Report7 November 2017Open Access Transparent process G-quadruplex-binding small molecules ameliorate C9orf72 FTD/ALS pathology in vitro and in vivo Roberto Simone Roberto Simone Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK Search for more papers by this author Rubika Balendra Rubika Balendra Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK Department of Genetics, Evolution and Environment, Institute of Healthy Ageing, University College London, London, UK Search for more papers by this author Thomas G Moens Thomas G Moens Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK Department of Genetics, Evolution and Environment, Institute of Healthy Ageing, University College London, London, UK Search for more papers by this author Elisavet Preza Elisavet Preza Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK Search for more papers by this author Katherine M Wilson Katherine M Wilson Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK Search for more papers by this author Amanda Heslegrave Amanda Heslegrave Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK Search for more papers by this author Nathan S Woodling Nathan S Woodling Department of Genetics, Evolution and Environment, Institute of Healthy Ageing, University College London, London, UK Search for more papers by this author Teresa Niccoli Teresa Niccoli Department of Genetics, Evolution and Environment, Institute of Healthy Ageing, University College London, London, UK Search for more papers by this author Javier Gilbert-Jaramillo Javier Gilbert-Jaramillo Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK Search for more papers by this author Samir Abdelkarim Samir Abdelkarim MRC Centre for Neuromuscular Disease, UCL Institute of Neurology, London, UK Search for more papers by this author Emma L Clayton Emma L Clayton Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK Search for more papers by this author Mica Clarke Mica Clarke Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK Search for more papers by this author Marie-Therese Konrad Marie-Therese Konrad Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK Search for more papers by this author Andrew J Nicoll Andrew J Nicoll Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK MRC Prion Unit at UCL, Institute of Prion Diseases, London, UK Search for more papers by this author Jamie S Mitchell Jamie S Mitchell Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK Search for more papers by this author Andrea Calvo Andrea Calvo 'Rita Levi Montalcini' Department of Neuroscience, ALS Centre, University of Turin, Turin, Italy Search for more papers by this author Adriano Chio Adriano Chio 'Rita Levi Montalcini' Department of Neuroscience, ALS Centre, University of Turin, Turin, Italy Search for more papers by this author Henry Houlden Henry Houlden Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK Search for more papers by this author James M Polke James M Polke Neurogenetics Unit, UCL Institute of Neurology, London, UK Search for more papers by this author Mohamed A Ismail Mohamed A Ismail Department of Chemistry, Georgia State University, Atlanta, GA, USA Search for more papers by this author Chad E Stephens Chad E Stephens Department of Chemistry, Georgia State University, Atlanta, GA, USA Search for more papers by this author Tam Vo Tam Vo Department of Chemistry, Georgia State University, Atlanta, GA, USA Search for more papers by this author Abdelbasset A Farahat Abdelbasset A Farahat Department of Chemistry, Georgia State University, Atlanta, GA, USA Search for more papers by this author W David Wilson W David Wilson Department of Chemistry, Georgia State University, Atlanta, GA, USA Search for more papers by this author David W Boykin David W Boykin Department of Chemistry, Georgia State University, Atlanta, GA, USA Search for more papers by this author Henrik Zetterberg Henrik Zetterberg Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK Clinical Neurochemistry Laboratory, Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden UK Dementia Research Institute at UCL, UCL Institute of Neurology, London, UK Search for more papers by this author Linda Partridge Linda Partridge orcid.org/0000-0001-9615-0094 Department of Genetics, Evolution and Environment, Institute of Healthy Ageing, University College London, London, UK Max Planck Institute for Biology of Ageing, Cologne, Germany Search for more papers by this author Selina Wray Selina Wray Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK Search for more papers by this author Gary Parkinson Gary Parkinson UCL School of Pharmacy, London, UK Search for more papers by this author Stephen Neidle