Review

TDP-43 and FUS in amyotrophic lateral sclerosis and

frontotemporal dementia
Ian R A Mackenzie, Rosa Rademakers, Manuela Neumann

Abnormal intracellular protein aggregates comprise a key characteristic in most neurodegenerative diseases, including Lancet Neurol 2010; 9: 995–1007
amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The seminal discoveries of accumulation of See In Context page 955
TDP-43 in most cases of ALS and the most frequent form of FTD, frontotemporal lobar degeneration with Department of Pathology and
ubiquitinated inclusions, followed by identification of FUS as the novel pathological protein in a small subset of Laboratory Medicine,
Vancouver General Hospital,
patients with ALS and various FTD subtypes provide clear evidence that these disorders are related. The creation of a
Vancouver, BC, Canada
novel molecular classification of ALS and FTD based on the identity of the predominant protein abnormality has, (Prof I R A Mackenzie MD);
therefore, been possible. The striking functional and structural similarities of TDP-43 and FUS, which are both Department of Neuroscience,
DNA/RNA binding proteins, imply that abnormal RNA metabolism is a pivotal event, but the mechanisms leading to Mayo Clinic College of
Medicine, Jacksonville, FL, USA
TDP-43 and FUS accumulation and the resulting neurodegeneration are currently unknown. Nonetheless, TDP-43
(R Rademakers PhD); and
and FUS are promising candidates for the development of novel biomarker assays and targeted therapies. Institute of Neuropathology,
University Hospital of Zurich,
Introduction patients with ALS due to SOD1 mutations (ALS-SOD), Zurich, Switzerland
(M Neumann MD)
The presence of abnormal protein aggregates in the but is not seen in inclusions in other ALS forms.7 This
Correspondence to:
cytoplasm of neurons is a key characteristic of most finding indicated that other pathological proteins
Dr Manuela Neumann, Institute
neurodegenerative diseases. In several common disorders remained to be discovered. of Neuropathology, University
the disease-specific pathological protein was identified FTD is the second most frequent cause of presenile Hospital of Zurich,
decades ago (eg, tau in neurofibrillary tangles of dementia and is a clinically, genetically, and Schmelzbergstrasse 12,
8091 Zurich, Switzerland
Alzheimer’s disease and α-synuclein in Lewy bodies of pathologically heterogeneous syndrome. The clinical
manuela.neumann@usz.ch
Parkinson’s disease), providing the logical basis for phenotypes include behavioural variant FTD, semantic
research into the underlying biological mechanisms, dementia, and progressive non-fluent aphasia.8 The
diagnosis, and treatment. The biochemical composition associated neuropathology of all phenotypes is
of the ubiquitinated inclusions in most cases of characterised by FTLD, but the microscopic features
amyotrophic lateral sclerosis (ALS) and the most frequent vary. Historically, FTLD subtypes were classified as
pathological form of frontotemporal dementia (FTD), those associated with abnormal accumulation of tau
frontotemporal lobar degeneration with ubiquitinated (FTLD-tau) and those with tau-negative, ubiquitin-
inclusions (FTLD-U), remained unknown until the TAR positive inclusions (FTLD-U).8 The observation that
DNA binding protein 43 (TDP-43)1 and the fused in some ALS patients develop cognitive deficits with
sarcoma protein ([FUS] also known as translocated in prominent frontal lobe features and that in those
liposarcoma)2,3 were identified as disease-related proteins patients the associated neuropathology resembles
in these two disorders. This Review highlights the novel FTLD-U led to the idea that ALS and FTD with FTLD-U
molecular classification of ALS and FTLD that emerged might be related.9–11
from these findings and summarises developments This hypothesis was confirmed in 2006 with the
in the clinicopathological and genetic knowledge of identification of the DNA/RNA binding protein TDP-43
disorders involving TDP-43 and FUS. Particular as the pathological protein in most cases of FTLD-U,1,12
emphasis is placed on the molecular features and (renamed as FTLD-TDP)13 and SOD1-negative ALS
their pathomechanistic implications. (ALS-TDP).1,14 This discovery prompted researchers to
prioritise genetic analysis of functionally related genes
Novel molecular classification in the chromosome 16 region that had previously been
ALS is the most frequent form of motor neuron disease, linked to some familial forms of ALS, and led to
in which the loss of motor neurons from the brain and the identification of mutations in FUS as the genetic
spinal cord leads to fatal paralysis and death, generally cause, and of FUS as the pathological protein in
within 1–5 years.4 A characteristic feature of degenerating TDP-43-negative, SOD1-negative ALS (ALS-FUS).2,3
neurons is the presence of cytoplasmic inclusions FUS was subsequently identified as the pathological
positive for ubiquitin. Approximately 5% of patients with protein in most of the remaining tau-negative,
ALS have a positive family history of the disorder. The TDP-43-negative FTLD subtypes (renamed as FTLD-
first pathological mutations identified in ALS were in FUS).13 A novel molecular classification was, therefore,
SOD1,5 and account for around 20% of familial ALS created probably reflecting distinct underlying
cases. That discovery has been the basis for most ALS pathological mechanisms in the different subtypes
research in the past decade, which has provided (figure 1).15–21 Moreover, the data provided clear molecular
important insights into SOD1-mediated neurotoxic evidence that ALS and FTD represent a clinico-
effects.6 Misfolded SOD1 is present in the inclusions in pathological spectrum of diseases.

