RNA toxicity in non‐coding repeat expansion disorders
2019; Springer Nature; Volume: 39; Issue: 1 Linguagem: Inglês
10.15252/embj.2018101112
ISSN1460-2075
AutoresBart Swinnen, Wim Robberecht, Ludo Van Den Bosch,
Tópico(s)Neurogenetic and Muscular Disorders Research
ResumoReview13 November 2019Open Access RNA toxicity in non-coding repeat expansion disorders Bart Swinnen Bart Swinnen orcid.org/0000-0002-8098-880X Department of Neurosciences, Experimental Neurology, Leuven Brain Institute (LBI), KU Leuven – University of Leuven, Leuven, Belgium Laboratory of Neurobiology, VIB, Center for Brain & Disease Research, Leuven, Belgium Department of Neurology, University Hospitals Leuven, Leuven, Belgium Search for more papers by this author Wim Robberecht Wim Robberecht Department of Neurosciences, Experimental Neurology, Leuven Brain Institute (LBI), KU Leuven – University of Leuven, Leuven, Belgium Laboratory of Neurobiology, VIB, Center for Brain & Disease Research, Leuven, Belgium Department of Neurology, University Hospitals Leuven, Leuven, Belgium Search for more papers by this author Ludo Van Den Bosch Corresponding Author Ludo Van Den Bosch [email protected] orcid.org/0000-0003-0104-4067 Department of Neurosciences, Experimental Neurology, Leuven Brain Institute (LBI), KU Leuven – University of Leuven, Leuven, Belgium Laboratory of Neurobiology, VIB, Center for Brain & Disease Research, Leuven, Belgium Search for more papers by this author Bart Swinnen Bart Swinnen orcid.org/0000-0002-8098-880X Department of Neurosciences, Experimental Neurology, Leuven Brain Institute (LBI), KU Leuven – University of Leuven, Leuven, Belgium Laboratory of Neurobiology, VIB, Center for Brain & Disease Research, Leuven, Belgium Department of Neurology, University Hospitals Leuven, Leuven, Belgium Search for more papers by this author Wim Robberecht Wim Robberecht Department of Neurosciences, Experimental Neurology, Leuven Brain Institute (LBI), KU Leuven – University of Leuven, Leuven, Belgium Laboratory of Neurobiology, VIB, Center for Brain & Disease Research, Leuven, Belgium Department of Neurology, University Hospitals Leuven, Leuven, Belgium Search for more papers by this author Ludo Van Den Bosch Corresponding Author Ludo Van Den Bosch [email protected] orcid.org/0000-0003-0104-4067 Department of Neurosciences, Experimental Neurology, Leuven Brain Institute (LBI), KU Leuven – University of Leuven, Leuven, Belgium Laboratory of Neurobiology, VIB, Center for Brain & Disease Research, Leuven, Belgium Search for more papers by this author Author Information Bart Swinnen1,2,3, Wim Robberecht1,2,3 and Ludo Van Den Bosch *,1,2 1Department of Neurosciences, Experimental Neurology, Leuven Brain Institute (LBI), KU Leuven – University of Leuven, Leuven, Belgium 2Laboratory of Neurobiology, VIB, Center for Brain & Disease Research, Leuven, Belgium 3Department of Neurology, University Hospitals Leuven, Leuven, Belgium *Corresponding author. Tel: +32 16330681; E-mail: [email protected] The EMBO Journal (2020)39:e101112https://doi.org/10.15252/embj.2018101112 See the Glossary for abbreviations used in this article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract 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. Glossary ALS amyotrophic lateral sclerosis ASO antisense oligonucleotide DPR dipeptide repeat protein ELISA enzyme-linked immunosorbent assay FTD frontotemporal dementia FTLD frontotemporal lobe degeneration FXTAS fragile X tremor ataxia syndrome HDL-2 Huntington's disease-like 2 hnRNP heterogeneous nuclear ribonucleoprotein HRE hexanucleotide repeat expansion iMNs induced motor neurons mRNP messenger ribonucleoprotein PET positron emission tomography RAN repeat-associated non-ATG RBP RNA-binding protein rRBP repeat RNA-binding protein SCA spinocerebellar ataxia UPS ubiquitin-proteasome system UTR untranslated region Introduction Non-coding repeat expansion disorders Several neurodegenerative disorders are caused by a non-coding repeat expansion and are referred