Corresponding Author Stephen Neidle [email protected] orcid.org/0000-0003-0622-6548 UCL School of Pharmacy, London, UK Search for more papers by this author Rickie Patani Corresponding Author Rickie Patani [email protected] orcid.org/0000-0002-3825-7675 Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK Search for more papers by this author Pietro Fratta Corresponding Author Pietro Fratta [email protected] orcid.org/0000-0002-8762-8188 MRC Centre for Neuromuscular Disease, UCL Institute of Neurology, London, UK Search for more papers by this author Adrian M Isaacs Corresponding Author Adrian M Isaacs [email protected] orcid.org/0000-0002-6820-5534 Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK UK Dementia Research Institute at UCL, UCL Institute of Neurology, London, UK Search for more papers by this author Roberto Simone Roberto Simone Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK Search for more papers by this author Rubika Balendra Rubika Balendra Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK Department of Genetics, Evolution and Environment, Institute of Healthy Ageing, University College London, London, UK Search for more papers by this author Thomas G Moens Thomas G Moens Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK Department of Genetics, Evolution and Environment, Institute of Healthy Ageing, University College London, London, UK Search for more papers by this author Elisavet Preza Elisavet Preza Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK Search for more papers by this author Katherine M Wilson Katherine M Wilson Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK Search for more papers by this author Amanda Heslegrave Amanda Heslegrave Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK Search for more papers by this author Nathan S Woodling Nathan S Woodling Department of Genetics, Evolution and Environment, Institute of Healthy Ageing, University College London, London, UK Search for more papers by this author Teresa Niccoli Teresa Niccoli Department of Genetics, Evolution and Environment, Institute of Healthy Ageing, University College London, London, UK Search for more papers by this author Javier Gilbert-Jaramillo Javier Gilbert-Jaramillo Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK Search for more papers by this author Samir Abdelkarim Samir Abdelkarim MRC Centre for Neuromuscular Disease, UCL Institute of Neurology, London, UK Search for more papers by this author Emma L Clayton Emma L Clayton Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK Search for more papers by this author Mica Clarke Mica Clarke Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK Search for more papers by this author Marie-Therese Konrad Marie-Therese Konrad Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK Search for more papers by this author Andrew J Nicoll Andrew J Nicoll Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK MRC Prion Unit at UCL, Institute of Prion Diseases, London, UK Search for more papers by this author Jamie S Mitchell Jamie S Mitchell Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK Search for more papers by this author Andrea Calvo Andrea Calvo 'Rita Levi Montalcini' Department of Neuroscience, ALS Centre, University of Turin, Turin, Italy Search for more papers by this author Adriano Chio Adriano Chio 'Rita Levi Montalcini' Department of Neuroscience, ALS Centre, University of Turin, Turin, Italy Search for more papers by this author Henry Houlden Henry Houlden Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK Search for more papers by this author James M Polke James M Polke Neurogenetics Unit, UCL Institute of Neurology, London, UK Search for more papers by this author Mohamed A Ismail Mohamed A Ismail Department of Chemistry, Georgia State University, Atlanta, GA, USA Search for more papers by this author Chad E Stephens Chad E Stephens Department of Chemistry, Georgia State University, Atlanta, GA, USA Search for more papers by this author Tam Vo Tam Vo Department of Chemistry, Georgia State University, Atlanta, GA, USA Search for more papers by this author Abdelbasset A Farahat Abdelbasset A Farahat Department of Chemistry, Georgia State University, Atlanta, GA, USA Search for more papers by this author W David Wilson W David Wilson Department of Chemistry, Georgia State University, Atlanta, GA, USA Search for more papers by this author David W Boykin David W Boykin Department of Chemistry, Georgia State University, Atlanta, GA, USA Search for more papers by this author Henrik Zetterberg Henrik Zetterberg Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK Clinical Neurochemistry