Abstract Amyotrophic lateral sclerosis (ALS) is a devastating and universally fatal neurodegenerative disease. Mutations in two related RNA-binding proteins, TDP-43 and FUS, that harbor prion-like domains, cause some forms of ALS. There are at least 213 human proteins harboring RNA recognition motifs, including FUS and TDP-43, raising the possibility that additional RNA-binding proteins might contribute to ALS pathogenesis. We performed a systematic survey of these proteins to find additional candidates similar to TDP-43 and FUS, followed by bioinformatics to predict prion-like domains in a subset of them. We sequenced one of these genes, TAF15, in patients with ALS and identified missense variants, which were absent in a large number of healthy controls. These disease-associated variants of TAF15 caused formation of cytoplasmic foci when expressed in primary cultures of spinal cord neurons. Very similar to TDP-43 and FUS, TAF15 aggregated in vitro and conferred neurodegeneration in Drosophila, with the ALS-linked variants having a more severe effect than wild type. Immunohistochemistry of postmortem spinal cord tissue revealed mislocalization of TAF15 in motor neurons of patients with ALS. We propose that aggregation-prone RNA-binding proteins might contribute very broadly to ALS pathogenesis and the genes identified in our yeast functional screen, coupled with prion-like domain prediction analysis, now provide a powerful resource to facilitate ALS disease gene discovery.

In the future, personalized genome sequencing will become routine, empowering us to define the genetic basis of many human diseases. Currently, however, complete genome sequencing for individuals to discover rare pathogenic mutations is still too costly and time consuming. Thus, more creative approaches are needed to accelerate the discovery of disease genes. Moreover, even once genes are revealed, the need for innovative approaches to elucidate causality remains critical.

ALS, also known as Lou Gehrig's disease, is a devastating adult-onset neurodegenerative disease that attacks upper and lower motor neurons (1). A progressive and ultimately fatal muscle paralysis ensues, usually causing death within 2–5 y of disease onset. ALS is mostly sporadic, but ∼10% of cases are familial. Pathogenic mutations in several genes have been linked to familial and sporadic ALS, including SOD1, TARDBP, FUS/TLS, VAPB, OPTN, VCP, and others (2). Two of these genes, TARDBP (TDP-43) and FUS/TLS (FUS) are notable because they encode related RNA-binding proteins that harbor a prion-like domain (3⇓⇓–6). Moreover, both of these proteins have been identified as components of pathological inclusions in neurons of patients with ALS (7⇓–9). Indeed, an emerging concept suggested by the association of FUS and TDP-43 to ALS is that defects in RNA metabolism might contribute to disease pathogenesis. These observations suggested an intriguing possibility: Could TDP-43 and FUS be just the tip of an iceberg? In other words, could other human RNA-binding proteins with properties similar to those of TDP-43 and FUS also contribute to ALS?

Here we report a simple yeast functional screen, followed by bioinformatics to predict prion-like domains, to identify human proteins with similar properties to TDP-43 and FUS. We then identify mutations in human patients with ALS in one gene from this screen, TAF15, which ranks with the highest prion-like domain score after FUS. Importantly, we show that TAF15 has similar in vitro and in vivo properties to TDP-43 and FUS. Moreover, the ALS-associated TAF15 mutations are more aggregation prone in vitro, have a more severe effect on lifespan than WT when expressed in Drosophila, and increase cytoplasmic mislocalization in mammalian spinal cord neurons. The identification of mutations in an additional RNA-binding protein harboring a prion-like domain further underscores a key role for RNA metabolism defects in ALS and suggests that this class of aggregation-prone RNA-binding proteins might contribute very broadly to ALS and perhaps other related neurodegenerative disorders.

