Cell 136, March 20, 2009 ©2009 Elsevier Inc. 1001
Amyotrophic lateral sclerosis (ALS) is an adult-onset neuro-
degenerative disorder in which premature loss of motor neu-
rons leads to fatal paralysis with a typical disease course of
1 to 5 years. Most forms of ALS are sporadic, but ~10% of
patients have an inherited familial form of the disease and
a clear family history. Understanding of ALS pathogenesis
began with the landmark discovery of dominant causative
mutations in the gene encoding copper/zinc superoxide dis-
mutase 1 (SOD1) in ~20% of familial ALS cases and ~1% of
sporadic cases. Subsequently, other even rarer familial cases
with atypical disease features were linked to mutations in sev-
eral other genes.
Most efforts to understand ALS pathogenesis over the past 15
years have focused on mutations in the ubiquitously expressed
SOD1. No consensus has yet emerged as to how SOD1 muta-
tions lead to selective premature death of motor neurons, except
that damage within motor neurons expressing mutant SOD1
drives disease onset, whereas damage within their glial cell neigh-
bors expressing mutant SOD1 accelerates disease progression
(Yamanaka et al., 2008). Multiple toxicity pathways have been
implicated including the ability of misfolded mutant SOD1 to trig-
ger aberrant mitochondrial function, endoplasmic reticulum stress
pathways, axonal transport defects, or excessive production of
extracellular superoxide radicals. Views about ALS pathogenesis
Rethinking ALS: The FUS about TDP-43
Clotilde Lagier-Tourenne1 and Don W. Cleveland1,*
1Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla,
CA 92093-0670, USA
Mutations in TDP-43, a DNA/RNA-binding protein, cause an inherited form of the neurodegenerative
disease amyotrophic lateral sclerosis (ALS). Two recent studies (Kwiatkowski et al., 2009; Vance
et al., 2009) now report that mutations in FUS/TLS, another DNA/RNA-binding protein, also trigger
premature degeneration of motor neurons. TDP-43 and FUS/TLS have striking structural and func-
tional similarities, implicating alterations in RNA processing as a key event in ALS pathogenesis.
Figure 1. TDP-43 and FUS/TLS Mutations in ALS
(A) Thirty dominant mutations in TDP-43 have been identified in sporadic (red) and familial (black) ALS patients, with most lying in the C-terminal glycine-rich
region of TDP-43. All are missense mutations, except for the truncating mutation TDP-43Y374X.
(B) Fourteen mutations in FUS/TLS have been identified in familial ALS cases, with most lying in the final 13 amino acids of this protein (R514S and G515S are found
in cis). (Data compiled from Kwiatkowski et al., 2009; Vance et al., 2009; domains from http://www.uniprot.org and http://www.cbs.dtu.dk/services/NetNES.)
1002 Cell 136, March 20, 2009 ©2009 Elsevier Inc.
are now undergoing a seismic shift triggered by the recent dis-
covery of mutations in a pair of DNA/RNA-binding proteins called
TDP-43 (Gitcho et al., 2008; Sreedharan et al., 2008; Kabashi et
al., 2008) and FUS/TLS (Kwiatkowski et al., 2009; Vance et al.,
2009) (Figure 1) as causes of familial and sporadic forms of ALS.