Laboratory, Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden UK Dementia Research Institute at UCL, UCL Institute of Neurology, London, UK Search for more papers by this author Linda Partridge Linda Partridge orcid.org/0000-0001-9615-0094 Department of Genetics, Evolution and Environment, Institute of Healthy Ageing, University College London, London, UK Max Planck Institute for Biology of Ageing, Cologne, Germany Search for more papers by this author Selina Wray Selina Wray Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK Search for more papers by this author Gary Parkinson Gary Parkinson UCL School of Pharmacy, London, UK Search for more papers by this author Stephen Neidle Corresponding Author Stephen Neidle [email protected] orcid.org/0000-0003-0622-6548 UCL School of Pharmacy, London, UK Search for more papers by this author Rickie Patani Corresponding Author Rickie Patani [email protected] orcid.org/0000-0002-3825-7675 Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK Search for more papers by this author Pietro Fratta Corresponding Author Pietro Fratta [email protected] orcid.org/0000-0002-8762-8188 MRC Centre for Neuromuscular Disease, UCL Institute of Neurology, London, UK Search for more papers by this author Adrian M Isaacs Corresponding Author Adrian M Isaacs [email protected] orcid.org/0000-0002-6820-5534 Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK UK Dementia Research Institute at UCL, UCL Institute of Neurology, London, UK Search for more papers by this author Author Information Roberto Simone1,‡, Rubika Balendra1,2,‡, Thomas G Moens1,2, Elisavet Preza3, Katherine M Wilson1, Amanda Heslegrave3, Nathan S Woodling2, Teresa Niccoli2, Javier Gilbert-Jaramillo1,13, Samir Abdelkarim4, Emma L Clayton1, Mica Clarke1, Marie-Therese Konrad1, Andrew J Nicoll1,5, Jamie S Mitchell1, Andrea Calvo6, Adriano Chio6, Henry Houlden3, James M Polke7, Mohamed A Ismail8, Chad E Stephens8, Tam Vo8, Abdelbasset A Farahat8, W David Wilson8, David W Boykin8, Henrik Zetterberg3,9,10, Linda Partridge2,11, Selina Wray3, Gary Parkinson12, Stephen Neidle *,12, Rickie Patani *,3, Pietro Fratta *,4 and Adrian M Isaacs *,1,10 1Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK 2Department of Genetics, Evolution and Environment, Institute of Healthy Ageing, University College London, London, UK 3Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK 4MRC Centre for Neuromuscular Disease, UCL Institute of Neurology, London, UK 5MRC Prion Unit at UCL, Institute of Prion Diseases, London, UK 6'Rita Levi Montalcini' Department of Neuroscience, ALS Centre, University of Turin, Turin, Italy 7Neurogenetics Unit, UCL Institute of Neurology, London, UK 8Department of Chemistry, Georgia State University, Atlanta, GA, USA 9Clinical Neurochemistry Laboratory, Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden 10UK Dementia Research Institute at UCL, UCL Institute of Neurology, London, UK 11Max Planck Institute for Biology of Ageing, Cologne, Germany 12UCL School of Pharmacy, London, UK 13Present address: Facultad de Ciencias de la Vida, Escuela Superior Politécnica del Litoral, ESPOL, Guayaquil, Ecuador ‡These authors contributed equally to this work *Corresponding author. Tel: +44 207 7535969; E-mail: [email protected] *Corresponding author. Tel: +44 203 796 0000 Ext 10369; E-mail: [email protected] *Corresponding author. Tel: +44 203 4484112; E-mail: [email protected] *Corresponding author. Tel: +44 207 8375470; E-mail: [email protected] EMBO Mol Med (2018)10:22-31https://doi.org/10.15252/emmm.201707850 See also: MH Schludi & D Edbauer (January 2018) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Intronic GGGGCC repeat expansions in C9orf72 are the most common known cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), which are characterised by degeneration of cortical and motor neurons, respectively. Repeat expansions have been proposed to cause disease by both the repeat RNA forming foci that sequester RNA-binding proteins and through toxic dipeptide repeat proteins generated by repeat-associated non-ATG translation. GGGGCC repeat RNA folds into a G-quadruplex secondary structure, and we investigated whether targeting this structure is a potential therapeutic strategy. We performed a screen that identified three structurally related small molecules that specifically stabilise GGGGCC repeat G-quadruplex RNA. We investigated their effect in C9orf72 patient iPSC-derived motor and cortical neurons and show that they significantly reduce RNA foci burden and the levels of dipeptide repeat proteins. Furthermore, they also reduce dipeptide repeat proteins and improve survival in vivo, in GGGGCC repeat-expressing