Investigation of repeat-associated non-AUG translation and dipeptide repeat proteins in C9orf72-associated ALS/FTD
Lead Research Organisation:
University College London
Department Name: Institute of Neurology
Abstract
A hexanucleotide (G4C2) repeat expansion in the gene C9orf72 is the commonest known cause of both familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (DeJesus-Hernandez et al., 2011; Renton et al., 2011). C9orf72 is expressed in neurons where the repeat expansion is transcribed bi-directionally into aberrant repeat-containing RNA (DeJesus-Hernandez et al., 2011). Both sense and antisense transcripts aggregate into cytoplasmic and intranuclear RNA foci (Mizielinska et al., 2014). Despite the lack of a conventional start codon, these transcripts are translated through repeat-associated non-AUG (RAN) translation (Ash et al., 2013; Green et al., 2017).
This produces five dipeptide repeat proteins (DPRs): glycine-alanine (GA), glycine-arginine (GR), proline-arginine (PR), proline-alanine (PA) and glycine-proline (GP) (Mori et al., 2013). The DPRs form neuronal inclusions in widespread brain regions (Ash et al., 2013). Additionally, most patient brains show truncation and translocation of the RNA-binding protein TDP-43 from the nucleus to cytoplasmic aggregates, a phenomenon also common in non-C9orf72 ALS and FTD (Neumann et al., 2006).
Both RNA foci and DPRs, particularly the arginine-rich GR and PR, were toxic to multiple model systems (Donnelly et al., 2013; Mizielinska et al., 2014; Lopez-Gonzalez et al., 2016). Recent work in our lab showed that inhibiting RAN-translation in a Drosophila model through inserting periodic stop codons into the repeat region retained mRNA foci, but ameliorated neurodegeneration (Mizielinska et al., 2014; Moens et al., 2018). This suggests that reducing the translation of DPRs may be protective against repeat-mediated disease.
Following this work, our lab has collaborated with an industry partner to screen 20,000 small molecules (SMs) to identify those that reduce RAN-translation in HeLa cells overexpressing G4C2 repeats. SMs have a chance of passing the blood-brain barrier and are cheaper to produce compared to antisense oligonucleotides, another therapeutic option currently under development (Schludi and Edbauer, 2017; Donnelly et al., 2013).
Techniques used
Tissue culture, ELISA, SIMOA, immunocytochemistry, confocal microscopy, Southern blotting, protein synthesis assay (Click-iT AHA), RNA FISH, transfection/transduction
This produces five dipeptide repeat proteins (DPRs): glycine-alanine (GA), glycine-arginine (GR), proline-arginine (PR), proline-alanine (PA) and glycine-proline (GP) (Mori et al., 2013). The DPRs form neuronal inclusions in widespread brain regions (Ash et al., 2013). Additionally, most patient brains show truncation and translocation of the RNA-binding protein TDP-43 from the nucleus to cytoplasmic aggregates, a phenomenon also common in non-C9orf72 ALS and FTD (Neumann et al., 2006).
Both RNA foci and DPRs, particularly the arginine-rich GR and PR, were toxic to multiple model systems (Donnelly et al., 2013; Mizielinska et al., 2014; Lopez-Gonzalez et al., 2016). Recent work in our lab showed that inhibiting RAN-translation in a Drosophila model through inserting periodic stop codons into the repeat region retained mRNA foci, but ameliorated neurodegeneration (Mizielinska et al., 2014; Moens et al., 2018). This suggests that reducing the translation of DPRs may be protective against repeat-mediated disease.
Following this work, our lab has collaborated with an industry partner to screen 20,000 small molecules (SMs) to identify those that reduce RAN-translation in HeLa cells overexpressing G4C2 repeats. SMs have a chance of passing the blood-brain barrier and are cheaper to produce compared to antisense oligonucleotides, another therapeutic option currently under development (Schludi and Edbauer, 2017; Donnelly et al., 2013).
