Expression of human FUS protein in
Drosophila leads to progressive
Yanbo Chen1,3*, Mengxue Yang2*, Jianwen Deng2, Xiaoping Chen3, Ye Ye2, Li Zhu2, Jianghong Liu2,
Haihong Ye2, Yan Shen1, Yan Li2,3, Elizabeth J. Rao3,5, Kazuo Fushimi3, Xiaohong Zhou3, Eileen H. Bigio4,
Marsel Mesulam4, Qi Xu1✉, Jane Y. Wu2,3✉
1National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Science
and Peking Union Medical College, Tsinghua University, Beijing 100730, China
2State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101,
3Department of Neurology, Center for Genetic Medicine, Lurie Cancer Center, Northwestern University Feinberg School of
Medicine, 303 E. Superior St., Chicago, IL 60611 USA
4The Cognitive Neurology & Alzheimer's Disease Center, Northwestern University Feinberg School of Medicine, 303 E. Chicago
Ave., Chicago, IL 60611, USA
5Trumbull College, Yale University, New Haven, CT 06511, USA
✉ Correspondence: firstname.lastname@example.org (Qi Xu), email@example.com (J. Y. Wu)
Received June 17, 2011Accepted June 23, 2011
Mutations in the Fused in sarcoma/Translated in liposar-
coma gene (FUS/TLS, FUS) have been identified among
patients with amyotrophic lateral sclerosis (ALS). FUS
protein aggregation is a major pathological hallmark of
FUS proteinopathy, a group of neurodegenerative dis-
eases characterized by FUS-immunoreactive inclusion
bodies. We prepared transgenic Drosophila expressing
either the wild type (Wt) or ALS-mutant human FUS
protein (hFUS) using the UAS-Gal4 system. When
expressing Wt, R524S or P525L mutant FUS in photo-
receptors, mushroom bodies (MBs) or motor neurons
(MNs), transgenic flies show age-dependent progressive
neural damages, including axonal loss in MB neurons,
morphological changes and functional impairment in
MNs. The transgenic flies expressing the hFUS gene
recapitulate key features of FUS proteinopathy, repre-
senting the first stable animal model for this group of
(FTLD), FUS proteinopathy, animal model, amyotrophic
lateral sclerosis, neurodegeneration
Since the landmark discovery of TAR-DNA binding protein of
43kDa (TDP-43) as a characteristic component of inclusion
bodies in amyotrophic lateral sclerosis (ALS) and tau-
negative/ubiquitin-positive frontotemporal lobar degeneration
(FTLD-U) (Neumann et al., 2006), a number of mutations
have been identified in ALS patients in two genes encoding
multi-functional RNA/DNA binding proteins, TDP-43 and FUS
(reviewed in Neumann et al., 2006, 2009; Mackenzie et al.,
2011). FUS-immunoreactive pathology hasbeen reported in a
range of neurodegenerative diseases, including ALS, FTLD-
U and polyglutamine diseases. Approximately 10% of FTLD
patients show FUS-immunoreactive pathology and are
classified as FTLD-FUS (Neumann et al., 2009; Mackenzie
et al., 2010). FUS mutations are recognized as the second
most common cause of familial ALS in a Spanish population
(Syriani et al., 2010).
The human FUS (also known as hnRNP P2) gene on
chromosome 16 encodes a 526-amino acid RNA/DNA
binding protein of versatile function, ranging from DNA repair,
transcriptional regulation, splicing modulation, RNA transport
to translational control (Crozat et al., 1993; Rabbitts et al.,
1993; Prasad et al., 1994; Aman et al., 1996; Zinszner et al.,
*These authors contributed equally to the work.
© Higher Education Press and Springer-Verlag Berlin Heidelberg 2011 477
Protein Cell 2011, 2(6): 477–486
Protein & Cell
1997; Yang et al., 1998; Baechtold et al., 1999). FUS proteins
are highly conserved during evolution, containing RNA
recognition motif (RRM) and zinc finger domains (see peptide
sequence alignment in supplementary Fig. S1).