to as "non-coding repeat expansion disorders". So far, ten non-coding repeat expansion disorders have been described (Table 1). Most of them are adult-onset disorders and are phenotypically characterized by variable syndromes that include ataxia, cognitive dysfunction, motor neuron symptoms, and extra-neuronal involvement (Table 1). The most frequent clinical syndromes are myotonic dystrophy, amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD), and spinocerebellar ataxia (SCA; Table 1). The repeat expansion can be located in the promoter region, the 5′UTR (untranslated region), an intron, an alternate exon, or the 3′UTR of the respective gene (Table 1). The repeat sequence is variable, ranging from trinucleotide to hexanucleotide repeats, and is in general characterized by a high GC content (except SCA10 and SCA31). The range of the repeat length in healthy individuals normally does not exceed 30 repeats (Table 1). However, the range of the (unambiguously) pathogenic repeat lengths is highly variable and can be subdivided into three classes. First, repeats in SCA12 and Huntington's disease-like 2 (HDL-2) are usually not longer than 100 repeats (Margolis et al, 2004; Dong et al, 2015). Second, repeats generally do not exceed a few hundred (± 200) in fragile X tremor ataxia syndrome (FXTAS; O'Donnell & Warren, 2002) although longer repeats are associated with fragile X syndrome due to FMR1 loss of function (Verkerk et al, 1991). Third, repeats in all other diseases are mostly in the range of many hundreds up to a few thousands (Table 1). A clear length–phenotype correlation (i.e., more aggressive phenotype with increasing repeat length) has only been described in myotonic dystrophy types 1 and 2 (Udd & Krahe, 2012). Table 1. Non-coding repeat expansion disorders Overview of key features of all non-coding repeat expansion disorders. Clinical features include age at onset (i.e., the main life phase(s)) and phenotypic presentation(s). Genetic features include inheritance pattern, gene containing the repeat expansion, location of the repeat in the respective gene, and sequence of the repeat. Data regarding repeat length include repeat length in healthy individuals, unambiguously pathogenic repeat lengths, and correlation between repeat length and phenotype. For each disease, all theoretical RAN proteins are described, both in sense and in antisense direction. Regarding possible mechanisms, rRBPs implicated in the disease are listed. Abbreviations: AD, autosomal dominant; FXTAS, fragile X tremor ataxia syndrome; HDL, Huntington disease-like; MND, motor neuron degeneration; rRBPs, repeat RNA-binding proteins; SCA, spinocerebellar ataxia; XL, X-linked. a Complex pentanucleotide (TAGAA, TAAAA, TAAAATAGAA). b "alteration" of function. c Impurity of repeat (associated with seizures). Three possible mechanisms Three non-mutually exclusive mechanisms have been linked to the pathogenesis of non-coding repeat expansion disorders (Fig 1; example is given for C9ORF72 ALS/FTD (C9 ALS/FTD)). Repeat RNA can cause toxicity by directly interacting with repeat RNA-binding proteins and thereby compromising their normal function (Miller et al, 2000). This is referred to as "RNA toxicity". The repeat RNA might also induce toxicity indirectly by being translated into toxic proteins (Zu et al, 2011). This translation occurs in a non-ATG-dependent way called "repeat-associated non-ATG" (RAN) translation and generates RAN proteins (Zu et al, 2011). This type of toxicity is called "RAN toxicity". Depending on the repeat type (i.e., tri-, penta-, or hexanucleotide) and the specific repeat code, the RAN proteins consist of iterations of one, two, four, or five amino acids (Table 1). RAN proteins consisting of two amino acid repeats are called "dipeptide repeat proteins" (DPRs; Mackenzie et al, 2013). The presence of the repeat expansion might also lead to loss of function of the respective protein. This can be caused by decreased transcription initiation (e.g., epigenetic