27 February 2009. Scientists have uncovered a fresh clue in the mystery that is amyotrophic lateral sclerosis. Unexpectedly, it comes from a gene first implicated in cancer, called fused in sarcoma (FUS). In back-to-back Science papers published today, researchers from Massachusetts General Hospital in Boston, and King’s College in London, U.K., report that 5 percent of people with familial ALS harbor a mutation in FUS. Its protein functions in RNA transcription, splicing and trafficking, and it is strikingly similar to the separate ALS-linked protein TDP-43. The parallels between the two proteins suggest to many scientists that ALS may be a disease of RNA mismanagement.
“This is the beginning of a new era, in which we need to look at RNA processing as a potential early pathogenic event in motor neuron death,” said Stanley Appel of the Methodist Hospital System in Houston, Texas, who was not involved in either study.
The U.S. and U.K. groups, led by Robert Brown and Christopher Shaw, respectively, have been collaborating in the hunt for this particular gene for the better part of a decade. Their combined data, from single nucleotide polymorphism analysis of two families with multiple cases of ALS, pointed to a mutation in chromosome 16. Thomas Kwiatkowski, first author on the U.S. paper, found a third family that allowed him to narrow down the locus and discover the FUS mutation. In London, Caroline Vance, joint first author along with Boris Rogelj, carried out the first in-vitro analysis that showed mutant FUS exits the nucleus to form abnormal aggregates in the cytoplasm of cultured cells.
This research took so long, in part, because false leads led the researchers down the wrong path before they were able to focus in on the FUS-containing locus. The target regions that SNP analysis identified in the first two families overlapped by 40 megabases (Ruddy et al., 2003; Sapp et al., 2003). That area was too large to sequence all the candidate genes. When Shaw and another collaborator each found another family with likely mutations in chromosome 16, they pooled the data to identify a much smaller region of overlap. But sequencing every gene in that region found no mutations, and the research dead-ended. Later, additional family members and further calculations showed that the third and fourth families’ mutations did not map to chromosome 16 after all, and the researchers were left with the impossibly long stretch of chromosome with which they started.
The break came when two sisters, both in the early stages of ALS, came to consult Kwiatkowski. They mentioned that their mother had had a similar condition, and that a third sister was developing muscle weakness. With 12 siblings, this Rhode Island family, originally from a small Cape Verde island off the western coast of Africa, provided a wealth of additional genetic samples. “This family showed a possible address which was exquisitely located on top of the chromosome 16 locus,” said Brown, who now works at the University of Massachusetts in Worchester. With the new data, the scientists were able to narrow their target region to four megabases, a manageable stretch of DNA.
Kwiatkowski identified 56 potentially relevant genes in that area. He included FUS, as a gene involved in genome stability, but did not consider it a likely candidate. The task of sequencing FUS, along with other less promising genes, fell to an undergraduate from Smith College, in Northampton, Massachusetts, who was working in the laboratory over the summer. The student, Alexandra Davis, returned to school before Kwiatkowski had time to analyze her results. When he finally looked at FUS, Kwiatkowski recalls, “I said, holy smokes, this is it!” After confirming his findings, he contacted Shaw’s lab, and the two groups sequenced the gene in their two families with chromosome 16 mutations, and in other people with familial ALS.
The Massachusetts group sequenced FUS in nearly 300 unrelated familial ALS cases, and the British scientists sequenced nearly 200 separate cases. All told, approximately 5 percent of people with familial ALS had a FUS mutation, Kwiatkowski said. Among people with familial ALS, approximately 20 percent have a mutation in superoxide dismutase 1, and an additional 5 percent have mutations in TARDBP, which encodes TDP-43. Other genes have also been linked to the disease, but only in a handful of cases.
The scientists found 15 different FUS mutations, mostly clustered in the carboxyl-terminal region of the protein. With rare exceptions, the mutant allele was dominant. Nearly 300 sporadic ALS cases contained no FUS mutations, the Brown group found. The two research groups sequenced FUS in nearly 2,000 control subjects, and found only one instance of a FUS mutation, in a person of Cape Verdean ancestry. “When you have back-to-back reports from different parts of the world and they come up with extremely similar findings…then you know for sure that it’s real,” Appel said.
FUS, also known as TLS (translated in liposarcoma), has many functions in healthy cells. It mends DNA breaks and binds to RNA to participate in transcription, splicing, and transporting mRNA out of the nucleus (reviewed by Law et al., 2006). None of these functions suggest an obvious connection to motor neuron viability. “No one, a priori, would have ever anticipated that it was related to ALS,” Appel said.
FUS protein primarily resides the nucleus, although it also transports mRNAs to other cellular destinations. Vance expressed mutant and wild-type forms of FUS in fibroblast and neuroblastoma cells, but did not expect to see a difference between the two. That’s because when she performed a similar experiment with TDP-43 mutants, their localization mirrored that of the wild-type. But the FUS mutant clearly shifted from the nucleus to the cytoplasm. Vance was so surprised, she recalls, that she asked a lab mate to confirm what she saw. Furthermore, FUS formed aggregates in the cytoplasm of motor neurons, the Shaw group discovered in postmortem tissue samples from three people who had FUS mutations.
What’s Next in FUS Research?