FUS-deficient mice show defects in B cell development,
chromosomal instability and perinatal lethality (Hicks et al.,
2000). FUS-null neurons show developmental defects in
dendritic spines (Fujii et al., 2005). However, no FUS
inclusion body-like pathology has been reported in FUS-
deficient mice. Thus far, no stable animal model for FUS
proteinopathy has been published.
We generated and characterized transgenic flies expres-
sing wild-type (Wt) or ALS-mutant hFUS in various sub-
populations of neurons. Such FUS transgenic flies show age-
dependent neurodegeneration with ALS-mutant FUS flies
exhibiting more severe phenotype than those expressing the
Wt FUS. These FUS transgenic flies recapitulate major
clinical and pathological features of FUS proteinopathy,
serving as a useful animal model for these debilitating
To build an animal model for FUS proteinopathy, we used the
Gal4-UAS system to express either Wt or ALS-associated
R524S and P525L mutant hFUS as red fluorescent protein
(RFP)-HA-tagged proteins. Flies expressing the RFP-HA
vector alone were used as negative controls. Multiple
transgenic fly lines were generated. Lines with comparable
levels of expression were further characterized after crossing
with different Gal4 drivers to express FUS in distinct neuronal
Expression of Wt or ALS-mutant hFUS in fly eyes causes
FUS expression in fly photoreceptors was achieved using a
GMR-Gal4 driver. Flies expressing either Wt or ALS-mutant
hFUS exhibited a range of eye abnormalities. Different from
transgenic flies expressing Wt TDP-43 that exhibited necro-
sis-like patches and ommatidial loss (Li et al., 2010), flies
expressing Wt hFUS showed a reduction in the red eye
pigment as compared with the controls, beginning at eclosion.
No necrosis-like darkened areas were observed (Fig. 1).
Ultrastructural abnormalities were detected by scanning
electron microscopy (SEM) in hFUS-expressing fly eyes.
Rough eye surfaces with disrupted ommatidia and bristle
organization were observed in hFUS-expressing eyes (Fig. 2,
panels B1–B3 to E1–E3), whereas the control fly eyes
remained normal (Fig. 2A1 and 2A2). SEM at higher
magnifications revealed ommatidial loss, ommatidial fusion,
bristle loss or ectopic bristles and aberrant bristle budding in
the inter-ommatidial spaces (Fig. 2B3–2E3). ALS-mutant
FUS expressing eyes showed more severe ommatidial
abnormalities. Furthermore, eye atrophy was observed in
two different lines expressing P525L mutant FUS (Fig. 2E1,
for example). It was reported that expression of Gal4 protein
might cause neurotoxicity and apoptosis in Drosophila
(Kramer and Staveley, 2003; Rezával et al., 2007). However,
our transgenic flies expressing RFP in photoreceptors
showed no detectable changes in eyes. In addition, no
neurodegeneration was observed in FUS transgenic flies
without Gal4 expression. These data indicate that photo-
receptor degeneration in hFUS expressing flies is not caused
by nonspecific cytotoxicity associated with either RFP tag or
SEM imaging was also performed at different time points,
day 5 and day 15 after eclosion (Fig. 3). Control RFP
expressing eyes remained normal at day 15. However, fly
eyes expressing hFUS showed significant progression in
degeneration by day 15, with a marked increase in the rough
eye area and more frequent findings of concave eye surfaces.
Notably, in a line expressing the P525L mutant FUS, the eye
surface appeared relatively normal at day 5, but remarkably
deteriorated by day 15 (Fig. 3D1 and 3D2). Only mild
progression of rough eye phenotype, on the other hand,
control or Wt or ALS mutant FUS expressing transgenic
flies (A–D) and a diagram illustrating the transgene
constructs use (E).
Light microscopic images of eyes from the
478© Higher Education Press and Springer-Verlag Berlin Heidelberg 2011
Yanbo Chen et al.
