27 Feb 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

Comments

1. • Robert Bowser
     University of Pittsburgh Sch Medicine
   • Posted: 27 Feb 2009

   These papers represent exciting work describing a new genetic mutation associated with familial ALS. The results further highlight the importance for RNA processing in at least familial forms of motor neuron disease. Much work remains to determine the exact mechanisms by which FUS modulates motor neuron survival. It may be related to that of TDP-43. However, the lack of cytoplasmic aggregation of TDP-43, and rare ubiquitin inclusions in the patients with FUS mutations, suggest the mechanisms may be distinct. It is interesting that FUS protein did not accumulate in the cytoplasm of motor neurons in sporadic ALS patients, again suggestive that the pathogenic mechanisms of mutant FUS-induced motor neuron degeneration may be distinct from that in sporadic ALS.

   View all comments by Robert Bowser
2. • Eric Frank
     Tufts University
   • Posted: 27 Feb 2009

   These studies raise interesting questions about whether one problem in ALS and perhaps other neurodegenerative diseases is that RNA trafficking proteins fail to properly deliver RNAs to dendritic spines. The paper by Kwiatkowski et al. reports evidence that wild-type FUS and TDP-43 may be involved in transporting RNA into dendrites, where it mediates local protein synthesis that can be stimulated by neural activity. The clumping of the mutant form described by both new papers could therefore perturb the transport of RNA. Local protein synthesis in dendrites plays a major role in the activity-dependent modulation of synaptic strength. Changes in synaptic activity have been recently reported in the mouse model of SOD1 mutation (van Zundert et al., 2008), so it will be worthwhile to examine this issue in the FUS mice that will certainly be developed by these investigators.

   References:

   van Zundert B, Peuscher MH, Hynynen M, Chen A, Neve RL, Brown RH, Constantine-Paton M, Bellingham MC. Neonatal neuronal circuitry shows hyperexcitable disturbance in a mouse model of the adult-onset neurodegenerative disease amyotrophic lateral sclerosis. J Neurosci. 2008 Oct 22;28(43):10864-74. PubMed.

   View all comments by Eric Frank
3. • Jeffrey D. Rothstein
     Johns Hopkins University
   • Posted: 02 Mar 2009

   This is an extremely exiting story in the understanding of ALS pathogenesis. It actually it dates back to 1998—with the first description of mRNA processing errors in sporadic ALS (Lin et al., 1998), which, interestingly, was made not in the SOD1 mouse model. At the same time, the spinal muscular atrophy gene was discovered. SMA is not unlike a childhood ALS, though predominately lower motor neurons are affected in that disease. The SMA gene defect is involved in RNA metabolism. So for the next 10 years, the SMA field has investigated the pathobiology of the defective protein. At the time it made the link between sporadic ALS and the SMA story intriguing. But there was no clear genetic link (or cause for the changes in sporadic ALS).

   Feed forward to 2008, when Chris Shaw and others found a true genetic defect in RNA metabolism-based protein TDP-43. (Of course more work needs to be done on that.) And now another gene by the Shaw group, and now verified by the group in Boston, does set a string of targets that all focus on RNA metabolism and (lower) motor neurons.

   By the way, all these cases appear to predominately involve a lower motor neuron form of ALS. The hint from genetics does suggest more of a loss of function rather than gain, but cell biology will ultimately sort that out. We certainly await the generation of mouse or fly models, which are now well underway for TDP-43. However, this may be a particularly difficult target for specific, non-toxic drug therapy.

   References:

   Lin CL, Bristol LA, Jin L, Dykes-Hoberg M, Crawford T, Clawson L, Rothstein JD. Aberrant RNA processing in a neurodegenerative disease: the cause for absent EAAT2, a glutamate transporter, in amyotrophic lateral sclerosis. Neuron. 1998 Mar;20(3):589-602. PubMed.