TDP-43 Mutations in Familial and Sporadic ALS
This seismic shift in our understanding of ALS pathogenesis began
with the identification (Arai et al., 2006; Neumann et al., 2006) of
the 43 kDa TAR DNA-binding protein (TDP-43) as a major compo-
nent of ubiquitinated protein aggregates found in many patients
with sporadic ALS or the most common form of frontotemporal
dementia called FTLD-U (frontotemporal lobar degeneration with
ubiquitinated inclusions). In ALS and FTLD-U patients, TDP-43
immunoreactive inclusions are observed in the cytoplasm and
nucleus of both neurons and glial cells. The brains and spinal
cords of patients with TDP-43 proteinopathy present a biochemi-
cal signature that is characterized by abnormal hyperphospho-
rylation and ubiquitination of TDP-43 and the production of ~25
kDa C-terminal fragments that are missing their nuclear targeting
domains (Arai et al., 2006; Neumann et al., 2006). TDP-43 is partly
cleared from the nuclei of neurons containing cytoplasmic aggre-
gates (Figure S1A available online) (e.g., Neumann et al., 2006;
Van Deerlin et al., 2008) supporting the notion that pathogenesis
of ALS in these cases may be driven, at least in part, by loss of
normal TDP-43 function in the nucleus. Combined with a flurry of
subsequent reports, TDP-43 inclusions are now recognized as
a common characteristic of most ALS patients, with the striking
exception of patients with familial ALS caused by SOD1 muta-
Although identification of TDP-43 aggregates proved to be a
breakthrough, the pathology alone left it unclear whether aggre-
gation of TDP-43 is a primary event in ALS pathogenesis or
whether it is a byproduct of the disease process. Accumulation of
intracellular or extracellular misfolded or misprocessed proteins
in the central nervous system is a feature of many neurodegen-
erative conditions. Rare mutations have been found in the genes
encoding misfolded proteins implicated in Alzheimer’s disease,
Parkinson’s disease, the tauopathies, and prion diseases. Thus,
the gene encoding TDP-43 on chromosome 1, TARDBP, consti-
tuted an excellent candidate for direct sequencing in search of
disease-causing mutations in cohorts of patients with motor neu-
ron disease or frontotemporal dementia.
Starting in early 2008, dominant mutations in the TARDBP
gene were reported by several groups as a primary cause of
ALS (e.g., Corrado et al., 2009; Daoud et al., 2009; Gitcho et al.,
2008; Kabashi et al., 2008; Sreedharan et al., 2008; Van Deerlin
et al., 2008; for review see Banks et al., 2008). These studies
collectively provided persuasive evidence that the aberrant form
of TDP-43 can directly trigger neurodegeneration. A total of 30
different mutations are now known in 22 unrelated families (~3%
of familial ALS cases) and in 29 sporadic cases of ALS (~1.5%
of sporadic cases) (Figure 1A). Given that linkage of familial ALS
to chromosome 1 had not been identified previously, key among
these genetic efforts was a retrospective analysis of a large
family in which direct sequencing of TDP-43 revealed a TDP-43
mutation M337V. This study (Sreedharan et al., 2008) identified
linkage between the disease and only one genomic region—an
8.2 Mb region on chromosome 1p36 containing the TARDBP
gene. Although unconventional, this approach provided strong
support for the pathogenic effect of the TDP-43M337V mutation.
Widely expressed and predominantly nuclear, TDP-43 is 414
amino acids long and is encoded by six exons. This protein con-
tains two RNA-recognition motifs (RRM1 and 2) and a C-terminal
glycine-rich region that may mediate interactions with other pro-
teins. All but one of the mutations identified so far are localized in
the C-terminal region encoded by exon 6 of TARDBP (Figure 1A).
All of these mutations are dominantly inherited missense changes
with the exception of a truncating mutation (Y374X) at the extreme
C terminus of the protein (Daoud et al., 2009). The pathogenic-
ity of these missense changes is strongly supported by several
lines of evidence. First, they affect amino acids that are highly
conserved throughout evolution. Second, they have not been
found in large cohorts of control individuals who do not have ALS.
Third, in familial ALS cases where DNA is available, the mutations
segregate with the disease and no mutations have been found in
unaffected family members (except those below the typical age
of disease onset). This indicates a high degree of penetrance for
TARDBP mutations in these families, although further studies are
needed as mutations in TDP-43 have been identified in patients
with apparent sporadic ALS. Collectively, the evidence is now
overwhelming that aberrant TDP-43 can trigger ALS.
Patients with TDP-43 mutations develop typical ALS with some
variability within families in the site and age of onset. Although
~50% of all ALS patients develop cognitive impairment of varying
severity, only one patient carrying a TDP-43 mutation has been
reported to develop cognitive deficits (Corrado et al., 2009). This
is despite the presence of TDP-43 inclusions in neurons and glial
cells within the spinal cords and throughout the brains of ALS
patients (e.g., Neumann et al., 2006; Van Deerlin et al., 2008). Dif-
fuse granular cytoplasmic staining of TDP-43 (which may repre-
sent an earlier stage of inclusion development) and nuclear clear-
ing of TDP-43 have also been described in the spinal cords and
brains of ALS patients carrying TDP-43 mutations (Figure S1).