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 Review

Ubiquitin-positive inclusions in FTLD-U and ALS

Disease
TDP-43 FUS SOD1 Unknown
protein

FTLD-TDP types 1–4 ALS-TDP FTLD-FUS ALS-FUS ALS-SOD FTLD-UPS

Clinico-
pathological Unclassifiable Atypical FTLD-U
subtypes NIFID
BIBD

Associated GRN TARDBP (FUS)‡ FUS SOD1 CHMP2B

genes Chromosome 9p (ANG)†
VCP
(TARDBP)*

Figure 1: New molecular classification of FTLD-U and ALS

Both disorders are characterised by ubiquitin-positive inclusions. The ubiquitinated protein in most cases of FTLD-U and ALS is TDP-43, and these cases should be
classified as FTLD-TDP and ALS-TDP, respectively. In a small subset of cases of FTLD-U and ALS, the pathological protein is FUS, and these should be classified as
FTLD-FUS and ALS-FUS, respectively. Misfolded SOD1 is the pathological protein in ubiquitin-positive inclusions in ALS with SOD1 mutations, and these are classified
as ALS-SOD. In a few cases of FTLD-U, the ubiquitinated protein is unknown, and these cases are classified as FTLD-UPS, to indicate that inclusions are currently
labelled only by markers of the ubiquitin-proteasome system. ALS=amyotrophic lateral sclerosis. BIBD=basophilic inclusion body disease. FTD=frontotemporal
dementia. FTLD=frontotemporal lobar degeneration. FTLD-U=FTLD with ubiquitinated inclusions. NIFID=neuronal intermediate filament inclusion disease.
UPS=ubiquitin-proteasome system. *Rare reports of TARDBP mutations in patients with FTD or ALS with FTD.15–17 †One case report.18 ‡Rare reports of FUS mutations
in patients with FTD or ALS with FTD.19–21

R495X R514G/S R521G/C/H and a Gly-rich C-terminal region that allow it to bind
R216C R234C/L G507D R514S;G515C R522G single-stranded DNA, RNA, and proteins.22,23 The exon
G225V R244C K510R/E H517Q*/P R524W/T/S skipping and splicing inhibitory activity requires the
G230C M254V S513P R518G/K P525L
C-terminal region, which interacts with other members
G187S S462F
S57del G156E G191S S402_P411delinsGGGG G466VfsX14 of the heterogeneous nuclear ribonucleoprotein family.24
TDP-43 can continuously shuttle between the nucleus
FUS QGSY-rich region Gly-rich RRM RGG-rich ZnF RGG-rich 526 aa
and cytoplasm—a process partly regulated by nuclear
NES NLS
localisation signal and nuclear export signal motifs.25,26
NLS NES
In addition to the well characterised role of TDP-43 in
TDP-43 RRM1 RRM2 Gly-rich 414 aa
transcription and splicing regulation, roles have been
D169G K263E suggested in other cellular processes, such as microRNA
N267S processing, apoptosis, cell division, and stabilisation of
G287S G294A/V M311V Q343R Y374X I383V messenger RNA.27–31 Early embryonic death in TDP-43
G290A G295R/S A315T N345K N378D G384R
S292N G298S A321V/G G348C/V S379P/C N390D/S knockout mice supports an essential role for this protein
Q331K N352T/S A382P/T S393L in normal development.32–34 In the brain, transient
S332N R361S
G335D P363A
redistribution to the cytoplasm could be a normal
M337V response to neuronal injury.35,36 Moreover, studies have
suggested a role in the regulation of neuronal plasticity37
Figure 2: Schematic overview of protein domains of FUS and TDP-43 and identified gene mutations
and maintenance of dendritic integrity.38
associated with ALS and FTLD
Mutations identified so far in FUS and TARDBP are summarised by their predicted protein changes and shown with
their relative positions in the protein domains of FUS (Genbank Accession number CAG33028.1) and TDP-43 Clinicopathological spectrum of TDP-43 proteinopathies
(Genbank Accession number NP_031401.1). Mutations shown in black were identified in patients with ALS only, TDP-43 proteinopathies are defined by the presence of
and mutations in blue were also associated with FTD phenotypes. ALS=amyotrophic lateral sclerosis.
inclusions composed of abnormal TDP-43. Antibodies
FTLD=frontotemporal lobar degeneration. NES=nuclear export signal. NLS=nuclear localisation signal.
QGSY=Gln-Gly-Ser-Tyr-rich region. RGG=Arg-Gly-Gly-rich motif. RRM=RNA recognition motif. against TDP-43 consistently label the inclusions in
ZnF=Cys2/Cys2-type zinc finger motif. FTLD-TDP and ALS-TDP, but not those in various other
neurodegenerative disorders.1,12,39,40 They also label
TDP-43 previously unrecognised ubiquitin-negative pathologies,
Normal function including glial cytoplasmic inclusions,41 neurons that
TDP-43 is a 414 aminoacid protein encoded by TARDBP showed diffuse granular cytoplasmic staining (so-called
on chromosome 1p36.2, and consists of five coding and preinclusions),42 and delicate neurites in the CA1 region.43
one non-coding exon (figure 2). The protein is highly Of potential functional importance, the presence of
conserved, widely expressed, and predominantly localised TDP-43-positive inclusions is consistently associated
to the nucleus.22 It contains two RNA recognition motifs with a substantial reduction in normal physiological