   View all comments by Jeffrey D. Rothstein
4. • P. Hande Ozdinler
     Northwestern University, Feinberg School of Medicine
   • Posted: 17 Mar 2009

   These back-to-back papers on the identification of FUS (fused in sarcoma) gene as a new genetic component of ALS open a new era of research and direct our attention to mRNA biology with respect to disease. After the first identification of mRNA processing errors in ALS patients (Lin, Bristol et al., 1998), the discovery of TDP-43 (Neumann, Sampathu et al., 2006) and now the FUS gene clearly indicate the importance of mRNA management in neurodegenerative diseases. Defects in RNA transcription, splicing, and trafficking may be the reason for cell-type-specific degeneration of motor neurons in ALS. Motor neurons both in the cortex and spinal cord are very large excitatory neurons that extend long axons to their targets and require high levels of energy and protein integrity for survival and function. Defects in transcriptional mechanisms may result in splicing defects, which could give rise to formation of non-functional proteins that would deplete the pool of required proteins for cellular function, and these non-functional proteins may form aggregates that are toxic to neurons. In addition, defects in the trafficking of mRNA may lead to depletion of key proteins that are in high demand locally for motor neuron function. But if FUS has a general function in mRNA transcription, splicing, and trafficking, why do mutations in this gene cause ALS and not other neurodegenerative diseases? What makes motor neurons more vulnerable in the presence of defective FUS? It could be true that in motor neurons FUS controls the transcription of a distinct set of mRNA that is expressed in a cell-type-specific manner in motor neurons, or that FUS controls the production of a key protein that is highly required in motor neurons when compared to other cell-types, and thus motor neurons may become vulnerable first. FUS seems to be the tip of the iceberg. Finding effectors, binding partners including mRNA, may lead to the identification of key components of both familial and sporadic ALS. More work is on the way!

   References:

   Kneussel M. Dynamic regulation of GABA(A) receptors at synaptic sites. Brain Res Brain Res Rev. 2002 Jun;39(1):74-83. PubMed.

   Lin CL, Bristol LA, Jin L, Dykes-Hoberg M, Crawford T, Clawson L, Rothstein JD. Aberrant RNA processing in a neurodegenerative disease: the cause for absent EAAT2, a glutamate transporter, in amyotrophic lateral sclerosis. Neuron. 1998 Mar;20(3):589-602. PubMed.

   Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, Chou TT, Bruce J, Schuck T, Grossman M, Clark CM, McCluskey LF, Miller BL, Masliah E, Mackenzie IR, Feldman H, Feiden W, Kretzschmar HA, Trojanowski JQ, Lee VM. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science. 2006 Oct 6;314(5796):130-3. PubMed.

   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 JM, Miller CC, Shaw CE. Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science. 2009 Feb 27;323(5918):1208-11. PubMed.

   View all comments by P. Hande Ozdinler

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References

Paper Citations

1. Ruddy DM, Parton MJ, Al-Chalabi A, Lewis CM, Vance C, Smith BN, Leigh PN, Powell JF, Siddique T, Meyjes EP, Baas F, de Jong V, Shaw CE. Two families with familial amyotrophic lateral sclerosis are linked to a novel locus on chromosome 16q. Am J Hum Genet. 2003 Aug;73(2):390-6. PubMed.
2. Sapp PC, Hosler BA, McKenna-Yasek D, Chin W, Gann A, Genise H, Gorenstein J, Huang M, Sailer W, Scheffler M, Valesky M, Haines JL, Pericak-Vance M, Siddique T, Horvitz HR, Brown RH. Identification of two novel loci for dominantly inherited familial amyotrophic lateral sclerosis. Am J Hum Genet. 2003 Aug;73(2):397-403. PubMed.
3. Law WJ, Cann KL, Hicks GG. TLS, EWS and TAF15: a model for transcriptional integration of gene expression. Brief Funct Genomic Proteomic. 2006 Mar;5(1):8-14. PubMed.
4. Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, Donaldson D, Goto J, O'Regan JP, Deng HX. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature. 1993 Mar 4;362(6415):59-62. PubMed.
5. Doi H, Okamura K, Bauer PO, Furukawa Y, Shimizu H, Kurosawa M, Machida Y, Miyazaki H, Mitsui K, Kuroiwa Y, Nukina N. RNA-binding protein TLS is a major nuclear aggregate-interacting protein in huntingtin exon 1 with expanded polyglutamine-expressing cells. J Biol Chem. 2008 Mar 7;283(10):6489-500. PubMed.
6. Khandjian EW, Huot ME, Tremblay S, Davidovic L, Mazroui R, Bardoni B. Biochemical evidence for the association of fragile X mental retardation protein with brain polyribosomal ribonucleoparticles. Proc Natl Acad Sci U S A. 2004 Sep 7;101(36):13357-62. PubMed.
7. Young JI, Hong EP, Castle JC, Crespo-Barreto J, Bowman AB, Rose MF, Kang D, Richman R, Johnson JM, Berget S, Zoghbi HY. Regulation of RNA splicing by the methylation-dependent transcriptional repressor methyl-CpG binding protein 2. Proc Natl Acad Sci U S A. 2005 Dec 6;102(49):17551-8. PubMed.
8. Simpson CL, Lemmens R, Miskiewicz K, Broom WJ, Hansen VK, van Vught PW, Landers JE, Sapp P, Van Den Bosch L, Knight J, Neale BM, Turner MR, Veldink JH, Ophoff RA, Tripathi VB, Beleza A, Shah MN, Proitsi P, Van Hoecke A, Carmeliet P, Horvitz HR, Leigh PN, Shaw CE, van den Berg LH, Sham PC, Powell JF, Verstreken P, Brown RH, Robberecht W, Al-Chalabi A. Variants of the elongator protein 3 (ELP3) gene are associated with motor neuron degeneration. Hum Mol Genet. 2009 Feb 1;18(3):472-81. PubMed.

