RNA toxicity in non-coding repeat expansion disorders.

Several neurodegenerative disorders like amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia (SCA) are caused by non-coding nucleotide repeat expansions. Different pathogenic mechanisms may underlie these non-coding repeat expansion disorders. While gain-of-function mechanisms, such as toxicity associated with expression of repeat RNA or toxicity associated with repeat-associated non-ATG (RAN) products, are most frequently connected with these disorders, loss-of-function mechanisms have also been implicated. We review the different pathways that have been linked to non-coding repeat expansion disorders such as C9ORF72-linked ALS/frontotemporal dementia (FTD), myotonic dystrophy, fragile X tremor/ataxia syndrome (FXTAS), SCA, and Huntington's disease-like 2. We discuss modes of RNA toxicity focusing on the identity and the interacting partners of the toxic RNA species. Using the C9ORF72 ALS/FTD paradigm, we further explore the efforts and different methods used to disentangle RNA vs. RAN toxicity. Overall, we conclude that there is ample evidence for a role of RNA toxicity in non-coding repeat expansion diseases.

Modeling muscle regeneration in RNA toxicity mice.

RNA toxicity underlies the pathogenesis of disorders such as myotonic dystrophy type 1 (DM1). Muscular dystrophy is a key element of the pathology of DM1. The means by which RNA toxicity causes muscular dystrophy in DM1 is unclear. Here, we have used the DM200 mouse model of RNA toxicity due to the expression of a mutant DMPK 3'UTR mRNA to model the effects of RNA toxicity on muscle regeneration. Using a BaCl2-induced damage model, we find that RNA toxicity leads to decreased expression of PAX7, and decreased numbers of satellite cells, the stem cells of adult skeletal muscle (also known as MuSCs). This is associated with a delay in regenerative response, a lack of muscle fiber maturation and an inability to maintain a normal number of satellite cells. Repeated muscle damage also elicited key aspects of muscular dystrophy, including fat droplet deposition and increased fibrosis, and the results represent one of the first times to model these classic markers of dystrophic changes in the skeletal muscles of a mouse model of RNA toxicity. Using a ligand-conjugated antisense (LICA) oligonucleotide ASO targeting DMPK sequences for the first time in a mouse model of RNA toxicity in DM1, we find that treatment with IONIS 877864, which targets the DMPK 3'UTR mRNA, is efficacious in correcting the defects in regenerative response and the reductions in satellite cell numbers caused by RNA toxicity. These results demonstrate the possibilities for therapeutic interventions to mitigate the muscular dystrophy associated with RNA toxicity in DM1.

Decoding ALS: from genes to mechanism.

Amyotrophic lateral sclerosis (ALS) is a progressive and uniformly fatal neurodegenerative disease. A plethora of genetic factors have been identified that drive the degeneration of motor neurons in ALS, increase susceptibility to the disease or influence the rate of its progression. Emerging themes include dysfunction in RNA metabolism and protein homeostasis, with specific defects in nucleocytoplasmic trafficking, the induction of stress at the endoplasmic reticulum and impaired dynamics of ribonucleoprotein bodies such as RNA granules that assemble through liquid-liquid phase separation. Extraordinary progress in understanding the biology of ALS provides new reasons for optimism that meaningful therapies will be identified.

RNA-mediated toxicity in C9orf72 ALS and FTD.

A GGGGCC hexanucleotide repeat expansion in the first intron of C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Compelling evidence suggests that gain of toxicity from the bidirectionally transcribed repeat expanded RNAs plays a central role in disease pathogenesis. Two potential mechanisms have been proposed including RNA-mediated toxicity and/or the production of toxic dipeptide repeat proteins. In this review, we focus on the role of RNA mediated toxicity in ALS/FTD caused by the C9orf72 mutation and discuss arguments for and against this mechanism. In addition, we summarize how G4C2 repeat RNAs can elicit toxicity and potential therapeutic strategies to mitigate RNA-mediated toxicity.

Site-Selective Nucleation and Size Control of Gold Nanoparticle Photothermal Antennae on the Pore Structures of a Virus.

