Search

Aims: Myotonic dystrophy type I (DM1) is one of the most frequent muscular dystrophies in adults. Although DM1 has long been considered mainly a muscle disorder, growing evidence suggests the involvement of peripheral nerves in the pathogenicity of DM1 raising the question of whether motoneurons (MNs) actively contribute to neuromuscular defects in DM1., Methods: By using micropatterned 96-well plates as a coculture platform, we generated a functional neuromuscular model combining DM1 and muscleblind protein (MBNL) knock-out human-induced pluripotent stem cells-derived MNs and human healthy skeletal muscle cells., Results: This approach led to the identification of presynaptic defects which affect the formation or stability of the neuromuscular junction at an early developmental stage. These neuropathological defects could be reproduced by the loss of RNA-binding MBNL proteins, whose loss of function in vivo is associated with muscular defects associated with DM1. These experiments indicate that the functional defects associated with MNs can be directly attributed to MBNL family proteins. Comparative transcriptomic analyses also revealed specific neuronal-related processes regulated by these proteins that are commonly misregulated in DM1., Conclusions: Beyond the application to DM1, our approach to generating a robust and reliable human neuromuscular system should facilitate disease modelling studies and drug screening assays., (© 2022 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society.)

Brain dysfunction in myotonic dystrophy type 1 (DM1), the prototype of toxic RNA disorders, has been mainly attributed to neuronal RNA misprocessing, while little attention has been given to non-neuronal brain cells. Here, using a transgenic mouse model of DM1 that expresses mutant RNA in various brain cell types (neurons, astroglia, and oligodendroglia), we demonstrate that astrocytes exhibit impaired ramification and polarization in vivo and defects in adhesion, spreading, and migration. RNA-dependent toxicity and phenotypes are also found in human transfected glial cells. In line with the cell phenotypes, molecular analyses reveal extensive expression and accumulation of toxic RNA in astrocytes, which result in RNA spliceopathy that is more severe than in neurons. Astrocyte missplicing affects primarily transcripts that regulate cell adhesion, cytoskeleton, and morphogenesis, and it is confirmed in human brain tissue. Our findings demonstrate that DM1 impacts astrocyte cell biology, possibly compromising their support and regulation of synaptic function., (© 2022. The Author(s).)

Myotonic dystrophy type 1 (DM1) is a severe neuromuscular disease mediated by a toxic gain of function of mutant RNAs. The neuropsychological manifestations affect multiple domains of cognition and behavior, but their etiology remains elusive. Transgenic DMSXL mice carry the DM1 mutation, show behavioral abnormalities, and express low levels of GLT1, a critical regulator of glutamate concentration in the synaptic cleft. However, the impact of glutamate homeostasis on neurotransmission in DM1 remains unknown. We confirmed reduced glutamate uptake in the DMSXL hippocampus. Patch clamp recordings in hippocampal slices revealed increased amplitude of tonic glutamate currents in DMSXL CA1 pyramidal neurons and DG granule cells, likely mediated by higher levels of ambient glutamate. Unexpectedly, extracellular GABA levels and tonic current were also elevated in DMSXL mice. Finally, we found evidence of synaptic dysfunction in DMSXL mice, suggestive of abnormal short-term plasticity, illustrated by an altered LTP time course in DG and in CA1. Synaptic dysfunction was accompanied by RNA foci accumulation in localized areas of the hippocampus and by the mis-splicing of candidate genes with relevant functions in neurotransmission. Molecular and functional changes triggered by toxic RNA may induce synaptic abnormalities in restricted brain areas that favor neuronal dysfunction.

Extracellular glutamate levels are maintained low by efficient transporters, whose dysfunction can cause neuronal hyperexcitability, excitotoxicity, and neurological disease. While many methods estimate glutamate uptake in vitro / ex vivo , a limited number of techniques address glutamate transport in vivo . Here, we used in vivo microdialysis in a two-in-one approach combining reverse dialysis of isotopic glutamate to measure uptake ability and zero-flow (ZF) methods to quantify extracellular glutamate levels. The complementarity of both techniques is discussed on methodological and anatomical basis. We used a transgenic mouse model of human disease, expressing low levels of the EAAT-2/GLT1 glutamate transporter, to validate our approach in a relevant animal model. As expected, isotopic analysis revealed an overall decrease in glutamate uptake, while the ZF method unveiled higher extracellular glutamate levels in these mice. We propose a sensitive and expedite two-in-one microdialysis approach that is sufficiently robust to reveal significant differences in neurotransmitter uptake and extracellular levels through the analysis of a relatively low number of animals.