Drosophila. Therefore, small molecules that target GGGGCC repeat G-quadruplexes can ameliorate the two key pathologies associated with C9orf72 FTD/ALS. These data provide proof of principle that targeting GGGGCC repeat G-quadruplexes has therapeutic potential. Synopsis Small molecules targeting G-quadruplex GGGGCC repeat RNA are effective at ameliorating disease phenotypes in C9orf72 patient neurons, and in vivo phenotypes in C9orf72 flies. Therefore, targeting expanded GGGGCC RNA could be an effective therapeutic strategy for C9orf72 ALS and FTD. FRET based screen identifies small molecules that specifically bind to C9orf72 repeat RNA G-quadruplexes. Small molecules reduce RNA foci and dipeptide repeat proteins (DPRs) in C9orf72 patient neurons. G-quadruplex GGGGCC binding small molecule improves survival and reduces levels of the toxic DPR poly-GR in C9orf72 flies. Provides proof of principle for targeting GGGGCC RNA G-quadruplexes in C9orf72 FTD/ALS. Introduction Expansions of a GGGGCC repeat within the first intron of the C9orf72 gene are the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two rapidly progressive and incurable neurodegenerative disorders (Dejesus-Hernandez et al, 2011; Renton et al, 2011). While the general population carries < 30 GGGGCC (G4C2) repeats, with approximately 90% of individuals carrying < 8 repeats, large hexanucleotide repeat expansions (HRE), typically between 800 to > 4,000, are causative of ALS and FTD (Beck et al, 2013; van Blitterswijk et al, 2013). HREs are transcribed and the resulting RNA forms nuclear foci and can also be translated in all reading frames into dipeptide repeat proteins (DPRs) through a non-canonical process termed repeat-associated non-ATG (RAN) translation (Ash et al, 2013; Gendron et al, 2013; Lagier-Tourenne et al, 2013; Mizielinska et al, 2013; Mori et al, 2013a,b; Zu et al, 2013). Both repeat RNA and DPRs have been proposed to drive pathogenesis: foci can sequester RNA-binding proteins (RBPs) and therefore impair their function (Haeusler et al, 2016), while DPRs have been proven to be toxic in numerous disease models (Mizielinska et al, 2014; Wen et al, 2014; Zhang et al, 2016). G4C2 RNA can fold to form the highly stable non-canonical G-quadruplex (G-Q) conformation (Fratta et al, 2012), a four-stranded structure formed by the stacking of planar tetrads of four non-sequential guanosine residues (G-quartets). RNA G-Qs are able to form in vivo (Biffi et al, 2014), are enriched in RNA 5′ and 3′ UTRs (Huppert et al, 2008) and are known to be involved in regulating numerous RNA functions, including splicing, RNA transport and translation (Simone et al, 2015). As G-Qs can directly affect translation (Bugaut & Balasubramanian, 2012), and G4C2 G-Qs have been shown to specifically sequester disease-relevant RBPs (Haeusler et al, 2014; Conlon et al, 2016), they may play an important role in both RNA foci and DPR toxicity. Small molecules binding to both DNA and RNA G-Qs have been identified (Di Antonio et al, 2012), and due to the different conformation of RNA and DNA G-Q molecules, ligands preferentially targeting RNA G-Qs have also been developed (Biffi et al, 2014). Identification of molecules that specifically target C9orf72 repeat RNA could have therapeutic potential by shielding pathogenic interactions of the C9orf72 expanded RNA with RBPs, and/or by interfering with RAN translation. We report here a screen identifying molecules with selectivity for the G4C2 G-Q RNA and show they are able to reduce both RNA foci formation and RAN translation in C9orf72 iPSC-neuron models and C9orf72 repeat-expressing flies. Results Identification of small molecules that preferentially stabilise RNA G4C2 G-quadruplexes In order to identify small molecules that preferentially stabilise RNA G4C2 G-Qs, we adapted a FRET-based G-Q melting assay (Guyen et al, 2004; Collie et al, 2012), to specifically report G4C2 G-Q stabilisation. We have previously identified several novel G-Q-binding chemotypes in the chemical library from the anti-parasitic drug discovery programme at Georgia State University based on non-conjugated aromatic diamidines (Ohnmacht et al, 2014). Here, we screened 138 small molecules, 104 from this library and 34 previously established G-Q-binding compounds (Schultes et al, 2004; Moore et al, 2006). We measured their ability to stabilise (G4C2)4 oligonucleotides composed of either RNA or DNA folded into G-Qs. 44/138 small molecules increased the melting temperature (Tm) of (G4C2)4 RNA by greater than 13°C. Twelve of these showed at least 5°C greater stabilisation of (G4C2)4 RNA than (G4C2)4 DNA (Fig 1A). Strikingly, three of these molecules (DB1246, DB1247, DB1273, green circles in Fig 1A) had very similar chemical structures, differing by only two atoms (Fig 1B), indicating a genuine structure–function relationship. No other compounds in the compound set have similar features of two linked five-membered rings. Figure 1. Identification and structure of small molecules that preferentially stabilise RNA G4C2 repeat G-quadruplexes A FRET assay was used to measure the difference in melting temperature (ΔTm) of (G4C2)4 RNA or DNA G-Qs in the presence of 2 μM of 138 different small molecules. An increase in ΔTm indicates stabilisation of the G-Q. Small molecules are ranked on the x-axis according to their increasing thermal stabilisation of the DNA (G4C2)4 G-Q. Small molecules that preferentially stabilise RNA over DNA (G4C2)4 G-Qs reside in the upper part of the scatter plot, above the blue curve. An arbitrary ΔTm threshold of 13°C greater than vehicle (grey line) and a differential binding to RNA over DNA (ΔTmRNA–ΔTmDNA ≥ 5°C) were used to select candidate small molecules. Structures of the three compounds (DB1246, DB1247, DB1273), highlighted by green circles in (A), that show preferential binding to RNA (G4C2)4 G-Qs and were further characterised. FRET dose response of DB1246, DB1247 and DB1273 on stabilisation of RNA or DNA (G4C2)4 G-Qs. Temperature unfold CD spectra for (G4C2)4 RNA alone (which shows a characteristic G-Q structure with minima at 237 nm, maxima at 264 nm and no additional signal), or in the presence of 2 μM DB1246, DB1247 or DB1273. A characteristic induced CD spectrum, in the 350–550 nm region, is observed only in the presence of each small molecule, confirming that each of these three compounds are binding to (G4C2)4 RNA G-Qs. Data information: Data in (A) represent mean ± SD, n = 1 with three technical replicates. Data in (C) represent mean ± SD, n = 3 independent experiments. Data in (D) represent mean ± SD, n = 3 independent experiments. Download figure Download PowerPoint We therefore took these three small molecules forward for further testing. A stabilisation dose response for both sense (G4C2)4 (Fig 1C) and antisense (Appendix Fig S1) (G2C4)4 RNA and DNA oligonucleotides confirmed the preferential stabilisation of RNA G4C2 G-Qs by DB1246, DB1247 and DB1273. Circular dichroism (CD) spectroscopy confirmed that the (G4C2)4 RNA formed the expected parallel G-Q structure, with a minimum at 236 nm and a maximum at 264 nm. Each of the three small molecules caused the appearance of a characteristic additional induced CD signal in a separate region of the spectrum (350–550 nm; Fig 1D), while the small molecules alone gave no CD signal (Appendix Fig S2), thus confirming direct binding of DB1246, DB1247 and DB1273 to (G4C2)4 RNA G-Qs. We derived the Tm from our CD denaturation curves, which confirmed that (G4C2)4 RNA G-Qs were stabilised in the presence of each small molecule (Appendix Fig S3). These compounds bind to the RNA G4C2 repeat G-Q with high affinities, with Kd values in the range ca 200–400 nM (measurements by fluorescence anisotropy, Appendix Fig S4). These data identify, using FRET, CD and fluorescence anisotropy techniques, three structurally related small molecules DB1246, DB1247 and DB1273 that bind and stabilise RNA G4C2 G-Qs. RNA G4C2 G-quadruplex-binding small molecules reduce RNA foci in patient iPSC-neurons We next determined whether the small molecules could alleviate the key pathologies associated with C9orf72 G4C2 repeat expansion in patient-derived iPSC-neurons. We first characterised three patient iPSC lines (described in Appendix Table S1). We confirmed the presence of G4C2 repeat expansions by Southern blotting, which were maintained following differentiation into either motor or cortical neurons (Appendix Fig S5). Cortical neurons were prepared using an established differentiation protocol (Shi et al, 2012; Sposito et al, 2015). Spinal motor neurons were generated using dual-SMAD and GSK3β inhibition followed by caudal and ventral patterning to the pMN domain and finally promoting cell cycle exit using a Notch antagonist (Hall et al, 2017), yielding 90% pure motor neuron cultures (Fig EV1). The efficiency of differentiation did not differ between C9orf72 and control lines (Fig EV1). We also confirmed that RNA foci were specifically observed in patient iPSC-motor and iPSC-cortical neurons (Appendix Fig S6). We next performed a dose response in one patient iPSC-cortical neuron line to investigate the effect of DB1246, DB1247 and DB1273 on RNA foci formation. For each of the small molecules, a 1 μM treatment for 4 days reduced RNA foci burden (Fig EV2). We therefore treated cortical neurons derived from all three