Discussion In an effort to streamline the identification of new ALS genetic risk factors, we devised a simple yeast functional screen to define additional RNA-binding proteins with properties shared by the known ALS disease genes FUS and TDP-43. This screen resulted in the enrichment of 38 proteins that behave like FUS and TDP-43 in yeast (cytoplasmic inclusions and toxicity), 13 of which contain a predicted prion-like domain (Table 1). Indeed, the combination of yeast screen and prion prediction algorithm enabled us to significantly focus our list of candidate genes ∼10-fold. As evidence of the usefulness of this approach to define genes with a role in ALS, we identified patient-specific missense variants in one of these genes, TAF15, in five unrelated patients with ALS (three variants from our initial cohort of North American Caucasian patients with ALS, a fourth variant from a cohort of Swedish patients with ALS, and a fifth variant from a cohort of Australian patients with ALS). Further, we provide in vitro and in vivo evidence that TAF15 has functional properties similar to those of TDP-43 and FUS: It is intrinsically aggregation prone and can confer neurodegeneration in Drosophila and the ALS-linked variants can increase aggregation in vitro, decrease lifespan in Drosophila, and alter protein subcellular localization in spinal cord neurons. Although familial segregation could not be assessed, the absence of the variants in a large number of healthy controls, the shared structural evidence with known ALS genes, and functional in vitro and in vivo data strongly support the notion that these variants in TAF15 represent pathogenic disease mutations for ALS. Future studies will be required to determine the relative contribution of TAF15 variants to ALS risk compared with known genetic risk factors such as TDP-43, FUS, SOD1, and others. Our initial analyses with TAF15 in patients with ALS and control populations, as well as recent studies by Ticozzi and colleagues with TAF15 and the related gene EWSR1 (37), suggest that if indeed TAF15 mutations contribute to ALS, they will likely be rarer than FUS and TDP-43 mutations. However, as for all complicated human diseases there will very likely be common genetic contributors as well as rare genetic risk factors. For ALS, we propose that there may be a delicate balance in RNA processing within motor neurons such that slight perturbations from any one of several different aggregation-prone RNA-binding proteins could lead to neurodegeneration. An interesting additional concept that emerges from our findings is that perhaps variants in multiple RNA-binding proteins could synergize with each other to contribute to ALS. There are likely to be some variants that are extremely damaging and thus fully penetrant and aggressive on their own. Case in point: P525L and R495X mutations in FUS lead to relatively severe ALS clinical phenotypes and very early age of disease onset (18, 23), whereas other FUS mutations are less severe (e.g., R521G) (18). This result seems to be due to the effect of the mutations on FUS nuclear localization, with variants having the strongest effect on nuclear localization resulting in the earliest age of onset of ALS (18). Those aggressive FUS variants might sit at one end of a spectrum, with weaker variants at the other. Perhaps then the accumulation of multiple weaker variants in two, three, or more different aggregation-prone RNA-binding proteins (e.g., the top candidates listed in Table 1) might be required to tip the balance in RNA metabolism toward ALS. Future studies will be required to test this hypothesis and to better resolve the complexities of the ALS genetic landscape. These findings predict that additional aggregation-prone RRM or other RNA-binding proteins, like TAF15, FUS, and TDP-43, could contribute to ALS. Notably, the prion-like domain algorithm ranked FUS and TAF15 first and second of 213 RRM proteins, respectively, and ranked TDP-43 10th. We suggest that genes ranked third through ninth should now be given top priority for genetic analysis in populations of patients with ALS, especially EWSR1, which ranked third and is a close relative of both FUS and TAF15 (38). In addition to ALS, these candidates should also be examined in related clinico-pathological disorders including FTLD and inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD). For example, mutations in ALS genes TARDBP and FUS have been identified in patients with FTLD and mutations in an IBMPFD gene, VCP, have been identified in patients with ALS (39⇓–41). Next-generation sequencing and exon capture approaches will eventually become routine in personalized medicine (42⇓–44) and promise to identify all genetic contributors to ALS. Meanwhile, the list of ALS candidate genes that we provide here (Table 1 and Dataset S1), generated by the combination of the yeast functional screen and prion-like domain prediction, will be a powerful resource to jumpstart efforts to identify new genetic risk factors for ALS and spur innovative new diagnostic and therapeutic approaches.

Acknowledgments We thank the patients and their families for their dedication and for their invaluable contributions to this research. We thank the Packard Center for ALS Research at Johns Hopkins for their generosity and collaborative spirit. We thank contributors, including the Alzheimer's Disease Centers who collected samples used in this study. We thank C. Cecere for assistance in sample collection. We thank Rose Li, Phoebe Leboy, and Marisa Bartolomei for helpful suggestions on the manuscript. This work was supported by National Institutes of Health Director's New Innovator Awards 1DP2OD004417 (to A.D.G.) and 1DP2OD002177-01 (to J.S.); National Institutes of Health Grants 1R01NS065317 (to A.D.G.), 5R21NS067354-02 (to J.S.), AG17586 (to V.V.D., J.Q.T., and R.G.), AG10124 (to V.V.D. and J.Q.T.), P01-AG-09215 (to N.M.B.), NS056070 and NS072561 (to Z.M.), T32-AG00255 [to F.I. and V.M.-Y.L. (program director)], R01 AG26251-03A1 (to R.R.), R01 NS065782 (to R.R.), and P50 AG16574 (to R.R.); the University of Pennsylvania Institute on Aging and Alzheimer's Disease Core Center Pilot Grant Program (AG10124) (to V.V.D.); the ALS Association (R.R); a grant from the Packard Center for ALS Research at Johns Hopkins (to A.D.G. and J.S.); and an Ellison Medical Foundation New Scholar in Aging Award (to J.S.). A.D.G. is a Pew Scholar in the Biomedical Sciences, supported by The Pew Charitable Trusts, and a Rita Allen Scholar, supported by the Rita Allen Foundation. J.Q.T. is the William Maul Measey–Truman G. Schnabel, Jr., Professor of Geriatric Medicine and Gerontology. N.M.B. is an Investigator of the Howard Hughes Medical Institute. Samples from the National Cell Repository for Alzheimer's Disease, which receives government support under a cooperative agreement grant (U24 AG21886) awarded by the National Institute on Aging, were used in this study. In Australia, the work was supported by the National Health and Medical Research Council of Australia (1004670 and 511941) and a Peter Stearne grant from the Motor Neurone Disease Research Institute of Australia. This research was conducted while J.C. was an Ellison Medical Foundation/AFAR Postdoctoral Fellow.

Footnotes This Feature Article is part of a series identified by the Editorial Board as reporting findings of exceptional significance.

Author contributions: J.C., M.P.H., J.S., M.D.-H., T.N., F.I., H.-J.K., G.A.N., I.P.B., N.M.B., V.V.D., R.R., Z.M., and A.D.G. designed research; J.C., M.P.H., J.S., M.D.-H., R.E., R.O., X.A.L., D.R., N.J., D.H., J.E., A.C., Z.D., T.N., F.I., H.-J.K., J.A.S., K.L.W., D.C.-F., L.E., L.M., R.G., N.M.B., V.V.D., R.R., Z.M., and A.D.G. performed research; J.M.-P., C.I., K.B., N.R.G.-R., D.W.D., R.G.K., V.M.-Y.L., J.Q.T., A.L., W.R., and P.M.A. contributed new reagents/analytic tools; J.C., M.P.H., J.S., M.D.-H., R.E., Z.D., T.N., F.I., H.-J.K., I.P.B., O.D.K., N.M.B., V.V.D., R.R., Z.M., and A.D.G. analyzed data; and A.D.G. wrote the paper.

Conflict of interest statement: A.D.G. is an inventor on patents and patent applications that have been licensed to FoldRx Pharmaceuticals.

This article is a PNAS Direct Submission.

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