alterations), defective transcription (e.g., abortion), or increased mRNA degradation of the host gene (Todd et al, 2010; Haeusler et al, 2014). In the next chapter, we will give an overview of the current state of evidence regarding which mechanism is at play (RNA toxicity, RAN toxicity, and loss of function) in C9 ALS/FTD and the other non-coding repeat expansion disorders. Figure 1. Three possible pathogenic mechanisms of non-coding repeat expansion disorders—example given for C9ORF72 ALS/FTDFirst, the repeat expansion might interfere with the normal transcription of the C9ORF72 gene, leading to loss of function of the C9orf72 protein. Second, repeat-containing mRNAs might bind to various RNA-binding proteins, hence disturbing their normal function. This is called "RNA toxicity". Third, the repeat RNA itself might unconventionally be translated into peculiar toxic RAN peptides. This is called "RAN toxicity". Download figure Download PowerPoint Mechanisms in C9 ALS/FTD Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons (Swinnen & Robberecht, 2014). Clinically, patients present with painless subacute focal muscle weakness. The disease is rapidly progressive, generally leading to death in 3–5 years after symptom onset, and is unfortunately still incurable (Swinnen & Robberecht, 2014). The genetic landscape of ALS has been redrawn significantly in recent years. In most (± 90%) cases, ALS does not run in the family and is hence called "sporadic ALS" (sALS). The remainder (± 10%) of ALS patients, however, has an affected first degree, which is called "familial ALS" (fALS). In the latter, a monogenetic cause is evidently suspected. Indeed, such monogenetic mutation is identified in the majority (± 70%) of patients, with C9ORF72, FUS, TARDBP, and SOD1 being the most frequent ones (Renton et al, 2014). Surprisingly, a monogenetic cause is identified in ± 10% of sALS patients, most likely reflecting both de novo mutations and incomplete penetrance of mutations. Frontotemporal dementia (FTD) is the clinical dementia syndrome caused by frontotemporal lobe degeneration (FTLD) and is the second most common dementia after Alzheimer's disease (AD) in patients younger than 65 years (Olney et al, 2017). Amyotrophic lateral sclerosis and FTD are considered to constitute the extremes of a disease spectrum (Swinnen & Robberecht, 2014). The most frequent cause of ALS/FTD is a repeat expansion in the C9ORF72 gene (DeJesus-Hernandez et al, 2011; Renton et al, 2011, 2014). Its structure at the DNA, RNA, and protein level is depicted in Fig 2. Post-mortem examinations of C9 ALS/FTD cases reveal TDP-43 pathology (Mackenzie et al, 2014; Saberi et al, 2015), reminiscent of sporadic ALS cases, but also RAN proteins (i.e., dipeptide repeat proteins (DPRs)) (Zu et al, 2013) and RNA foci (DeJesus-Hernandez et al, 2011). Figure 2. C9ORF72 gene structure, transcription, and translationFour potentially pathogenic RNA species can be discerned. (1) At the pre-mRNA level, transcription of v1 and v3 might stall at the repeat region, resulting in the generation of abortive transcripts. (2) Transcription of the repeat region in the antisense direction generates antisense transcripts. (3) Ineffective splicing of intron 1 in transcripts v1 and v3 might result in intron 1-retaining transcripts. (4) Effective splicing of intron 1 in transcripts v1 and v3 might generate repeat-containing spliced-out intron 1. Download figure Download PowerPoint C9 ALS/FTD is mainly a gain-of-function disease C9 ALS/FTD is considered to be mainly driven by a gain-of-function mechanism, based on several observations. First, patients homozygous for the repeat expansion do not have an excessively aggressive clinical nor pathological phenotype which would have been expected in case of a loss-of-function mechanism (Cooper-Knock et al, 2013; Fratta et al, 2013). Second, while there is one study reporting a C9ORF72 coding mutation (Liu et al, 2016), none were found in a large cohort of ALS patients (Harms et al, 