In this Article, we show that the surface of the bacteriophage Qß is equipped with natural ligands for the synthesis of small gold nanoparticles (AuNPs). By exploiting disulfides in the protein secondary structure and the geometry formed from the capsid quaternary structure, we find that we can produce regularly arrayed patterns of ∼6 nm AuNPs across the surface of the virus-like particle. Experimental and computational analyses provide insight into the formation and stability of this composite. We further show that the entrapped genetic material can hold upward of 500 molecules of the anticancer drug Doxorubicin without leaking and without interfering with the synthesis of the AuNPs. This direct nucleation of nanoparticles on the capsid allows for exceptional conduction of photothermal energy upon nanosecond laser irradiation. As a proof of principle, we demonstrate that this energy is capable of rapidly releasing the drug from the capsid without heating the bulk solution, allowing for highly targeted cell killing in vitro.

NKX2-5, a modifier of skeletal muscle pathology due to RNA toxicity.

RNA toxicity is implicated in a number of disorders; especially those associated with expanded repeat sequences, such as myotonic dystrophy (DM1). Previously, we have shown increased NKX2-5 expression in RNA toxicity associated with DM1. Here, we investigate the relationship between NKX2-5 expression and muscle pathology due to RNA toxicity. In skeletal muscle from mice with RNA toxicity and individuals with DM1, expression of Nkx2-5 or NKX2-5 and its downstream targets are significantly correlated with severity of histopathology. Using C2C12 myoblasts, we show that over-expression of NKX2-5 or mutant DMPK 3'UTR results in myogenic differentiation defects, which can be rescued by knockdown of Nkx2-5, despite continued toxic RNA expression. Furthermore, in a mouse model of NKX2-5 over-expression, we find defects in muscle regeneration after induced damage, similar to those seen in mice with RNA toxicity. Using mouse models of Nkx2-5 over-expression and depletion, we find that NKX2-5 levels modify disease phenotypes in mice with RNA toxicity.

Repeat expansion disease: progress and puzzles in disease pathogenesis.

Repeat expansion mutations cause at least 22 inherited neurological diseases. The complexity of repeat disease genetics and pathobiology has revealed unexpected shared themes and mechanistic pathways among the diseases, such as RNA toxicity. Also, investigation of the polyglutamine diseases has identified post-translational modification as a key step in the pathogenic cascade and has shown that the autophagy pathway has an important role in the degradation of misfolded proteins--two themes that are likely to be relevant to the entire neurodegeneration field. Insights from repeat disease research are catalysing new lines of study that should not only elucidate molecular mechanisms of disease but also highlight opportunities for therapeutic intervention for these currently untreatable disorders.

Reversible model of RNA toxicity and cardiac conduction defects in myotonic dystrophy.

Myotonic dystrophy (DM1), the most common muscular dystrophy in adults, is caused by an expanded (CTG)n tract in the 3' UTR of the gene encoding myotonic dystrophy protein kinase (DMPK), which results in nuclear entrapment of the 'toxic' mutant RNA and interacting RNA-binding proteins (such as MBNL1) in ribonuclear inclusions. It is unclear if therapy aimed at eliminating the toxin would be beneficial. To address this, we generated transgenic mice expressing the DMPK 3' UTR as part of an inducible RNA transcript encoding green fluorescent protein (GFP). We were surprised to find that mice overexpressing a normal DMPK 3' UTR mRNA reproduced cardinal features of myotonic dystrophy, including myotonia, cardiac conduction abnormalities, histopathology and RNA splicing defects in the absence of detectable nuclear inclusions. However, we observed increased levels of CUG-binding protein (CUG-BP1) in skeletal muscle, as seen in individuals with DM1. Notably, these effects were reversible in both mature skeletal and cardiac muscles by silencing transgene expression. These results represent the first in vivo proof of principle for a therapeutic strategy for treatment of myotonic dystrophy by ablating or silencing expression of the toxic RNA molecules.

RNA toxicity in human disease and animal models: from the uncovering of a new mechanism to the development of promising therapies.

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.

RNA toxicity is a component of ataxin-3 degeneration in Drosophila.