Protein & Cell
was found in eyes expressing the Wt hFUS protein. Knocking
down the endogenous Drosophila FUS homologue, cabeza,
did not show detectable effects on the eye morphology.
Expression of the Wt or ALS-mutant hFUS in MB neurons
leads to progressive axonal loss
To model FTLD-FUS, we investigated the effects of hFUS
the OK107-Gal4 driver and visualizing the MBs by a
membrane-localized GFP in MB neurons (CD8::GFP, mGFP
in the text). Z-stack images of MB lobes (α/α′, β/β′, and γ/γ′
lobes) formed by axon bundles of different subtypes of MB
neurons were generated by confocal microscopy. Young (1-
day-old) flies expressing either Wt or mutant FUS showed
decreased size of MBs and thinner lobes as compared with
the control flies (Fig. 4). Similar to TDP-43 expressing flies (Li
et al., 2010), such axonal loss and lobe involvement were not
symmetric, but often in a “group” fashion. That is, any of the
lobes may be affected. However, once a particular lobe was
involved, that lobe progressively degenerated. In many
cases, one of the MBs may exhibit more degeneration than
the MB on the opposite side.
Expressing the Wt or ALS-mutant hFUS results in
morphological changes of MNs
Wecarefully examined the MNs expressing hFUS. Significant
morphological changes in cell bodies and neuromuscular
junctions (NMJ) were detected by fluorescent microscopy
when OK371-Gal4 was used to drive the transgene expres-
sion in glutamatergic neurons of the ventral nerve cord (VNC)
with neuronal morphology visualized by co-expression of
mGFP. In control larvae, MNs in the VNC are assembled into
well-organized clusters. Expression of hFUS led to disruption
in MN clusters (Fig. 5B–D) with obvious cell body swelling in a
fraction of MNs, especially in the posterior segment (arrow-
heads in corresponding panels of Fig. 5). To quantify the cell
electron microscopy (SEM). (A–E) SEM images of fly eyes expressing red fluorescent protein (RFP) (Ctr) or Wt or mutant FUS.
(A1 and A2) SEM images of fly eyes expressing RFP, showing normal morphology of ommatidia and inter-ommatidial bristles. (B–E)
Flies expressing Wt (B), R524S (C) or P525L mutant FUS (E) show rough eyes (B1–E1), ectopic bristles and malformed ommatidia
(B2–E2). Atrophic eyes were observed in a line expressing P525L FUS (E1). (B3–E3) Higher magnification images show detailed
structural changes. Arrows mark ectopic bristles, with more than one bristles existing in a single inter-ommatidial space. Thin arrows
indicate loss of normal bristles or aberrant budding of the inter-ommatidial bristles. Arrowheads mark fused ommatidia. Scale bar:
A1–E1: 100μm; A2–E2: 20μm; B3–E3: 20μm. All flies were raised at 25°C, with at least10 flies from each line examined by SEM.
Genotypes: A1, A2: Ctr; GMR-Gal4/UAS-RFP; B1–B3: Wt, GMR-Gal4/UAS-Wt-FUS-RFP; C1–C3: R524S, GMR-Gal4/UAS-
R524S-FUS-RFP; D1–D3 and E1–E3: two lines of P525L, GMR-Gal4/UAS-P525L-FUS-RFP.
Expression of wild-type or ALS-mutant FUS in fly eyes leads to retinal degeneration as detected by scanning
© Higher Education Press and Springer-Verlag Berlin Heidelberg 2011479
Drosophila model for FUS proteinopathy
Protein & Cell
swelling phenotype, we measured the cross-section areas of
MN cell bodies. MNs expressing hFUS showed significant
increases in MN cell body size as compared to controls
(p<0.001; Fig. 5E). The majority of the cell bodies were
between 20 and 30μm2in all groups. However, fly larvae
expressing hFUS, either Wt, R524S or P525L, contained
increased fractions of large MNs with cross section areas of
35μm2or larger (Fig. 5F).
