DataSheet2_Expanded CUG Repeat RNA Induces Premature Senescence in Myotonic Dystrophy Model Cells.PDF

Myotonic dystrophy type 1 (DM1) is a dominantly inherited disorder due to a toxic gain of function of RNA transcripts containing expanded CUG repeats (CUGexp). Patients with DM1 present with multisystemic symptoms, such as muscle wasting, cognitive impairment, cataract, frontal baldness, and endocrine defects, which resemble accelerated aging. Although the involvement of cellular senescence, a critical component of aging, was suggested in studies of DM1 patient-derived cells, the detailed mechanism of cellular senescence caused by CUGexp RNA remains unelucidated. Here, we developed a DM1 cell model that conditionally expressed CUGexp RNA in human primary cells so that we could perform a detailed assessment that eliminated the variability in primary cells from different origins. Our DM1 model cells demonstrated that CUGexp RNA expression induced cellular senescence by a telomere-independent mechanism. Furthermore, the toxic RNA expression caused mitochondrial dysfunction, excessive reactive oxygen species production, and DNA damage and response, resulting in the senescence-associated increase of cell cycle inhibitors p21 and p16 and secreted mediators insulin-like growth factor binding protein 3 (IGFBP3) and plasminogen activator inhibitor-1 (PAI-1). This study provides unequivocal evidence of the induction of premature senescence by CUGexp RNA in our DM1 model cells.</p

Table1_Expanded CUG Repeat RNA Induces Premature Senescence in Myotonic Dystrophy Model Cells.XLSX

Myotonic dystrophy type 1 (DM1) is a dominantly inherited disorder due to a toxic gain of function of RNA transcripts containing expanded CUG repeats (CUGexp). Patients with DM1 present with multisystemic symptoms, such as muscle wasting, cognitive impairment, cataract, frontal baldness, and endocrine defects, which resemble accelerated aging. Although the involvement of cellular senescence, a critical component of aging, was suggested in studies of DM1 patient-derived cells, the detailed mechanism of cellular senescence caused by CUGexp RNA remains unelucidated. Here, we developed a DM1 cell model that conditionally expressed CUGexp RNA in human primary cells so that we could perform a detailed assessment that eliminated the variability in primary cells from different origins. Our DM1 model cells demonstrated that CUGexp RNA expression induced cellular senescence by a telomere-independent mechanism. Furthermore, the toxic RNA expression caused mitochondrial dysfunction, excessive reactive oxygen species production, and DNA damage and response, resulting in the senescence-associated increase of cell cycle inhibitors p21 and p16 and secreted mediators insulin-like growth factor binding protein 3 (IGFBP3) and plasminogen activator inhibitor-1 (PAI-1). This study provides unequivocal evidence of the induction of premature senescence by CUGexp RNA in our DM1 model cells.</p

DataSheet1_Expanded CUG Repeat RNA Induces Premature Senescence in Myotonic Dystrophy Model Cells.PDF

Myotonic dystrophy type 1 (DM1) is a dominantly inherited disorder due to a toxic gain of function of RNA transcripts containing expanded CUG repeats (CUGexp). Patients with DM1 present with multisystemic symptoms, such as muscle wasting, cognitive impairment, cataract, frontal baldness, and endocrine defects, which resemble accelerated aging. Although the involvement of cellular senescence, a critical component of aging, was suggested in studies of DM1 patient-derived cells, the detailed mechanism of cellular senescence caused by CUGexp RNA remains unelucidated. Here, we developed a DM1 cell model that conditionally expressed CUGexp RNA in human primary cells so that we could perform a detailed assessment that eliminated the variability in primary cells from different origins. Our DM1 model cells demonstrated that CUGexp RNA expression induced cellular senescence by a telomere-independent mechanism. Furthermore, the toxic RNA expression caused mitochondrial dysfunction, excessive reactive oxygen species production, and DNA damage and response, resulting in the senescence-associated increase of cell cycle inhibitors p21 and p16 and secreted mediators insulin-like growth factor binding protein 3 (IGFBP3) and plasminogen activator inhibitor-1 (PAI-1). This study provides unequivocal evidence of the induction of premature senescence by CUGexp RNA in our DM1 model cells.</p

Intracerebral Distribution of CAG Repeat-Binding Small Molecule Visualized by Whole-Brain Imaging

Understanding the pharmacokinetics of drug candidates of interest in the brain and evaluating drug delivery to the brain are important for developing drugs targeting the brain. Previously, we demonstrated that a CAG repeat-binding small molecule, naphthyridine-azaquinolone (NA), resulted in repeat contraction in mouse models of dentatorubral–pallidoluysian atrophy and Huntington’s disease caused by aberrant expansion of CAG repeats. However, the intracerebral distribution and drug deliverability of NA remain unclear. Here, we report three-dimensional whole-brain imaging of an externally administered small molecule using tissue clearing and light sheet fluorescence microscopy (LSFM). We designed and synthesized an Alexa594-labeled NA derivative with a primary amine for whole-brain imaging (NA-Alexa594-NH2), revealing the intracerebral distribution of NA-Alexa594-NH2 after intraparenchymal and intracerebroventricular administrations by whole-brain imaging combined with tissue clearing and LSFM. We also clarified that intranasally administered NA-Alexa594-NH2 was delivered into the brain via multiple nose-to-brain pathways by tracking the time-dependent change in the intracerebral distribution. Whole-brain imaging of small molecules by tissue clearing and LSFM is useful for elucidating not only the intracerebral distribution but also the drug delivery pathways into the brain

Accumulation of nuclear RNA foci in DM1 hiNeurons at 10 DPI and their reduction by ACT treatment.