Polyglutamine (polyQ) diseases are a class of dominantly inherited neurodegenerative disorders caused by the expansion of a CAG repeat encoding glutamine within the coding region of the respective genes. The molecular and cellular pathways underlying polyQ-induced neurodegeneration are the focus of much research, and it is widely considered that toxic activities of the protein, resulting from the abnormally long polyQ tract, cause pathogenesis. Here we provide evidence for a pathogenic role of the CAG repeat RNA in polyQ toxicity using Drosophila. In a Drosophila screen for modifiers of polyQ degeneration induced by the spinocerebellar ataxia type 3 (SCA3) protein ataxin-3, we isolated an upregulation allele of muscleblind (mbl), a gene implicated in the RNA toxicity of CUG expansion diseases. Further analysis indicated that there may be a toxic role of the RNA in polyQ-induced degeneration. We tested the role of the RNA by altering the CAG repeat sequence to an interrupted CAACAG repeat within the polyQ-encoding region; this dramatically mitigated toxicity. In addition, expression of an untranslated CAG repeat of pathogenic length conferred neuronal degeneration. These studies reveal a role for the RNA in polyQ toxicity, highlighting common components in RNA-based and polyQ-protein-based trinucleotide repeat expansion diseases.

Perturbation of U2AF65/NXF1-mediated RNA nuclear export enhances RNA toxicity in polyQ diseases.

Expanded CAG RNA has recently been reported to contribute to neurotoxicity in polyglutamine (polyQ) degeneration. In this study, we showed that RNA carrying an expanded CAG repeat progressively accumulated in the cell nucleus of transgenic Drosophila that displayed degeneration. Our gene knockdown and mutant analyses demonstrated that reduction of U2AF50 function, a gene involved in RNA nuclear export, intensified nuclear accumulation of expanded CAG RNA and resulted in a concomitant exacerbation of expanded CAG RNA-mediated toxicity in vivo. We found that the human U2AF50 ortholog, U2AF65, interacted directly and specifically with expanded CAG RNA via its RRM3 domain. We further identified an RNA/protein complex that consisted of expanded CAG RNA, U2AF65 and the NXF1 nuclear export receptor. The U2AF65 protein served as an adaptor to link expanded CAG RNA to NXF1 for RNA export. Finally, we confirmed the nuclear accumulation of expanded CAG RNA in symptomatic polyQ transgenic mice and also observed a neurodevelopmental downregulation of U2AF65 protein levels in mice. Altogether, our findings demonstrate that the cell nucleus is a site where expanded CAG RNA exerts its toxicity. We also provide a novel mechanistic explanation to how perturbation of RNA nuclear export would contribute to polyQ degeneration.

Genes and pathways affected by CAG-repeat RNA-based toxicity in Drosophila.

Spinocerebellar ataxia type 3 is one of the polyglutamine (polyQ) diseases, which are caused by a CAG-repeat expansion within the coding region of the associated genes. The CAG repeat specifies glutamine, and the expanded polyQ domain mutation confers dominant toxicity on the protein. Traditionally, studies have focused on protein toxicity in polyQ disease mechanisms. Recent findings, however, demonstrate that the CAG-repeat RNA, which encodes the toxic polyQ protein, also contributes to the disease in Drosophila. To provide insights into the nature of the RNA toxicity, we extracted brain-enriched RNA from flies expressing a toxic CAG-repeat mRNA (CAG100) and a non-toxic interrupted CAA/G mRNA repeat (CAA/G105) for microarray analysis. This approach identified 160 genes that are differentially expressed specifically in CAG100 flies. Functional annotation clustering analysis revealed several broad ontologies enriched in the CAG100 gene list, including iron ion binding and nucleotide binding. Intriguingly, transcripts for the Hsp70 genes, a powerful suppressor of polyQ and other human neurodegenerative diseases, were also upregulated. We therefore tested and showed that upregulation of heat shock protein 70 mitigates CAG-repeat RNA toxicity. We then assessed whether other modifiers of the pathogenic, expanded Ataxin-3 polyQ protein could also modify the CAG-repeat RNA toxicity. This approach identified the co-chaperone Tpr2, the transcriptional regulator Dpld, and the RNA-binding protein Orb2 as modifiers of both polyQ protein toxicity and CAG-repeat RNA-based toxicity. These findings suggest an overlap in the mechanisms of RNA and protein-based toxicity, providing insights into the pathogenicity of the RNA in polyQ disease.

Expandable DNA repeats and human disease.