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 Review

A B I

Control FTLD-TDP FTLD-TDP

100

C D 75 ***

MW (kDa)
50
**

E F
37

25 *

Pan TDP Phospho-

G H specific TDP

Figure 3: Pathological features in ALS-TDP and FTLD-TDP

TDP-43 immunohistochemistry on paraffin-embedded tissue showing (A) TDP-43-immunoreactive skein-like and (B) round inclusions in motor neurons in ALS-TDP.
FTLD-TDP pathology is subdivided into four distinct morphological subtypes: (C) type 1 is characterised by compact neuronal cytoplasmic inclusions and short
neurites; (D) type 2 is characterised by long neurites; (E) type 3 is characterised by compact and granular cytoplasmic inclusions; and (F) type 4 is characterised by
numerous neuronal intranuclear inclusions. (G) Cytoplasmic inclusions in the dentate granule cells of the hippocampus. Nuclear staining is absent in inclusion-bearing
cells. (H) Glial cytoplasmic inclusions. Scale bars: 40 μm (D), 20 μm (A, C, G), 15 μm (E, F), and 10 μm (B, H). (I) Immunoblot analysis of urea fractions isolated from
brain tissue, showing the highly characteristic biochemical signature of TDP-43 in FTLD-TDP, with pathological bands of approximately 25 kDa (*) and 45 kDa (**), and
a high-molecular-weight smear (***) that are not detected in controls. The arrow indicates the wild-type 43 kDa TDP-43 band present in controls and in patients with
FTLD-TDP. Notably, only pathological TDP-43 is detected by a phosphorylation-specific TDP-43 antibody against phosphorylated serine residues 409 and 410.45
ALS=amyotrophic lateral sclerosis. ALS-TDP=TDP-43-positive ALS. FTLD-TDP=TDP-43-positive frontotemporal lobar degeneration.

diffuse nuclear staining.1,39 Pathological forms of TDP-43
show evidence of abnormal processing, being hyper-
phosphorylated, ubiquitinated, and N-terminally
truncated (figure 3).1,44–46
ALS-TDP
In most cases of sporadic ALS, the neuropathology is
characterised by abnormal cytoplasmic accumulation of
TDP-43 in neurons and glia of the primary motor cortex,
brainstem motor nuclei, spinal cord, and the associated
white matter tracts.14 Neuronal cytoplasmic inclusions
in motor neurons vary in morphology and include
small granules, compact Lewy-body-like inclusions, and
filamentous skeins (figure 3). No obvious correlation
between the site of disease onset and the spread of
TDP-43 pathology in the spinal cord has been found.47
ALS with dementia also involves accumulations of
TDP-43 in the extramotor neocortex and hippocampus.14,48
The presence of pathological TDP-43 in a wide variety of
subcortical regions beyond the pyramidal system might
correlate with other clinical features, such as
parkinsonism.42,49,50 Pathological TDP-43 seems to be a
less consistent feature of other motor neuron disease
phenotypes, such as primary lateral sclerosis and
progressive muscular atrophy,51 but further studies are
needed to confirm the degree of contribution.