Furthermore, cytoplasmic FUS-RFP signals were detected
in hFUS transgenic flies expressing ALS-mutant hFUS as
revealed by biochemical fractionation experiments (data not
shown) or by counterstaining MNs with a nuclear dye (Fig. 6).
Low levels of cytoplasmic RFP signals were also detected in
larvae expressing Wt hFUS.
MN axons project to their target muscles in each segment
and form synapses with muscle cells within the varicosities
termed as boutons. Two types of nerve processes were
defined based on their morphology and the bouton sizes
abnormalities in fly eyes expressing hFUS protein.
Expression of Wt or mutant FUS driven by GMR-Gal4 leads
to rough eye surfaces and disruption in the pattern of inter-
ommatidial bristles. (A) Flies expressing the RFP control show
normal eye morphology. Organization of hexagonal ommatidia
and interommatida bristles are not changed with the age in the
control flies. (B) Fly eyes expressing Wt FUS show abnormal
ommatida organization and shape as well as irregular
appearance of the bristles, although no remarkable differences
were observed at days 5 and 15 after eclosion. (C) Fusion of
ommatidia was frequently observed in flies expressing R524S
FUS, andthe affected areabecamelargerwithaging.(D) In the
P525La line expressing P525L hFUS, the eyes appeared to be
almost normal by day 5, whereas pervasive ommatidia fusion
was discovered at day 15. (E) In another line, P525Lb, more
severe retinal degeneration was detected with eye atrophy. A
few ommatidia were still recognizable at day 5, but none was
visible at day 15. Scale bar: 100μm. All flies were raised at
25°C, and ten flies from each line were examined by SEM.
Progressive and age-dependent ultrastructural
body (MB) neurons leads to axonal degeneration. Axonal
bundles of MB neurons are visualized by membrane GFP (CD8::
GFP). Confocal images were obtained in 20 Z-stacks to include
all lobes in each brain and projected as single images. (A)
Control flies (OK107-Gal4/UAS-RFP) showed normal MB lobe
structures: α/α’ (marked by the arrow), β/β’ (arrowhead) and γ/γ’
(star), at both day 1 and day 25. (B–D) Flies expressing Wt,
R524S or P525L FUS (OK107-Gal4/UAS-Wt-FUS-RFP, OK107-
Gal4/UAS-R524S-FUS-RFP or OK107-Gal4/UAS-R524S-FUS-
RFP) showed a mild reduction in the size of all 3 lobes.
Moreover, P525L FUS expressing flies showed a significant
axonal loss by day 25. Some MBs show asymmetric degenera-
tion. For example, the right side MB in panel D2 shows more
severe axonal loss. Scale bar: 20μm.
Expression of Wt or mutant FUS in mushroom
480 © Higher Education Press and Springer-Verlag Berlin Heidelberg 2011
Yanbo Chen et al.
Protein & Cell
(Johansen et al., 1989). NMJs in the third instar larvae were
revealed by fluorescent microscopy, with the short type I
processes visualized and the total numbers of boutons in
each axon terminal quantified (Fig. 7). A significant decrease
control larvae, the dorsal clusters were well organized. MNs are visualized by membrane localized GFP (mGFP). (B–D) The 3rd
instar larvae expressing Wt, R524S or P525L FUS show anomalous arrangement of motor neurons in dorsal clusters. Wt FUS
protein is localized predominantly in the nucleus, with weak RFP signals detected in the cytoplasm. Remarkable cytoplasmically
localized FUS protein is detected in MNs of both mutant groups with P525L showing stronger cytoplasmic signals in a punctate
pattern. Some motor neurons in Wt or mutant FUS expressing larvae appear enlarged as compared to those in the control flies. An
enlarged MN is indicated by arrowheads in panel D. Larval genotypes, in panel A: OK371-Gal4, mGFP/UAS-RFP; B: OK371-Gal4,
mGFP/UAS-Wt-FUS-RFP; C: OK371-Gal4, mGFP/UAS-R524S-RFP; D: OK371-Gal4, mGFP/UAS-P525L-RFP. (E) Quantitative
analysis of the projection area of motor neuron cell body. Wt, R524S or P525L FUS expressing larvae showed significant increase in
cell size compared with control. More than 60 cells were measured in each group (???: p<0.001). (F) Frequency distribution of
projection area of MN cell bodies. The cross-section areas of the majority of cells in all groups are between 20 and 30μm2. Wt,
R524S and P525L groups, however, exhibit increased frequencies of cells larger than 30μm2.