Techniques used
Tissue culture, ELISA, SIMOA, immunocytochemistry, confocal microscopy, Southern blotting, protein synthesis assay (Click-iT AHA), RNA FISH, transfection/transduction
| Title | "Inner Cinema": iPSC-neuron immunocytochemistry art prints |
| Description | In January 2020, I decided to make artwork by digitally manipulating immunocytochemistry images I had produced from patient iPSC-motor neurons I had differentiated in the lab. Friends, family and colleagues gave me a lot of encouragement and ordered prints from me right away. I have since got a UCL Business Advisor and I plan to develop this further into a for-profit business, with the aim of selling art prints, exhibiting in galleries, making pattern designs for fashion designers, etc. An important part of this endeavour will be to donate a share of the profits back into research. Another motivation behind this project is to use the appeal of the artwork to increase public understanding and interest in neurodegenerative disease research. |
| Type Of Art | Artwork |
| Year Produced | 2020 |
| Impact | This is a new endeavour which has not been extensively publicised yet. However, the plan is to grow this into a business to exhibit and sell works internationally. A portion of potential profits will eventually be donated back to neurodegenerative disease research. |
| URL | http://innercinema.bigcartel.com |
| Description | IBRO-RIKEN CBS Summer Program 2019 Travel Award |
| Amount | £523 (GBP) |
| Organisation | RIKEN |
| Sector | Public |
| Country | Japan |
| Start | 07/2019 |
| End | 07/2019 |
| Description | MRC-DTP Conference Travel Award for attending Society for Neuroscience Meeting 2019 |
| Amount | £1,309 (GBP) |
| Organisation | Medical Research Council (MRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 10/2019 |
| End | 10/2019 |
| Title | Screen development for measuring dipeptide repeat proteins in C9orf72 ALS/FTD patient iPSC-motor neurons |
| Description | As part of my PhD work, I miniaturised an already existing methodological pipeline for assaying dipeptide repeat proteins produced by repeat associated non-AUG (RAN) translation in C9orf72 ALS/FTD patient iPSC-motor neurons. I transported new patient iPSC lines from a collaboration with the Chandran lab at the University of Edinburgh to our lab in London, and after motor neuron differentiation, I established which of our available patient lines had the highest dipeptide repeat protein expression. I subsequently plated motor neurons from this line in a 96-well format tissue culture plate, which is conducive to screening, and then developed a method for reliably extracting enough protein from this cell line and plate format to run a screen. I initially developed this method to run a secondary screen on iPSC-neurons, to test small molecules which could inhibit RAN translation in a primary screen on transfected immortalised cells, though the primary screen did not produce any viable hits. This method would still be useful in the future as a small molecule or genetic screen. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2019 |
| Provided To Others? | No |
| Impact | The Isaacs lab now has the resources to run a RAN translation screen in patient iPSC motor neurons, though it has not been used with notable impact yet. |
| Title | Collection and characterisation of C9orf72 ALS/FTD patient iPSC lines |
| Description | I have collected C9orf72 patient iPSC lines with isogenic controls for the Isaacs lab from another lab at the University of Edinburgh as part of a collaboration (described under Collaborations). These lines, and other lines obtained from different groups, are now part of a sample collection of C9orf72 patient iPSC-derived motor neuron progenitor cells made by me. I have characterised these cell lines and assembled data on measures such as baseline dipeptide repeat protein production (a pathological feature associated with the C9orf72 mutation) from iPSC to neuronal progenitor cell. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2019 |
| Provided To Others? | No |
| Impact | This collection of samples and characteristics data is a significant resource in the Isaacs lab, and the basis of my experimental work on C9orf72 repeat associated non-AUG translation. A substantial sample size of patient iPSCs and neurons derived from them is important in a translational lab such as the Isaacs lab. C9orf72 patient cells provide a window in vitro into the actual developmental C9orf72 phenotype, and allows the study of endogenous repeat-associated non-AUG translation, which is not very well understood yet, and which cannot be completely recapitulated and studied using artificial overexpression constructs in other model organisms. |
| Description | SRSF1 cell permeable peptide collaboration |
