Investigating Roles for C9orf72 in Regulating FIS1-mediated Cell Death and Survival

Details

Mutation in the non-coding region of C9orf72 gene represents by far the most common genetic cause identified for amyotrophic lateral sclerosis (ALS), a rare, lethal, devastating but currently incurable, unstoppable and irreversible motor neuron disease. Moreover, reduced levels of C9orf72 transcript and C9orf72 (C9) expression have been associated with ALS patients, and C9orf72 is important for initiating autophagy (1), suggesting a mechanistic link between impaired autophagy and ALS pathogenesis. Intriguingly mitochondrial fission 1 protein (FIS1) is identified as a genetic interacting partner for C9 (2). FIS1 is a protein key to mitochondrial quality control and cell death induction (3, 4), and we have recently demonstrated that essential role for FIS1 for mitochondrial autophagy (mitophagy) induced by iron chelation drug deferiprone (DFP)(5, 6), which is under clinical trials (NCT02164253 and NCT03293069) for ALS treatment (7). This has led two questions: i) is C9 involved in initiating DFP-induced mitophagy? and ii) does C9 have a role in cell death mediated by FIS1?

This project aims to answer the two questions. In addition to capitalising on the molecular biology, protein chemistry, imaging tools (e.g., mito-pHfluorin probe) and procedures, cell survival and death assays as well as model cell lines (WT and FIS1 knockout (KO) MEFs and WT and FIS1 KO HeLa cells) available in our lab, for manipulating cellular C9 levels, we will use validated C9 RNAi and constructs encoding C9 and C9 KO human cells as well as C9 and FIS1 binding defective mutants. With all these resources we are confident in determining the functional consequence due to the loss of C9orf72-Fis1 interaction in DFP-induced mitophagy and FIS1-induced cell death. To test the role of C9orf72 hexanucleotide in mitophagy induction by DFP and FIS1-mediated cell death, human C9 KO cells expressing C9orf72 WT and disease-causing C9orf72 mutant will be used through collaboration with Dr Hautbergue (8). Our mechanistic findings will be validated in motor-neuron-like NSC-34 cells and iPS-derived motor neurons. Outcomes from this project would reveal novel roles for C9 in mitophagy and cell death regulation and their relevance to ALS disease mechanisms.

For more information about the project, or to discuss a potential application, please contact Dr Chun Guo (https://www.sheffield.ac.uk/biosciences/people/academic-staff/chun-guo; c.guo@sheffield.ac.uk)

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Please apply for this project using this link: https://www.sheffield.ac.uk/postgraduate/phd/apply/applying

Funding Notes

Available for self or externally funded students only.

References

1. C. P. Webster et al., The C9orf72 protein interacts with Rab1a and the ULK1 complex to regulate initiation of autophagy. Embo j 35, 1656-1676 (2016). DOI: 10.15252/embj.201694401

2. N. Chai et al., Genome-wide synthetic lethal CRISPR screen identifies FIS1 as a genetic interactor of ALS-linked C9ORF72. Brain research 1728, 146601 (2020). DOI: 10.1016/j.brainres.2019.146601

3. R. Iwasawa, A. L. Mahul-Mellier, C. Datler, E. Pazarentzos, S. Grimm, Fis1 and Bap31 bridge the mitochondria-ER interface to establish a platform for apoptosis induction. The EMBO journal 30, 556-568 (2011). DOI: 10.1038/emboj.2010.346

4. Q. Shen et al., Mutations in Fis1 disrupt orderly disposal of defective mitochondria. Molecular biology of the cell 25, 145-159 (2014). DOI: 10.1091/mbc.E13-09-0525

5. E. Waters et al., The SUMO protease SENP3 regulates mitochondrial autophagy mediated by Fis1. EMBO reports 23, e48754 (2022). DOI: 10.15252/embr.201948754

6. K. A. Wilkinson, C. Guo, Iron chelation promotes mitophagy through SENP3-mediated deSUMOylation of FIS1. Autophagy 18, 1743-1745 (2022). DOI: 10.1080/15548627.2022.2046898

7. C. Petillon et al., The Relevancy of Data Regarding the Metabolism of Iron to Our Understanding of Deregulated Mechanisms in ALS; Hypotheses and Pitfalls. Front Neurosci 12, 1031 (2018). DOI: 10.3389/fnins.2018.01031

8. L. M. Castelli et al., SRSF1-dependent inhibition of C9ORF72-repeat RNA nuclear export: genome-wide mechanisms for neuroprotection in amyotrophic lateral sclerosis. Mol Neurodegener 16, 53 (2021). DOI: 10.1186/s13024-021-00475-y