independent C9orf72 repeat expansion iPSC lines with 1 μM of DB1246, DB1247 or DB1273 for 4 days. Each small molecule significantly reduced RNA foci burden by approximately 50% (Fig 2). The same treatment on iPSC-motor neurons derived from the three independent patient lines showed that DB1246 and DB1273 reduced RNA foci burden, again by approximately 50%, while DB1247 did not significantly reduce RNA foci burden. These data show that small molecules that bind RNA G4C2 G-Qs can reduce RNA foci in both iPSC-motor and iPSC-cortical neurons. Click here to expand this figure. Figure EV1. Highly efficient differentiation of iPSCs into motor neurons is not affected by C9orf72 repeat expansion C9orf72 iPSC-motor neurons express choline acetyltransferase (ChAT) and beta-tubulin (TUJ1). Scale bar represents 10 μm. The percentage of total cells positive for ChAT was quantified after differentiation into motor neurons. No difference was observed between control and C9orf72-derived iPSC-motor neurons, with approximately 90% of all cells converted to ChAT-positive motor neurons. Two independent control iPSC lines and three independent C9orf72 lines were analysed, with one to two independent differentiations per line and > 100 cells quantified per line. Bars show the average and SEM. P > 0.05, Mann–Whitney U-test. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. G4C2 repeat G-quadruplex binding small molecules reduce RNA foci in C9orf72 patient iPSC-cortical neuronsG4C2 repeat RNA foci were detected by FISH and automatically quantified using image analysis software (Volocity, PerkinElmer). Representative images of RNA foci (red) within iPSC-cortical neurons; nuclei are visualised with DAPI (blue). Scale bar represents 10 μm. Quantification shows RNA foci are significantly reduced by all three small molecules, DB1246, DB1247 and DB1273, at a concentration of 1 μM for 4 days. Data are shown as the average and SD of the percentage of neurons containing RNA foci in 5–10 40× fields of view for one C9orf72 patient iPSC-cortical neuron line. ***P = 0.0006 (DB1246, 0.5 μM), ***P = 0.0001 (DB1247, 1 μM), ***P = 0.0001 (DB1273, 1 μM), one-way ANOVA with Dunnett's post hoc test versus DMSO. Download figure Download PowerPoint Figure 2. G4C2 repeat G-quadruplex binding small molecules reduce RNA foci in patient iPSC-cortical and iPSC-motor neurons Representative images of C9orf72 iPSC-cortical neurons treated with DMSO (vehicle control) or 1 μM of DB1246, DB1247 or DB1273, for 4 days. RNA foci are shown in red and nuclei (DAPI) in blue. Scale bar represents 10 μm. Quantification shows RNA foci are significantly reduced by all three compounds in iPSC-cortical neurons and by DB1246 and DB1273 in iPSC-motor neurons. Data are shown as the average percentage of neurons containing RNA foci relative to vehicle (DMSO). N = 3 independent C9orf72 patient lines with two to three inductions per line and at least 70 neurons counted per induction, data are shown as mean and SEM. *P < 0.05, **P < 0.01, ***P < 0.001, one-sample two-tailed t-test versus normalised control. For cortical neurons, *P = 0.0124 (DB1246), **P = 0.0065 (DB1247), **P = 0.0096 (DB1273). For motor neurons, ***P = 0.0004 (DB1246), **P = 0.0030 (DB1273). Download figure Download PowerPoint RNA G4C2 G-quadruplex-binding small molecules reduce dipeptide repeat proteins in patient iPSC-neurons without causing toxicity We next addressed whether DB1246, DB1247 or DB1273 could reduce the other major pathology in C9FTD/ALS—dipeptide repeat proteins. We established an MSD ELISA for poly(GP) and showed that poly(GP) is specifically detected in C9orf72 repeat expansion iPSC-motor and iPSC-cortical neurons (Appendix Fig S7). Treatment with 1 or 4 μM of DB1246, DB1247 or DB1273 for 4 days did not reduce poly(GP) levels in iPSC-motor neurons (Appendix Fig S8), indicating a differential response of RNA foci and poly(GP) to the small molecules. We therefore investigated 7-day treatments with a range of concentrations (8, 12 and 16 μM). We focused on iPSC-motor neurons due to their shorter differentiation protocol compared to cortical neurons. We found that the two small molecules that reduced RNA foci in iPSC-motor neurons, DB1246 and DB1273, also significantly reduced poly(GP) levels (Fig 3A). DB1273 was the most effective, significantly reducing poly(GP) at all concentrations, with greater than 50% reduction at 16 μM. Importantly, expression levels of C9orf72 transcripts were not affected by the same treatment (Fig 3B), indicating a direct effect on G4C2 repeat G-Q RNA, rather than a more general effect on transcription. We also measured the expression levels of MCM2 as it has a
