Molecular Neurobiology

, Volume 54, Issue 4, pp 3062–3077 | Cite as

C9ORF72 Regulates Stress Granule Formation and Its Deficiency Impairs Stress Granule Assembly, Hypersensitizing Cells to Stress

  • Niran Maharjan
  • Christina Künzli
  • Kilian Buthey
  • Smita SaxenaEmail author


Hexanucleotide repeat expansions in the C9ORF72 gene are causally associated with frontotemporal lobar dementia (FTLD) and/or amyotrophic lateral sclerosis (ALS). The physiological function of the normal C9ORF72 protein remains unclear. In this study, we characterized the subcellular localization of C9ORF72 to processing bodies (P-bodies) and its recruitment to stress granules (SGs) upon stress-related stimuli. Gain of function and loss of function experiments revealed that the long isoform of C9ORF72 protein regulates SG assembly. CRISPR/Cas9-mediated knockdown of C9ORF72 completely abolished SG formation, negatively impacted the expression of SG-associated proteins such as TIA-1 and HuR, and accelerated cell death. Loss of C9ORF72 expression further compromised cellular recovery responses after the removal of stress. Additionally, mimicking the pathogenic condition via the expression of hexanucleotide expansion upstream of C9ORF72 impaired the expression of the C9ORF72 protein, caused an abnormal accumulation of RNA foci, and led to the spontaneous formation of SGs. Our study identifies a novel function for normal C9ORF72 in SG assembly and sheds light into how the mutant expansions might impair SG formation and cellular-stress-related adaptive responses.


C9ORF72 ALS Motor neuron degeneration Stress granules Cell recovery 



This work was supported by the Synapsis Foundation, Frick foundation for ALS research, and Swiss National Science Foundation Professorship grant (150756), to S.S. We thank Gabor Morotz, Kings College London, UK, for help with cortical neuron cultures.

Supplementary material

12035_2016_9850_MOESM1_ESM.pdf (894 kb)
Fig. S1 a) Examination of the phosphorylation status of eIF2a (peIF2a) revealed that C9-CRISPR cells with or without DTT treatment displayed significantly lower amounts of phosphorylated eIF2a as compared to control condition. b) Quantification of peIF2a intensity in C9-CRISPR positive and negative cells in the presence or absence of DTT treatment. Each value represents the mean of ± SEM of 15 cells each from 3 independent experiments, ***p=<0.001. c) and d) Immunostaining of n2a cells overexpressing C9(LF) myc construct with TDP-43 and FUS/TLS, displayed normal nuclear localization and expression levels as in GFP transfected cells. (Scale bar = 4 μm for all image panels) (PDF 893 kb)
12035_2016_9850_MOESM2_ESM.pdf (682 kb)
Fig. S2 Transfection of cortical neurons with GFP construct having long repeat expansions led to the formation of TIA-1 positive SGs (arrow), (Scale bar = 4 μm) (PDF 682 kb)
12035_2016_9850_MOESM3_ESM.pdf (434 kb)
Fig. S3 a) Representative image of FISH against hexanucleotide repeat showing formation of RNA foci with increment in repeat length, (Scale bar = 4 μm). b) Western blot analysis of n2a cells transfected with GFP constructs with different hexanucleotide repeat expansion shows formation of dipeptide-repeat protein with higher repeat expansion (shown by arrow). c) Cell viability assay using Propidium Iodide (PI) after transfection of cortical neurons with GFP construct with different hexanucleotide repeat expansion length shows increase in dead cells with increment in repeat length. Each value represents the mean of ± SEM of 15 cells each from 3 independent experiments (PDF 433 kb)


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Institute of Cell BiologyUniversity of BernBernSwitzerland
  2. 2.Graduate School for Cellular and Biomedical SciencesUniversity of BernBernSwitzerland

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