However, it is unclear whether TDP-43 mutations lead to motor
neuron loss through a gain of one or more toxic properties or a
loss of normal function arising from sequestration of the protein in
nuclear or cytoplasmic inclusions and the corresponding disrup-
tion of its interactions with protein partners or RNA targets.
FUS/TLS Mutations in Familial ALS
The identification of TDP-43 in ALS pathogenesis fueled the dis-
covery recently reported by Kwiatkowski et al. (2009) and Vance
et al. (2009) in Science of additional ALS mutations in a gene
encoding another DNA/RNA-binding protein called FUS (fused in
sarcoma) or TLS (translocation in liposarcoma). Previous reports
had identified linkage between chromosome 16 and a familial
form of ALS, but the underlying mutations were not known. Based
on the knowledge that TDP-43 is a DNA/RNA-binding protein,
Vance et al. (2009) prioritized sequencing of genes within the link-
age region identified in a large British family with familial ALS so
as to target genes encoding DNA/RNA-binding proteins. This led
to their identification of a dominant missense mutation (R521C) in
the FUS/TLS gene. A survey of 197 familial ALS cases identified
the same R521C mutation in four additional families, as well as
two additional missense mutations in another four families.
Cell 136, March 20, 2009 ©2009 Elsevier Inc. 1003
Independently, Kwiatkowski et al. (2009) used a linkage study
in an ALS family originating from the Cape Verde islands in which
disease transmission was compatible with an autosomal reces-
sive inheritance pattern. A region of homozygosity by descent
shared by all affected members of this family was identified on
chromosome 16. This region overlapped with the previously
reported ALS locus and contained the FUS/TLS gene. Again,
homing in on genes encoding DNA/RNA-binding proteins led to
screening of the FUS/TLS locus for mutations, resulting in identifi-
cation of a homozygous missense mutation (H517Q) in all affected
members. Although three healthy siblings were also homozygous
for this mutation, they were younger than the typical age of dis-
ease onset. None of the individuals heterozygous for the mutation
developed ALS, confirming autosomal recessive inheritance of
this particular mutation. Subsequent screening in 292 familial ALS
cases identified 12 dominant mutations in 16 families including
two large families previously shown to have linkage to chromo-
some 16 (Kwiatkowski et al., 2009). No FUS/TLS mutations were
found in a survey of 293 patients with sporadic ALS.
Combining the efforts of both teams, FUS/TLS mutations
were detected in ~4% of familial ALS (~0.4% of all ALS). As is
the case for TDP-43 mutations, all patients developed classical
ALS with no cognitive deficits. Except for the recessive mutation
in the family of Cape Verdean origin, the inheritance pattern is
dominant (albeit with an incomplete penetrance reported for the
R521G mutation) (Kwiatkowski et al., 2009).
The FUS/TLS protein is 526 amino acids long and is encoded
by 15 exons. It is characterized by an N-terminal domain
enriched in glutamine, glycine, serine, and tyrosine residues
(QGSY region), a glycine-rich region, an RNA-recognition motif
(RRM), multiple RGG repeats implicated in RNA binding, a
C-terminal zinc finger motif, and a highly conserved extreme
C-terminal region (Figure 1B). The vast majority of ALS-linked
mutations are clustered in the extreme C terminus, with reports
of mutations in all five arginine residues in this region. All muta-
tions are missense changes except for two, both of which are
located in the glycine-rich region and correspond to an inser-
tion or a deletion of two glycines in a 10 glycine-long tract.
Like TDP-43, FUS/TLS is almost ubiquitously expressed. It is
mainly localized in the nucleus, but cytoplasmic accumulation
has been detected in most cell types. Analysis of the brains and
spinal cords of ALS patients with FUS/TLS mutations revealed
normal staining of FUS/TLS in the nuclei of many neurons and
glial cells but aggregates of FUS/TLS in the cytoplasm of neu-
rons (Kwiatkowski et al., 2009; Vance et al., 2009) (Figure S1C).