| Organisation | University of Sheffield |
| Department | Sheffield Institute for Translational Neuroscience (SITraN) |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | I set up a collaboration with the Hautbergue lab at the University of Sheffield, to assay SRSF1-mimicking cell permeable peptides (CPPs) which were hypothesised to have an effect on repeat associated non-AUG (RAN) translation associated with C9orf72 mutation ALS/FTD. I added the CPPs to C9orf72 patient iPSC-motor neurons and subsequently immunoassayed the cell lysates for dipeptide repeat proteins produced through RAN translation. |
| Collaborator Contribution | The Hautbergue lab provided me with their SRSF1-mimicking cell permeable peptides (CPPs) and they advised on the final concentration to add to the cell medium. |
| Impact | The results have not been published but may become part of my PhD thesis, if the Hautbergue lab agrees. |
| Start Year | 2019 |
| Description | Sharing of C9orf72 ALS/FTD patient iPSC lines |
| Organisation | University College London |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Around October 2018, the Isaacs lab entered a collaboration with other research groups around the UK who are also working on the C9orf72 mutation associated with ALS/FTD and who use patient iPSCs to model the disease. We have invited them to send samples to us for immunoassaying of dipeptide repeat proteins produced through the mutation, which we carry out using our sensitive MSD assays developed in the lab. |
| Collaborator Contribution | Our partners have shared with us their iPSC lines with isogenic controls, expanding our patient iPSC line sample size. |
| Impact | This collaboration has not yet resulted in a publication, though my work on characterising the lines' baseline expression levels of C9orf72 ALS/FTD-associated dipeptide repeat proteins, from iPSC stage to motor neuron, is a part of my PhD thesis. |
| Start Year | 2018 |
| Description | Sharing of C9orf72 ALS/FTD patient iPSC lines |
| Organisation | University of Edinburgh |
| Department | Edinburgh Neuroscience |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Around October 2018, the Isaacs lab entered a collaboration with other research groups around the UK who are also working on the C9orf72 mutation associated with ALS/FTD and who use patient iPSCs to model the disease. We have invited them to send samples to us for immunoassaying of dipeptide repeat proteins produced through the mutation, which we carry out using our sensitive MSD assays developed in the lab. |
| Collaborator Contribution | Our partners have shared with us their iPSC lines with isogenic controls, expanding our patient iPSC line sample size. |
| Impact | This collaboration has not yet resulted in a publication, though my work on characterising the lines' baseline expression levels of C9orf72 ALS/FTD-associated dipeptide repeat proteins, from iPSC stage to motor neuron, is a part of my PhD thesis. |
| Start Year | 2018 |
| Description | Sharing of C9orf72 ALS/FTD patient iPSC lines |
| Organisation | University of Oxford |
| Department | Oxford Neuroscience |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Around October 2018, the Isaacs lab entered a collaboration with other research groups around the UK who are also working on the C9orf72 mutation associated with ALS/FTD and who use patient iPSCs to model the disease. We have invited them to send samples to us for immunoassaying of dipeptide repeat proteins produced through the mutation, which we carry out using our sensitive MSD assays developed in the lab. |
| Collaborator Contribution | Our partners have shared with us their iPSC lines with isogenic controls, expanding our patient iPSC line sample size. |
| Impact | This collaboration has not yet resulted in a publication, though my work on characterising the lines' baseline expression levels of C9orf72 ALS/FTD-associated dipeptide repeat proteins, from iPSC stage to motor neuron, is a part of my PhD thesis. |
| Start Year | 2018 |
| Description | Charitable donor visit to research institution |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Supporters |
| Results and Impact | I have on two occasions taken part in demonstrating the work we do in the Isaacs lab to charitable donors who have either already donated, or were interested in donating funds to the UK Dementia Research Institute at UCL where we are based. I explained C9orf72-mutation ALS/FTD in lay terms and demonstrated iPSC and neuronal cell cultures under brightfield microscope, and showed neuron immunocytochemistry on a confocal microscope. The donors seemed interested and impressed. While I have not spoken to the donors after the visit, I have received positive feedback from my PI Prof. Adrian Isaacs. |
| Year(s) Of Engagement Activity | 2019,2020 |