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a non-coding CTG repeat expansion in the DMPK gene. This mutation generates a toxic CUG RNA that interferes with the RNA processing of target genes in multiple tissues. Despite debilitating neurological impairment, the pathophysiological cascade of molecular and cellular events in the central nervous system (CNS) has been less extensively characterized than the molecular pathogenesis of muscle/cardiac dysfunction. Particularly, the contribution of different cell types to DM1 brain disease is not clearly understood. We first used transcriptomics to compare the impact of expanded CUG RNA on the transcriptome of primary neurons, astrocytes and oligodendrocytes derived from DMSXL mice, a transgenic model of DM1. RNA sequencing revealed more frequent expression and splicing changes in glia than neuronal cells. In particular, primary DMSXL oligodendrocytes showed the highest number of transcripts differentially expressed, while DMSXL astrocytes displayed the most severe splicing dysregulation. Interestingly, the expression and splicing defects of DMSXL glia recreated molecular signatures suggestive of impaired cell differentiation: while DMSXL oligodendrocytes failed to upregulate a subset of genes that are naturally activated during the oligodendroglia differentiation, a significant proportion of missplicing events in DMSXL oligodendrocytes and astrocytes increased the expression of RNA isoforms typical of precursor cell stages. Together these data suggest that expanded CUG RNA in glial cells affects preferentially differentiation-regulated molecular events. This hypothesis was corroborated by gene ontology (GO) analyses, which revealed an enrichment for biological processes and cellular components with critical roles during cell differentiation. Finally, we combined exon ontology with phosphoproteomics and cell imaging to explore the functional impact of CUG-associated spliceopathy on downstream protein metabolism. Changes in phosphorylation, protein isoform expression and intracellular localization in DMSXL astrocytes demonstrate the far-reaching impact of the DM1 repeat expansion on cell metabolism. Our multi-omics approaches provide insight into the mechanisms of CUG RNA toxicity in the CNS with cell type resolution, and support the priority for future research on non-neuronal mechanisms and proteomic changes in DM1 brain disease., Competing Interests: AG-B was a former employee of BioMarin Pharmaceutical Inc., and holds shares of the company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 González-Barriga, Lallemant, Dincã, Braz, Polvèche, Magneron, Pionneau, Huguet-Lachon, Claude, Chhuon, Guerrera, Bourgeois, Auboeuf, Gourdon and Gomes-Pereira.)

Background: Studies have shown an association between depression and frailty, even though the literature has not reached a consensus regarding how these syndromes interact. Although prospective cohort studies on this topic are still scarce, they could contribute to understanding this relationship. We aimed to observe whether depressive symptoms are risk factors for the onset of frailty among older adults living in São Paulo, Brazil., Methods: Prospective cohort study using the "Health, Well-being and Aging" (SABE) Study databases of 2006 and 2010. The sample was representative of the community-dwelling older adults living in São Paulo, and it is composed of non-frail men and women aged 60 years and older without cognitive decline. We calculated the frailty incidence rate between older adults with and without depressive symptoms and calculated the incidence rate ratio. Multiple analysis was carried out through Poisson regression with robust variance estimation., Results: The initial sample (n = 1,109) presented a mean age of 72 years (from 60 to 96) and 61.1% were women. The final sample was composed of 830 individuals, and the mean follow-up time in the study was 3.8 years. After adjusting the model, depressive symptoms did not predict the onset of frailty at follow-up., Conclusion: Depressive symptoms were not shown to be a risk factor for frailty among older adults living in São Paulo in this study., Competing Interests: The authors have declared that no competing interests exist.