(a) FISH with 5’ Texas red 2-O-methyl- CAG RNA probe and immunostaining with TUJ1 and DAPI revealed punctate and discrete nuclear RNA foci (red) in DM1 hiNeurons (a2) but not in control hiNeurons (a1). Nuclear RNA foci were abundant in DM1 hiNeurons (a2) but treatment with 100 nM (a3) or 200 nM (a4) ACT reduced them remarkably (left: FISH, middle: merged images of FISH and DAPI staining nuclei (blue), right: merged images of FISH, DAPI and TUJ1 antibody staining neurons (green)). Scale bar, 20 μm. (b) Scatter plot shows percentage of nuclei containing RNA foci in DM1 hiNeurons. Each sample is presented in different color. Each symbol represents the percentage of nuclei containing RNA foci for each sample replicate. Line represents the mean. Note that nuclear RNA foci were present in most of DM1 hiNeurons. (c) Box and whisker plot shows number of RNA foci per nucleus in DM1 hiNeurons. Each sample is presented in different color. Line and (+) sign inside the box represent median and mean of replicates (outcome analyzed), respectively. Whiskers show minimum & maximum values. Counting was performed manually. n = 3 for each group, a total of 324 nuclei were analyzed per sample. ****PEq (1). (f) Scatter plot shows percentage of nuclei containing RNA foci in placebo and ACT treated DM1 hiNeurons. Each sample is presented in different color. Each symbol represents the percentage of nuclei containing RNA foci for each sample replicate. Line represents the mean. ACT treatment reduced the percentage of nuclei with RNA foci by 10.3% or 16.5% at 100 nM or 200 nM ACT, respectively. Counting was performed manually. n = 3 for each group, a total of 324 nuclei were analyzed per sample. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05 and ns, not significant compared by repeated measures one-way ANOVA test. P, placebo (same amount of diluent without drug).</p

Direct reprogramming of DM1 patients’ skin fibroblasts into neuronal like cells.

(a) Differentiation process of DM1 skin fibroblasts into neuronal like cells. The top left image shows DM1 skin fibroblasts before adding lentivirus co-expressing shRNA against PTBP and puromycin resistant gene. The top middle image shows lentivirus transduced cells successfully passing the selection process by adding 5 μg/mL puromycin to the culture medium for 2 days. The top right image shows expansion of cell bodies at 6 DPI. The bottom left image shows cells with neuronal like morphology of expanded cell bodies and distinct neurites at 8 DPI. The bottom right image shows bipolar and multipolar neurons with extended neurites at 10 DPI. Scale bar, 50 μm. (b) Co-immunostaining of control (top panel) and DM1 (bottom panel) hiNeurons with a neuron specific β-III tubulin antibody (TUJ1) (green) and MAP2 (red) at 10 DPI. DAPI was used to stain nuclei (blue). On the right: merged images of immunostaining with TUJ1, MAP2 and DAPI. Scale bar, 50 μm. (c) Graph shows the percentage of positive TUJ1-stained cells (TUJ1+) at 10 DPI, quantified by dividing the number of TUJ1+ cells by the total number of nuclei stained by DAPI. Data are shown as mean ± SEM; n = 3 for each group, a total of 90 images were analyzed per group (11,510 nuclei were analyzed per ctrl group and 14,668 nuclei per DM1 group). ns, not significant compared to control group by unpaired t-test. (d) Graph displays the percentage of MAP2 positive cells at 10 DPI, quantified by dividing the number of MAP2+ cells by the total number of TUJ1+ cells. Data are shown as mean ± SEM; n = 3 for each group, a total of 90 images were analyzed per group (9,941 TUJ1+ cells were analyzed per ctrl group and 16,286 TUJ1+ cells per DM1 group). ns, not significant compared to control group by unpaired t-test. (e) Additional staining for neural markers. Fluorescent images display positive co-staining of DM1 hiNeurons with TUJ1 (green) and anti GABA or glutamate or NeuN (red) antibodies at 15 DPI. The bottom panel shows lack of synapse formation at 10 DPI in DM1 hiNeurons co-stained with TUJ1 (green) and SYN1 (red) antibodies. DAPI was used to stain nuclei (blue). On the right merged images. Scale bar, 50 μm. Ctrl, control; DM1, myotonic dystrophy type 1; hiN, human induced neurons; DPI, days post viral-infection; TUJ1, β-III tubulin antibody; MAP2, microtubule-associated protein 2; SEM, standard error of the mean; GABA, gamma-aminobutyric acid; NeuN, neuronal nuclear antibody; SYN1, synapsin 1 antibody.</p

Rescue of <i>MAPT</i> e2, MPRIP e9 and CSNK1D e9 mis-splicing by erythromycin lactobionate treatment at 10 DPI.