Nearly 30 hereditary disorders in humans result from an increase in the number of copies of simple repeats in genomic DNA. These DNA repeats seem to be predisposed to such expansion because they have unusual structural features, which disrupt the cellular replication, repair and recombination machineries. The presence of expanded DNA repeats alters gene expression in human cells, leading to disease. Surprisingly, many of these debilitating diseases are caused by repeat expansions in the non-coding regions of their resident genes. It is becoming clear that the peculiar structures of repeat-containing transcripts are at the heart of the pathogenesis of these diseases.

Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways.

RNA interference (RNAi) is a universal and evolutionarily conserved phenomenon of post-transcriptional gene silencing by means of sequence-specific mRNA degradation, triggered by small double-stranded RNAs. Because this mechanism can be efficiently induced in vivo by expressing target-complementary short hairpin RNA (shRNA) from non-viral and viral vectors, RNAi is attractive for functional genomics and human therapeutics. Here we systematically investigate the long-term effects of sustained high-level shRNA expression in livers of adult mice. Robust shRNA expression in all the hepatocytes after intravenous infusion was achieved with an optimized shRNA delivery vector based on duplex-DNA-containing adeno-associated virus type 8 (AAV8). An evaluation of 49 distinct AAV/shRNA vectors, unique in length and sequence and directed against six targets, showed that 36 resulted in dose-dependent liver injury, with 23 ultimately causing death. Morbidity was associated with the downregulation of liver-derived microRNAs (miRNAs), indicating possible competition of the latter with shRNAs for limiting cellular factors required for the processing of various small RNAs. In vitro and in vivo shRNA transfection studies implied that one such factor, shared by the shRNA/miRNA pathways and readily saturated, is the nuclear karyopherin exportin-5. Our findings have fundamental consequences for future RNAi-based strategies in animals and humans, because controlling intracellular shRNA expression levels will be imperative. However, the risk of oversaturating endogenous small RNA pathways can be minimized by optimizing shRNA dose and sequence, as exemplified here by our report of persistent and therapeutic RNAi against human hepatitis B virus in vivo.

Muscleblind participates in RNA toxicity of expanded CAG and CUG repeats in Caenorhabditis elegans.

We have utilized Caenorhabditis elegans as a model to investigate the toxicity and underlying mechanism of untranslated CAG repeats in comparison to CUG repeats. Our results indicate that CAG repeats can be toxic at the RNA level in a length-dependent manner, similar to that of CUG repeats. Both CAG and CUG repeats of toxic length form nuclear foci and co-localize with C. elegans muscleblind (CeMBL), implying that CeMBL may play a role in repeat RNA toxicity. Consistently, the phenotypes of worms expressing toxic CAG and CUG repeats, including shortened life span and reduced motility rate, were partially reversed by CeMbl over-expression. These results provide the first experimental evidence to show that the RNA toxicity induced by expanded CAG and CUG repeats can be mediated, at least in part, through the functional alteration of muscleblind in worms.

Responses of increasingly complex intestinal epithelium in vitro models to bacterial toll-like receptor agonists.

The intestine fulfills roles in the uptake of nutrients and water regulation and acts as a gatekeeper for the intestinal microbiome. For the latter, the intestinal gut barrier system is able to respond to a broad range of bacterial antigens, generally through Toll-like receptor (TLR) signaling pathways. To test the capacity of various in vitro intestinal models, we studied IL-8 secretion, as a marker of pro-inflammatory response through the TLR pathway, in a Caco-2 monoculture, Caco-2/HT29-MTX di-culture, Caco-2/HT29-MTX/HMVEC-d tri-culture and in a HT29-p monoculture in response to exposure to various TLR agonists. Twenty-one-day-old differentiated cells in Transwells were exposed to Pam3CSK4 (TLR1/2), lipopolysaccharide (TLR4), single-stranded RNA (TLR7/8), Poly(i:C) (TLR3) and flagellin (TLR5) for 24 h. In all systems IL-8 secretion was increased in response to flagellin exposure, with HT29-p cells also responding to Poly(I:C) exposure. All other agonists did not induce an IL-8 response in the tested in vitro models, indicating that the specific TLRs are either not present or not functional in these models. This highlights the need for careful selection of in vitro models when studying intestinal immune responses and the need for improved in vitro models that better recapitulate intestinal immune responses.

Neurotoxic effect of the complex of the ovine prion protein (OvPrP(C)) and RNA on the cultured rat cortical neurons.