More than 30 human disorders are caused by the expansion of simple sequence DNA repeats, among which triplet repeats remain the most frequent. Most trinucleotide repeat expansion disorders affect primarily the nervous system, through mechanisms of neurodysfunction and/or neurodegeneration. While trinucleotide repeat tracts are short and stably transmitted in unaffected individuals, disease-associated expansions are highly dynamic in the germline and in somatic cells, with a tendency toward further expansion. Since longer repeats are associated with increasing disease severity and earlier onset of symptoms, intergenerational repeat size gains account for the phenomenon of anticipation. In turn, higher levels of age-dependent somatic expansion have been linked with increased disease severity and earlier age of onset, implicating somatic instability in the onset and progression of disease symptoms. Hence, tackling the root cause of symptoms through the control of repeat dynamics may provide therapeutic modulation of clinical manifestations. DNA repair pathways have been firmly implicated in the molecular mechanism of repeat length mutation. The demonstration that repeat expansion depends on functional DNA mismatch repair (MMR) proteins, points to MMR as a potential therapeutic target. Similarly, a role of DNA base excision repair (BER) in repeat expansion has also been suggested, particularly during the removal of oxidative lesions. Using a well-characterized mouse cell model system of an unstable CAG•CTG trinucleotide repeat, we tested if expanded repeat tracts can be stabilized by small molecules with reported roles in both pathways: cadmium (an inhibitor of MMR activity) and a variety of antioxidants (capable of neutralizing oxidative species). We found that chronic exposure to sublethal doses of cadmium and antioxidants did not result in significant reduction of the rate of trinucleotide repeat expansion. Surprisingly, manganese yielded a significant stabilization of the triplet repeat tract. We conclude that treatment with cadmium and antioxidants, at doses that do not interfere with cell survival and cell culture dynamics, is not sufficient to modify trinucleotide repeat dynamics in cell culture., Competing Interests: DGM had been a scientific consultant and/or received an honoraria/stock options from AMO Pharma, Biogen Idec, Charles River, LoQus23, Small Molecule RNA, Triplet Therapeutics and Vertex Pharmaceuticals, and held research contracts with AMO Pharma and Vertex Pharmaceuticals. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gomes-Pereira and Monckton.)

Proper brain function requires the coordinated and intricate interaction between neuronal and glial cells. Like many other neurological conditions, trinucleotide repeat expansion disorders are likely initiated by the synergistic combination of abnormalities hitting different brain cell types, which ultimately disrupt brain function and lead to the onset of neurological symptoms. Understanding how trinucleotide repeat expansions affect the phenotypes and physiology of neurons and glia is fundamental to improve our understanding of disease mechanisms in the brain and shape the design of future therapeutic interventions.Here we describe a protocol for semiautomated videomicroscopy analysis of cultured brain cells, maintained under suitable and controlled conditions. Through real-time monitoring of basic cell phenotypes (such as proliferation, cell morphology, differentiation, and migration) this method provides an accurate primary assessment of the impact of the repeat expansion on the physiology of neurons and glia. The versatility of the system, the automated image acquisition and the semiautomated processing of the data collected allow rapid phenotypic analysis of individual cell types, as well as the investigation of cell-cell interactions. The stability of the acquisition system provides reproducible and robust results. The raw data can be easily exported to other software to perform more sophisticated imaging analysis and statistical tests. In summary, the methods described offer versatile, reproducible, and time-effective means to dissect the impact of the repeat expansion on different brain cell types and on intercellular interactions.

Intensive effort has been directed toward the modeling of myotonic dystrophy (DM) in mice, in order to reproduce human disease and to provide useful tools to investigate molecular and cellular pathogenesis and test efficient therapies. Mouse models have contributed to dissect the multifaceted impact of the DM mutation in various tissues, cell types and in a pleiotropy of pathways, through the expression of toxic RNA transcripts. Changes in alternative splicing, transcription, translation, intracellular RNA localization, polyadenylation, miRNA metabolism and phosphorylation of disease intermediates have been described in different tissues. Some of these events have been directly associated with specific disease symptoms in the skeletal muscle and heart of mice, offering the molecular explanation for individual disease phenotypes. In the central nervous system (CNS), however, the situation is more complex. We still do not know how the molecular abnormalities described translate into CNS dysfunction, nor do we know if the correction of individual molecular events will provide significant therapeutic benefits. The variability in model design and phenotypes described so far requires a thorough and critical analysis. In this review we discuss the recent contributions of mouse models to the understanding of neuromuscular aspects of disease, therapy development, and we provide a reflective assessment of our current limitations and pressing questions that remain unanswered.