2013). Third, C9ORF72 promoter hypermethylation, associated with gene silencing, is neuroprotective as observed using cross-sectional and longitudinal neuroimaging data (McMillan et al, 2015). Fourth, several in vitro observations are not in line with a loss-of-function hypothesis. Most importantly, C9ORF72 transcript-directed antisense oligonucleotide (ASO) treatment resulting in decreased or dysfunctional C9ORF72 transcripts rescued the phenotype (e.g., glutamate-induced cell death (Donnelly et al, 2013) and transcriptional changes (Sareen et al, 2013), cfr. Table 5) in patient-derived induced motor neurons (iMNs). Furthermore, ASO-mediated C9ORF72 knockdown has no effect in control iMNs and neuronal primary cultures (Sareen et al, 2013; Sellier et al, 2016). Fifth, none of the C9orf72 knockout murine models develop a neurodegenerative phenotype (Lagier-Tourenne et al, 2013; Koppers et al, 2015; Atanasio et al, 2016; Burberry et al, 2016; Jiang et al, 2016; O'Rourke et al, 2016; Sudria-Lopez et al, 2016). Altogether, these data support the conclusion that C9ORF72 loss-of-function is not the main pathogenic driver suggesting mainly a gain-of-function mechanism; i.e., RNA and/or RAN toxicity. RNA toxicity in C9 ALS/FTD The exact nature of the repeat RNA present in RNA foci is still unclear. Four RNA species can be proposed (Fig 2). At the pre-mRNA level, transcription of transcripts v1 and v3 might stall at the repeat region, resulting in the generation of abortive transcripts. Transcription of the repeat region in the antisense direction also generates antisense transcripts. Ineffective splicing of intron 1 in transcripts v1 and v3 might result in intron 1-retaining transcripts. Finally, effective splicing of intron 1 in transcripts v1 and v3 might generate repeat-containing spliced-out intron 1. In general, repeat RNA is thought to form RNA foci that contain a cluster of repeat RNAs in complex with several RNA-binding proteins (Kumar et al, 2017). RNA foci are not restricted to neurons and are also found in astrocytes, microglia, and oligodendrocytes (Mizielinska et al, 2013). While RNA foci are mostly intranuclear, cytoplasmic RNA foci as well as RNA foci at the edge of the nucleus have also been observed (Mizielinska et al, 2013; Cooper-Knock et al, 2015b). RNA foci do not follow a rostrocaudal anatomical distribution as they are equally prevalent in the frontal cortex and in the spinal cord (DeJesus-Hernandez et al, 2011, 2017). RAN (DPR) toxicity in C9 ALS/FTD Both sense DPRs (GA, GR, GP) and antisense DPRs (PR, PA, GP) are formed with sense DPRs being more abundant than antisense and GA being the most frequently observed one (Mori et al, 2013a; Mackenzie et al, 2015). DPRs are exclusively present in neurons and are mainly detected as cytoplasmic aggregates (Ash et al, 2013; Mackenzie et al, 2013). All DPRs have a similar anatomical distribution and are most abundant in cortical and cerebellar regions and almost absent in brainstem and spinal cord (Davidson et al, 2016; Mackenzie et al, 2015). The toxic potential of the different DPRs has been examined comprehensively both in in vitro and in vivo disease models (Table 2). The potential mechanisms of this DPR toxicity have recently been reviewed (Freibaum & Taylor, 2017). Altogether, these data indicate that the arginine-rich DPRs can be highly toxic, at least in overexpression systems. Data also support the notion that GA can be toxic, while GP and PA are probably harmless (at least in the currently available disease models). Despite these in vitro and in vivo findings, it remains to be determined whether DPRs contribute to the pathogenesis of C9 ALS/FTD in humans. One should note that obtaining post-mortem support for DPR toxicity might be difficult as toxic DPR species might kill vulnerable motor neurons, hence leaving no trace to be uncovered. However, recent post-mortem data favor an association between DPRs and pathology as GR aggregates correlate with neurodegeneration and even colocalize with