It has not been reported whether FUS/TLS inclusions are also
present in the cytoplasm of glial cells. Cell fractionation experi-
ments after expression of tagged wild-type or mutant FUS/TLS
confirmed an increase in the cytoplasmic accumulation of this
mutant protein (Kwiatkowski et al., 2009; Vance et al., 2009).
A very curious aspect of mutant TDP-43 pathology is its partial
clearance from the nucleus of either neuronal or glial cells when
there are aggregates of TDP-43 in the cytoplasm. In a minority of
neurons from ALS patients with FUS/TLS mutations (Figure S1C)
or cells transfected to express fluorescently tagged mutant FUS/
TLSR521G, a uniquely cytoplasmic pattern of FUS/TLS aggregates
has been reported. Cytoplasmic inclusions containing the FUS/
TLS protein are absent in normal individuals, in ALS patients
with SOD1 mutations, and in sporadic ALS patients who pre-
sumably are positive for TDP-43 aggregates. Importantly, TDP-
43-positive inclusions are absent in ALS patients with FUS/TLS
mutations, implying that neurodegenerative processes driven
by FUS/TLS mutations are independent of TDP-43 aggrega-
tion (Vance et al., 2009). It will now be essential to assess FUS/
TLS accumulation and localization in ALS patients with TDP-43
mutations, as well as in patients with other neurodegenerative
diseases, especially those with mislocalized TDP-43.
TDP-43 and FUS/TLS in Gene Regulation
The precise roles of TDP-43 and FUS/TLS have not been fully
elucidated, but both are multifunctional proteins that have been
implicated in several steps of gene expression regulation includ-
ing transcription, RNA splicing, RNA transport, and translation
(for review see Buratti and Baralle, 2008; Janknecht, 2005). They
might also be involved in the processing of microRNAs, and
FUS/TLS may play a role in the maintenance of genomic integ-
rity. Both proteins contain RNA-binding motifs and are struc-
turally close to a family of heterogeneous ribonucleoproteins
(hnRNPs). Indeed, FUS/TLS is sometimes referred to as hnRNP
P2. Consistently, both TDP-43 and FUS/TLS directly bind to
RNA, as well as to single- and double-stranded DNA.
TDP-43 was initially proposed to repress transcription by
binding to the TAR DNA sequence of human immunodeficiency
virus type-1 and to the mouse SP-10 gene promoter, but little is
known about the mechanisms and selectivity of transcriptional
repression. The normal function of FUS/TLS has been studied
more extensively following its identification as a fusion protein
generated by chromosomal translocations in human cancers.
It is a member of the TET protein family that also includes
the Ewing’s sarcoma protein and the TATA-binding protein-
associated factor (TAFII68). Wild-type FUS/TLS associates
with both general and more specialized factors, presumptively
influencing transcription initiation. Indeed, FUS/TLS interacts
with several nuclear hormone receptors and with gene-specific
transcription factors. It also associates with the general tran-
scriptional machinery, interacting with RNA polymerase II and
the TFIID complex.
Recently, a very interesting and unexpected mechanism of
transcriptional regulation was described for FUS/TLS (Wang et
al., 2008). In response to DNA damage, FUS/TLS is recruited
by sense and antisense noncoding RNAs transcribed in the 5′
regulatory region of the gene encoding cyclin D1. Then FUS/
TLS binds and inhibits CREB-binding protein and p300 histone
acetyltransferase activities leading to the repression of cyclin
D1 transcription (Wang et al., 2008). This provides a direct link
between the RNA-binding properties of FUS/TLS and a role
in transcriptional regulation. Moreover, this kind of regulation
might be more general in light of four recent reports in Science
demonstrating that production of short sense and antisense
noncoding RNAs upstream of the active transcription start site
occurs in other contexts (e.g., Core et al., 2008).