Prion diseases are conformational diseases, many factors are involved in altering the conformation of prion, such as RNA, DNA, pH, and copper etc. However the neurotoxic mechanism of prion diseases is not clear yet. The aim of this study is to investigate the effect of the nucleoprotein complex of RNA and recombinant ovine prion protein (OvPrP(C)) on the cultured rat cortical neurons in vitro. Our previous study revealed that the nucleoprotein complex (OvPrP(C)-RNA) is characterized with high ß sheet conformation and proteinase K resistance. Here we found that the OvPrP(C)-RNA induced marked neuronal cell death by the MTT (3-(4,5-dimethyl-thiazole -2-yl)-2,5-diphenyl -tetrazolium bromide) and TUNEL (TdT mediated biotin-dUTP nicked-end labeling) assay, and the neurotoxic effects were confirmed by testing the content of Bcl-2 Associated X protein (Bax) in the immunoprecipitation assay and Western blot assay. Compared to the control group, there is no significant difference of active Bax or total Bax after RNA alone treatment or OvPrP(C) alone treatment, but the OvPrP(C)-RNA induced significant increases of active Bax level, while the contents of total Bax had no obvious changes after OvPrP(C)-RNA treatment. The results suggested that OvPrP(C)-RNA is neurotoxic in vitro, which added further evidence to the current understanding of mechanism of cellular injury by RNA molecules for transformation of the PrP(C) to PrP(Sc).

Degradation of Toxic RNA in Myotonic Dystrophy Using Gapmer Antisense Oligonucleotides.

Myotonic dystrophy (DM) types 1 (DM1) and 2 (DM2) are caused by autosomal dominant gain-of-function RNA which are, in turn, created by the expansion of repeat sequences in the DMPK and ZNF9 genes, respectively. The expansions are highly unstable and biased for further expansion in somatic cells and across generations. Despite the different genes involved, DM1 and DM2 share several clinical features due to having the similar underlying mechanism of repetitive RNA-mediated toxicity. Both disorders manifest as multisystemic conditions with features including myotonia, cataract development, and abnormalities in cardiac conduction. At present, there is no cure for DM and treatments mostly aim at symptom management. Among the therapeutics being developed, antisense therapy using gapmers is one of the most promising. Compared to other antisense oligonucleotides, gapmers maintain the ability to induce RNase H cleavage while having enhanced target binding affinity and nuclease resistance. This chapter will consolidate the different strategies studied thus far to develop a treatment for DM1 through the targeting of toxic repetitive RNA using gapmers.

Interferon mediated neuroinflammation in polyglutamine disease is not caused by RNA toxicity.

Polyglutamine diseases are neurodegenerative diseases that occur due to the expansion of CAG repeat regions in coding sequences of genes. Previously, we have shown the formation of large protein aggregates along with activation of the interferon pathway leading to apoptosis in a cellular model of SCA17. Here, we corroborate our previous results in a tetracycline-inducible model of SCA17. Interferon gamma and lambda were upregulated in 59Q-TBP expressing cells as compared to 16Q-TBP expressing cells. Besides interferon-stimulated genes, the SCA17 model and Huntington's mice brain samples showed upregulation of RNA sensors. However, in this improved model interferon pathway activation and apoptosis preceded the formation of large polyglutamine aggregates, suggesting a role for CAG repeat RNA or soluble protein aggregates. A polyglutamine minus mutant of TBP, expressing polyCAG mRNA, was created by site directed mutagenesis of 10 potential start codons. Neither this long CAG embedded mRNA nor short polyCAG RNA could induce interferon pathway genes or cause apoptosis. polyQ-TBP induced the expression of canonical RNA sensors but the downstream transcription factor, IRF3, showed a muted response. We found that expanded CAG repeat RNA is not sufficient to account for the neuronal apoptosis. Neuronal cells sense expanded CAG repeats embedded in messenger RNAs of protein-coding genes. However, polyglutamine containing protein is responsible for the interferon-mediated neuroinflammation and cell death seen in polyglutamine disease. Thus, we delineate the inflammatory role of CAG repeats in the mRNA from the resulting polyglutamine tract in the protein. Embedded in messenger RNAs of protein-coding regions, the cell senses CAG repeat expansion and induces the expression of RNA sensors and interferon-stimulated genes.