phospho-TDP-43, albeit with some variability (Saberi et al, 2018; Sakae et al, 2018). Moreover, whereas DPR load generally does not correlate with clinical severity (Gendron et al, 2015), cerebellar GP levels inversely correlate with cognitive scores and GA burden is inversely related with age at onset (Davidson et al, 2014). Nevertheless, several post-mortem observations are difficult to reconcile with DPR toxicity being the main culprit. Anatomical distribution of DPR aggregation post-mortem does not obviously correlate with neurodegeneration. In short, DPR load is highest in unaffected tissue (i.e., cerebellum) and lowest in affected tissue (i.e., spinal motor neurons; Gomez-Deza et al, 2015; Mackenzie et al, 2015). Moreover, coexistence of DPR and TDP-43 aggregates in a given cell is very rare (Mackenzie et al, 2013; Davidson et al, 2014). In addition, their predominant appearance in disease models is not in line with post-mortem findings (Mackenzie et al, 2015), especially the supposed nucleolar localization of GR and PR in disease models (Kwon et al, 2014; Wen et al, 2014). Nevertheless, the in vitro and in vivo models where GA forms cytoplasmic aggregates recapitulate post-mortem findings in C9 ALS/FTD patients (Mackenzie et al, 2015). Altogether and despite the DPR toxicity observed in both in vitro and in vivo systems, its pathogenic involvement in ALS is still an open question. Table 2. In vitro and in vivo toxicity of individual DPRs Numbers indicate the repeat lengths used. Abbreviations: ATP, adenosine triphosphate; LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PI, propidium iodide; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. a Motor neuron driver (OK371 or D42). b Pan-neuronal driver (elav or tubulin). c Eye driver (GMR). Loss of function in C9 ALS/FTD Despite C9 ALS/FTD being considered as mainly having a gain-of-function disease mechanism, some data indicate that C9ORF72 loss-of-function might contribute to disease pathogenesis and could enhance the gain-of-function mechanisms. In C9 ALS/FTD post-mortem brain tissue, C9ORF72 transcript levels are decreased by 50% (DeJesus-Hernandez et al, 2011; Gijselinck et al, 2012; van Blitterswijk et al, 2015). Similarly, decreased levels of the long C9orf72 protein isoform have been observed in the frontal and temporal cortex of C9 ALS/FTD patients (Waite et al, 2014; Xiao et al, 2015; Saberi et al, 2018). Knockdown of C9ORF72 in in vitro models is associated with autophagic dysfunction, including p62 accumulation, perinuclear clustering of swollen lysosomes, and TDP-43 aggregation (Sellier et al, 2016; Webster et al, 2016; Yang et al, 2016; Amick & Ferguson, 2017; Aoki et al, 2017). In patient-derived cells, the glutamate hypersensitivity phenotype is rescued by C9ORF72 overexpression as well as being recapitulated by C9ORF72 knockout in control cells (Shi et al, 2018). The mechanism of C9ORF72 loss of function as well as its contribution to disease pathogenesis has recently been reviewed in detail (Balendra & Isaacs, 2018). Essentially, C9ORF72 loss-of-function might contribute to pathology via its role in autophagy (Balendra & Isaacs, 2018; Webster et al, 2018). Mechanisms in other repeat expansion diseases Myotonic dystrophy type 1 Myotonic dystrophy type 1 is caused by CTG repeats in the 3′ UTR of DMPK and is mainly driven by RNA toxicity. CUG repeat RNA adopts a stable hairpin conformation (Tian et al, 2000) that forms nuclear RNA foci (Taneja et al, 1995; Davis et al, 1997). The RNA foci can sequester muscleblind-like (MBNL) proteins (Miller et al, 2000; Mankodi et al, 2001) leading to an imbalance between MBNL proteins and CUGBP1 (Lin et al, 2006; Kuyumcu-Martinez et al, 2007). This imbalance causes altered splicing of several mRNAs (e.g., the insulin receptor IR2, the chloride channel CLC2, and the cardiac troponin cTNNT2) in a tissue-dependent manner (Philips et al, 1998; Charlet-B et al, 2002; Mankodi et al, 2002; Fugier et al, 2011) explaining the various multisystemic phenotypic