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 Review
ALS associated with mutations in TARDBP is reported
to have TDP-43 pathology indistinguishable from that
found in most sporadic cases.17,52–55 Several other causative
genes and genetically linked chromosomal loci have been
identified for familial ALS.56 TDP-43-positive ALS
pathology in combination with FTLD-TDP has been
described in affected members of families with FTD and
ALS linked to chromosome 9p.40 One case report of ALS
associated with a mutation in ANG described TDP-43
pathology but with several atypical features.18 Mutations
in SOD1 and FUS are not associated with pathological
TDP-43.2,3,14,19,57–60
FTLD-TDP
FTLD-TDP is the most frequent pathological FTLD
subtype (~50% of cases) and can present with any of the
major clinical phenotypes of the FTD syndrome,
including behavioural variant FTD, semantic dementia,
or progressive non-fluent aphasia.39,40 Extrapyramidal
movement disorders comprise a frequently seen minor
feature, and FTLD-TDP rarely presents as Parkinson’s
disease or corticobasal syndrome.61–63 TDP-43 is also the
pathological substrate of most cases of dementia
combined with ALS.64,65 Although amnestic features are
generally mild or emerge late in the course of FTD, some
cases of FTLD-TDP present with an Alzheimer-like
phenotype.62
TDP-43-positive cytoplasmic inclusions and neurites
in the frontotemporal neocortex and dentate granule
cells of the hippocampus characterise FTLD-TDP
(figure 3).1,12,39,40 Lentiform neuronal intranuclear inclu-
sions are seen more frequently in familial cases than in
sporadic cases,39,66 and glial TDP-43 pathology is present
in the subcortical white matter in all cases but to varying
degrees.41 TDP-43 pathology is also present in a wide
range of subcortical neuroanatomical sites.48,67 Four
different patterns of TDP-43 pathology are recognised in
FTLD-TDP, based on the anatomical distribution,
morphology, and relative proportion of distinct types of
inclusions (table).68–70 The relevance of this heterogeneity
is supported by clinical and genetic correlations: subtype 1
presents with either behavioural variant FTD or
progressive non-fluent aphasia and represents all cases
Type 1*
Neuronal cytoplasmic inclusions +++
Dystrophic neurites +++
Neuronal intranuclear inclusions –/++
Major clinical phenotypes bvFTD; PNFA
Associated gene defects GRN mutations
Semiquantative grading is used: –, absent; +, few; ++, moderate; +++, numerous. ALS=amyotrophic lateral sclerosis. bvFTD=behavioural variant frontotemporal dementia.
FTD=frontotemporal dementia. FTLD=frontotemporal lobar degeneration. FTLD-U=FTLD with ubiquitinated inclusions. FTLD-TDP=TDP-43-positive FTLD. IBMPFD=inclusion
body myopathy associated with Paget disease of bone and frontotemporal dementia. PNFA=progressive non-fluent aphasia. SD=semantic dementia. *Numbering of types
according to MacKenzie et al.69 The same three patterns of FTLD-U pathology were independently described by Sampathu et al,68 but with a different numbering system.
Table: Neuropathological subtypes of FTLD-TDP
998
with GRN mutations; subtype 2 generally presents with
sporadic semantic dementia; subtype 3 is frequently
associated with ALS with FTD, including familial cases
linked to chromosome 9p; and subtype 4 is found only in
cases associated with VCP mutations.39,40,67,69,71–74
Other disorders with TDP-43 pathology
Perry syndrome is a rare autosomal dominant disorder
characterised by parkinsonism, depression, weight loss,
and hypoventilation and is associated with mutations in
exon 2 of DCTN1 encoding p150glued, the major subunit of
the dynactin protein complex.75 TDP-43 pathology is
largely restricted to the substantia nigra and other nuclei
of the rostral brainstem and the basal ganglia.76 Mutations
in other regions of DCTN1 have previously been reported
in ALS with and without FTD,77–79 but it is unknown
whether these disorders show TDP-43 pathology.
Concomitant TDP-43 pathology has been reported in
several other neurodegenerative disorders. This feature
is consistently seen in Guamanian ALS-parkinsonism-
dementia complex,80,81 in some cases of hippocampal
sclerosis,82,83 and in 20–60% of cases of the most common
neurodegenerative disorders, including Alzheimer’s
disease and Lewy body disorders where TDP-43 pathology
is mainly found in the mesial lobe.83–86
Genes and risk factors for TDP-43 proteinopathies
TARDBP
In early 2008, mutations in TARDBP were identified in
a subset of patients with ALS.87–89 So far, 38 different
TARDBP mutations have been reported in
79 genealogically unrelated families in the USA, Europe,
Australia, and Asia. Mutations in this gene account for
4% of all familial and around 1·5% of sporadic ALS
cases (figure 2). Some evidence indicates that
frequencies of TARDBP mutations are higher among
patients from southern Europe than among patients
from other regions.90
Most TARDBP mutations are missense changes in
exon 6, encoding the Gly-rich region and C-terminus of
TDP-43, and are suggested to interfere with normal
protein-protein interactions of TDP-43 (eg, by alteration
of phosphorylation or increase of aggregation tendency).
Type 2* Type 3* Type 4
+ ++ +
+++ + +++
Generally absent Generally absent +++
SD bvFTD; FTD with ALS IBMPFD
.. Linkage to chromosome 9p VCP mutations
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These effects might lead to impaired nuclear import,
altered exon skipping, or transcriptional repression
activity.91 Two exceptions are the Asp169Gly mutation,
which is located in exon 4, and the truncation mutation
Tyr374X. These two mutations have, however, each been
identified in only one patient with sporadic ALS and,
therefore, their pathological roles remain unproven.88,92
Despite extensive screening, TARDBP multiplications
have not been identified.93–95 The clinical phenotype
associated with TARDBP mutations is classic adult-onset
ALS with bulbar, limb, or respiratory symptoms,
although age of onset and duration vary.88,90,92,96 Three
different TARDBP mutations have been reported in
patients with FTD: Gly295Ser in a patient with FTD
and ALS, Asn267Ser in a patient with behavioural
variant FTD, and Lys263Gln in a patient with
behavioural variant FTD with supranuclear gaze palsy
and chorea. TARDBP mutations, therefore, might rarely
cause FTD.15–17
GRN
Mutations in the gene encoding the secreted growth
factor progranulin (GRN) are the most frequent cause
of familial FTLD identified so far (up to 20%; 5–10% of
all FTLD cases).97–99 68 different GRN mutations have
already been identified in more than 200 families.
Despite their dominant nature, GRN mutations all
cause disease through a haploinsufficiency/loss-of-
function mechanism.98,99 Although other types of
pathogenic mutations have been reported, most are
small out-of-frame insertions or deletions, splice-site, or
nonsense mutations that introduce a premature
termination codon and result in the degradation of the
mutant messenger RNA via nonsense-mediated
decay.98–102 Of clinical relevance, concentrations of
progranulin in plasma, serum, and CSF are predictive
of GRN mutation status in mutation carriers with and
without symptoms.103–105 As a result, ELISA is expected to
become an inexpensive alternative to GRN sequence
analysis, having the advantage of identifying patients
with rare mutations, such as single-exon deletions or
complete gene rearrangements, that would otherwise
be difficult to detect.105
FTLD patients with GRN mutations show notable
variability in clinical phenotypes, including age of onset
and disease duration, even within individual families.106–109
The most frequent presentations are behavioural variant
FTD and progressive non-fluent aphasia. Memory
impairment and extrapyramidal features are also
frequently seen, but ALS is rare. Finally, common variants
in GRN have been reported as genetic risk factors for
TDP-43 proteinopathies.110–114 The exact mechanism by
which a loss of progranulin leads to TDP-43 pathology is
unknown. GRN knockdown in cell culture was reported
to lead to increased cleavage of TDP-43 into potentially
pathogenic C-terminal fragments,115 but this finding was
not confirmed in other studies.116
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VCP
Mutations in the valosin containing protein gene (VCP)
cause a rare familial syndrome with TDP-43 pathology, in
which the clinical features of inclusion body myopathy,
Paget’s disease of bone, and behavioural variant FTD