Brain function is compromised in myotonic dystrophy type 1 (DM1), but the underlying mechanisms are not fully understood. To gain insight into the cellular and molecular pathways primarily affected, we studied a mouse model of DM1 and brains of adult patients. We found pronounced RNA toxicity in the Bergmann glia of the cerebellum, in association with abnormal Purkinje cell firing and fine motor incoordination in DM1 mice. A global proteomics approach revealed downregulation of the GLT1 glutamate transporter in DM1 mice and human patients, which we found to be the result of MBNL1 inactivation. GLT1 downregulation in DM1 astrocytes increases glutamate neurotoxicity and is detrimental to neurons. Finally, we demonstrated that the upregulation of GLT1 corrected Purkinje cell firing and motor incoordination in DM1 mice. Our findings show that glial defects are critical in DM1 brain pathophysiology and open promising therapeutic perspectives through the modulation of glutamate levels., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The abnormal expansion of unstable simple sequence DNA repeats can cause human disease through a variety of mechanisms, including gene loss-of-function, toxic gain-of-function of the encoded protein and toxicity of the repeat-containing RNA transcript. Disease-associated unstable DNA repeats display unusual biophysical properties, including the ability to adopt non-B-DNA structures. CAG•CTG trinucleotide sequences, in particular, have been most extensively studied and they can fold into slipped-stranded DNA structures, which have been proposed as mutation intermediates in repeat size expansion. Here, we describe a simple assay to detect unusual DNA structures generated by PCR amplification, based on their slow electrophoretic migration in agarose and on the effects of ethidium bromide on the mobility of structural isoforms through agarose gels. Notably, the inclusion of ethidium bromide in agarose gels and running buffer eliminates the detection of additional slow-migrating DNA species, which are detected in the absence of the intercalating dye and may be incorrectly classified as mutant alleles with larger than actual expansion sizes. Denaturing and re-annealing experiments confirmed the slipped-stranded nature of the additional DNA species observed in agarose gels. Thus, we have shown that genuine non-B-DNA conformations are generated during standard PCR amplification of CAG•CTG sequences and detected by agarose gel electrophoresis. In contrast, ethidium bromide does not change the multi-band electrophoretic profiles of repeat-containing PCR products through native polyacrylamide gels. These data have implications for the analysis of trinucleotide repeat DNA and possibly other types of unstable repetitive DNA sequences by standard agarose gel electrophoresis in diagnostic and research protocols. We suggest that proper sizing of CAG•CTG PCR products in agarose gels should be performed in the presence of ethidium bromide.

Genetically unstable expanded CAG·CTG trinucleotide repeats are causal in a number of human disorders, including Huntington disease and myotonic dystrophy type 1. It is still widely assumed that DNA polymerase slippage during replication plays an important role in the accumulation of expansions. Nevertheless, somatic mosaicism correlates poorly with the proliferative capacity of the tissue and rates of cell turnover, suggesting that expansions can occur in the absence of replication. We monitored CAG·CTG repeat instability in transgenic mouse cells arrested by chemical or genetic manipulation of the cell cycle and generated unequivocal evidence for the continuous accumulation of repeat expansions in non-dividing cells. Importantly, the rates of expansion in non-dividing cells were at least as high as those of proliferating cells. These data are consistent with a major role for cell division-independent expansion in generating somatic mosaicism in vivo. Although expansions can accrue in non-dividing cells, we also show that cell cycle arrest is not sufficient to drive instability, implicating other factors as the key regulators of tissue-specific instability. Our data reveal that de novo expansion events are not limited to S-phase and further support a cell division-independent mutational pathway., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Many human diseases are associated with the abnormal expansion of unstable trinucleotide repeat sequences. The mechanisms of trinucleotide repeat size mutation have not been fully dissected, and their understanding must be grounded on the detailed analysis of repeat size distributions in human tissues and animal models. Small-pool PCR (SP-PCR) is a robust, highly sensitive and efficient PCR-based approach to assess the levels of repeat size variation, providing both quantitative and qualitative data. The method relies on the amplification of a very low number of DNA molecules, through sucessive dilution of a stock genomic DNA solution. Radioactive Southern blot hybridization is sensitive enough to detect SP-PCR products derived from single template molecules, separated by agarose gel electrophoresis and transferred onto DNA membranes. We describe a variation of the detection method that uses digoxigenin-labelled locked nucleic acid probes. This protocol keeps the sensitivity of the original method, while eliminating the health risks associated with the manipulation of radiolabelled probes, and the burden associated with their regulation, manipulation and waste disposal.