features. Missplicing has been confirmed in patient tissue and correlates with clinical features (Savkur et al, 2001; Fugier et al, 2011; Freyermuth et al, 2016), and MBNL1 dysfunction is regarded as the key mechanism involved in myotonic dystrophy 1. Observations in several mouse models [i.e., Mbnl1 knockout (Kanadia et al, 2003), Mbnl2 knockout (Hao et al, 2008), Cugbp1 overexpressing (Ho et al, 2005; Ward et al, 2010), and (CUG)n expressing (Mahadevan et al, 2006)] are consistent with this view. However, RNA toxicity might encompass more than missplicing alone. Several additional modes of action of CUG repeat RNA toxicity have been proposed, including miRNA misprocessing (Perbellini et al, 2011), transcriptional dysregulation (Botta et al, 2007), global translational inhibition through stress granule induction (Onishi et al, 2008; Huichalaf et al, 2010), and use of alternative polyadenylation sites (Batra et al, 2014). In addition to RNA toxicity, RAN toxicity has been suggested as well. While polyQ, derived from antisense CAG repeat RNA, has been found in patient material (Zu et al, 2011), its pathogenic contribution is still not clear. DMPK loss of function is unlikely given the absence of a clear relevant phenotype in Dmpk knockout mice (Jansen et al, 1996; Reddy et al, 1996). Myotonic dystrophy type 2 Myotonic dystrophy type 2 is caused by CCTG repeats in the intron of ZNF9 and resembles myotonic dystrophy type 1 in many regards. As a consequence, the underlying mechanism is believed to be very similar as well. Essentially, the CCUG repeat RNA leads to an MBNL-CUGBP1 imbalance (Salisbury et al, 2009; Jones et al, 2011), making RNA toxicity the prevailing mechanism. However, as both sense (LPAC) and antisense (QAGR) RAN peptides are present in post-mortem tissue and display in vitro toxicity (Zu et al, 2017), they might contribute to certain aspects of the disease as well. Additionally, CNBP loss of function might also play a role, as Cnbp-deficient mice develop key features of myotonic dystrophy (Chen et al, 2007). Fragile X tremor ataxia syndrome FXTAS is caused by CGG repeats in the 5′ UTR of the FMR1 gene. Pathological hallmarks of FXTAS consist of Purkinje cell loss and intranuclear ubiquitin-positive inclusions containing a polyglycine RAN peptide (Buijsen et al, 2014; Boivin et al, 2018). Loss of function is excluded as patients with Fragile X syndrome, caused by FMR1 loss-of-function due to very long (> 200) CGG repeats, do not develop any FXTAS features (Boivin et al, 2018). Moreover, FMR1 mRNA levels are even increased in FXTAS patients (Kenneson et al, 2001; Allen et al, 2004) and expression of CGG repeat RNA induces in vitro and in vivo neurotoxicity (Jin et al, 2003; Willemsen et al, 2003; Hukema et al, 2014), suggesting a primary gain-of-function mechanism. The CGG repeat RNA is able to adopt secondary structures (i.e., G-quadruplexes, duplexes and hairpins; Malgowska et al, 2014), which might compromise the function of various RNA-binding proteins like Pur-alpha (Jin et al, 2007), hnRNPA2/B1 (Sofola et al, 2007), CUGBP1 (Sofola et al, 2007), Sam68 (Sellier et al, 2010), and Drosha-DGCR8 (Sellier et al, 2013). The observation that overexpression of most of these proteins can rescue the phenotype in CGG Drosophila (Jin et al, 2007; Sofola et al, 2007; Sellier et al, 2013) supports a functional role of these proteins in FXTAS pathogenesis. However, RNA toxicity seems to be insufficient to explain FXTAS pathogenesis because of the following three observations. First, the repeat size is relatively short, compared to (mainly) RNA toxicity driven diseases (e.g., myotonic dystrophy—cfr. Table 1). Second, the large ubiquitin-positive intranuclear inclusions in FXTAS are reminiscent of aggregates typically seen in protein-mediated neurodegenerative disorders (e.g., Huntington's disease). Third, the toxicity of CGG constructs in Drosophila and mouse models seems to depend on FMRpolyG production (Todd et al, 2013; Sellier et al, 2017) suggesting