Deciphering the C9ORF72-DPR aggregation pathology in neurodegeneration using optogenetic and in vitro analyses of modifiers

Background

C9ORF72 hexanucleotide repeat expansion is a frequent genetic cause of frontotemporal lobar degeneration/amyotrophic lateral sclerosis (FTD/ALS), and yet the mechanisms of DPR-mediated neurodegeneration remain debated and not fully understood. Both sense and antisense repeated-associated RNA transcripts can be translated in all reading frames via non-canonical repeat-associated non-AUG (RAN) translation, resulting in the synthesis of five dipeptide-repeat (DPR) proteins: poly-GA, -GR, -PR, -PA and -GP. Proteinaceous inclusions in neurons consisting of aggregated DPRs is a histopathological hallmark in FTD/ALS-C9 cases. Poly-PR and -GR, the arginine-containing DPRs (R-DPRs), are believed to be the most toxic DPR species. DPRs in patient tissue are highly insoluble, and inclusion load correlates with insolubility. In line with this, some DPRs readily undergo condensation/aggregation in vitro. DPR aggregate/inclusion formation have proven difficult to achieve in cellular models. In the multitude of cellular models reported to date, including neuronal ones, only poly-GA aggregation could be reproduced, whereas the remaining four DPR species typically displayed diffuse distribution. More recently, production of “chimeric” DPRs, due to ribosomal frameshifting, was described. However differential properties of these DPR species as compared to “pure DPRs”, remain to be characterised.

Several DPR toxicity modifiers have been reported. Positively charged R-DPRs can bind RNA which is negatively charged and affect properties of RNA-rich complexes, such as ribosomes. R-DPRs can be methylated on arginines, and this modification can alter their aggregation properties and modify toxicity – evidence for this was obtained in patients. Despite these recent insights, our knowledge of the modifiers of DPR aggregation and toxicity is still rudimentary, largely due to the lack of appropriate cellular models. Recently, the primary supervisor’s lab developed an optogenetic approach to modeling DPR aggregation in cells (manuscript in preparation). In addition, the lab is currently establishing two in vitro approaches for the analysis of DPR aggregation in cell-free systems.

In this PhD project, we propose to use these in vitro and cellular state-of-the-art platforms to unveil the relationships between DPR aggregation and its cellular effects, to answer the question as to whether or not DPR aggregation contributes to neurodegeneration.

Project hypothesis

We hypothesise that R-DPR aggregation exacerbates their toxicity and is significantly modulated by RNA and arginine methylation. We also hypothesise that chimeric and pure DPRs have different aggregation properties and that their aggregation profile is linked to their toxicity.

Aim, objectives and approaches

The aim of this project is to comprehensively characterise the profiles of pure and chimeric DPR aggregation in vitro and in cells, at high resolution, linking them to the known modifiers and cellular readouts of toxicity.

Objectives:

1. To refine and extend the optoDPR platform available in the lab via the use of longer repeats, a less aggregate-prone optogenetic module, inducible expression and inclusion of chimeric DPRs. Genetic constructs (codon-optimised) with longer repeat lengths (60-100) will be generated and tested. A less oligomerisation-prone optogenetic module will be utilised to eliminate potential background from baseline aggregation. Chimeric DPR constructs will be prepared and tested in cells in parallel with the pure DPRs. Light stimulation paradigms (laser, coupled with high-content imaging, and blue-light array) will be optimised. Stable cell lines with dox-inducible DPR expression will be generated.
2. To characterise the aggregation properties and downstream cellular effects of the aggregation of chimeric vs. pure DPRs. Detailed analysis of pure vs. chimeric DPR aggregation and the neurodegeneration-relevant RNA-binding proteins will be first performed in vitro, using an assay established in the lab (ImmuCon, manuscript submitted), and subsequently in cells. The following readouts will be analysed: TDP-43 condensation (nucleus and cytoplasm), nuclear pore integrity, cell survival and cryptic splicing. Super-resolution imaging will be performed to analyse the aggregate structure.
3. To determine the impact of putative DPR aggregation and toxicity modifiers, for pure and chimeric DPRs, in vitro and in cells. The role of arginine methylation in R-DPR aggregation will be studied using a two-pronged approach: i) in vitro with synthetic R-DPRs and ii) in cells, using optoDPRs and specific inhibitors of methyltransferases. Similarly, the modifying effect of RNA will be studied in vitro using synthetic oligonucleotides and in cells using transcription and splicing inhibitors.
4. To investigate the in vitro behaviour of pure vs. chimeric DPRs using flow induced dispersion analysis (FIDA). A novel in-solution in vitro approach for the analysis of protein assemblies, FIDA, will be applied to pure and chimeric DPRs (recombinant peptides) in the presence of modifiers. Subsequently, using the setup optimised for DPRs, the effect of these species on recombinant RNA-binding proteins will be analysed.

Training and development opportunities for the student

This project will provide a unique training opportunity to learn a well-rounded set of techniques for in-depth analysis of disease-linked proteins and their higher-order assemblies in vitro and in cellulo: optogenetics (cells); immune-based in vitro analysis of biological phase separation/condensation/aggregation; advanced imaging; as well as a novel analytical approach for in-solution studies of molecular interaction using flow induced dispersion (FIDA). The primary supervisor is a UKRI Future Leaders Fellow based in SITraN – a centre with an outstanding reputation for training PhD students. Co-supervisors on this project are two established researchers (professors) – experts in the field of C9ORF72 pathology/therapeutic targeting and protein/RNA biomolecular interactions, including post-translational protein modifications. The student will have an opportunity to attend lab meetings in all three labs and present at internal seminars at both departments.

Interviews are likely to be held between 4 – 15 March. Students must be able to start by October 2024.

Applications are open to students from both the UK and overseas. We anticipate competition for these studentships to be very intense. We would expect applicants to have an excellent undergraduate degree in a relevant discipline. We would also expect applicants to have completed or be undertaking a relevant master’s degree to a similar very high standard (or have equivalent research experience).

Please ensure you pick the Department/Division of Neuroscience when filling in your application form, regardless of where your first supervisor sits.