Mutant ribonucleic acid (RNA) molecules can be toxic to the cell, causing human disease through trans-acting dominant mechanisms. RNA toxicity was first described in myotonic dystrophy type 1, a multisystemic disorder caused by the abnormal expansion of a non-coding trinucleotide repeat sequence. The development of multiple and complementary animal models of disease has greatly contributed to clarifying the complex disease pathways mediated by toxic RNA molecules. RNA toxicity is not limited to myotonic dystrophy and spreads to an increasing number of human conditions, which share some unifying pathogenic events mediated by toxic RNA accumulation and disruption of RNA-binding proteins. The remarkable progress in the dissection of disease pathobiology resulted in the rational design of molecular therapies, which have been successfully tested in animal models. Toxic RNA diseases, and in particular myotonic dystrophy, clearly illustrate the critical contribution of animal models of disease in translational research: from gene mutation to disease mechanisms, and ultimately to therapy development. This article is part of a Special Issue entitled: Animal Models of Disease., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The toxicity of expanded transcripts in myotonic dystrophy type 1 (DM1) is mainly mediated by the disruption of alternative splicing. However, the detailed disease mechanisms in the central nervous system (CNS) have not been fully elucidated. In our recent study, we demonstrated that the accumulation of mutant transcripts in the CNS of a mouse model of DM1 disturbs splicing in a region-specific manner. We now discuss that the spatial- and temporal-regulated expression of splicing factors may contribute to the region-specific spliceopathy in DM1 brains. In the search for disease mechanisms operating in the CNS, we found that the expression of expanded CUG-containing RNA affects the expression and phosphorylation of synaptic vesicle proteins, possibly contributing to DM1 neurological phenotypes. Although mediated by splicing regulators with a described role in DM1, the misregulation of synaptic proteins was not associated with missplicing of their coding transcripts, supporting the view that DM1 mechanisms in the CNS have also far-reaching implications beyond the disruption of a splicing program.

Myotonic dystrophy type 1 is a complex multisystemic inherited disorder, which displays multiple debilitating neurological manifestations. Despite recent progress in the understanding of the molecular pathogenesis of myotonic dystrophy type 1 in skeletal muscle and heart, the pathways affected in the central nervous system are largely unknown. To address this question, we studied the only transgenic mouse line expressing CTG trinucleotide repeats in the central nervous system. These mice recreate molecular features of RNA toxicity, such as RNA foci accumulation and missplicing. They exhibit relevant behavioural and cognitive phenotypes, deficits in short-term synaptic plasticity, as well as changes in neurochemical levels. In the search for disease intermediates affected by disease mutation, a global proteomics approach revealed RAB3A upregulation and synapsin I hyperphosphorylation in the central nervous system of transgenic mice, transfected cells and post-mortem brains of patients with myotonic dystrophy type 1. These protein defects were associated with electrophysiological and behavioural deficits in mice and altered spontaneous neurosecretion in cell culture. Taking advantage of a relevant transgenic mouse of a complex human disease, we found a novel connection between physiological phenotypes and synaptic protein dysregulation, indicative of synaptic dysfunction in myotonic dystrophy type 1 brain pathology.

