GEN-01: An Interval Look at Early Markers of Disease in Familial ALS

Ten of thousands of ORFs are hidden within genomes (1). They have eluded annotations because they are either small or within unsuspected locations. Thus, novel functional coding sequences (altORFs) are camouflaged within annotated ones (CDS) in a different reading frame (2). With the mechanism of the wild-type or ALS-linked mutated FUS toxicity still left unclear (3), we hypothesized an overlooked protein within the FUS gene may be at play. Methods: We used the proteogenomic resource, OpenProt (1), to query the unbiased coding potential of the FUS gene. We then combined proteomics, ribosome profiling, genomics, bioinformatics and cellular assays to functionally characterize a novel protein. We further validated our findings in a fruit fly model of ALS. Results: We discovered an altORF nested in the FUS CDS encoding a conserved 170 amino acid protein, altFUS. AltFUS is endogenously expressed in human tissues, notably in the motor cortex and motor neurons. Overexpression of wild-type FUS and/or amyotrophic lateral sclerosis-linked FUS mutants is known to trigger toxic mechanisms in different models. These include an inhibition of autophagy, loss of mitochondrial potential, and accumulation of cytoplasmic aggregates (4). We show here that altFUS, not FUS, is responsible for the inhibition of autophagy. AltFUS is also pivotal in the mechanisms leading to the mitochondrial potential loss and accumulation of cytoplasmic aggregates. Suppression of altFUS expression in a Drosophila model of FUS-related toxicity protects against neurodegeneration. Some mutations found in ALS patients are overlooked because of their synonymous effect on the FUS protein. Yet we showed they exert a deleterious effect via their missense consequence on the overlapping altFUS protein. These findings demonstrate that FUS is a bicistronic gene and suggest that both proteins, FUS and altFUS, cooperate in toxic mechanisms. Acknowledgements: This research was supported by CIHR grants MOP137056 and MOP-136962, by an ALS Canada Project Grant, and by a Canada Research Chair in Functional Proteomics and Discovery of Novel Proteins. References: (1) Brunet, M. A. et al. Nucleic Acids Res. (2019) PMID: 30299502. (2) Brunet, M. A., et al. Genome Res. (2018) PMID: 29626081. (3) Taylor, J. P., et al. Nature (2016) PMID: 27830784. (4) Nolan, M., et al. Acta Neuropathol. Commun. (2016) PMID: 27600654. 31st International Symposium on ALS/MND Theme 02 – Genetics and Genomics 6 GEN-07: Genetic and epigenetic disease modifiers in a C9orf72 family presenting with ALS, FTD or PD phenotypes PhD Antonia Ratti1,2, Dr Silvia Peverelli1, MD Elisabetta D'Adda3, PhD Claudia Colombrita1, MD Michele Gennuso3, MD PhD Alessandro Prelle4, MD PhD Vincenzo Silani1,5 1Irccs Istituto Auxologico Italiano, Cusano Milanino, Italy, 2Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Milano, Italy, 3UOC Neurologia-Stroke Unit, ASST Crema, Crema, Italy, 4UOC di Neurologia e Stroke Unit, ASST Ovest milanese, , Legnano (Mi), Italy, 5Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Milano, Italy Live Poster Session A, December 9, 2020, 5:10 PM 5:50