-100896, Santa Cruz biotechnologies, dilution 1/500; RED, PA5-101789, Thermo Fisher, 1/500). Next day, following 3× 10 min washes in 1× PBS, cells were incubated with secondary antibody diluted in permeabilization solution for 1 h at room temperature. Excess antibody was washed 3× 10 min in 1× PBS, and incubated with 0.0002% DAPI (10236276001, Sigma) for 15 min at room temperature. Cells were finally washed 3× 10 min in 1× PBS and mounted with Dako Fluorescent Mounting Medium (S302380, Dako) prior to observation. Images were acquired with a laser apotome (Zeiss, AxioObserver), using a 40× objective and Zeiss Zen software.

Statistical analysis
For comparisons between three or more sample groups, an analysis of variance (one-way ANOVA) with Tukey post-hoc test was performed. For pair-wise comparisons, Student’s t-test (two-tailed) was used. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All histograms depict mean ± S.E.M. All experiments were performed at least three times independently.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Despite decades of research, the neuropathological mechanisms underlying Myotonic dystrophy type 1 (DM1) are still not\nfully understood. In this study, the authors model DM1 neuropathology by expressing untranslated expanded CUG repeats\nat the neuromuscular junction in Drosophila larvae. They observed that both presynaptic (motoneuron) and postsynaptic\n(muscle) expression of CUG repeats led to a reduction in synaptic boutons, increased arbor disassembly, and impaired\nlarval locomotion. The reported findings further indicate that CUG repeat expression elevated levels of the cell adhesion\nmolecule FasII (analogous to NCAM1 in mammals) in both motor neurons and body wall muscles. They report that reducing\nfasII expression did restore bouton numbers and improved locomotor activity. Further analysis suggested that the\nupregulation of the FasII-C isoform was primarily responsible for these phenotypic changes. Additionally, overexpressing the\nFasII-A-PEST+ isoform helped mitigate synaptic and behavioral abnormalities, likely by outcompeting the excess FasII-C.\nThrough their research, they provided insights into synapse dysregulation in DM1, offering a foundational understanding of\nthe molecular mechanisms involved in this disorder.\nIn this manuscript, Koon et al. explored the effects of expressing CUG repeats in the developing larval neuromuscular\njunction (NMJ), demonstrating some neuropathological aspects of a previously well-established Drosophila model of\nmyotonic dystrophy type 1 (DM1). The authors mainly used NMJ bouton number and larval locomotor activity as readouts,\nand dissected the effects of pre- and/or postsynaptic CUG repeats expression. They show that either pre- or postsynaptic\nCUG repeats expression resulted in locomotor deficits, without affecting the gross NMJ structure when analyzed as NMJ\nbouton numbers. Furthermore, simultaneous pre- and postsynaptic CUG repeats expression triggered both locomotor and\nNMJ structural defects. They then show that increased FasII level might contribute to these structural and behavioral defects.\nWhile the link between the Drosophila DM1 model and FasII might per se interesting, this study has notable conceptual and\nmethodological shortcomings. The argument for specific distinct molecular mechanisms connecting DM1 pathology to FasII\nisoforms lacks convincing evidence. The authors appear to over-interpret their data, particularly regarding the claimed\n“outcompetition of FasII-C by FasII-A,” which does not seem convincingly supported by their findings. Additionally, it is\nsurprising that the authors opted to test only FasII-C overexpression in electrophysiological experiments, leaving the DM1\nmodel unexamined in this context. This choice significantly limits the study’s comprehensiveness and the robustness of its\nconclusions. To strengthen their findings, the authors should include a broader range of electrophysiological tests in the\nDM1 model and provide clearer evidence to support their claims about the molecular mechanisms involving FasII isoforms.\nFurther major concerns:\n1. The authors chose to utilize the more accessible larval neuromuscular junction (NMJ) to investigate the neuropathology of\nDM1. However, they did not incorporate any functional readouts, such as electrophysiology, to assess potential changes in\nneurotransmission associated with DM1. Instead, they focused solely on the electrophysiological phenotypes of FasII-C\noverexpression (OE), which cannot be considered a valid proxy for DM1. This approach significantly limits the study’s ability\nto provide comprehensive insights into DM1 neuropathology and its impact on neurotransmission.\n2. 2. The authors did not investigate potential signaling pathways known to mediate NMJ bouton structure, such as BMP and\nWnt signaling. There are numerous established tools available in the field for studying these pathways, and the authors\nshould utilize these to elucidate the mechanisms regulating NMJ structural phenotypes in DM1. Currently, the connection\nbetween DM1 and FasII does not sufficiently explain the observed NMJ structural phenotypes. By exploring these signaling\npathways, the authors could provide a more comprehensive understanding of the molecular mechanisms underlying the\nstructural abnormalities seen in DM1.\n3. The authors proposed the hypothesis of “FasII isoform competition to bind to isoform A” based on the distinct phenotypes\nobserved in NMJ boutons and larval locomotion after FasII isoform knockdown or overexpression. This hypothesis appears\nto be speculative and lacks robust support from the provided data and existing literature. Additionally, the potential differing\nstrengths of the C380 and C57 Gal4 lines and fasII RNAi lines, as shown in Figure S1b, may further complicate the situation.\n4. Using the number of NMJ boutons as the primary and nearly exclusive indicator of synaptic abnormalities is superficial,\nalso in the context of DM1. For instance, in Figure 4, although FasII-A-PEST+ overexpression does not affect NMJ bouton\nnumbers, it does result in a gain-of-function in locomotion. This discrepancy suggests that the NMJ bouton count alone may\nnot fully capture the synaptic changes in DM1. Furthermore, while the authors mention the use of anti-FasII (1D4) in\nimmunostaining methods, it is surprising that they did not perform FasII staining using isoform-specific antibodies like 1D4\nversus 34B3. Conducting NMJ immunostaining against FasII would provide critical spatial information regarding FasII\ndistribution in DM1 and FasII overexpression models. Such information would be essential for understanding the precise\nrole of FasII isoforms in NMJ structural phenotypes and could significantly enhance the mechanistic insights derived from\nthe study.\nMinor points:\n1. In the abstract the authors state, “In this study, we characterized a novel model of DM1 neuropathology by expressing\nuntranslated expanded CUG repeats at the Drosophila larval neuromuscular junction.” This in my eyes is misleading, as the\nuse of CUG repeat expression to model DM1 in Drosophila was proposed over 15 years ago. Thus, the model itself is not\nthat novel. Am I right here? If yes the authors should clarify this point to avoid overstating the novelty of their approach and to\naccurately reflect the existing body of research.\n2. In Figure 2, was elav-GeneSwitch/C57-driven DM1 model also sufficient to cause NMJ bouton phenotypes like with\nC380/C57 ?\n3. Quantification is missing in Figure 3a,b.\n4. In Figure 3c,d, the NMJ bouton phenotypes and larval locomotion of fasII-A and A&C RNAi are shown only in DM1\nbackground, the effects of fasII-A and A&C RNAi alone are missing\n5. In Figure 4b, CUG-60 expression is not the correct control for FasII OE\n6. Similar in Figure 4d,e, CUG-60 expression is not the correct control for FasII-C OE\n7. In Figure 5, the authors differentiated pre- and post- OE of FasII-C in electrophysiology. However, the authors did do the\nsame in Figure 4.\n8. In Figure 6, did the authors also performed fasII-C OE in DM1? Were there additive effects in NMJ bouton number and\nlocomotion, and electrophysiology\n9. Except for DM1 model flies, genetic controls for all the UAS lines including RNAi lines and cDNA OE line are missing.\nGiven that genetic background affects NMJ bouton phenotypes, it will be important to include those UAS controls.\n10. It is also a bit surprising that, given the per se limited electrophysiological data of Figure 5, the authors did not show any\nsample traces for minis and evoked.", "reviewer_2": "Kun et al., report CUG repeat dependent upregulation of FasII in Drosophila model of DM1 showing that it causes\nneuromuscular junction (NMJ) defects and affected mobility. Interestingly, FasII ortholog NCAM1 expression is also\nupregulated in muscular and neural tissues of DM1 mouse model (DMXL) and in DM1 patients. Authors take advantage of\nthe well characterised Drosophila NMJ system in 3rd instar larva to test influence of toxic CUG repeats on presynaptic\n(motor neurons) and post-synaptic (body wall muscles) components of NMJ. They apply genetic tools available in fly model\nto rescue observed DM1-associated NMJ phenotypes and larva mobility. Authors propose a model in which the expression\nof FasII isoforms is affected in DM1 context with aberrant upregulation of FasII C isoform that impacts synapse integrity.\nThe originality of the manuscript resides in assessing effects of toxic repeats in pre- and post-synaptic components of the\nNMJ and how they impact the neuromuscular synapse.\nThis experimental setup allows identification of FasII/NCAM1gene deregulations conserved between fly and human which\ncould inspire future investigations in the field.\nThe manuscript suffers however from some inconsistencies. It would also benefit from applying image analyses tools to\nbetter describe NMJ phenotypes and from linking at molecular levels toxic repeats expression with the deregulation of FasII.\nInconsistencies :\n1. Authors refer in the title and in the text to neuropathological and behavioral defects in DM1 but they analyse phenotypes of\nneuromuscular junctions and larval mobility. Whether dysregulation of FasII influences fly behavior and neurological CNS\nfunctions is not shown.