Morphological changes in motor neurons (MNs) of FUS transgenic flies. (A) In the ventral nerve cords (VNC) of the
© Higher Education Press and Springer-Verlag Berlin Heidelberg 2011 481
Drosophila model for FUS proteinopathy
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in the bouton number was detected in the hFUS-expressing
flies, as compared to the controls (Fig. 7A–E). Remarkably, in
flies expressing Wt hFUS that survived to the third instar,
boutons were frequently enlarged as compared to the control
larvae expressing RFP control (p<0.001; Fig. 7B and 7F).
The bouton size in P525L mutant FUS larvae was signifi-
cantly reduced as compared to controls (P<0.05; Fig. 7D and
7F), although no significant difference was found in R524S
mutant FUS expressing larvae (p>0.05).
Expression of hFUS in MNs impaires locomotive function
of transgenic flies
We examined the locomotive ability of the FUS transgenic
flies. The larval movement in flies expressing hFUS either the
Wt or ALS-mutant (R524S or P525L) was significantly
reduced as compared with the controls (Fig. 7G). These
FUS-expressing larvae moved more slowly with their tails
lifted ratherthan anchored on the supporting surface, possibly
as a result of paralysis of the posterior segments (Fig. 7H).
We surveyed multiple lines expressing ALS-mutant FUS. In
larvae expressing R524S or P525L, (for example, in the fly
line FUS-P525L2b that exhibited atrophic eyes), some
showed such severe locomotion defects that they were
unable to climb up the wall or hardly moving. There were no
pupae hatched in most P525L mutant expressing fly lines
tested, with one exception, in which the fly line survived to
adulthood. This particular P525L mutant fly line remains to be
further characterized to understand its differences from other
lines. Overall, flies expressing R524S or P525L mutant show
more severe MN damage and locomotive impairment. It
should be noted that the mobility index in Fig. 7G was
measured from those fly larvae that were able to move,
therefore, representing an underestimate of motor impairment
in R524S- or P525L-mutant FUS flies. No locomotive defects
were observed in the flies in which FUS fly homolog cabeza
was knocked down in MNs by RNA interference (RNAi).
FUS proteinopathy has recently emerged as a syndrome with
shared neuropathological features but heterogeneous clinical
manifestations. FUS proteinopathy may occur in patients with
mutations in the coding region of the FUS gene or in patients
without detectable FUS mutations (Neumann et al., 2009).
The pathogenic mechanisms underlying FUS proteinopathy
remain largely unknown, although it is clear that FUS
proteinopathy not only affects motor neurons but also other
neuronal populations such as cortical neurons. Data pre-
sented in this study provide strong evidence for a pathogenic
role of hFUS and its ALS-associated mutants in neurode-
generation. Expression of either Wt or ALS-mutant FUS in
different neuronal subpopulations, including photoreceptors,
mushroom bodies and motor neurons, leads to age-
dependent progressive degeneration and functional deficits.
MN denervation is an important aspect in motor neuron
diseases, including ALS (Jokic et al., 2006; Blijham et al.,
2007). The presence of chromatolytic and swollen neurons is
among the early pathological signs of MN degeneration in
ALS (Okamoto et al., 1990; Sasaki and Maruyama, 1994). We
systematically examined the effects of hFUS expression on
the morphology and function of MNs in hFUS transgenic flies.
Expression of two ALS-mutants, P525L and R524S, caused
marked MN changes, including swollen cell bodies in ventral
Hoechst nuclear dye.
