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Generalized displacement of DNA- and RNA-binding factors mediates the toxicity of arginine-rich cell-penetrating peptides

V. Lafarga, O. Sirozh, I. Díaz-López, M. Hisaoka, E. Zarzuela, J. Boskovic, B. Jovanovic, R. Fernandez-Leiro, J. Muñoz, G. Stoecklin, I. Ventoso, O. Fernandez-Capetillo

Preprint posted on July 10, 2019 https://www.biorxiv.org/content/10.1101/441808v2.full

Arginine-rich peptides, including those associated with ALS, may result in toxicity through a reduction in RNA- and DNA- binding factors from mRNA and chromatin.

Selected by Emily Graves

Background

Cell penetrating peptides (CPPs) are used widely as a transfection agent when introducing nucleic acids, proteins or viruses in vitro. Early studies indicated that these peptides are more effective when they are arginine-rich, however this comes with a risk of toxicity of which the mechanism is unknown.

Researchers working on the neurodegenerative diseases Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal dementia (FTD) have established that one of the most common mutations responsible for causing these conditions occurs within the C9ORF72 gene (DeJesus-Hernandez et al. 2011; Renton et al. 2011). This mutation causes anywhere from a hundred to a thousand-fold expansion of the GGGGCC hexanucleotide found within the gene’s first intron. This expansion can be translated through non-AUG (RAN) translation within the cytoplasm, resulting in the production of arginine-rich dipeptide repeat (DPR) proteins. These DPR proteins have been shown to be toxic in both cell culture and animal models, as seen previously with the arginine rich CPPs.

This preprint evaluates this toxicity caused by arginine rich peptides further and aims to establish the mechanism through which this occurs.

 

Key findings

Addition of the arginine rich (PR)20 peptide negatively affects the binding of DNA and RNA substrates to mRNA

This study looks at how processes involving both RNA and DNA templates are negatively affected by the presence of (PR)20 peptides in vitro in a dose dependent manner. The authors hypothesised that if the arginine-rich peptides were coating the RNA and thereby inhibiting translation, as has been observed in the presence of C9ORF72 repeat expansion DPRs, then a similar effect would be observable in multiple different pathways involving an RNA intermediate (such as splicing or mRNA export). They demonstrate that this is indeed the case by adding (PR)20 repeats in a dose-dependent manner and measuring the effect by looking at outputs from certain pathways: firstly by measuring the rate of reverse transcription via the cDNA levels and also by tracking the percentage of RNA remaining following RNase A digestion.

The presence of (PR)20  also had a similar effect on DNA-based reactions, as shown by impairment of the DNA double strand break repair pathway in the presence of (PR)20 repeats.

 

Addition of non-coding nucleic acids in vitro can rescue some of the effects of (PR)20 repeat peptides

The authors showed that addition of non-coding RNA alongside the (PR)20 repeat peptides acts as a competitive inhibitor of the repeats, blocking them from interacting with RNA- and DNA- binding factors. Therefore improving cell viability as shown by both clonogenic assays and cell culture survival assays.

 

PROTAMINE, a protein found specifically in sperm which is naturally arginine rich, behaves in a similar way to (PR)20 repeat peptides

PROTAMINE has been commonly used in the past as a transfection agent, however it also causes high levels of toxicity. In this study they demonstrate that as an arginine-rich peptide PROTAMINE behaves in vitro in a very similar way to the (PR)20 repeat peptides; inhibiting both DNA and RNA based processes and showing similar nucleolar accumulation. This indicates that these properties are shared between all arginine rich peptides.

 

Exposing cells to arginine-rich CPPs can result in RNA- and DNA- binding factors moving away from cellular RNA and chromatin

The authors here have demonstrated through proteomic analysis that, upon addition of either PROTAMINE or (PR)20   repeat peptides, there is a significant decrease in the number of chromatin-bound factors. This trend could also be seen in RNA-bound factors. Together this data indicates a possible mechanism by which these arginine-rich CPPs are causing such extensive problems through the displacement of RNA- and DNA-binding factors.

 

Why I think the research is important

Dipeptide repeats are a major factor in disease pathogenesis in ALS, and the C9ORF72 repeat expansion is one of the most common known causes of both familial and sporadic ALS. Research into how these DPR proteins affect cellular pathways and function can give us insight into how they may be causing disease and ultimately, a means by which this can be treated. The authors indeed mention that there is precedence for the use of non-coding antisense oligonucleotides through injection into the cerebrospinal fluid within Spinal Muscular Atrophy therapies (Wood, Talbot, and Bowerman 2017), thereby pointing out the potential for such therapies to be adopted in ALS research.