The RNA-mediated disease model for myotonic dystrophy (DM) proposes that microsatellite C(C)TG expansions express toxic RNAs that disrupt splicing regulation by altering MBNL1 and CELF1 activities. While this model explains DM manifestations in muscle, less is known about the effects of C(C)UG expression on the brain. Here, we report that Mbnl2 knockout mice develop several DM-associated central nervous system (CNS) features including abnormal REM sleep propensity and deficits in spatial memory. Mbnl2 is prominently expressed in the hippocampus and Mbnl2 knockouts show a decrease in NMDA receptor (NMDAR) synaptic transmission and impaired hippocampal synaptic plasticity. While Mbnl2 loss did not significantly alter target transcript levels in the hippocampus, misregulated splicing of hundreds of exons was detected using splicing microarrays, RNA-seq, and HITS-CLIP. Importantly, the majority of the Mbnl2-regulated exons examined were similarly misregulated in DM. We propose that major pathological features of the DM brain result from disruption of the MBNL2-mediated developmental splicing program., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Myotonic dystrophy type 1 (DM1) is caused by an unstable CTG repeat expansion in the 3'UTR of the DM protein kinase (DMPK) gene. DMPK transcripts carrying CUG expansions form nuclear foci and affect splicing regulation of various RNA transcripts. Furthermore, bidirectional transcription over the DMPK gene and non-conventional RNA translation of repeated transcripts have been described in DM1. It is clear now that this disease may involve multiple pathogenic pathways including changes in gene expression, RNA stability and splicing regulation, protein translation, and micro-RNA metabolism. We previously generated transgenic mice with 45-kb of the DM1 locus and >300 CTG repeats (DM300 mice). After successive breeding and a high level of CTG repeat instability, we obtained transgenic mice carrying >1,000 CTG (DMSXL mice). Here we described for the first time the expression pattern of the DMPK sense transcripts in DMSXL and human tissues. Interestingly, we also demonstrate that DMPK antisense transcripts are expressed in various DMSXL and human tissues, and that both sense and antisense transcripts accumulate in independent nuclear foci that do not co-localize together. Molecular features of DM1-associated RNA toxicity in DMSXL mice (such as foci accumulation and mild missplicing), were associated with high mortality, growth retardation, and muscle defects (abnormal histopathology, reduced muscle strength, and lower motor performances). We have found that lower levels of IGFBP-3 may contribute to DMSXL growth retardation, while increased proteasome activity may affect muscle function. These data demonstrate that the human DM1 locus carrying very large expansions induced a variety of molecular and physiological defects in transgenic mice, reflecting DM1 to a certain extent. As a result, DMSXL mice provide an animal tool to decipher various aspects of the disease mechanisms. In addition, these mice can be used to test the preclinical impact of systemic therapeutic strategies on molecular and physiological phenotypes., Competing Interests: The authors have declared that no competing interests exist.

To study the effect of DM1-associated CTG repeats on neuronal function, we developed a PC12 cell-based model that constitutively expresses the DMPK gene 3'-untranslated region with 90 CTG repeats (CTG90 cells). As CTG90 cells exhibit impaired neurite outgrowth and as microtubule-associated proteins (MAPs) are crucial for microtubule stability, we analyzed whether MAPs are a target of CTG repeats. NGF induces mRNA expression of Map2, Map1a and Map6 in control cells (PC12 cells transfected with the empty vector), but this induction is abolished for Map2 and Map1a in CTG90 cells. MAP2 and MAP6/STOP proteins decrease in NGF-treated CTG90 cells, whereas MAP1A increases. Data suggest that CTG repeats might alter somehow the expression of MAPs, which appears to be related with CTG90 cell-deficient neurite outgrowth. Decreased MAP2 levels found in the hippocampus of a DM1 mouse model indicates that targeting of MAPs expression by CTG repeats might be relevant to DM1.

DNA repeat expansions can result in the production of toxic RNA. RNA toxicity has been best characterised in the context of myotonic dystrophy. Nearly 20 mouse models have contributed significant and complementary insights into specific aspects of this novel disease mechanism. These models provide a unique resource to test pharmacological, anti-sense, and gene-therapy therapeutic strategies that target specific events of the pathobiological cascade. Further proof-of-principle concept studies and preclinical experiments require critical and thorough analysis of the multiple myotonic dystrophy transgenic lines available. This review provides in-depth assessment of the molecular and phenotypic features of these models and their contribution towards the dissection of disease mechanisms, and compares them with the human condition. More importantly, it provides critical assessment of their suitability and limitations for preclinical testing of emerging therapeutic strategies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Trinucleotide expansions cause disease by both protein- and RNA-mediated mechanisms. Unexpectedly, we discovered that CAG expansion constructs express homopolymeric polyglutamine, polyalanine, and polyserine proteins in the absence of an ATG start codon. This repeat-associated non-ATG translation (RAN translation) occurs across long, hairpin-forming repeats in transfected cells or when expansion constructs are integrated into the genome in lentiviral-transduced cells and brains. Additionally, we show that RAN translation across human spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1) CAG expansion transcripts results in the accumulation of SCA8 polyalanine and DM1 polyglutamine expansion proteins in previously established SCA8 and DM1 mouse models and human tissue. These results have implications for understanding fundamental mechanisms of gene expression. Moreover, these toxic, unexpected, homopolymeric proteins now should be considered in pathogenic models of microsatellite disorders.