\n2. On page 4 authors say that they overexpressed control UAS-CUG60 construct (which should rather be UAS-CTG60) with\nthe number of CTG repeats that is lower than the disease treshold. This is not correct as the number of CTG repeats in dmpk\ngene higher than 37 is already classified as potentially DM1 pathogenic.\n3. On page 6 authors comment that « Previous studies suggested that the expression of CUG480 in the postsynaptic BWMs\nalone was sufficient to produce locomotor defects in Drosophila larvae ». They refer to Picchio et al., 2013.\nThis is not correct. In this paper larval locomotion was analysed using inducible UAS-240, UAS-600 and UAS-960 CTG\nrepeat lines but not UAS-480. Also in Picchio et al., repeats expression was driven by Mef-GAL4 showing much earlier\nmuscle expression than C57-GAL4 used by authors.\nGeneral comment :\nAuthors hypothesize that FasII could be dysregulated by CUG repeat transcripts,\nUnderlying question is whether this dysregulation is dependent on Mbnl1, major RNA-binding protein sequestered by toxic\nCUG repeats.\nMuscle targeted knockdown of Muscleblind (Mbl), Drosophila ortolog of Mbnl1, mimics partial loss of Mbnl1 and could be\nused to test whether NMJ phenotypes and FasII deregulation is Mbl-dependent.\nSpecific comments:\nFig. 1\n- Fig.1a - In the presented view the reduction of button number in CUG480 versus CUG60 is not evident to see….A larger\nview showing NMJs of muscle 6 and 7 would help to appreciate the reduction in button numbers and the retraction of arbors.\n- It is surprising that an increase in arbor disassembly observed in C380-driven presynaptic overexpression of CUG480 does\nnot reduce the button number.\n- Criteria for quantification of arbors disassembly are not clear. Segmentation and image analyses tools allowing to mesure\narbor’s surface/extensions need to be applied to assess NMJ arborisations in different genetic contexts.\n- Fig. 1d – Dlg signal is saturated. A larger view of NMJs is required.\n- It would be appropriate to present expression pattern of the two drivers (C57 and C380) used in this study (in a\nsupplementary data).\nFig. 2\nConsidering that several FasII isoforms are produced and differentially dysregulated in DM1 context, western blot would\nallow to visualize isoform specific dysregulation. Using slot blot and not western blot when assessing FasII protein\nexpression in Drosophila is surprising.\nFasII ortholog NCAM1 in DM1 mouse models and in DM1 patients is also upregulated. Is NCAM1 presenting several\nisoforms that are differentially dysregulated in DM1?\nFig. 3\nThis figure would benefit from a scheme of FasII gene with different FasII isoforms. According to Flybase, FasII encodes 7\ndifferent isoforms. Why authors focus on FasII A and FasII C only ? If alternative splicing is in play, how FasII splice variants\nare expressed in DM1 contexts.\nFig.4 Simultanous overexpression of FasII C in MNs and BWMs leads to arbors disassembly.\nDid authors check effects of FasII C overexpression in MNs only ?\nFig.6 – Link between buttons number and locomotor capacities is unclear. Why increase in buttons number in FasII A-PEST-\nrescue context does not result in mobility improvement ?", "reviewer_3": "This is a very nice paper with interesting phenotypes and genetics. In this study, the authors reveal the function of fasII in\ndisassembly and impairment of locomotor activity in the Drosophila DM1 model. While the manuscript is well-written and\nlikely to contribute to the field, it could be further improved with additional data and analyses, which are crucial for\nstrengthening the conclusions.\n1) The connection to DM1 is tenuous. NCAM1 changes in DM1 seem variable depending on what tissue or dataset they are\nlooking at. Also, there is no clarity to what these changes mean. Similar things can be said about the data from DMSXL\nmice. The data doesn’t say anything about where the NCAM is being expressed (what cells, etc) and so has no relevance in\nthe context of this manuscript.\n2) There is no data on the expression level of the CUG transgene in the various conditions. This is essential to ensure that\nthis element of the experiment is controlled and assessed.\n3) Do the CUG repeats get expressed in the NMJ and which cells if any have RNA foci? For example, in Figure 1, although\nit has been shown previously that CUG480 RNA is expressed in this model, fluorescence in situ hybridization (FISH)\nanalysis will help show that CUG480 RNA is also expressed in the pre-synaptic MNs and post-synaptic BWMs. Does Mbl\nget sequestered in those cells? Is Mbl expressed in the pre-synaptic and post-synaptic cells? Does the RNA get expressed\nthere?\n4) Does an Mbl knockout recapitulate the CUG480 phenotype? Does it or does it not? Answer would be interesting either\nway.\n5) There is no description of how the quantification of FasII RNA levels were carried out. Was it slot blot or quantitative real-\ntime RT-PCR. The first method is inadequate. This study's findings depend on the upregulation of fasII (cell adhesion\nmolecule) in different models. If done quantitatively, the upregulation of of fasII data could be more convincing (Figure 2).\nAuthors need to verify the upregulation of fasII by different methodologies, too.\n6) There is an implication in this study that there is an imbalance in the isoforms of FasII. No clear studies were done to\nassess the relative levels of the isoforms at both the levels of the RNA and protein. This would be essential.\n7) FasII protein expression at the NMJ/boutons should have been done using isoform specific and total FasII antibodies, as\ndescribed in reference 39. For example, Figure 3 shows the rescues of NMJs in the Drosophila DM1 model, but no data\nshows how much knockdown was achieved.\n8) Similarly, western blots should have been done to assess the relative protein levels of the various isoforms.\n9) The studies done by Beck et al (ref 39), identifying beag and dsmu1 mutants and the essential role of these proteins in\nsplicing of FasII, are highly relevant to this current study. They aren’t even mentioned, other than a passing reference to the\nfact that the flies used in that paper were used in this study. The phenotypes mentioned in that paper are highly reminiscent\nof the results from the current study. This includes the importance of the FasII-A-PEST+ isoform for regulation of NMJ growth\nand bouton number. This can’t be ignored. The authors are trying to make a connection to splicing defects in DM1, but\nignoring the splicing factors that have already been reported to be relevant in DM1 and in FasII splicing.\n10) Are there antibodies for Beag? If so, what are the protein levels of Mb1, Elav (the fly homolog of ELAV proteins in\nmammals), Beag and dsmu1 in the current experiments? Does Beag co-localize with the RNA foci and Mbl? Does FLAG-\ntagged Beag (see Ref 39) co localize with the RNA foci or Mbl? What are the expression levels of Beag and dsmu1 in the\nCUG480 model, at the RNA and protein level? The mammalian homolog of Beag is called RED (if I am correct) and there\nare antibodies for that.\nThere are a couple of grammatical errors in the abstract section and elsewhere.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports as part of the Nature\nCommunications initiative to facilitate training in peer review and appropriate recognition for co-reviewers."}, "author_response": "POINT-BY-POINT RESPONSE TO REVIEWER COMMENTS\nDear Reviewers,\nFor your convenience, we have included low resolution versions of the main figures (and the\nfigure legends) in the manuscript where the figures are being cited. But for the high resolution\nversions of the figures, please kindly refer to the attached figures at the end of the manuscript.\nThank you for your attention.\nReviewer #1 (Remarks to the Author):\nWhile the link between the Drosophila DM1 model and FasII might per se interesting, this study\nhas notable conceptual and methodological shortcomings. The argument for specific distinct\nmolecular mechanisms connecting DM1 pathology to FasII isoforms lacks convincing evidence.\nThe authors appear to over-interpret their data, particularly regarding the claimed\n“outcompetition of FasII-C by FasII-A,” which does not seem convincingly supported by their\nfindings.\nResponse: We thank the reviewer for this pointing out, and we apologize for over-interpreting\nthis data. Since we don’t have additional support for this hypothesis, we have decided to remove\nthis interpretation from the Results section but to introduce this possible hypothesis in the\nDiscussion section.\nAdditionally, it is surprising that the authors opted to test only FasII-C overexpression in\nelectrophysiological experiments, leaving the DM1 model unexamined in this context. This\nchoice significantly limits the study’s comprehensiveness and the robustness of its conclusions.\nTo strengthen their findings, the authors should include a broader range of electrophysiological\ntests in the DM1 model and provide clearer evidence to support their claims about the molecular\nmechanisms involving FasII isoforms.\nResponse: We completely agree with the reviewer's viewpoint. We have conducted the\nelectrophysiological test on the DM1 model. For the detailed results of our electrophysiological\ntests, please refer to our response for Further Major Concerns #1 below.\nFurther major concerns:\n1. The authors chose to utilize the more accessible larval neuromuscular junction (NMJ) to\ninvestigate the neuropathology of DM1. However, they did not incorporate any functional\nreadouts, such as electrophysiology, to assess potential changes in neurotransmission associated\nwith DM1. Instead, they focused solely on the electrophysiological phenotypes of FasII-C\noverexpression (OE), which cannot be considered a valid proxy for DM1. This approach\nsignificantly limits the study’s ability to provide comprehensive insights into DM1\nneuropathology and its impact on neurotransmission.\nResponse: We totally agree with the reviewer’s comments. Therefore, we performed the\nrequested electrophysiological analysis for the revision (Fig. 2), and it has helped us to refine\nour model in several aspects. We are grateful for the experimental suggestion.