Further Reading

Papers

1. Geser F, Martinez-Lage M, Robinson J, Uryu K, Neumann M, Brandmeir NJ, Xie SX, Kwong LK, Elman L, McCluskey L, Clark CM, Malunda J, Miller BL, Zimmerman EA, Qian J, Van Deerlin V, Grossman M, Lee VM, Trojanowski JQ. Clinical and pathological continuum of multisystem TDP-43 proteinopathies. Arch Neurol. 2009 Feb;66(2):180-9. PubMed.
2. Sreedharan J, Blair IP, Tripathi VB, Hu X, Vance C, Rogelj B, Ackerley S, Durnall JC, Williams KL, Buratti E, Baralle F, de Belleroche J, Mitchell JD, Leigh PN, Al-Chalabi A, Miller CC, Nicholson G, Shaw CE. TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science. 2008 Mar 21;319(5870):1668-72. Epub 2008 Feb 28 PubMed.
3. Furukawa Y, O'Halloran TV. Posttranslational modifications in Cu,Zn-superoxide dismutase and mutations associated with amyotrophic lateral sclerosis. Antioxid Redox Signal. 2006 May-Jun;8(5-6):847-67. PubMed.
4. Chiò A, Schymick JC, Restagno G, Scholz SW, Lombardo F, Lai SL, Mora G, Fung HC, Britton A, Arepalli S, Gibbs JR, Nalls M, Berger S, Kwee LC, Oddone EZ, Ding J, Crews C, Rafferty I, Washecka N, Hernandez D, Ferrucci L, Bandinelli S, Guralnik J, Macciardi F, Torri F, Lupoli S, Chanock SJ, Thomas G, Hunter DJ, Gieger C, Wichmann HE, Calvo A, Mutani R, Battistini S, Giannini F, Caponnetto C, Mancardi GL, La Bella V, Valentino F, Monsurrò MR, Tedeschi G, Marinou K, Sabatelli M, Conte A, Mandrioli J, Sola P, Salvi F, Bartolomei I, Siciliano G, Carlesi C, Orrell RW, Talbot K, Simmons Z, Connor J, Pioro EP, Dunkley T, Stephan DA, Kasperaviciute D, Fisher EM, Jabonka S, Sendtner M, Beck M, Bruijn L, Rothstein J, Schmidt S, Singleton A, Hardy J, Traynor BJ. A two-stage genome-wide association study of sporadic amyotrophic lateral sclerosis. Hum Mol Genet. 2009 Apr 15;18(8):1524-32. PubMed.
5. Rutherford NJ, Zhang YJ, Baker M, Gass JM, Finch NA, Xu YF, Stewart H, Kelley BJ, Kuntz K, Crook RJ, Sreedharan J, Vance C, Sorenson E, Lippa C, Bigio EH, Geschwind DH, Knopman DS, Mitsumoto H, Petersen RC, Cashman NR, Hutton M, Shaw CE, Boylan KB, Boeve B, Graff-Radford NR, Wszolek ZK, Caselli RJ, Dickson DW, Mackenzie IR, Petrucelli L, Rademakers R. Novel mutations in TARDBP (TDP-43) in patients with familial amyotrophic lateral sclerosis. PLoS Genet. 2008 Sep 19;4(9):e1000193. PubMed.

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