Myotonic dystrophy (DM1) is a dominant autosomal multisystemic disorder caused by the expansion of an unstable CTG trinucleotide repeat in the 3' untranslated region of the DMPK gene. Nuclear accumulation of the enlarged CUG-containing DMPK transcripts has a deleterious effect on the regulation of alternative splicing of some RNAs and has a central role in causing the symptoms of DM1. In particular, Insulin Receptor (IR) mRNA splicing defects have been observed in the muscle of DM1 patients. In this study, we have investigated IR splicing in insulin-responsive tissues (i.e. skeletal muscles, adipose tissue, liver) and pancreas and we have studied glucose metabolism in mice carrying the human genomic DM1 region with expanded (>350 CTG) or normal (20 CTG) repeats and in wild-type mice. Mice carrying DM1 expansions displayed a tissue- and age-dependent abnormal regulation of IR mRNA splicing in all the tissues that we investigated. Furthermore, these mice showed a basal hyperglycemia and glucose intolerance which disappeared with age. Our findings show that deregulation of IR splicing due to the DM1 mutation can occur in different mouse tissues, suggesting that CTG repeat expansions might also result in IR misplicing not only in muscles but also in other tissues in DM1 patients.

Trinucleotide repeat expansions are the genetic cause of numerous human diseases, including fragile X mental retardation, Huntington disease, and myotonic dystrophy type 1. Disease severity and age of onset are critically linked to expansion size. Previous mouse models of repeat instability have not recreated large intergenerational expansions ("big jumps"), observed when the repeat is transmitted from one generation to the next, and have never attained the very large tract lengths possible in humans. Here, we describe dramatic intergenerational CTG*CAG repeat expansions of several hundred repeats in a transgenic mouse model of myotonic dystrophy type 1, resulting in increasingly severe phenotypic and molecular abnormalities. Homozygous mice carrying over 700 trinucleotide repeats on both alleles display severely reduced body size and splicing abnormalities, notably in the central nervous system. Our findings demonstrate that large intergenerational trinucleotide repeat expansions can be recreated in mice, and endorse the use of transgenic mouse models to refine our understanding of triplet repeat expansion and the resulting pathogenesis., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

A mounting number of inherited human disorders, including Huntington disease, myotonic dystrophy, fragile X syndrome, Friedreich ataxia and several spinocerebellar ataxias, have been associated with the expansion of unstable simple sequence DNA repeats. Despite a similar genetic basis, pathogenesis in these disorders is mediated by a variety of both loss and gain of function pathways. Thus, therapies targeted at downstream pathology are likely to be disease specific. Characteristically, disease-associated expanded alleles in these disorders are highly unstable in the germline and somatic cells, with a tendency towards further expansion. Whereas germline expansion accounts for the phenomenon of anticipation, tissue-specific, age-dependent somatic expansion may contribute towards the tissue-specificity and progressive nature of the symptoms. Thus, somatic expansion presents as a novel therapeutic target in these disorders. Suppression of somatic expansion should be therapeutically beneficial, whilst reductions in repeat length could be curative. It is well established that both cis- and trans-acting genetic modifiers play key roles in the control of repeat dynamics. Importantly, recent data have revealed that expanded CAG.CTG repeats are also sensitive to a variety of trans-acting chemical modifiers. These data provide an exciting proof of principle that drug induced suppression of somatic expansion might indeed be feasible. Moreover, as our understanding of the mechanism of expansion is refined more rational approaches to chemical intervention in the expansion pathway can be envisioned. For instance, the demonstration that expansion of CAG.CTG repeats is dependent on the Msh2, Msh3 and Pms2 genes, highlights components of the DNA mismatch repair pathway as therapeutic targets. In addition to potential therapeutic applications, the response of expanded simple repeats to genotoxic assault suggests such sequences could also have utility as bio-monitors of environmentally induced genetic damage in the soma.