show variable penetrance.117 A total of 14 different
missense mutations have been identified in 30 families.118
The link between VCP dysfunction and TDP-43
accumulation is unsolved, but transgenic mice expressing
mutant forms of VCP develop pathology in muscle,
brain, and bone, recapitulating some features of the
symptoms in humans.119
Chromosome 9p
An association with variation on the chromosomal locus
9p13.2–21.3 has been consistently identified in genome-
wide linkage studies of autosomal dominant families in
which individual patients present with behavioural variant
FTD, ALS, or a combined phenotype.120–126 Despite extensive
genetic screening of the shared candidate region, the
disease-associated gene has not yet been identified.
Notably, a two-stage genome-wide association study that
included data from nearly 20 000 patients with sporadic
ALS identified a significant genome-wide association with
UNC13A and a significant association in the replication
phase with a region on chromosome 9p, overlapping with For the FTD mutation database
the locus associated with familial ALS with FTD.127 The see http://www.molgen.ua.ac.
be/FTDmutations/
association with the chromosome 9p locus has been
confirmed in two independent genome-wide association
studies.128,129 These findings strongly suggest that the
disease gene is also a risk factor for sporadic ALS.
TMEM106B
Large cohorts of patients with FTD are rare, and the in-
herent molecular heterogeneity had previously prohibited
genome-wide association studies. The identification of a
pathological role for TDP-43, however, prompted a large
collaborative effort in which a homogeneous population
of patients with pathologically confirmed FTLD-TDP
was identified for such a study. Genome-wide associations
were identified between FTLD-TDP and variants in
TMEM106B.130 The function of the TMEM106B protein is
currently unknown, but increased expression is suggested
to be a risk factor for developing FTLD-TDP.
Pathogenesis of TDP-43 proteinopathies
Little is known about the role of TARDBP mutations or
TDP-43 pathology in neurodegeneration. The abnormal
intracellular redistribution of TDP-43 from the nucleus
to the cytoplasm in inclusion-bearing neurons suggests a
pathogenic mechanism associated with the loss of normal
nuclear TDP-43 function.27,28,31 In support of this
hypothesis, knock down of TDP-43 in human cell lines
induces morphological nuclear defects, altered neurite
outgrowth, dysregulation of cell cycle, and increased cell
death.131,132 Reduced TDP-43 expression in drosophila,
zebrafish, and mouse models resulted in motor deficits,
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some with structural abnormalities of motor
neurons.32,133,134 In one model, this phenotype could be
rescued by coexpression of wild-type TDP-43.133
Sequestration of TDP-43 in cellular inclusions could
induce a toxic gain of function independently of the
basic biological role of this protein. A study in which
TDP-43 was overexpressed in yeast suggested that only
aggregated TDP-43 was toxic.135 Several in-vitro studies
have shown that C-terminal fragments of TDP-43 are
more likely than full-length TDP-43 to form insoluble
cytoplasmic aggregates that become ubiquitinated,
phosphorylated, and toxic to cells.136–139 Although one
study suggested that these cytoplasmic aggregates bind
the endogenous full-length protein and deplete it from
the nucleus,137 another showed retention of normal
nuclear expression, suggesting a pure toxic effect.139
Some in-vivo studies, in which wild-type human
TDP-43 was overexpressed in drosophila,38,140 mice,141
and by viral expression in rats,142 have shown the
formation of TDP-43 immunoreactive neuronal cyto-
plasmic inclusions with associated neurodegeneration
and motor dysfunction. The findings, however, are
inconsistent, because at least one study found no effect
in transgenic rats overexpressing the wild-type human
protein.143 The two basic disease mechanisms are not
mutually exclusive, because factors that affect the
normal intracellular trafficking of TDP-43 might
predispose to the formation of abnormal aggregates and
the loss of nuclear localisation.26
The identification of TARDBP mutations in ALS has
provided additional clues to the possible pathogenic
mechanisms involved in TDP-43 proteinopathies. So
far, no consequences on splicing activity, messenger
RNA processing, or protein binding have been
described.30,137,144 Expression of mutant TDP-43 in
cultured cells is claimed to result in increased gen-
eration of C-terminal fragments, with even more
cytoplasmic aggregation and toxic effects than wild-type
TDP-43.88,89,133,137,138,145,146 Results from transgenic animal
models overexpressing mutant forms of human TDP-43
are somewhat inconsistent and difficult to interpret. In
one study, transgenic rats overexpressing TDP-43 with
the Met337Val mutation developed progressive weakness
and early death.143 Neurodegeneration was widespread
but was most severe in the spinal cord. The human
TDP-43, however, was diffusely distributed in neuronal
nuclei and cytoplasm, with cytoplasmic aggregates
being rare and present only in the cortex. Transgenic
mice expressing the human Ala315Thr mutation have
also been reported to develop progressive motor
abnormalities with widespread neurodegeneration.147
Although some neuronal populations were found to
have cytoplasmic inclusions immunoreactive for
ubiquitin, these did not contain aggregated TDP-43 and
loss of nuclear TDP-43 was inconsistent.
The inconsistencies among the various in-vitro and
in-vivo models currently available, and the fact that none
1000
fully replicates the features of human disease, indicate
that the pathogenic mechanism in TDP-43 proteinopathy
is still unresolved. Given the multifaceted role of TDP-43,
however, multiple disease mechanisms might be involved.
FUS
Normal function
FUS is a ubiquitously expressed 526 aminoacid protein
encoded by 15 exons that belongs to the FET/TET family
of multifunctional DNA/RNA binding proteins (together
with Ewing’s sarcoma protein and the TATA-binding
protein associated factor TAF15),148 and was originally
discovered as a component of fusion oncogenes in
human cancers.149 It contains an N-terminal Gln-Gly-
Ser-Tyr-rich domain, a Gly-rich domain, an RNA
recognition motif, multiple Arg-Gly-Gly repeats, a zinc
finger motif,149 and a highly conserved extreme
C-terminus that encodes for a non-classic nuclear
localisation signal that is recognised by transportin
(figure 2).150–152 FUS shows nuclear and cytoplasmic
expression,153 and continuously shuttles between the
nucleus and the cytoplasm.154 It is implicated in
numerous cellular processes, including cell proliferation,
DNA repair, transcription regulation, and RNA and
microRNA processing,28,149,155 although its precise
function is poorly characterised. This protein might be
involved in neuronal plasticity and maintenance of
dendritic integrity by transporting messenger RNA to
dendritic spines for local translation.156,157 FUS knockout
mice show variable phenotypes with perinatal lethality
on an inbred genetic background158 but viability on an
outbred genetic background.159
Clinicopathological spectrum of FUS proteinopathies
FUS proteinopathies are characterised mainly by the
presence of inclusions immunoreactive for FUS in
neuronal and glial cells, but morphology and distribution
of inclusions vary between entities (figure 4). Re-
distribution of FUS from the nucleus to the cytoplasm is
seen to a lesser degree than for TDP-43, with at least
some of the nuclear staining being preserved in neurons
with FUS inclusions.57,160 Despite increased FUS con-
centrations in insoluble protein fractions, no obvious
disease-associated modifications of this protein, such as
truncation, phosphorylation, or ubiquitination, have yet
been identified.160
ALS-FUS
Mutations in the FUS gene were identified as the
primary cause of familial ALS linked to chromosome 16.2,3
Patients with these mutations present with classic ALS
or might present with atypical phenotypes. Pathological
data on FUS mutation carriers are available for only a
few cases and only for single mutations.2,3,19,57,59,60,161 Initial
reports described motor neuron loss in the spinal cord
and to a lesser degree in the brainstem, with inconsistent
demyelination of corticospinal tracts and the presence
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A B C D E