\nOur initial analysis showed that the DM1 model had both locomotion defects and blunted\nsynaptic development. Considering just DM1, the prediction from those results would be that\nneurotransmission would be diminished too. However, overexpression of CUG caused some\n480\nfunctional phenotypes that are different from that of FasII-C overexpression. Pre+postsynaptic\nexpression of CUG resulted in increased excitatory postsynaptic potential (EPSP) amplitude\n480\nand quantal content (QC) (Fig. 2), whereas pre+postsynaptic expression of UAS-FasII-C\n1\nresulted in decreased miniature EPSP (mEPSP) amplitude, EPSP amplitude, QC and mEPSP\nfrequency (Fig. 6). In our Discussion section, we hypothesized that the phenotypes observed in\nCUG -expressing animals could be due to the upregulation of other FasII isoforms, such as\nThis idea makes sense considering some prior work. Specifically, our co-authors (C. Andrew\nFrank) previously published a study examining overexpression of various FasII isoforms and\ntheir effects on synaptic plasticity (Spring et al., 2016). The results showed that when either of\nthe two FasII-A isoforms was overexpressed pre- and postsynaptically, QC was increased.\nFurthermore, when Total-FasII was overexpressed, QC was also increased. These results\nindicate that the effects observed in CUG -expressing animals may represent a combinatorial\n480\neffect of multiple upregulated FasII isoforms.\nWe note that the increased QC in the DM1 model does not necessarily indicate increased muscle\nactivity. Rather, the increase in QC represents more vesicles being released per evoked response,\nwhich could be a compensatory mechanism to counter the functional impairment of the CUG -\n480\nexpressing animals’ reduced locomotor activities. This idea matches prior evidence supporting\nthe existence of robust homeostatic regulatory systems at the NMJ.\n2. 2. The authors did not investigate potential signaling pathways known to mediate NMJ bouton\nstructure, such as BMP and Wnt signaling. There are numerous established tools available in the\nfield for studying these pathways, and the authors should utilize these to elucidate the\nmechanisms regulating NMJ structural phenotypes in DM1. Currently, the connection between\nDM1 and FasII does not sufficiently explain the observed NMJ structural phenotypes. By\nexploring these signaling pathways, the authors could provide a more comprehensive\nunderstanding of the molecular mechanisms underlying the structural abnormalities seen in\nDM1.\nResponse: We thank the reviewer for these insightful comments. In this revision, we have\nexamined the impact of BMP signaling on the Drosophila DM1 model NMJ (Supplementary\nFig. 13). In the Drosophila BMP signaling pathway, Glass Bottom Boat (Gbb) is a retrograde\nsignaling ligand molecule that is necessary for normal synaptic growth. At the presynaptic\nmotorneuron terminal, a receptor complex formed by Wishful Thinking (Wit) and Thickveins\n(Tkv) is known to be activated by Gbb to regulate this retrograde signal (Smith et al., 2012). In\nour experiment, we attempted to rescue the DM1 model by expressing UAS-Gbb-GFP or UAS-\nTkv.CA. Tkv.CA is a constitutively active form of the Tkv receptor. Expression of neither of\nthese constructs could rescue the decreased boutons in the DM1 background (Supplementary\nFig. 13a and 13b). It is possible that Wnt is involved rather than Gbb. It is also possible that\nspecific levels of Gbb signal are required for synapse integrity.\nWe appreciate the reviewer's suggestion on investigating the BMP and Wnt pathways. Since\nthese are pathways that involve complex molecular mechanisms, it will take a lot more\nexperiments to decipher which and how they participate in the DM1 model. Although we are\nunable to demonstrate a rescue involving the downstream retrograde signaling at the moment,\nwe intend to further investigate these pathways in our next study. In the current study, we would\nlike to focus on the synergistic effect of the adhesion molecule FasII in the model.\n3. The authors proposed the hypothesis of “FasII isoform competition to bind to isoform A”\nbased on the distinct phenotypes observed in NMJ boutons and larval locomotion after FasII\nisoform knockdown or overexpression. This hypothesis appears to be speculative and lacks\nrobust support from the provided data and existing literature. Additionally, the potential\n2\ndiffering strengths of the C380 and C57 Gal4 lines and fasII RNAi lines, as shown in Figure\nS1b, may further complicate the situation.\nResponse: We apologize for the lack of support for this speculation once again and thank the\nreviewer for pointing this out. As the reviewer suggested, the potential differing strengths of\nGal4 lines and FasII-RNAi lines may further complicate the situation. Thus, we decided to\nremove this interpretation from the results section but to introduce this as a possible hypothesis\nin the discussion section instead.\n4. Using the number of NMJ boutons as the primary and nearly exclusive indicator of synaptic\nabnormalities is superficial, also in the context of DM1. For instance, in Figure 4, although\nFasII-A-PEST+ overexpression does not affect NMJ bouton numbers, it does result in a gain-of-\nfunction in locomotion. This discrepancy suggests that the NMJ bouton count alone may not\nfully capture the synaptic changes in DM1.\nResponse: We thank the reviewer’s comments and agree with the reviewer's concerns. We have\nnow added electrophysiological analysis of CUG overexpression in motorneurons and/or\n480\nmuscles (Fig. 2), which further revealed the synaptic functional abnormalities of the model\n(Response to Further Major Concerns #1).\nApart from using NMJ boutons as the primary indicator of synaptic abnormalities, we have also\nused arbor disassembly to quantify synaptic abnormalities (Fig. 1d, 1e, 5c, 5d and 5h). For your\nconvenience, we have cropped up parts of these figures below for your perusal (see below). In\nsome cases, this phenotype appears to be more sensitive to expanded CUG RNA toxicity than\nbouton numbers, since expression of CUG in motorneurons alone is sufficient to induce a\n480\nphenotype (Fig. 1e and 5h).\nit is surprising that they did not perform FasII staining using isoform-specific antibodies like\n1D4 versus 34B3. Conducting NMJ immunostaining against FasII would provide critical spatial\ninformation regarding FasII distribution in DM1 and FasII overexpression models. Such\ninformation would be essential for understanding the precise role of FasII isoforms in NMJ\nstructural phenotypes and could significantly enhance the mechanistic insights derived from\nthe study.\nResponse: We completely agree with the reviewer’s comments and suggestions. There are two\navailable Drosophila FasII antibodies that have been previously characterized: 1D4 and 34B3.\n1D4 can only detect the two FasII-A isoforms (Hummel et al., 2000), while 34B3 can potentially\ndetect all FasII isoforms (Beck et al., 2012; Grenningloh et al., 1991) (Supplementary Fig. 8c).\nWe have conducted the NMJ immunostaining against FasII using 1D4 and 34B3 antibodies\nrespectively as suggested by the reviewer (Supplementary Fig. 9 and Fig. 10). Unfortunately,\nresults from 1D4 immunostaining showed no detectable changes in the spatial distribution of\nFasII-A in CUG -expressing animals (Supplementary Fig. 9). As for 34B3, no\n480\nimmunostaining was detected in any boutons of the NMJs at muscle 6 and 7 (Supplementary\nFig. 10a). However, we did observe very faint signals occasionally from the terminal boutons of\nthe NMJs at muscle 15 and 16 of segment A5 and A6 of CUG -expressing animals\n480\n(Supplementary Fig. 10b and 10c). Such signals were not observed in the control CUG -\n60\nexpressing animals, which indicated an upregulation of FasII in the disease model. This result\nwas consistent with our findings on fasII upregulation (Fig. 4a-d).\n1. In the abstract the authors state, “In this study, we characterized a novel model of DM1 neuropathology by expressing\nuntranslated expanded CUG repeats at the Drosophila larval neuromuscular junction.” This in my eyes is misleading, as the\nuse of CUG repeat expression to model DM1 in Drosophila was proposed over 15 years ago. Thus, the model itself is not\nthat novel. Am I right here? If yes the authors should clarify this point to avoid overstating the novelty of their approach and to\naccurately reflect the existing body of research.\nResponse: We apologize for the inappropriate wording here and thank the reviewer for pointing\nthis out. We have amended the sentence to, \"Building on an established Drosophila model of\nDM1, we studied the neuropathological features of the disease by expressing untranslated\nexpanded CUG repeats at the Drosophila larval neuromuscular junction.\"\n2. In Figure 2, was elav-GeneSwitch/C57-driven DM1 model also sufficient to cause NMJ\nbouton phenotypes like with C380/C57 ?\nResponse: We thank the reviewer for raising this question. We performed this experiment and\ndemonstrated that elavGS/C57-driven DM1 model is indeed sufficient to cause NMJ bouton\nphenotypes, like that of C380/C57. This data has now been included in Supplementary Fig. 2.\n3. Quantification is missing in Figure 3a,b.\nResponse: We apologize for this missing part and thank the reviewer for pointing this out. The\noriginal Fig. 3 has become Fig. 4 in the revised manuscript. We have now included the\nquantifications (Fig. 4b and 4d) for this figure.\n4. In Figure 3c,d, the NMJ bouton phenotypes and larval locomotion of fasII-A and A&C RNAi\nare shown only in DM1 background, the effects of fasII-A and A&C RNAi alone are missing\nResponse: We thank the reviewer for this pointing out. We apologize for the missing part and\nhave included the NMJ bouton phenotypes and larval locomotion analysis of overexpressing\nfasII-A-RNAi and fasII-A&C-RNAi in the wild type background now (Supplementary Fig. 5).\n5. In Figure 4b, CUG-60 expression is not the correct control for FasII OE\n6. Similar in Figure 4d,e, CUG-60 expression is not the correct control for FasII-C OE\nResponse: We apologize for not having the correct controls and thank the reviewer very much\nfor pointing this out. We have now added the controls (depicted as white bars on the graphs) into\nthe revised Fig. 5b, 5d and 5e (the original Fig. 4 has become Fig. 5 in the revised manuscript).