The expansion of CAG.CTG repeat sequences is the cause of several inherited human disorders. Longer alleles are associated with an earlier age of onset and more severe symptoms, and are highly unstable in the germline and soma with a marked tendency towards repeat length gains. Germinal expansions underlie anticipation; whereas age-dependent, tissue-specific, expansion-biased somatic instability probably contributes toward the progressive nature and tissue-specificity of the symptoms. The mechanism(s) of repeat instability is not known, but recent data have implicated mismatch-repair (MMR) gene mutS homologues in driving expansion. To gain further insight into the expansion mechanism, we have determined the levels of somatic mosaicism of a transgenic expanded CAG.CTG repeat in mice deficient for the Pms2 MMR gene. Pms2 is a MutL homologue that plays a critical role in the downstream processing of DNA mismatches. The rate of somatic expansion was reduced by approximately 50% in Pms2-null mice. A higher frequency of rare, but very large, deletions was also detected in these animals. No significant differences were observed between Pms2(+/+) and Pms2(+/-) mice, indicating that a single functional Pms2 allele is sufficient to generate normal levels of somatic mosaicism. These findings reveal that as well as MMR enzymes that directly bind mismatched DNA, proteins that are subsequently recruited to the complex also play a central role in the accumulation of repeat length changes. These data suggest that somatic expansion results not by replication slippage, single stranded annealing or simple MutS-mediated stabilization of secondary structures, but by inappropriate DNA MMR.

Somatic mosaicism of repeat length is prominent in repeat expansion disorders such as Huntington disease and myotonic dystrophy. Somatic mosaicism is age-dependent, tissue-specific and expansion-biased, and likely contributes toward the tissue-specificity and progressive nature of the symptoms. We propose that therapies targeted at somatic repeat expansion may have general utility in these disorders. Specifically, suppression of somatic expansion would be expected to be therapeutic, whilst reversion of the expanded mutant repeat to within the normal range would be predicted to be curative. However, the effects of genotoxic agents on the mutational properties of specific nuclear genes are notoriously difficult to define. Nonetheless, we have determined that chronic exposure over a three month period to a number of genotoxic agents can alter the rate of triplet repeat expansion in whole populations of mammalian cells. Interestingly, high doses of caffeine increased the rate of expansion by approximately 60%. More importantly, cytosine arabinoside, ethidium bromide, 5-azacytidine and aspirin all significantly reduced the rate of expansion by from 35 to 75%. These data establish that drug induced suppression of somatic expansion is possible. These data also suggest that highly unstable expanded simple sequence repeats may act as sensitive reporters of genotoxic assault in the soma.

Small-pool polymerase chain reaction (PCR) constitutes the PCR amplification of a trinucleotide repeat in multiple small pools of input DNA containing in the order of from 0.5 to 200 genome equivalents. Products are resolved by agarose gel electrophoresis and detected by Southern blot hybridization under conditions that allow the identification of products derived from single-input molecules. The method allows the detailed quantification of the degree of repeat-length variation in a given sample, including the detection of common variants and those alleles present only in a small subset of cells. Detailed analysis of repeat dynamics is essential for a complete understanding of the molecular mechanisms that generate diversity and lead to disease in the unstable trinucleotide DNA repeat disorders.

Once into the expanded disease-associated range, trinucleotide repeat alleles become dramatically unstable in the germline and in somatic cells. The molecular mechanism(s) that underlie this unique form of dynamic mutation are poorly understood. Numerous transgenic mouse models of unstable trinucleotide repeats, which reconstitute the dynamic nature of somatic mosaicism observed in humans, have been generated. Given their easy accessibility, tissues from these mice can be collected to establish homogenous cell culture models of trinucleotide repeat dynamics. This chapter describes how such cultures can be established and maintained. Such in vitro systems may be useful to study relevant biological questions concerning fundamental triplet repeat metabolism. In particular, monitoring of repeat stability in cells growing under controlled conditions could help to clarify the relationship among the accumulation of repeat length variation, cell division rates, and DNA replication.