G H I J

Figure 4: Pathological features in ALS-FUS and FTLD-FUS

FUS immunohistochemistry on paraffin-embedded tissue showing FUS-immunoreactive cytoplasmic inclusions in lower motor neurons of a familial ALS case with
the Arg521Cys FUS mutation, with (A) filamentous or (B) granular morphology. Nuclear FUS staining is present in the inclusion-bearing neuron (B). Three distinct
pathological entities of FTLD-FUS can be delineated. In atypical FTLD-U, FUS-immunoreactive cytoplasmic inclusions are detected in (C) the dentate granule cells and
(D) the frontal cortex, and (E, F) characteristic vermiform intranuclear inclusions are visible. Numerous FUS-immunoreactive cytoplasmic inclusions are detected with
variable morphology in the frontal cortex of (G) basophilic inclusion body disease and (H) neuronal intermediate filament inclusion disease. (I) Many inclusions in the
latter disorder are labelled only for FUS, and neurons with α-internexin-positive inclusions always show FUS pathology, with labelling of separate inclusion
components as demonstrated by double-label immunofluorescence for FUS (red) and α-internexin (green). (J) FUS-immunoreactive glial cytoplasmic inclusions are
present in ALS-FUS and FTLD-FUS. Scale bars: 30 μm (D, G, H), 20 μm (C), 15 μm (A, B), 8 μm (E, F, I, J). ALS=amyotrophic lateral sclerosis. FTLD=frontotemporal lobar
degeneration. FTLD-U=FTLD with ubiquitinated inclusions.