It is interesting to see that arginine-rich polypeptides – which are naturally occurring within the genome – also possess these toxic traits associated with the DPRs observed in ALS patients. Perhaps the endogenous activity of these proteins could provide us with a different perspective on DPR production and toxicity, giving us a deeper understanding of the disease pathogenesis.

 

Future directions and questions for the authors

Future work could perhaps look at whether these observations are conserved in current models of C9ORF72-associated ALS/FTD and whether the same rescue phenotype can be observed upon the addition of non-coding oligonucleotides. It would be interesting to see whether the same effects of the shorter ssDNA oligonucleotides could be replicated in these models or even in vivo, or whether the longer sequences are needed in these conditions. This could be very interesting in terms of future therapies for ALS-FTD cases.

Another question I would have is whether any of these properties can also be observed in the other DPR populations which are known to be toxic (e.g. poly-GA). Although the arginine containing DPRs are known to be the most toxic but it could be interesting to see whether these properties are purely specific to the arginine-rich CPPs. Differences in the pathogenic mechanisms across multiple DPR subpopulations may ultimately make finding one universal treatment difficult.

 

 

References

DeJesus-Hernandez, Mariely, Ian R. Mackenzie, Bradley F. Boeve, Adam L. Boxer, Matt Baker, Nicola J. Rutherford, Alexandra M. Nicholson, et al. 2011. “Expanded GGGGCC Hexanucleotide Repeat in Noncoding Region of C9ORF72 Causes Chromosome 9p-Linked FTD and ALS.” Neuron 72 (2): 245–56. https://doi.org/10.1016/j.neuron.2011.09.011.

Renton, Alan E., Elisa Majounie, Adrian Waite, Javier Simón-Sánchez, Sara Rollinson, J. Raphael Gibbs, Jennifer C. Schymick, et al. 2011. “A Hexanucleotide Repeat Expansion in C9ORF72 Is the Cause of Chromosome 9p21-Linked ALS-FTD.” Neuron 72 (2): 257–68. https://doi.org/10.1016/j.neuron.2011.09.010.

Wood, Matthew J.A., Kevin Talbot, and Melissa Bowerman. 2017. “Spinal Muscular Atrophy: Antisense Oligonucleotide Therapy Opens the Door to an Integrated Therapeutic Landscape.” Human Molecular Genetics 26 (R2): R151–59. https://doi.org/10.1093/hmg/ddx215.

Tags: amyotrophic lateral sclerosis, arginine-rich peptides, c9orf72

Posted on: 30th July 2019

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  • Author's response

    Oscar Fernandez-Capetillo shared

    What we have learned is that the toxicity of oligoarginines is due to their extreme affinity for nucleic acids, which out-competes the majority of DNA- and RNA-binding factors. We think this model explains why virtually all reactions using nucleic acids (translation, transcription, export, splicing…) have been found to be altered in patients. We show that this can be alleviated by oligonucleotides that can bind to the peptides and therefore reduce the effective dose of the oligoarginines that is free to mess up with endogenous nucleic acids. While this works in vitro, taking this into an actual therapy seems like a big endeavour with many questions. Which oligos? What would be the effect of having too many unspecific oligos around? Will they be taken by the proper cells? Not trivial challenges.

    Nevertheless, our work is not focused on the therapy angle, it just tries to provide a simple model to explain why these arginine-rich peptides are toxic (and I think we suceed!). To share some more thoughts with you, I must admit that once we verified that our model seems to be correct, it was somewhat sad. If the toxicity of these peptides is due to competition with nucleic-acid binding proteins, my gut feeling tells me that this will be extremely challenging to overcome with chemicals. Thus, I am pessimistic about the prospect of a chemical molecule capable or reducing DPR toxicity, and I think the efforts should be placed in preventing its expression. To end with a positive note though, there are other things that could be tried to limit DPR toxicity in vivo, and we are definitely going to explore some of them…

    As for the toxicity, I think that considering all the evidence out-there, while some attention is still placed on poly-GAs and other models such as RNA toxicity, there are a few manuscripts that -in my opinion- clearly discard those models and strongly suggest that the toxicity linked to C9ORF72 mutations is due to the arginine-rich dipeptide repeats.

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