\n7. In Figure 5, the authors differentiated pre- and post- OE of FasII-C in electrophysiology.\nHowever, the authors did do the same in Figure 4.\nResponse: We thank the reviewer for his/her comments. We have now performed NMJ bouton\nphenotypical and larval locomotor analysis on pre- and post-overexpression of FasII-C animals.\nThe data is now shown in the revised Fig. 5f, 5g and 5h. Our results showed that overexpression\nof FasII-C using a single driver (either C380 or C57 alone) was not sufficient to cause\nsignificant bouton changes at the NMJ (Fig. 5g). However, C380 alone was sufficient to cause\narbor disassembly (Fig. 5h), and either C380 or C57 alone was sufficient to cause locomotor\ndefects (Fig. 5i). These results very closely resemble overexpression of CUG (Fig. 1).\n5\n8. In Figure 6, did the authors also performed fasII-C OE in DM1? Were there additive effects in\nNMJ bouton number and locomotion, and electrophysiology\nResponse: We thank the reviewer for raising this concern. We have now performed NMJ bouton\nphenotypical and larval locomotor analysis to investigate the effect of FasII-C overexpression in\nDM1 background. Our results showed that the combined effect of FasII-C and CUG\n480\noverexpression resulted in significant decreases of number of boutons and locomotor activity in\ncomparison to CUG overexpression alone (Fig. 7a-c) (the original Fig. 6 has become Fig. 7 in\n480\nour revised manuscript). This suggests that the upregulation of FasII-C in the DM1 model may\nnot be at saturation point yet, and that further increase of FasII-C results in more severe\nphenotypes.\nAfter performing electrophysiological analysis of CUG overexpression (Fig. 2), we showed\n480\nthat the transmission phenotypes of these animals are in fact opposite to FasII-C overexpression.\nThus, it would not be possible to have an observable additive effect using this method of\nanalysis.\n9. Except for DM1 model flies, genetic controls for all the UAS lines including RNAi lines and\ncDNA OE line are missing. Given that genetic background affects NMJ bouton phenotypes, it\nwill be important to include those UAS controls.\nResponse: We agree with the reviewer and thank him/her for this comment. We have now\nincluded the bouton phenotype analysis of these genetic controls in Supplementary Fig. 21. No\nsignificant differences were observed among the analyzed control genotypes.\n10. It is also a bit surprising that, given the per se limited electrophysiological data of Figure 5,\nthe authors did not show any sample traces for minis and evoked.\nResponse: We thank the reviewer for pointing this out and apologize for the missing part. We\nhave now added the sample traces into the revised Fig. 6 (Fig. 6m, 6n and 6o) (the original Fig.\n5 has become Fig. 6 in the revised manuscript). Representative traces have also been included in\nthe new Fig. 2 (electrophysiological result of CUG -expressing DM1 model). In summary,\n480\noverexpression of FasII-C results in decreased transmission, while overexpression of CUG\n480\n(which causes upregulation of FasII-A-PEST+, FasII-A-PEST- and FasII-C) results in increased\ntransmission.\nReviewer #2 (Remarks to the Author):\nInconsistencies :\n1. Authors refer in the title and in the text to neuropathological and behavioral defects in DM1\nbut they analyse phenotypes of neuromuscular junctions and larval mobility. Whether\ndysregulation of FasII influences fly behavior and neurological CNS functions is not shown.\nResponse: We thank the reviewer for raising concern about possible inconsistencies. In response\nto this concern, we have amended our paper’s title to “Pre- and postsynaptic upregulation of\nFasII synergistically underlies neuropathological and behavioral phenotypes in a Drosophila\nmodel of myotonic dystrophy”. In the text of the manuscript, we have also substituted the word\n“defects” with “phenotypes”.\nwe have shown that the knockdown of FasII in the DM1 model fully rescued both NMJ and\nbehavioral phenotypes (Fig. 4e-i). Also, the overexpression of FasII-C causes NMJ phenotypes\nand behavior impairment that resemble the DM1 model (Fig. 5a-i). Furthermore, the\noverexpression of FasII-C directly influences synaptic transmission from motorneurons\n(motorneurons are part of the CNS, so motorneuron functions are CNS functions) (Fig. 6a-o).\nLast but not least, the NMJ and behavior phenotypes of the DM1 model could be fully rescued\nby overexpressing FasII-A-PEST+ (Fig. 7a-g). All these experiments strongly suggest that the\ndysregulation of FasII influences Drosophila behavior and CNS functions.\n2. On page 4 authors say that they overexpressed control UAS-CUG60 construct (which should\nrather be UAS-CTG60) with the number of CTG repeats that is lower than the disease treshold.\nThis is not correct as the number of CTG repeats in dmpk gene higher than 37 is already\nclassified as potentially DM1 pathogenic.\nResponse: We thank the reviewer for his/her comments. While it is true that the constructs we\nused were UAS-CTG and UAS-CTG , we would like to emphasize to the readers that CUG\n60 480 60\nand CUG were the untranslated transcriptional products (RNA) in our experiments that drove\n480\nour results. Thus, we used “CUG ” or “CUG ” in most parts of the manuscript. We are glad\n60 480\nthat the reviewer reminded us to clarify this difference in naming more clearly.\nAs for the disease threshold of DM1, even though 37 is already classified as “potential for\nDM1”, we were unable to detect any phenotype in our experiments. As shown in Fig. 5b and\n5e, CUG -expressing animals showed no phenotypes in comparison to driver controls regarding\n60\nboth NMJ morphology and locomotor behavior. The UAS-CTG line was previously\n60\ncharacterized and published (Garcia-Lopez et al., 2008), and we have not found any\nabnormalities in CUG -expression animals in our study.\n60\n3. On page 6 authors comment that « Previous studies suggested that the expression of CUG480\nin the postsynaptic BWMs alone was sufficient to produce locomotor defects in Drosophila\nlarvae ». They refer to Picchio et al., 2013.\nThis is not correct. In this paper larval locomotion was analysed using inducible UAS-240,\nUAS-600 and UAS-960 CTG repeat lines but not UAS-480. Also in Picchio et al., repeats\nexpression was driven by Mef-GAL4 showing much earlier muscle expression than C57-GAL4\nused by authors.\nResponse: We apologize for this error and thank the reviewer so much for pointing this out. We\nhave now amended the sentence to, \"A previous study demonstrated that the expression of\nexpanded CUG repeats in the postsynaptic BWMs alone was sufficient to produce locomotor\ndefects in Drosophila larvae (Picchio et al., 2013).\"\nIn addition, we cited this landmark paper again in the Introduction section to highlight its\nsignificant contribution to the development of DM1 models in Drosophila.\nGeneral comment :\nAuthors hypothesize that FasII could be dysregulated by CUG repeat transcripts,\nUnderlying question is whether this dysregulation is dependent on Mbnl1, major RNA-binding\nprotein sequestered by toxic CUG repeats.\nMuscle targeted knockdown of Muscleblind (Mbl), Drosophila ortolog of Mbnl1, mimics partial\nloss of Mbnl1 and could be used to test whether NMJ phenotypes and FasII deregulation is Mbl-\ndependent.\nunderlying question regarding the involvement of Mbl in the dysregulation of FasII. Therefore,\nwe conducted the analysis on Mbl in our fly model based on the advice of the reviewer.\nIn Drosophila, we analyzed mbl E27 mutants to determine whether they recapitulated the CUG\n480\nphenotypes. mbl E27 is a homozygous lethal null mutation (Garcia-Lopez et al., 2008). Therefore,\nheterozygous mbl E27 mutants were analyzed in the investigation. The heterozygous mbl E27\nmutants have a bouton reduction phenotype and an arbor disassembly phenotype that indeed\nrecapitulated CUG (Supplementary Fig. 14a-c).\nof fasII. Our results showed that the heterozygous mbl E27 mutants exhibited upregulated fasII,\nsimilar to CUG overexpression (Supplementary Fig. 14d and 14e). These data suggested\n480\nthat the partial loss of Mbl functions in the heterozygous mbl E27 mutant might reflect the\nphysiological conditions of the DM1 model. Furthermore, these data also supported Mbl's role\nin fasII processing and NMJ morphology in the DM1 neuropathological context.\nSpecific comments:\nFig. 1\n- Fig.1a - In the presented view the reduction of button number in CUG480 versus CUG60 is not\nevident to see….A larger view showing NMJs of muscle 6 and 7 would help to appreciate the\nreduction in button numbers and the retraction of arbors.\nResponse: We thank the reviewer for the comment. We would like to explain that an image of\nthe entire NMJ (lower magnification) would help the reader to appreciate the reduction in\nbouton numbers, whereas a magnified (larger) view would help the reader to appreciate the\nretraction of arbors. Fig. 1a aimed to present the entire NMJ of muscle 6 and 7, so that the\nreader can more easily observe the reduction of total bouton numbers. On the other hand, a\nmagnified (larger) view of the boutons is presented in Fig. 1d to allow the reader to more clearly\nsee the retraction of arbors.\n- It is surprising that an increase in arbor disassembly observed in C380-driven presynaptic\noverexpression of CUG480 does not reduce the button number.\nResponse: We thank the reviewer for his/her comment. This may be due to the methods of\nquantification. When quantifying the number of NMJs with disassembling arbors, an NMJ with\neven just one disassembling arbor is regarded as an NMJ with disassembly. However, since each\nNMJ has many arbors, it is possible that in some cases, one disassembling arbor is not sufficient\nto impact on overall bouton numbers.\n- Criteria for quantification of arbors disassembly are not clear. Segmentation and image\nanalyses tools allowing to mesure arbor’s surface/extensions need to be applied to assess NMJ\narborisations in different genetic contexts.\nResponse: We apologize for oversighting this and thank the reviewer for pointing this out. We\nhave now added the details of our quantification method in the Methods section. In brief, a\ndisassembling arbor was defined as a stretch of at least two boutons that are severed from the\narbor and from one another (immunostained by anti-HRP), in which at least one of these\nboutons showed DLG staining on the postsynaptic muscle. would be regarded as a\n\"disassembling arbor\", while any NMJ with at least one disassembling arbor would be counted\nas an \"NMJ with disassembling arbors\".