a contribution of RAN toxicity. FMRpolyG has been found in patient-derived cells (Sellier et al, 2017), mouse models (Hukema et al, 2015; Sellier et al, 2017) and in post-mortem tissue (Todd et al, 2013; Buijsen et al, 2014), where it colocalizes with the ubiquitin-positive intranuclear inclusions (Todd et al, 2013). In several models, FMRpolyG displays a length-dependent propensity to aggregate in the nucleus (Todd et al, 2013; Sellier et al, 2017), and it is suggested to be neurotoxic by disturbing the ubiquitin-proteasome system (UPS) and the nuclear lamina structure (Oh et al, 2015; Sellier et al, 2017). Interestingly, antisense RAN proteins have also been observed in patient material (Krans et al, 2016). However, their pathogenic contribution has not yet been characterized. SCA8 The 3′UTR repeat expansion in SCA8 is bidirectionally transcribed (i.e., (CTG.CAG)n), complicating the quest for the underlying mechanism. In the ATXN8 strand, the CUG repeat RNA is believed to cause RNA toxicity via MBNL1 dysfunction, similar to what is seen in myotonic dystrophy. Supporting this, nuclear CUG RNA foci colocalize with MBNL1 in molecular layer interneurons of SCA8 patients and mouse models, and loss of Mbnl1 exacerbates the phenotype of SCA8 mice (Daughters et al, 2009). Additionally, splicing changes in a target of MBNL1 (i.e., GAT4) have been established in post-mortem tissue, validating the pathogenic relevance of RNA toxicity (Daughters et al, 2009). In the ATXN8OS strand, the CAG repeat RNA is translated into toxic polyglutamine and intranuclear polyglutamine inclusions have been seen in Purkinje cells and brainstem neurons of SCA8 mice and post-mortem tissue (Moseley et al, 2006). Moreover, in post-mortem tissue polyserine derived from the ATXN8OS strand by RAN translation has been discovered in degenerating white matter regions (Ayhan et al, 2018). Loss of function of the host gene(s) seems unlikely, as individuals harboring a genomic deletion in the SCA8 region do not exhibit cerebellar degeneration (Mandrile et al, 2016). Moreover, CTG expression in Drosophila and mouse models leads to neurodegenerative phenotypes further supporting a gain-of-function mechanism (Moseley et al, 2006; Tripathi et al, 2016). Therefore, SCA8 seems to be mainly driven by two gain-of-function mechanisms, being RNA and RAN toxicity, arising from bidirectional repeat RNAs. SCA10 SCA10 is caused by ATTCT repeats in the intron of ATXN10, and a gain-of-function mechanism has been proposed due to two main observations. First, an ATXN10 loss-of-function mechanism is unlikely as ATXN10 transcript levels are unaltered in SCA10 patients (Wakamiya et al, 2006), as heterozygous Atxn10 knockout mice display no abnormalities (Wakamiya et al, 2006) and as loss-of-function ATXN10 mutations do not give rise to a SCA10 phenotype in humans (Keren et al, 2010). Second, in vitro and in vivo (mainly mouse) models overexpressing ATTCT repeat constructs exhibit phenotypes resembling SCA10 (White et al, 2010; White et al, 2012). The exact nature of this gain of function is still unclear, but current data suggest RNA toxicity as the prevailing mechanism. The repeat expansion is spliced out and adopts a hairpin structure (Handa et al, 2005; Park et al, 2015) that binds hnRNPK in vitro and forms nuclear and cytoplasmic RNA foci in patient-derived cells which colocalize with hnRNPK (White et al, 2010). Furthermore, hnRNPK overexpression rescues in vitro ATTCT toxicity (White et al, 2010), indicating that hnRNPK dysfunction is a key factor in SCA10. However, post-mortem examinations have not been performed yet and RAN peptides (i.e., poly(ILFYS)) have not been assessed leaving the role of RAN toxicity in SCA10 unclear. SCA12 SCA12 is caused by CAG repeats in the promoter region of PPP2R2B that encodes a subunit of the phosphatase PP2A. The repeat is located in the promoter region of one (of many) protein isoforms, leading to increased promoter ac
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