TDP-43 and FUS/TLS in RNA Splicing and Localization
Beyond transcription, TDP-43 and FUS/TLS have been impli-
cated in RNA maturation and splicing. Only a few of their respec-
tive RNA targets have been identified, and a comprehensive map
1004 Cell 136, March 20, 2009 ©2009 Elsevier Inc.
of their RNA targets is a crucial next goal. Recent technologies
using high-throughput sequencing have demonstrated that a
single RNA-binding protein can affect many alternatively spliced
transcripts (Licatalosi et al., 2008). Such approaches will be nec-
essary to understand the role of TDP-43 and FUS/TLS in neuro-
degeneration. Indeed, observation of a widespread mRNA splic-
ing defect in diseases characterized by aggregation of TDP-43 or
FUS/TLS would reinforce the crucial role of splicing regulation in
neuronal integrity and potentially could identify candidate genes
whose altered splicing is central to ALS pathogenesis. It should
not be overlooked that TDP-43 and FUS/TLS also may be involved
in microRNA processing as both have been found by mass spec-
trometry to associate with Drosha (Gregory et al., 2004).
Despite their enrichment in the nucleus and potential roles in
nuclear RNA maturation, TDP-43 and FUS/TLS shuttle between
the nucleus and cytosol. In addition, both are found in granules
associated with RNA transport in neurons, with translocation to
dendritic spines following different neuronal stimuli (e.g., Fujii et
al., 2005). Moreover, abnormal spine morphology is observed
in cultured neurons from FUS/TLS knockout mice (Fujii et al.,
2005). These results suggest that both proteins could play a role
in the modulation of neuronal plasticity or other properties by
altering mRNA transport and local RNA translation in neurons.
Beyond ALS: The Next Steps
The identification of causative TDP-43 and FUS/TLS mutations,
along with TDP-43 pathology in most cases of ALS, represents a
dramatic shift in our understanding of this disease. Now we need
to define the normal roles of TDP-43 and FUS/TLS and to deter-
mine whether mutant forms of these proteins and their abnormal
aggregation lead to general or specific alterations in gene expres-
sion. Both cellular and animal models will be essential to define the
link between mutations in TDP-43 and FUS/TLS and disease.
The lessons to be learned for TDP-43 and FUS/TLS in trig-
gering neurodegenerative disease will not be unique to ALS.
TDP-43 aggregation is present in most sporadic and familial
FTLD-U patients including those with mutations in progranulin
and valosin-containing protein. Moreover, abnormal TDP-43
inclusions have been reported for several other neurodegen-
erative conditions, including in ~30% of Alzheimer’s disease
patients (reviewed in Banks et al., 2008). Wild-type FUS/TLS
was recently identified as a major component of polyQ aggre-
gates in cellular models of spinal cerebellar ataxia type 3 and
Huntington’s disease (Doi et al., 2008). The latter observation
was confirmed by the finding of intranuclear inclusions in neu-
rons from Huntington’s disease patients, provoking the proposal
that the protein binds directly to polyQ aggregates at an early
stage of disease (Doi et al., 2008).
Discovery of the involvement of TDP-43 and FUS/TLS in
ALS and other neurodegenerative diseases reinforces the role
of altered RNA processing in neurodegeneration. Earlier well-
established examples of altered RNA processing in neurode-
generation include errors in RNA metabolism due to loss of the
SMN (survival of motor neuron) protein in spinal muscular atro-
phy and of FMRP in fragile-X mental retardation. In addition, an
RNA gain-of-function mechanism has been implicated in a set of
diseases including the myotonic dystrophies, where a transcript
with an abnormal repeat expansion alters the function and local-
ization of alternative splicing regulators. The emerging TDP-43
and FUS/TLS stories add considerable support to the proposal
that defects in RNA processing play a central role in neurode-
Supplemental Data contain one Figure and can be found with this article on-
line at http://www.cell.com/supplemental/S0092-8674(09)00263-3.
Arai, T., Hasegawa, M., Akiyama, H., Ikeda, K., Nonaka, T., Mori, H., Mann, D.,
Tsuchiya, K., Yoshida, M., Hashizume, Y., et al. (2006). Biochem. Biophys. Res.
Commun. 351, 602–611.
Banks, G.T., Kuta, A., Isaacs, A.M., and Fisher, E.M. (2008). Mamm. Genome
Buratti, E., and Baralle, F.E. (2008). Front. Biosci. 13, 867–878.