Some 90 percent of people with ALS have a sporadic form that is not inherited. Even so, the data about FUS, TDP-43, and SOD1 mutations may ultimately help scientists solve sporadic ALS as well. “Anytime we learn any other clue for ALS, it’s good for the field,” said Merit Cudkowicz, a neurologist specializing in ALS who is also at Massachusetts General Hospital but was not involved in the current research. Although the FUS mutation does not apply to many of her patients, she said, “it seems that it fits into a general theme of pathways that could be abnormal in ALS.” The 1993 discovery of SOD1 mutations (Rosen et al., 1993), Cudkowicz noted, led to an explosion of research on ALS, with new rodent models and new therapies to try. The recent TDP-43 and current FUS discoveries will likely do the same, she said, recruiting people who study those genes to consider ALS.
Next, Kwiatkowski and colleagues hope to develop model systems based on FUS mutations. A new mutant mouse would be a welcome addition for a field frustrated by drug candidates that look promising in mSOD1 mice but later fail in human trials. Cell culture models, Kwiatkowski said, will also provide an important method to screen for potential drugs.
In addition, Kwiatkowski plans to address the biological mechanism of FUS mutations. Any of its functions, when disrupted, might somehow lead to disease. Alternatively, one of FUS’s RNA targets could be required for motor neuron health. The FUS aggregates seen in motor neurons could be toxic, or could simply be a byproduct of the cell’s attempts to deal with the rogue protein.
At this point, it is not yet certain if FUS mutations reflect a loss or gain of function. “I think you’ve got to go with gain of function; it’s an autosomal-dominant disease,” Vance said. But the cell biology does not seem to follow that standard genetic rule. “It is a loss of function, because [FUS] suddenly goes from the nucleus to the cytoplasm,” suggested Lucie Bruijn, senior vice president for research and development at the ALS Association, headquartered in Calabasas Hills, California, which provided funding for the current studies. Mislocalized, the protein might not get its job done.
Compounding this question is the finding that the FUS mutation in the Cape Verde family that cracked the case is recessive. The parents came from the same village, and the maternal grandparents were first cousins. The three sisters who have the disease are homozygous for the mutation, whereas three elderly aunts and the father, who are heterozygous, show no symptoms. The Cape Verde mutation also causes atypical disease. The mother lived for 14 years with her condition and eventually died of a heart attack, not the respiratory failure that usually ends the life of people with ALS. Typically, a person with ALS is unlikely to live more than a few years after diagnosis. The presence of both dominant and recessive FUS mutations confounds any simple explanation. “We’ve got both, so I’ve been scratching my head about this,” Kwiatkowski said. “Maybe the different mutations disturb different functions.”
FUS Fits into Bigger RNA Picture
RNA-shuttling proteins have been implicated in other conditions. FUS itself forms a part of the abnormal huntingtin-containing aggregates in Huntington disease, although the significance of its presence there is uncertain (Doi et al., 2008). The Fragile X Mental Retardation Protein (FMRP) associates with RNA (Khandjian et al., 2004), and methyl-CpG binding protein 2 (MeCP2), which is associated with Rett syndrome, is involved in RNA splicing (Young et al., 2005). And in ALS, Brown and colleagues recently found that the elongator protein 3 (ELP3) gene, which encodes part of RNA polymerase II, is associated with disease (Simpson et al., 2009). Then, of course, there is TDP-43. “This raises the possibility that there’s something fundamentally important about RNA metabolism that is important for the viability of the neuron,” Brown said.
At the same time, there are differences in the pathology between FUS mutations and TDP-43 proteinopathies. The cells of people with FUS mutations do not contain aggregated TDP-43, as do the majority of sporadic ALS cases, although they do have ubiquitin inclusions in the nucleus. Those features “suggest the mechanisms may be distinct,” wrote Robert Bowser of the University of Pittsburgh, Pennsylvania, in an e-mail to ARF (see full comment below).
The current results mean that ALS scientists will carefully scrutinize the role of RNA management, in familial as well as sporadic forms of the disease. Looking for FUS pathology in sporadic ALS cases is high on Brown’s to-do list, he said. And two-thirds of familial cases still remain unexplained, raising the question of whether more RNA regulators are waiting to be discovered. The search goes on: loci on chromosomes 9 and 20 have been linked to familial ALS, and may be next to give up their secrets.—Amber Dance.
Kwiatkowski TJ Jr, Bosco DA, LeClerc AL, Tamrazian E, Vanderburg CR, Russ C, Davis A, Gilchrist J, Kasarskis EJ, Munsat T, Valdmanis P, Rouleau GA, Hosler BA, Cortelli P, de Jong PJ, Yoshinaga Y, Haines JL, Pericak-Vance MA, Yan J, Ticozzi N, Siddique T, McKenna-Yasek D, Sapp PC, Horvitz HR, Landers JE, Brown RH Jr. Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science 27 Feb 2009 323:1205-1208. Abstract
Vance C, Rogelj B, Hortobágyi T, De Vos KJ, Nishimura AL, Sreedharan J, Hu X, Smith B, Ruddy D, Wright P, Ganesalingam J, Williams KL, Tripathi V, Al-Saraj S, Al-Chalabi A, Leigh PN, Blair IP, Nicholson G, de Belleroche J, Gallo J-M, Miller CC, Shaw CE. Mutations in FUS, and RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science 27 Feb 2009 323:1208-1211. Abstract