of FUS-positive inclusions3 or increased concentrations
of cytoplasmic FUS2 in lower motor neurons. More
detailed pathological analysis of several cases with the
Arg521Cys mutation revealed more-widespread FUS
pathology, involving glial cells and regions outside the
pyramidal motor system, such as motor nuclei in the
brainstem, striatum, thalamus, and substantia nigra.
The importance of extramotor pathology in ALS-FUS,
as well as variability of pathological features among
different FUS mutations, needs to be assessed in
clinicopathological correlative studies.
FTLD-FUS
FTLD-FUS consists of three distinct clinicopathological
entities (figure 1).13 Although all FTLD-U cases were
thought to involve TDP-43, studies in large series of
patients identified up to 20% of cases that were TDP-43
negative. This FTLD subtype was termed atypical
FTLD-U162,163 and is characterised by sporadic early-onset
behavioural variant FTD. Motor symptoms are not
prominent, but if present they are typically limited to
mild rigidity, intermittent hyperkinesias, or both.162–165
In addition to frontotemporal atrophy severe caudate
atrophy, and hippocampal sclerosis are characteristic
features; caudate atrophy on neuroimaging has been
proposed as a useful clinical predictor of this FTLD
subtype.164,166 FUS pathology in atypical FTLD-U is most
abundant in the frontotemporal neocortex and the
hippocampus but is also present in the striatum,
thalamus, and brainstem. Despite the absence of obvious
clinical features of motor neuron disease, most cases
reveal at least some FUS inclusions in lower motor
neurons.160,162–164 The most intriguing lesions in atypical
FTLD-U are neuronal intranuclear inclusions, which
appear as elongated straight, curved, or twisted
(vermiform) filaments, or sometimes as ring-like
structures at the periphery of the nucleus and are most
easily recognised in the hippocampus (figure 4).160
Basophilic inclusion body disease is a rare and clinically
heterogeneous disorder named for the presence of
basophilic inclusions that are visible with haematoxylin
and eosin staining. It can present with behavioural
variant FTD,167–169 juvenile or adult-onset ALS,170–174 or a
combination of both.167,168,175,176 FUS immunohistochemistry
labels the basophilic inclusions and much more
widespread, previously unrecognised neuronal and glial
pathology, predominantly in the frontal cortex, basal
ganglia, brainstem, cerebellum, and spinal cord. Intra-
nuclear inclusions are usually absent.177
Neuronal intermediate filament inclusion disease is
an uncommon neurodegenerative disorder that typically
presents as early-onset sporadic behavioural variant
FTD that is associated with pyramidal or extrapyramidal
movement disorders, or both. This subtype was initially
defined as a unique entity owing to the presence of
neuronal inclusions immunoreactive for all class IV
intermediate filaments (light, medium, and heavy
neurofilament subunits and α-internexin).178–180 The role
of intermediate filaments in the pathogenesis of
neuronal intermediate filament inclusion disease, how-
ever, is uncertain, because only a subset of inclusions
are positive for these proteins. Antibodies against
FUS label many more inclusions than do those
against intermediate filaments.181 A large proportion of

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 Review
inclusions are only detected by FUS staining. By
contrast, aggregates positive for intermediate filaments
are always found in association with FUS pathology in
the same cell (figure 4),181 thereby suggesting that FUS
accumulation has a more central role in neuronal
intermediate filament inclusion disease than does
accumulation of intermediate filaments, which might
be a secondary phenomenon, possibly caused by altered
messenger RNA processing or trafficking.
Other disorders with FUS pathology
FUS immunoreactivity has been described in the
intranuclear inclusions in a wide spectrum of
polyglutamine diseases, such as Huntington’s disease
and spinocerebellar ataxias.182,183 The functional relevance
of FUS accumulation in these disorders, however,
remains to be determined.
Spectrum of FUS gene mutations
FUS was deemed to be a candidate gene for familial ALS
linked to chromosome 16 on the basis of its homologous
domain structure to TDP-43.2,3 In only 2 years, 35 different
pathogenic FUS mutations have been identified that
account for about 4% of familial ALS cases, but less than
1% of sporadic ALS cases (figure 2).2,3,19,20,57,59,60,161,184–192
Mutation carriers have been reported in the USA, Europe,
Australia, Africa, and Asia. The mutation frequency in
familial ALS cohorts has varied from 0 to 10%, and might
depend on the geographical origin of patients, the
numbers screened, or the fact that only a subset of exons
was analysed.161,185–187,191,192 Mutations in FUS are mostly
missense changes that cluster in two regions: most are
located in exons 13–15, which encode the Arg-Gly-Gly-rich
region and the nuclear localisation signal of FUS, and
about a third of the mutations are located in exons 3, 5,
and 6, which encode the Gln-Gly-Ser-Tyr-rich and Gly-rich
regions. Mutations affecting the C-terminus are clearly
pathogenic, with several of them leading to disruption of
the nuclear localisation signal motif, which impairs
transportin-mediated nuclear import.152 C-terminal
mutations have so far been found in 54 patients with
familial ALS, but in only four patients with sporadic
ALS.2,3,19,20,57,59,60,161,184–192 By contrast, seven of 11 patients with
mutations affecting exons 3, 5, or 6 had sporadic disease
and no segregation with disease was seen for the other
four familial cases.2,20,57,189,191 Although several of these
mutations in exons 3, 5, and 6 affect evolutionary
conserved aminoacids and are predicted to be harmful on
the basis of findings of in-silico analysis, no functional
consequences have yet been shown.152 Moreover, no
autopsy data showing FUS pathology are yet available for
these mutations, which leaves open the possibility that
they might be risk factors rather than true causal
mutations. In addition to missense mutations, several
in-frame insertions and deletions affect the Gly stretches
encoded by exons 5 and 6. Although initially suggested to
cause disease, subsequent studies identified similar
1002
variants in controls, which indicates a benign role for
these polymorphisms.2,21,59,90
Two FUS mutations have been reported that completely
truncate the C-terminus: a splice-site mutation inducing
skipping of exon 14 (Gly466ValfsX14) and a nonsense
mutation in exon 14 (Arg495X).187,192 The mutation
Gly466ValfsX14 has occurred de novo, leading to sporadic
disease.187 Because relatives of sporadic FUS mutation
carriers have been unavailable in most studies, the true
frequency of de-novo FUS mutations remains to be
determined.
Patients with FUS mutations generally present with
classic ALS, although some develop very little or no upper
motor neuron signs. Some studies have associated
specific phenotypes with certain FUS mutations
(eg, symmetric weakness of proximal muscle groups
with only late involvement of distal muscles and
minimum evidence of upper motor neuron involvement
for the common Arg521Cys mutation).19,20,57,189,193 The
identification of additional families carrying the same
mutation should provide more insight into the
correlations between genotypes and phenotypes. Onset
age varies strikingly from early 20s to late 70s, even
within families.57 For some mutations non-penetrance
has been described with mutation carriers surviving to
age 80 years without signs of ALS,2,59,185 a finding that
should be taken into consideration when providing
genetic counselling for FUS mutation carriers.
Reports of FTD associated with FUS mutations are
rare. Two patients had ALS with FTD features.19,20
Although one patient with the Met254Val mutation
presented with behavioural variant FTD, both parents
were still alive and asymptomatic, and no autopsy data
were available to confirm FUS pathology.21 Additional
studies will, therefore, be needed to determine whether
FUS is a true cause of FTD.
In sharp contrast to ALS-FUS, where genetic
sequencing has shown FUS mutations in almost all
cases, extensive analysis of genomic DNA, complementary
DNA, and messenger RNA in patients with FTLD-FUS
has thus far excluded FUS mutations.160,165,181 Although
this finding supports the sporadic nature of FTLD-FUS
in most patients, the presence of recessive or de-novo
mutations in FUS or other genes cannot be excluded.
Pathogenesis of FUS proteinopathies
Understanding of the underlying pathomechanisms
leading to FUS accumulation and FUS-mediated
neurodegeneration is still limited. As for TDP-43
proteinopathies, a toxic gain-of-function mechanism, a
loss-of-function mechanism by depletion of physiological
FUS and maybe co-sequestration of other vital factors, or
both is plausible. So far, no in-vivo models for FUS
proteinopathies have been reported. Expression of
C-terminal FUS missense and truncation mutations
in vitro have revealed variable increases in cytoplasmic
concentrations of FUS2,3,187 that are compatible with
www.thelancet.com/neurology Vol 9 October 2010