\nanalyses tools, such as IMARIS or AIVIA. However, these tools are unable to definitively\nrecognize disassembling arbors according to our criteria. Thus, we performed our quantification\nmanually.\n- Fig. 1d – Dlg signal is saturated. A larger view of NMJs is required.\nResponse: We thank the reviewer for his/her comment. It is known that the Dlg signal of type I\nsmall boutons (type Is) is much fainter than that of type I big boutons (type Ib) (Koh et al.,\n2000). In Fig. 1d, we had to saturate the signals on type Ib boutons to visualize the dim Dlg\nsignals of type Is of both genotypes. In particular, the remaining Dlg signals from type Is in\nC380+C57>CUG animals are what we would like to show the readers. Therefore, we cannot\n480\nreduce this saturation on the image. The confocal images shown in Fig. 1d are already\nmagnified images of the NMJs. Whole NMJ view of the same genotypes are shown in Fig. 1a.\n- It would be appropriate to present expression pattern of the two drivers (C57 and C380) used in\nthis study (in a supplementary data).\nResponse: We thank the reviewer’s comment. We have now included confocal micrographs to\nshow the expression pattern of C380 and C57 (Supplementary Fig. 20). Further details\nregarding the expression pattern of C380-Gal4 can be found in Sanyal in 2009 (Sanyal, 2009). In\nshort, in C380-Gal4, Gal4 is expressed in most larval motor neurons, dorsal medial cluster and\nlarval brain lobes. Similar to C380, the expression pattern of C57 has also been presented in\nseveral previous studies (Budnik et al., 1996; Gorczyca et al., 2007; Zhao & Geisbrecht, 2025).\nIn brief, in C57-Gal4, Gal4 is expressed in all larval muscles, from mid-first to third instar stage.\nFig. 2\nConsidering that several FasII isoforms are produced and differentially dysregulated in DM1\ncontext, western blot would allow to visualize isoform specific dysregulation. Using slot blot\nand not western blot when assessing FasII protein expression in Drosophila is surprising.\nResponse: We thank the reviewer for raising this concern. There are only two available FasII\nantibodies: 1D4 and 34B3. 1D4 only detects the two FasII-A isoforms (Hummel et al., 2000),\nwhile 34B3 is predicted to detect all FasII isoforms (Beck et al., 2012; Grenningloh et al., 1991)\n(Supplementary Fig. 8c). Since 1D4 only detects FasII-A, only 34B3 has the potential to detect\nand differentiate different FasII isoforms in WB. The possible band sizes for the different FasII\nisoforms are: FasII-A-PEST+ (96 kDa), FasII-A-PEST- (93 kDa), FasII-C (90 kDa), FasII-B (86\nkDa). However, we found that 34B3 could only allow us to effectively visualize a single band in\nDrosophila CNS and muscles even when we over-exposed the blots (Supplementary Fig. 11).\nThis band likely represented FasII-A-PEST+, which is the major isoform known to be expressed\nin the CNS (Wright & Copenhaver, 2001). Even though 34B3 might have recognized other\nisoforms, there were just insufficient signals for other isoforms to be visualized via WB.\nTherefore, we resorted to the slot blot approach to detect all isoforms together (Fig. 3e and 3f),\nwhile relying on RT-PCR instead to analyze individual fasII isoforms at the transcript level.\nFasII ortholog NCAM1 in DM1 mouse models and in DM1 patients is also upregulated. Is\nNCAM1 presenting several isoforms that are differentially dysregulated in DM1?\nResponse: We thank the reviewer for raising this question. To determine whether NCAM1\nisoforms are dysregulated in DM1, we performed semi-quantitative RT-PCR analysis of\nalternative splicing using cultured astrocytes and frontal cortex tissues of DMSXL mice. Our\nresults showed that there was indeed splicing dysregulation of Ncam1 transcripts in cultured\nastrocytes and in the frontal cortex of DMSXL mice (Supplementary Fig. 17).\nFig. 3\nThis figure would benefit from a scheme of FasII gene with different FasII isoforms. According\nto Flybase, FasII encodes 7 different isoforms. Why authors focus on FasII A and FasII C only ?\nIf alternative splicing is in play, how FasII splice variants are expressed in DM1 contexts.\nResponse: We thank the reviewer for raising this concern. Although there are 7 different\nisoforms in Flybase (Fas2-RA, Fas2-RB, Fas2-RC, Fas2-RD, Fas2-RF, Fas2-RG and Fas2-RH),\nmost are only predicted, and have not been shown to be expressed in neuronal or muscular\ntissues of the larva. We did not dismiss the possibility that there could be other isoforms\nexpressed at the NMJ. But instead of investigating all potential isoforms, it would be more\nreasonable to initially focus on FasII-A-PEST+, FasII-A-PEST- and FasII-C, as they have been\ncharacterized and tools are available (Wright & Copenhaver, 2001). We have now included a\nschematic diagram of these FasII isoforms (Supplementary Fig. 8). Since we were able to\nrescue the DM1 phenotypes by either knocking down Total-FasII or FasII-A&C, but not by\nknocking down FasII-A, it was more logical to further pursue studying FasII-C rather than\ninvestigating other uncharacterized isoforms.\nAs a side note, out of the 7 predicted isoforms in Flybase (RA, RB, RC, RD, RF, RG and RH),\nFas2-RA, Fas2-RB and Fas2-RC are NOT alternatively names for FasII-A, FasII-B and FasII-C.\nThe actual translations are as follows:\nFasII-A-PEST+ = Fas2-RA\nFasII-A-PEST- = Fas2-RC\nFasII-C = Fas2-RB\nWe have now clarified this in Supplementary Fig. 8.\nIn contrast to FasII-A-PEST+ and FasII-A-PEST- which are known to be expressed at the\nDrosophila nervous system and neuromuscular junction to mediate intercellular interactions\n(Wright & Copenhaver, 2001), FasII-C was proposed to act as homotypic bridging\nproteins within the same renal Malpighian tubule cell to stabilize the microvillar brush border of\nagainst shear stress (Halberg et al., 2016).\nFig.4 Simultanous overexpression of FasII C in MNs and BWMs leads to arbors disassembly.\nDid authors check effects of FasII C overexpression in MNs only ?\nResponse: We thank the reviewer for his/her insightful comment. We have now performed\nthe arbor disassembly analysis on animals expressing FasII-C in either motorneurons or body\nwall muscles and included it in our revised Fig. 5. Like expressing CUG , FasII-C\n480\noverexpression in motorneurons only (C380) resulted in a small but significant increase of arbor\ndisassembly, while FasII-C overexpression in body wall muscles only (C57) did not have any\nimpact (Fig. 5h) (the original Fig. 4 has become Fig. 5 in the revised manuscript). For your\nconvenience, we have cropped the corresponding part of the figure below for your perusal (see\nbelow).\nbuttons number in FasII A-PEST- rescue context does not result in mobility improvement ?\nResponse: We thank the reviewer for raising this question. Usually, the number of boutons\ncorrelate well with locomotor activity of an animal. However, it also depends on whether\nthe morphology of the boutons is normal. We analyzed the boutons' morphology of the FasII-A-\nPEST- rescue animals and found abnormalities (Fig. 7h and 7i) (the original Fig. 6 has become\nFig. 7 in the revised manuscript). Normal boutons are supposed to have the well-known \"beads-\non-a-string\" morphology, which can be seen in the FasII-A-PEST+ rescue animals. However,\nmany boutons of the FasII-A-PEST- rescue animals do not have such round shape. We speculate\nthat these abnormalities might have contributed to abnormal functions in the FasII-A-PEST-\nanimals.\nReviewer #3 (Remarks to the Author):\nThis is a very nice paper with interesting phenotypes and genetics. In this study, the authors\nreveal the function of fasII in disassembly and impairment of locomotor activity in the\nDrosophila DM1 model. While the manuscript is well-written and likely to contribute to the\nfield, it could be further improved with additional data and analyses, which are crucial for\nstrengthening the conclusions.\nResponse: We would like to thank the reviewer for the compliment. We appreciate the time and\neffort of the reviewer to help us improve the manuscript.\n1a) The connection to DM1 is tenuous. NCAM1 changes in DM1 seem variable depending on\nwhat tissue or dataset they are looking at.\nResponse: We thank the reviewer for raising this concern. While NCAM1 could be\ndownregulated in certain tissues of DM1 patients (Supplementary Fig. 12), it was upregulated\nin most of the mammalian tissues that we have examined (Fig. 3i-n). Furthermore, regardless of\nwhether NCAM1 is upregulated or downregulated, as long as NCAM1 is dysregulated, it is\n11\nlikely to impair synapse functions and integrity, which may reflect the synaptopathology in\nDM1.\n1b) Also, there is no clarity to what these changes mean.\nResponse: We thank the reviewer for this comment. Since we found that NCAM1 is consistently\ndysregulated in DMSXL mice and DM1 patients (across different types of CNS and muscular\ntissues), and this dysregulation could impair neuronal and muscular functions, it is likely that the\nNCAM1 changes we observed will provide insights into the neural pathogenesis of DM1.\nOur findings also bridge the well-known “upstream” pathology of ribonuclear foci sequestration\nof splicing factors over to the largely unexplored “downstream” pathology of synapse\ndysfunction in the disease.\nIn our study, we have proposed a working model of synaptopathology in the NMJ model of\nDM1 (Fig. 8), which explained how the changes in FasII/NCAM1 contribute to the loss of\nsynapses.\n1c) Similar things can be said about the data from DMSXL mice. The data doesn’t say anything\nabout where the NCAM is being expressed (what cells, etc) and so has no relevance in the\ncontext of this manuscript.\nResponse: We thank the reviewer for raising this question. Regarding cell types that express\nNCAM1, in our additional experiments performed in this revision, we were able to demonstrate\nNcam1 splicing dysregulation in cultured mouse astrocytes of DMSXL mice (Supplementary\nFig. 17a-c and 18a-b), implying that NCAM1 is at least expressed in this cell type. In general,\nNCAM1 is known to be expressed in most neuronal cell types, thus we mostly used mouse\ntissues such as the spinal cord or the frontal cortex to examine NCAM1 changes (Fig. 3i, 3k,\n3m, Supplementary Fig. 17 and 18). These results demonstrated the relevance of NCAM1\ndysregulation in the context of neuropathology of DM1.