VNC motor neurons as counterstained with the
482 © Higher Education Press and Springer-Verlag Berlin Heidelberg 2011
Yanbo Chen et al.
Protein & Cell
of FUS transgenic flies. (A–D) NMJ images from larvae expressing RFP vector control (Ctr) (A), Wt FUS (B), R524S FUS (C) or
P525L FUS (D) in motor neurons. Numbers of type I bouton in each terminus were counted, and projected areas of boutons were
measured. Enlarged boutons are marked by arrows in panel B. (E) Numbers of boutons per motor terminus were significantly
reduced in FUS-expressing flies as compared to the control group (F). The projection areas of boutons in flies expressing Wt FUS
were increased, whereas that in flies expressing P525L mutant FUS was decreased as compared to control flies. Genotypes of
larvae in A: OK371-Gal4, mGFP/UAS-RFP; B: OK371-Gal4, mGFP/UAS-Wt-FUS-RFP; C: OK371-Gal4, mGFP/UAS-R524S-FUS-
RFP; D: OK371-Gal4, mGFP/UAS-P525L-FUS-RFP. (G) Locomotive deficits in flies expressing FUS in MNs. Third instar larvae
expressing Wt or mutant FUS (R524S or P525L-line#a) showed significant decrease in mobility index. More than 20 larvae were
scored each group in each experiment. The data represent three independent experiments. All data were analyzed using one-way
ANOVA (?: p<0.05;???: p<0.001). (H) The tail lifting phenotype observed in FUS expressing larvae. Control or FUS expressing
larvae were crawling on an agar surface with their side view images taken. In the control group, larvae moved with their tails
anchored on the surface, whereas FUS-expressing larvae lifted their tails while moving.
Morphological changes at the neuromuscular junctions (NMJs) and functional deficits in motor neurons (MNs)
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Drosophila model for FUS proteinopathy
Protein & Cell
nerve cords, and reduced NMJ boutons. FUS transgenic flies
also show signs of motor denervation, with a significant
reduction of mobility and viability of the larvae, mimicking
clinical features of ALS. In the locomotion assay, the
functional deficits were accompanied by tail paralysis,
whereas the anterior body segment appeared relatively
normal. This is consistent with the distal-to-proximal progres-
sion of motor neuron failure observed in ALS patients.
Although reduced numbers of MN boutons were found in
flies expressing either the Wt or ALS-mutant FUS expressing
larvae, enlarged boutons were frequently observed in flies
expressing the Wt FUS. Interestingly, reductions in bouton
numbers accompanied by bouton enlargement and increased
active-zone density at the synapses have been reported in
flies carrying metro or Fas II mutations (Stewart et al., 1996;
Bachmann et al., 2010). Bouton enlargement has been
proposed as a compensatory mechanism to increase func-
tional active zones in the presence of motor denervation. In
metro and Fas II mutants, such reciprocal correlation may
result from an active redistribution of synaptic components to
compensate for the reduction in bouton numbers, or
alternatively, from a passive accumulation of continuously
delivered synaptic materials. It remains to be investigated
whether there is an increase in the active-zone density in the
FUS transgenic flies. No obvious bouton enlargement was
observed in larvae expressing ALS mutant FUS. This is
possibly due to more rapid progression of neurodegeneration
that exceeds the compensatory mechanism(s), and thus only
residual varicosities could be detected in the 3rd instar larvae.
Other possibilities include axonal transport impairment in
MNs expressing mutant FUS, resulting in defects in bouton
formation or axonal repair. It remains to be determined
whether there was a transient bouton enlargement event in
mutant FUS flies. Interestingly, mammalian FUS has been
implicated in dendritic spine development (Belly et al., 2005;
Fujii et al., 2005). FUS-null hippocampal pyramidal neurons
showed abnormal spine morphology and reduced spine
density (Fujii et al., 2005). The underlying mechanisms are
not clear, though mRNA transport and local protein synthesis
might be involved.