Core, L.J., Waterfall, J.J., and Lis, J.T. (2008). Science 322, 1845–1848.
Corrado, L., Ratti, A., Gellera, C., Buratti, E., Castellotti, B., Carlomagno, Y.,
Ticozzi, N., Mazzini, L., Testa, L., Taroni, F., et al. (2009). Hum. Mut. Published
online February 17, 2009. 10.1002/humu.20950.
Daoud, H., Valdmanis, P.N., Kabashi, E., Dion, P., Dupre, N., Camu, W., Mein-
inger, V., and Rouleau, G.A. (2009). J. Med. Genet. 46, 112–114.
Doi, H., Okamura, K., Bauer, P.O., Furukawa, Y., Shimizu, H., Kurosawa, M.,
Machida, Y., Miyazaki, H., Mitsui, K., Kuroiwa, Y., et al. (2008). J. Biol. Chem.
Fujii, R., Okabe, S., Urushido, T., Inoue, K., Yoshimura, A., Tachibana, T., Ni-
shikawa, T., Hicks, G.G., and Takumi, T. (2005). Curr. Biol. 15, 587–593.
Gitcho, M.A., Baloh, R.H., Chakraverty, S., Mayo, K., Norton, J.B., Levitch, D.,
Hatanpaa, K.J., White, C.L., 3rd, Bigio, E.H., Caselli, R., et al. (2008). Ann. Neu-
rol. 63, 535–538.
Gregory, R.I., Yan, K.P., Amuthan, G., Chendrimada, T., Doratotaj, B., Cooch, N.,
and Shiekhattar, R. (2004). Nature 432, 235–240.
Janknecht, R. (2005). Gene 363, 1–14.
Kabashi, E., Valdmanis, P.N., Dion, P., Spiegelman, D., McConkey, B.J., Vande
Velde, C., Bouchard, J.P., Lacomblez, L., Pochigaeva, K., Salachas, F., et al.
(2008). Nat. Genet. 40, 572–574.
Kwiatkowski, T.J., Bosco, J.D., LeClerc, A.D., Tamrazian, E., Van den Berg,
C.R., Russ, C., Davis, A., Gilchrist, J., Kasarskis, E.J., Munsat, T., et al. (2009).
Science 323, 1205–1208.
Licatalosi, D.D., Mele, A., Fak, J.J., Ule, J., Kayikci, M., Chi, S.W., Clark, T.A.,
Schweitzer, A.C., Blume, J.E., Wang, X., et al. (2008). Nature 456, 464–469.
Neumann, M., Sampathu, D.M., Kwong, L.K., Truax, A.C., Micsenyi, M.C.,
Chou, T.T., Bruce, J., Schuck, T., Grossman, M., Clark, C.M., et al. (2006). Sci-
ence 314, 130–133.
Sreedharan, J., Blair, I.P., Tripathi, V.B., Hu, X., Vance, C., Rogelj, B., Ackerley, S.,
Durnall, J.C., Williams, K.L., Buratti, E., et al. (2008). Science 319, 1668–1672.
Vance, C., Rogelj, B., Hortobagyi, T., De Vos, K.J., Nishimura, A.L., Sreedha-
ran, J., Hu, X., Smith, B., Ruddy, D.M., Wright, P., et al. (2009). Science 323,
Van Deerlin, V.M., Leverenz, J.B., Bekris, L.M., Bird, T.D., Yuan, W., Elman, L.B.,
Clay, D., Wood, E.M., Chen-Plotkin, A.S., Martinez-Lage, M., et al. (2008). Lan-
cet Neurol. 7, 409–416.
Wang, X., Arai, S., Song, X., Reichart, D., Du, K., Pascual, G., Tempst, P., Rosen-
feld, M.G., Glass, C.K., and Kurokawa, R. (2008). Nature 454, 126–130.
Yamanaka, K., Chun, S.J., Boillee, S., Fujimori, N., Yamashita, H., Gutmann,
D.H., Misawa, H., Takahashi, R., and Cleveland, D.W. (2008). Nat. Neurosci.