\n2) There is no data on the expression level of the CUG transgene in the various conditions. This\nis essential to ensure that this element of the experiment is controlled and assessed.\nResponse: We thank the reviewer for raising this concern. We conducted RT-PCR to investigate\nthe CUG transgene expression level in the CNS and muscle tissues of Drosophila 3rd instar\nlarvae respectively (Supplementary Fig. 19). Since forward primers containing CTG repeats\nand reverse primers containing CAG repeats would freely bind to anywhere along the region of\nCTG repeats, resulting in variable band sizes, we used primers against the heat shock promoter\n(hs) and primers against the simian vacuolating virus 40 (SV40) instead. These sequences are\npresent in the expressed transcripts of UAS-CTG and UAS-CTG constructs. The hs primers\n60 480\nwere used to detect the CUG transgene expression in the CNS tissues because hs was absent in\nelavGS-GAL4. Meanwhile the SV40 primers were used to detect the CUG transgene expression in\nthe muscle tissues, because SV40 was absent in C57-GAL4. The forward and reverse primer\nsequences of the hs and SV40 were described in the Methods section. Both CUG and CUG\n60 480\nwere well-expressed in the CNS and muscle respectively (Supplementary Fig. 19). Further\ncharacterizations of the UAS-CTG and UAS-CTG fly lines could be found in Garcia-Lopez\n60 480\net al., 2008 and Luu et al., 2016 (Garcia-Lopez et al., 2008; Luu et al., 2016).\n3) Do the CUG repeats get expressed in the NMJ and which cells if any have RNA foci? For\nexample, in Figure 1, although it has been shown previously that CUG480 RNA is expressed in\nthis model, fluorescence in situ hybridization (FISH) analysis will help show that CUG480 RNA\n12\nis also expressed in the pre-synaptic MNs and post-synaptic BWMs. Does Mbl get sequestered\nin those cells? Is Mbl expressed in the pre-synaptic and post-synaptic cells? Does the RNA get\nexpressed there?\nResponse: We thank the reviewer for his/her insightful comment. We have now performed\nFluorescence In Situ Hybridization (FISH) analysis on Drosophila larvae according to the\nreviewer’s suggestion. The result has shown that CUG RNA is present as foci in the nuclei of\n480\nboth pre-synaptic motorneurons and post-synaptic body wall muscles (Supplementary Fig. 1).\nA previous study demonstrated that exogenous MblC:GFP forms nuclear inclusions with the\nribonuclear foci in the muscle nuclei of the Drosophila DM1 model (Garcia-Lopez et al., 2008).\nAnother study also demonstrated that endogenous Mbl co-localizes with CUG-containing\nribonuclear foci (de Haro et al., 2006). Unfortunately, due to the limitation of the antibodies,\nwe were unable to perform immunostaining of Mbl in our samples. However, according to Li et al.,\n2022, Mbl is highly expressed in both motorneurons and body wall muscles (Li et al., 2022).\n4) Does an Mbl knockout recapitulate the CUG480 phenotype? Does it or does it not? Answer\nwould be interesting either way.\nResponse: We thank the reviewer for raising this insightful question. We agree with the answer\nwould be interesting in either way. Hence, we conducted the analysis on Mbl using our fly\nmodel.\nIn Drosophila, we analyzed mbl E27 mutant to determine whether they recapitulated the CUG\n480\nphenotypes. mbl E27 is a homozygous lethal null mutation (Garcia-Lopez et al., 2008). Therefore,\nheterozygous mbl E27 mutants were analyzed in the investigation. The heterozygous mbl E27\nmutants have a bouton reduction phenotype and an arbor disassembly phenotype that indeed\nrecapitulated CUG (Supplementary Fig. 14a-c).\nof fasII. Our results showed that the heterozygous mbl E27 mutants exhibited upregulated fasII,\nsimilar to CUG overexpression (Supplementary Fig. 14d and 14e). These data suggested\n480\nthat the partial loss of Mbl functions in the heterozygous mbl E27 mutant might reflect the\nphysiological conditions of the DM1 model. Furthermore, these data also supported Mbl's role\nin fasII processing and NMJ morphology in the DM1 neuropathological context.\n5) There is no description of how the quantification of FasII RNA levels were carried out. Was it\nslot blot or quantitative real-time RT-PCR. The first method is inadequate. This study's findings\ndepend on the upregulation of fasII (cell adhesion molecule) in different models. If done\nquantitatively, the upregulation of of fasII data could be more convincing (Figure 2). Authors\nneed to verify the upregulation of fasII by different methodologies, too.\nResponse: We apologize for this missing part and thank the reviewer for pointing this out. The\noriginal Fig. 3 has become Fig. 4 in the revised manuscript. We have now included the\nquantifications (Fig. 4b and 4d) for this figure, and the quantification method has now been\nincluded in the Methods section.\nFor RT-PCR, we chose not to perform quantitative PCR, but to perform semi-quantitative RT-\nPCR instead. The reason for this is that fasII-A-PEST- is only missing a very small PEST\ndomain compared to fasII-A-PEST+ (Supplementary Fig. 8a), so the primers for fasII-A-PEST-\nwill always pick up fasII-A-PEST+ as well (Beck et al., 2012). Since fasII-A-PEST+ and fasII-\nA-PEST- are always amplified in the same reaction, it is difficult to investigate using real-time\nisoforms.\nIn summary, we verified the upregulation of FasII in Drosophila with 2 methods: (1) by semi-\nquantitative RT-PCR (mRNA) (Fig. 3a-d and Fig. 4a-d), and (2) by slot-blot (protein) (Fig. 3e-\nh).\n6) There is an implication in this study that there is an imbalance in the isoforms of FasII. No\nclear studies were done to assess the relative levels of the isoforms at both the levels of the RNA\nand protein. This would be essential.\nResponse: We thank the reviewer for raising this concern. To access the relative levels of the\nfasII isoforms in the context of RNA, we have performed semi-quantitative RT-PCR in the\nDrosophila CNS and muscle tissues respectively. In Fig. 4a and 4b, the Total fasII and three\ntargeted fasII isoforms, fasII-A-PEST+, fasII-A-PEST- and fasII-C, are upregulated in the\nCUG -expressing Drosophila CNS. Consistent with the CNS, the Total fasII and all the three\n480\ntargeted fasII isoforms are upregulated in the Drosophila muscle as showed in Fig. 4c and 4d.\nThese results have shown that the fasII isoforms are dysregulated in the CUG -expressing fly\n480\nmodel at RNA level.\nWe tried to assess the FasII isoforms protein levels using Western blot (WB). There are only two\navailable FasII antibodies: 1D4 and 34B3. 1D4 only detects the two FasII-A isoforms (Hummel et al., 2000),\nwhile 34B3 is predicted to detect all FasII isoforms (Beck et al., 2012; Grenningloh et al., 1991)\n(Supplementary Fig. 8c). Since 1D4 only detects FasII-A, only 34B3 has the potential to detect\nand differentiate different FasII isoforms in WB. The possible band sizes for the different FasII\nisoforms are: FasII-A-PEST+ (96 kDa), FasII-A-PEST- (93 kDa), FasII-C (90 kDa), FasII-B (86\nkDa). However, we found that 34B3 could only allow us to effectively visualize a single band in\nDrosophila CNS and muscles even when we over-exposed the blots (Supplementary Fig. 11).\nThis band likely represented FasII-A-PEST+, which is the major isoform known to be expressed\nin the CNS (Wright & Copenhaver, 2001). Even though 34B3 might have recognized other\nisoforms, there were just insufficient signals for other isoforms to be visualized via WB.\nTherefore, we resorted to the slot blot approach to detect all isoforms together (Fig. 3e and 3f),\nwhile relying on RT-PCR instead to analyze individual fasII isoforms at the transcript level in\nDrosophila (Fig. 4a-d).\n7) FasII protein expression at the NMJ/boutons should have been done using isoform specific\nand total FasII antibodies, as described in reference 39. For example, Figure 3 shows the rescues\nof NMJs in the Drosophila DM1 model, but no data shows how much knockdown was achieved.\nResponse: We thank the reviewer for his/her insightful comment. Since we were unable to use\n34B3 to pick up different FasII isoforms in our WB, we opted for the alternative method of RT-\nPCR to demonstrate the level of knockdown achieved with different RNAi constructs. The\nlevels of FasII knockdown for each isoform by FasII-A&C-RNAi was shown in Supplementary\nFig. 3 (the line FasII-C-RNAi#38 was eventually renamed as FasII-A&C-RNAi).\nFor Total-FasII-RNAi and FasII-A-RNAi, the lines were characterized in reference 39 (Beck et\nal., 2012).\nOn the other hand, we performed additional NMJ immunostaining of FasII using 1D4 and 34B3\nantibodies respectively (Supplementary Fig. 9 and 10). The results from 1D4 (detecting FasII-\nA only) immunostaining showed no detectable changes of FasII-A in the NMJ of CUG -\n480\nexpressing animals (Supplementary Fig. 9). For 34B3, no immunostaining was detected in any\nboutons of the NMJs at muscle 6 and 7 (Supplementary Fig. 10a). However, we did observe\n14\nvery faint signals occasionally from the terminal boutons of the NMJs at muscle 15 and 16 in\nsegment A5 and A6 of CUG -expressing animals\n480\n(Supplementary Fig. 10b and 10c). Such signals were not observed in the control CUG -\n60\nexpressing animals, which indicated an upregulation of FasII in the disease model. This result\nwas consistent with our findings on FasII\nupregulation using RT-PCR and slot blot (Fig. 3a-h and Fig. 4a-d).\n8) Similarly, western blots should have been done to assess the relative protein levels of the\nvarious isoforms.\nResponse: We thank the reviewer for his/her comment. As mentioned previously (in Point #6),\nit was unfortunate that we were unable to assess the relative protein levels of the various FasII\nisoforms. Therefore, we resorted to the slot blot approach to detect all isoforms together, while\nrelying on RT-PCR instead to analyze individual fasII isoforms at the transcript level (Fig. 4a-\nd).