Search strategy and selection criteria
We searched PubMed to identify full-text articles published
up to June, 2010, with the terms “frontotemporal dementia”,
“amyotrophic lateral sclerosis”, “TDP-43”, “TARDBP”, “FUS”,
and “TLS”. Articles were also identified through searches of
the authors’ own article collections. Only articles published in
English were reviewed.
disruption or deletion of the nuclear localisation signal
and impaired transportin-mediated nuclear import,152
thereby implicating altered nuclear import as a key event
in disease pathogenesis. Notably, the two reported
truncation mutations led to very aggressive and early-
onset forms of ALS,187,192 which might reflect their severe
impairment of nuclear import. Further studies are needed
to address whether and how additional domains and post-
translational modifications modulate nuclear-cytoplasmic
shuttling of FUS. Modifications of FUS under specific
conditions include phosphorylation194,195 and arginine
methylation.196,197 Although the associated functional
consequences have not yet been elucidated for FUS,
similar modifications of other RNA binding proteins
affect protein-protein or protein-RNA interactions that
are crucial for nuclear-cytoplasmic shuttling.198,199 Although
the ways in which impaired nuclear import of FUS
mediates neurodegeneration remain unclear, recruitment
of FUS into stress granules upon interference with
nuclear import in vitro implicates formation of stress
granules in disease pathogenesis.152 This hypothesis is
supported by the presence of stress granule markers in
human FUS inclusions.152,173
Conclusions and future directions
The seminal discoveries of two novel key players, TDP-43
and FUS, have led to a notable shift in the understanding
of pathomechanisms in ALS and FTD, and have opened
new avenues for research. The structural and functional
similarities of these proteins implicate alterations in
processing of RNA and microRNA as key events in
disease development, although the underlying functional
mechanisms remain unresolved. So far, all studies except
one200 have shown TDP-43 and FUS pathology to be
mutually exclusive, thereby implying independent disease
pathways.3,59,160,164 However, the possibility of interactions
between TDP-43 and FUS and their associations with the
protein optineurin, which has been related to familial
ALS,201 needs to be addressed in more detail. Future efforts
should focus on the generation and establishment of valid
in-vivo models, the identification of disease-relevant post-
translational modifications, and brain-specific messenger
RNA and microRNA targets of both proteins. From the
clinical perspective, TDP-43 and FUS might be promising
candidates for the development of novel biomarker assays
for the diagnosis202,203 and progression of disease, and for
the development of targeted therapies.
www.thelancet.com/neurology Vol 9 October 2010
Review
Contributors
All authors contributed equally to the conception, design, literature
search, and writing of this Review.
Conflicts of interest
We declare that we have no conflicts of interest.
Acknowledgments
We thank Richard Crook, Department of Neuroscience, Mayo Clinic
College of Medicine, Jacksonville, FL, USA, for help on figure 2.
Work relevant to this Review was funded by grants from the National
Institutes of Health/National Institute on Aging (AG26251-03A1 to
RR), the ALS Association (RR), the German Federal Ministry of
Education and Research (MN, grant number 01GI0704), the Synapsis
Foundation (MN), Canadian Institutes of Health Research (IRAM,
grant number 74580), and the Pacific Alzheimer Research
Foundation (IRAM).
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