(a-b) Electropherograms plot the results of RT-PCR products for MAPT e2 (a) and CSNK1D e9 (b) in ctrl (top) and DM1 hiNeurons (bottom). Note the increased inclusion of MAPT e2 (+e2, -e3 and +e2, +e3) and CSNK1D e9 (+e9) in DM1 hiNeurons treated with 35 or 65 μM erythromycin for 48 h relative to placebo treated DM1 hiNeurons. RT-PCR products were analyzed by MultiNA automated microchip electrophoresis system. (c) Scatter plots show inclusion percentage of MAPT e2 (left), CSNK1D e9 (middle) and MPRIP e9 (right) in control and DM1 hiNeurons treated with placebo or erythromycin. n = 3 for each group. Each sample is presented in different color. Each symbol represents percentage of exon inclusion for one sample replicate. Line represents mean. *PMAPT e2 (left), CSNK1D e9 (middle) and MPRIP e9 (right) in DM1 hiNeurons by erythromycin treatment. Note that erythromycin rescued MAPT exon 2 mis-splicing by 38.4% or 46.4% at a concentration of 35 or 65 μM, respectively. Data are presented as mean ± SEM. P, placebo (same amount of diluent without drug).</p

Tolerability of ctrl and DM1 hiNeurons to the studied doses of ACT.

(a and c) Live cell images of untreated ctrl and DM1 hiNeurons at 9 DPI, respectively. (b and d) Live cell images of ctrl and DM1 hiNeurons after 24 h treatment with placebo (left), 100 nM ACT (middle) or 200 nM ACT (right). Good tolerability was observed at 100 nM ACT in ctrl and DM1 hiNeurons whereas some cytotoxicity was observed in 200 nM ACT treated cells. Scale bar, 50 μm. (TIF)</p

Rational Design of Bioactive, Modularly Assembled Aminoglycosides Targeting the RNA that Causes Myotonic Dystrophy Type 1

Myotonic dystrophy type 1 (DM1) is caused when an expanded r­(CUG) repeat (r­(CUG)^exp) binds the RNA splicing regulator muscleblind-like 1 protein (MBNL1) as well as other proteins. Previously, we reported that modularly assembled small molecules displaying a 6′-<i>N</i>-5-hexynoate kanamycin A RNA-binding module (<b>K</b>) on a peptoid backbone potently inhibit the binding of MBNL1 to r­(CUG)^exp. However, these parent compounds are not appreciably active in cell-based models of DM1. The lack of potency was traced to suboptimal cellular permeability and localization. To improve these properties, second-generation compounds that are conjugated to a d-Arg₉ molecular transporter were synthesized. These modified compounds enter cells in higher concentrations than the parent compounds and are efficacious in cell-based DM1 model systems at low micromolar concentrations. In particular, they improve three defects that are the hallmarks of DM1: a translational defect due to nuclear retention of transcripts containing r­(CUG)^exp; pre-mRNA splicing defects due to inactivation of MBNL1; and the formation of nuclear foci. The best compound in cell-based studies was tested in a mouse model of DM1. Modest improvement of pre-mRNA splicing defects was observed. These studies suggest that a modular assembly approach can afford bioactive compounds that target RNA

Combination Treatment of Erythromycin and Furamidine Provides Additive and Synergistic Rescue of Mis-splicing in Myotonic Dystrophy Type 1 Models

Myotonic dystrophy type 1 (DM1) is a multisystemic disease that presents with clinical symptoms including myotonia, cardiac dysfunction, and cognitive impairment. DM1 is caused by a CTG expansion in the 3′ UTR of the DMPK gene. The transcribed expanded CUG-repeat RNA sequester the muscleblind-like (MBNL) and up-regulate the CUG-BP Elav-like (CELF) families of RNA-binding proteins leading to global mis-regulation of RNA processing and altered gene expression. Currently, there are no disease-targeting treatments for DM1. Given the multistep pathogenic mechanism, combination therapies targeting multiple aspects of the disease mechanism may be a viable therapeutic approach. Here, as proof-of-concept, we studied a combination of two previously characterized small molecules, erythromycin and furamidine, in two DM1 models. In DM1 patient-derived myotubes, the rescue of mis-splicing was observed with little to no cell toxicity. In a DM1 mouse model, a combination of erythromycin and the prodrug of furamidine (pafuramidine), administered orally, displayed both additive and synergistic mis-splicing rescue. Gene expression was only modestly affected, and over 40% of the genes showing significant expression changes were rescued back toward WT expression levels. Further, the combination treatment partially rescued the myotonia phenotype in the DM1 mouse. This combination treatment showed a high degree of mis-splicing rescue coupled with low off-target gene expression changes. These results indicate that combination therapies are a promising therapeutic approach for DM1