\n9) The studies done by Beck et al (ref 39), identifying beag and dsmu1 mutants and the essential\nrole of these proteins in splicing of FasII, are highly relevant to this current study. They aren’t\neven mentioned, other than a passing reference to the fact that the flies used in that paper were\nused in this study. The phenotypes mentioned in that paper are highly reminiscent of the results\nfrom the current study. This includes the importance of the FasII-A-PEST+ isoform for\nregulation of NMJ growth and bouton number. This can’t be ignored. The authors are trying to\nmake a connection to splicing defects in DM1, but ignoring the splicing factors that have already\nbeen reported to be relevant in DM1 and in FasII splicing.\nResponse: We thank the reviewer very much for raising this important matter. Indeed, Beck et\nal. has shown that Beag and Dsmu1 are involved in the splicing of FasII-A, and they showed the\nimportance of FasII-A-PEST+ in regulating NMJ bouton numbers. We have now included this\nin our Discussion section to recognize their findings and compare them to our results.\n10a) Are there antibodies for Beag? If so, what are the protein levels of Mb1, Elav (the fly\nhomolog of ELAV proteins in mammals), Beag and dsmu1 in the current experiments?\nResponse: We are grateful to the reviewer for his/her insightful comments. We contacted our\ncoauthors of Beck et al. (Ref 39), but unfortunately their Beag antibody is no longer available.\nSimilarly, no antibodies are available for Dsmu1. As for Mbl, although we were able to acquire\nantibodies from our coauthor, the Artero Lab, in Spain, we were unable to produce satisfactory\nimmunostaining.\nAlthough we were unable to assess the protein levels, we assessed the transcriptional level by\nperforming semi-quantitative RT-PCR as an alternative to examine elav, mbl, beag and dsmu1 in\nthe CUG fly model. Our results showed that the expression of beag and dsmu1 were upregulated\n480\nin CUG -expressing animals, while the expression of elav and mbl had no significant changes\n480\n(Supplementary Fig. 15).\n10b) Does Beag co-localize with the RNA foci and Mbl? Does FLAG-tagged Beag (see Ref 39)\nco localize with the RNA foci or Mbl? What are the expression levels of Beag and dsmu1 in the\nCUG480 model, at the RNA and protein level? The mammalian homolog of Beag is called RED\n(if I am correct) and there are antibodies for that.\nResponse: We thank the reviewer for the suggestions on investigating colocalization of these\nsplicing factors with RNA foci. Although we were unable to acquire Beag and Dsmu1 antibodies\nto conduct this experiment in flies, nor could we acquire UAS-Beag-FLAG or UAS-Dsmu1-FLAG\n15\nfly lines from our coauthors of Beck et al., we were able to acquire RED and SMU1 antibodies as\nthe reviewer suggested. RED and SMU1 are the mammalian homolog of Beag and Dsmu1 in\nDrosophila. Thus, as an alternative, we performed immuno-FISH on primary astrocytes of\nDMSXL mice to examine if RED and SMU1 co-localized with CUG RNA foci. Our results\nshowed that RED and SMU1 do not co-localize with RNA foci (Supplementary Fig. 16).\nSummarizing the above results, although Beag and Dsmu1 are upregulated in the Drosophila DM1\nmodel, RED and SMU1 do not co-localize with RNA foci in the mouse DM1 model. It is possible\nthat the expanded CUG RNA caused an abnormal upregulation of these genes, which contributes\nto part of the pathogenesis. It is also possible that the upregulation of Beag and Dsmu1 may serve\nas a compensatory mechanism to alleviate the damaging effect of Mbl and FasII dysregulation.\nSince RED and SMU1 do not co-localize with RNA foci in the nuclei, the latter explanation seems\nmore likely.\nThere are a couple of grammatical errors in the abstract section and elsewhere.\nResponse: We thank the reviewer for pointing this out and apologies for the grammatical errors.\nThey have been fixed in this revised version.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports as part of\nthe Nature Communications initiative to facilitate training in peer review and appropriate\nrecognition for co-reviewers.\nResponse: We appreciate the effort of all reviewers in facilitating the review process of our paper.\nBeck, E. S., Gasque, G., Imlach, W. L., Jiao, W., Jiwon Choi, B., Wu, P. S., Kraushar, M. L., &\nMcCabe, B. D. (2012). 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PLoS One, 3(2), e1595. https://doi.org/10.1371/journal.pone.0001595\nGorczyca, D., Ashley, J., Speese, S., Gherbesi, N., Thomas, U., Gundelfinger, E., Gramates, L.\nS., & Budnik, V. (2007). Postsynaptic membrane addition depends on the Discs-Large-\ninteracting t-SNARE Gtaxin. J Neurosci, 27(5), 1033-1044.\nhttps://doi.org/10.1523/JNEUROSCI.3160-06.2007\nGrenningloh, G., Rehm, E. J., & Goodman, C. S. (1991). Genetic analysis of growth cone\nguidance in Drosophila: fasciclin II functions as a neuronal recognition molecule. Cell,\n67(1), 45-57. https://doi.org/10.1016/0092-8674(91)90571-f\nHalberg, K. A., Rainey, S. M., Veland, I. R., Neuert, H., Dornan, A. J., Klambt, C., Davies, S. A.,\n& Dow, J. A. (2016). The cell adhesion molecule Fasciclin2 regulates brush border length\nand organization in Drosophila renal tubules. Nat Commun, 7, 11266.\nhttps://doi.org/10.1038/ncomms11266\nHummel, T., Krukkert, K., Roos, J., Davis, G., & Klambt, C. (2000). Drosophila Futsch/22C10 is\na MAP1B-like protein required for dendritic and axonal development. Neuron, 26(2), 357-\n370. https://doi.org/10.1016/s0896-6273(00)81169-1\nKoh, Y. H., Gramates, L. S., & Budnik, V. (2000). Drosophila larval neuromuscular junction:\nmolecular components and mechanisms underlying synaptic plasticity. Microsc Res Tech,\n49(1), 14-25. https://doi.org/10.1002/(SICI)1097-0029(20000401)49:1<14::AID-\nJEMT3>3.0.CO;2-G\nLi, H., Janssens, J., De Waegeneer, M., Kolluru, S. S., Davie, K., Gardeux, V., Saelens, W., David,\nF. P. A., Brbic, M., Spanier, K., Leskovec, J., McLaughlin, C. N., Xie, Q., Jones, R. C.,\nBrueckner, K., Shim, J., Tattikota, S. G., Schnorrer, F., Rust, K., . . . Zinzen, R. P. (2022).\nFly Cell Atlas: A single-nucleus transcriptomic atlas of the adult fruit fly. Science,\n375(6584), eabk2432. https://doi.org/10.1126/science.abk2432\nLuu, L. M., Nguyen, L., Peng, S., Lee, J., Lee, H. Y., Wong, C. H., Hergenrother, P. J., Chan, H.\nY., & Zimmerman, S. C. (2016). A Potent Inhibitor of Protein Sequestration by Expanded\nTriplet (CUG) Repeats that Shows Phenotypic Improvements in a Drosophila Model of\nMyotonic Dystrophy. ChemMedChem, 11(13), 1428-1435.\nhttps://doi.org/10.1002/cmdc.201600081\nPicchio, L., Plantie, E., Renaud, Y., Poovthumkadavil, P., & Jagla, K. (2013). Novel Drosophila\nmodel of myotonic dystrophy type 1: phenotypic characterization and genome-wide view\nof altered gene expression. Hum Mol Genet, 22(14), 2795-2810.\nhttps://doi.org/10.1093/hmg/ddt127\nSanyal, S. (2009). Genomic mapping and expression patterns of C380, OK6 and D42 enhancer\ntrap lines in the larval nervous system of Drosophila. Gene Expr Patterns, 9(5), 371-380.\nhttps://doi.org/10.1016/j.gep.2009.01.002\nsynaptogenic signals through axonal transport of BMP receptors. J Cell Sci, 125(Pt 16),\n3752-3764. https://doi.org/10.1242/jcs.094292\nSpring, A. M., Brusich, D. J., & Frank, C. A. (2016). C-terminal Src Kinase Gates Homeostatic\nSynaptic Plasticity and Regulates Fasciclin II Expression at the Drosophila Neuromuscular\nJunction. PLoS Genet, 12(2), e1005886. https://doi.org/10.1371/journal.pgen.1005886\nWright, J. W., & Copenhaver, P. F. (2001). Cell type-specific expression of fasciclin II isoforms\nreveals neuronal-glial interactions during peripheral nerve growth. Dev Biol, 234(1), 24-\n41. https://doi.org/10.1006/dbio.2001.0247\nZhao, Z., & Geisbrecht, E. R. (2025). Stage-specific modulation of Drosophila gene expression\nwith muscle GAL4 promoters. Fly (Austin), 19(1), 2447617.\nhttps://doi.org/10.1080/19336934.2024.2447617\nReviewer #1 (Remarks to the Author):\nI appreciate the considerable effort the authors have put into revising their manuscript. It is clear that they took the\ncomments seriously and have added a number of new experiments, including electrophysiological analyses,\nimmunostainings, and initial pathway tests. The presentation has also improved, and the revised version reads more\nclearly than before. I fully acknowledge and value this commitment and the amount of work that went into the\nrevision.\nRESPONSE: We thank the reviewer for giving us helpful suggestions in the previous round of revision, which\nhelped us greatly improve the study, and we are very happy that the reviewer acknowledges our effort.\nThat said, I must admit that I still remain with my main reservations. My initial concerns were not about a single\nmissing experiment, but rather about the overall mechanistic depth of the study. While the new data extend the\ndescriptive part of the work, they do not substantially strengthen the causal or mechanistic link between CUG-\nrepeat toxicity and FasII dysregulation. The proposed pre- and postsynaptic “synergistic” effects also remain\ninterpretative rather than experimentally dissected. In this sense, the revision does not really change the conceptual\nlevel of the study.\nFrom my own perspective as someone working on synaptic mechanisms, the study remains primarily correlative\nand lacks the mechanistic resolution that would turn it into a conceptual advance. It is also a little difficult for me to\njudge the specific relevance for the DM1 field. If my expert colleagues in that area felt that the work represents a\nclear and important step forward, I would not object to publication. However, from the viewpoint of synaptic\nbiology, I am sorry to say that I do not see the added value or conceptual advance that would justify publication in\nNature Communications.\nRESPONSE: We sincerely thank the reviewer for the thoughtful and detailed comments, as well as for highlighting\nthe mechanistic aspects that merit further investigation. We acknowledge that our current study primarily advances\ndescriptive and correlative findings regarding CUG-repeat toxicity and FasII dysregulation, and that additional\nexperiments would be required to fully elucidate the underlying presynaptic and postsynaptic mechanisms.