The C9ORF72 Gene, Implicated in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia, Encodes a Protein That Functions in Control of Endothelin and Glutamate Signaling

ABSTRACT

A GGGGCC repeat expansion in the C9ORF72 (C9) gene is the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Several mechanisms have been proposed to account for its toxicity, including the possibility that reduced C9 protein levels contribute to disease. To investigate this possibility, we examined the effects of reduced C9 levels in several cell systems. We first showed that C9 knockdown (KD) in U87 glioblastoma cells results in striking morphological changes, including vacuolization and alterations in cell size. Unexpectedly, RNA analysis revealed changes in expression of many genes, including genes involved in endothelin (EDN) signaling and immune system pathways and multiple glutamate cycling genes (e.g., EAAT2), which were verified in several cell models, including astrocytes and brain samples from C9-positive patients. Consistent with deregulation of the glutamate cycling genes, elevated intracellular glutamate was detected in both KD cells and patient astrocytes. Importantly, levels of mRNAs encoding EDN1 and its receptors, known to be elevated in ALS, were sharply increased by C9 KD, likely resulting from an observed activation of NF-κB signaling and/or a possible role of a C9 isoform in gene control.

KEYWORDS:

• amyotrophic lateral sclerosis
• endothelin
• glutamate
• glutamine
• NF-κB signaling
• transcription

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/MCB.00155-18.

ACKNOWLEDGMENTS

We give special thanks to Tristan Coady for providing plasmids encoding the long and short C9 isoforms. We also thank Ella Freulich and Veronica Li for their technical help. We thank Neil Shneider, Lei Lu (Department of Neurology, Center for Motor Neuron Biology and Disease, Columbia University, New York, NY), and the New York Brain Bank at Columbia University for providing valuable patient samples and patient-derived astrocytes. Additionally, we thank the Manley and Prives lab members for insightful discussions and help. We also thank Rachel Giesler (Novus Biologicals) for providing us with the C9 antibody and Bethyl Laboratories for providing the C9L- and C9S-specific antibodies.

V.F. was supported in part by NIH training grant T32 GM008798. J.L.M. acknowledges support from NIH grant R35 GM118136. B.T. was supported by NIH grant GM084089.

V.F. designed, performed, and analyzed the data for most of the experiments. P.R. performed cell fractionation and C9 ChIP assays and contributed to the manuscript writing process. M.H. and B.T. designed and performed the RNA-seq experiment. C.L., Z.G., and M.F.-O. assisted with experiments. J.L.M. and C.P. supervised the project. V.F. and J.L.M. conceived of the project, designed the studies, and wrote the manuscript. All authors edited and approved the final version of the manuscript.

We have no conflicts of interest to report.