\nOur intention has been to lay the groundwork for future mechanistic dissection by identifying reproducible synaptic\nphenotypes and implicating FasII as a potential mediator in the context of DM1 models. We agree that while our\ndata do not yet resolve all causal links, these findings provide a necessary framework and important entry points for\nfurther mechanistic studies. Moreover, we believe that the synaptic phenotypes described here, and their possible\nconnection to DM1 pathogenesis, will be of relevance to researchers in both the DM1 field and the wider synaptic\nbiology community, helping to bridge these domains.\nWe are grateful for the reviewer’s openness to publication if the DM1 community considers these advances\nsignificant, and we hope that by situating our work as a foundation for future research, it can be of value to the field.\nThank you again for the constructive feedback and consideration.\nReviewer #2 (Remarks to the Author):\nAuthors in response to my and other reviewer's comments improved significantly their manuscript.\nRESPONSE: We thank the reviewer for giving us helpful suggestions that significantly improved our manuscript.\nOne of important supplementary data provided in the revised version is the analysis of a reduced Mbl level The\nauthors show that the heterozygous Mbl mutant context mimics toxic CTG repeats and FasII OE effects on NMJ.\nThis finding reinforces authors conclusions. However, when using heterozygous Mbl mutant authors do not address\nspecific pre- versus post-synaptique roles of Mbl attenuation (analysed in the contexts of CTG repeats and Fas2). It\nwould be thus appropriate and of interest to the field to provide this analysis (using UAS-MblRNAi line(s)).\nRESPONSE: We agree with the reviewer that RNAi would have provided us with the tools to address the pre-\nversus post-synaptic roles of Mbl attenuation. However, as Mbl is not our study’s focus, we hope the reviewer\nwould kindly let us round-up the study this time without adding this additional experiment.\nRegarding author's response to my first comment and to be more precise: As the DM1 neuropathology includes\nbrain impairment authors need to clarify which aspects of neuropathology they analyse.\nOne possibility could be by modifying the last sentence in the introduction.\nFor example:\n1\n\"These findings provide important new insights into the mechanisms underlying motor defects associated with DM1\nneuropathology\".\nRESPONSE: We thank and agree with the reviewer. We have modified the last sentence in the introduction\naccording to the reviewer’s suggestion.\nReviewer #3 (Remarks to the Author):\nThe authors have made a genuine effort to address the queries of all three reviews. The manuscript is all the better\nfor it.\nI still have a question that remains unanswered. The authors should clarify if they think that NCAM1 expression is\nupregulated in DMSXL and in DM1.\nRESPONSE: We thank the reviewer for raising this point and apologise for any confusion. Yes, our data support\nthat NCAM1 expression is upregulated in DMSXL mice and in DM1, although the direction and magnitude of the\nchange vary across tissues. As shown in Figure 3, slot blot analyses revealed a significant increase in total NCAM1\nprotein in the spinal cord and tibialis anterior of DMSXL mice, as well as in the frontal cortex of patients with DM1\n(with expected inter-individual variability). To clarify this in the manuscript, we have revised slightly the Results\nsection as follows:\nPage 12: “Using the slot blot technique, we found that NCAM1 protein was indeed upregulated in the spinal cord\nand tibialis anterior of DMSXL mice (Fig. 3i–l), confirming that our findings in Drosophila are relevant to\nmammals.”\nPage 12: “We again observed the upregulation of NCAM1 protein in these human tissues (Fig. 3m and 3n),\nconfirming that our findings are truly relevant to patients with DM1.”\nIn contrast, NCAM1 levels were reduced in DM1 deltoids. As discussed in the manuscript, the slot blot method\ndoes not distinguish between isoforms, and NCAM1 regulation may differ by tissue or cell type. A detailed\nisoform-specific analysis would be required to fully resolve this. The point is discussed in the final section of the\nmanuscript (changes are bolded below):\nPage 30: “Nevertheless, dysregulation of NCAM1 was still detected in human deltoids and is likely to have resulted\nin synaptic dysfunction at the NMJ. Alternatively, mature human muscle cells may have compensatory mechanisms\nthat could explain the high level of NCAM1 in transdifferentiated myotubes but low level in deltoid muscles.”\nIn summary, our results consistently show NCAM1 dysregulation in DM1, with upregulation in DMSXL neural\nand skeletal muscle tissues and in DM1 frontal cortex, and reduced levels in deltoids, supporting a model in\nwhich tissue-specific NCAM1 changes contribute to DM1 pathogenesis.\nThe other thing is a technical issue. I am not sure that the way the assay seems to be described for supplemental\nfigures 17 and 18 is the correct way to assess alternatively spliced isoforms of NCAM. It seems like they used\nprimers situated in exon 16 and 17 for the isoform that includes exon 17 and a primer pair located in exon 16 and 18\nfor the other. But, if exon 17 is alternatively spliced, two products should have shown up in the exon 16-18 product;\none representing exons 16,17,18 and the other being exons16 and 18 (i.e. exon 17 is spliced out). But that is not\nwhat the data shows. The way the results appear, it looks like there is no alternative splicing of exon 17. The\nSashimi plot confirms that. If you look at it, it looks like there are transcripts that contain exon 16 and 17 that seem\nto terminate there (i.e. no 3' exons are joined to this transcript), and there are no transcripts that contain exon 16, 17\nand 18. This is not an alternative splicing pattern.\nIf the implication of the data is that MBNL proteins are causing alternative splicing defects in NCAM in the mouse\nsituation, as the MBNL proteins do for 100s of other transcripts in DM1, and that is how we tie the effects on Fas in\nflies to the spliceopathy model of RNA toxicity in DM1, then the conclusion is incorrect.\nPlease correct your discussion and don't imply splicing defects in NCAM for the human or mmouse situation. Your\ndata doesn't show it.\nIt is more likely a transcriptional upregulation.\nRESPONSE: We thank the reviewer for this valuable and detailed comment, which helped us clarify the nature of\nthe abnormal RNA event observed in Ncam1 transcripts. Based on the literature and public transcript annotations,\nisoforms. One major isoform includes exon 16 followed by exon 17 only (labelled E16+E17 in Supplementary Fig.\n17), while another includes exon 16 followed by a distal block of exons (exons 18–21) (labelled\nE16+E18+E19+E21). Using a multiplex semi-quantitative RT-PCR strategy with primer pairs spanning exon 16 to\neither exon 17 or exon 18, we detected a shift in terminal exon usage in both DMSXL mice and DM1 samples.\nSpecifically, the DM1 condition was associated with increased inclusion of distal exons 18, 19 and 21, with exon 20\nbeing excluded in the samples analysed. We agree with the reviewer that this mechanism should be described as\naltered terminal exon usage rather than “classical” exon skipping of exon 17, and we have revised the text\naccordingly to avoid any implication of an exon-17 splicing defect that is not supported by our data. The changes\nare listed below (changes are bolded below).\nDiscussion, page 31-32:\nAs we have demonstrated that the expression of total NCAM1 is dysregulated in neural and muscular tissues from\nDMSXL mice (Fig. 3i–l), an important question concerns whether Ncam1 RNA isoforms are also dysregulated in\nthese animals. To answer this question, we subjected primary astrocytes and frontal cortex of DMSXL mice to RT-\nPCR analysis of distal exon selection. As expected, we indeed observed dysregulated usage of distal Ncam1\nterminal exons in these cells and tissues (Supplementary Fig. 17). In addition, we examined whether dysregulated\nNcam1 splicing could be caused by Mbnl1/Mbnl2 double knockdown or knockout. Our results showed that Ncam1\nterminal exon splicing was indeed dysregulated in cultured astrocytes subjected to Mbnl1/Mbnl2 double\nknockdown (Supplementary Fig. 18a and 18b) and in the frontal cortex of Mbnl1/Mbnl2 conditional double-\nknockout mice (Mbnl1-/-; Mbnl2c/c; Nestin-Cre+) 61 (Supplementary Fig. 18c and 18d). As these defects parallel\nthose seen in DMSXL mice (Supplementary Fig. 17), the shift in Ncam1 terminal exon usage in DM1 is likely\nlinked to functional depletion of MBNL proteins.\nMethods, page 36-37:\nSemi-quantitative RT-PCR for the analysis of alternative exon usage. RNA extraction was performed with the\nRNeasy Mini kit (QIAGEN; 74104) following the manufacturer’s protocol, including a DNAse digestion step\n(RNase-Free DNase Set; QIAGEN; 79254) after the first wash with RW1 buffer. RNA concentration was assessed\nusing the NanoDrop (Thermo Scientific) and RNA quality was verified by electrophoresis on agarose gel. cDNA\nsynthesis and semi-quantitative reverse-transcriptase PCR analysis of alternative exon usage were performed as\npreviously described 66, 67. Oligonucleotide primers used for RT-PCR analysis of Ncam1 were the following: Ncam1\nE16 (forward): ACGTCATGCTCAAGTCCCTG. Ncam1 E17 (reverse): AGTCACCGCAGAGAAAAGCA.\nNcam1 E18 (reverse): ATGAGCAGGCCACACTTGTT.\nSupplementary figure 17.\nPanels b and d. The amplified isoforms are now labelled as E16+E17 and E16+E18+E19+E21.\nPanels c and e. The y-axis of the graph now reads: Percentage distal exon usage\nThe legends now reads:\n(c) Quantification of the Ncam1 RNA isoform containing the distal exons E18, E19 and E21 in (b).\n(e) Quantification of the Ncam1 RNA isoform containing the distal exons E18, E19 and E21 in (d).\nSupplementary figure 18.\nPanels a and c. The amplified isoforms are now labelled as E16+E17 and E16+E18+E19+E21.\nPanels b and d. The y-axis of the graph now reads: Percentage distal exon usage\nThe legends now reads:\n(b) Quantification of the Ncam1 RNA isoform containing the distal exons E18, E19 and E21 in (a).\n(d) Quantification of the Ncam1 RNA isoform containing the distal exons E18, E19 and E21 in (c).\nOtherwise, you have done a commendable job and I have no other issues.\nRESPONSE: We greatly appreciate the reviewer for giving us helpful suggestions to improve our study.\n3"}]

Decision letter