It is still an open question whether FUS proteinopathy is
caused by haploinsufficiency (a loss of function of FUS gene
products) or by gain-of-function neurotoxicity. In our study,
down-regulating the cabeza gene, the Drosophila homolog of
FUS, by RNAi in the eye or MNs, did not affect either the eye
morphology or the larval locomotion. On the other hand,
expression of Wt FUS was sufficient to produce the
phenotypes similar to ALS-mutant FUS expressing lines,
albeit less severe. This suggests that abnormal accumulation
or decreased clearance of otherwise normal FUS protein
product(s) could contribute to the pathogenesis of FUS
proteinopathy, especially among patients without detectable
FUS mutations. It will be interesting to investigate potential
rolesof both transcriptionaland post-transcriptional
mechanisms in the pathogenesis of FUS proteinopathy. It is
possible that nucleotide sequence variations or mutations in
the non-coding regions, including promoter, intronic or
translational regulatory regions may contribute to both clinical
and genetic heterogeneity of this group of neurodegenerative
disorders, especially among patients without mutations in the
coding region of the FUS gene.
Previous studies show mutant FUS protein is redistributed
from the nucleus to the cytoplasm (Kwiatkowski et al., 2009;
Vance et al., 2009). The C-terminal region of FUS is critical for
its nuclear retention by interacting with Ran guanosine
triphosphatase-dependent transport machinery (Dormann et
al., 2010; Ito et al., 2010). Together, our data are consistent
with the gain-of-toxicity hypothesis but do not exclude the
possible involvement of impaired or loss of FUS function in
the pathogenesis of the disease.
The two mutations studied in this report, R524S and
P525L, are both located in the carboxyl terminal of the FUS
protein (Fig. S1). Several reports of either sporadic or familial
ALS patients show that patients with P525L mutation show
very early onset and rapid disease progression (Chiò et al.,
2009; Kwiatkowski et al., 2009; Bäumer et al., 2010; Huang et
al., 2010). This mutation may contribute to juvenile ALS
(Table S1). In contrast, clinical phenotypes associated with
R524S mutation seem relatively milder with later onset and
slower progression than that of P525L. Consistent with these
observations, similar results were seen in our transgenic fly
model, with flies expressing P525L mutant exhibiting more
severe phenotypes as compared to ones expressing compar-
able levels of R524S mutant FUS protein.
The cytoplasmic sequestration of a normally shuttling
nuclear protein and the formation of insoluble protein
aggregates have been proposed as pathogenic mechanisms
in TDP-43 proteinopathy, and formation of insoluble aggre-
gates may count for this redistribution. Similar to TDP-43,
FUS is a shuttling protein predominantly localized in the
nucleus. Elevated levels of cytoplasmically localized FUS
protein have been demonstrated by immunohistochemistry in
postmortem ALS samples and in cell cultures. Although in
cultured cells, only ALS-related mutations led to subcellular
mislocalization, cytoplasmic FUS immunoreactivity was also
detected in atypical FTLD-U (aFTLD-U) patients without
detectable FUS gene mutations. FUS immunoreactivity has
often been detected in basophilic inclusions in motor neuron
diseases and aFTLD-U, which were tau-negative. It was
noticed that a greater number of FUS-positive inclusion
bodies (IBs) than ubiquitin- or p62-immunoreactive IBs were
seen in both TDP-43 and FUS proteinopathies (Munoz et al.,
2009; Fujita et al., 2010). This suggests that the formation of
FUS containing basophilic inclusions is not necessarily
ubiquitin-dependent. It remains unclear whether the protein
mislocalization is a cause or consequence in these diseases.
The molecular nature of the FUS gene products that are
responsible for neurodegeneration in FUS proteinopathy
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Yanbo Chen et al.
Protein & Cell
needs to be determined in future studies.
Taken together, our transgenic Drosophila model recapitu-
lates key clinical and pathological features of FUS proteino-
pathy, including ALS and FTLD-FUS. It is a powerful animal
model for studying mechanisms of FUS proteinopathy and
can be used in our future search for genetic modifiers or
therapeutic agents that may alter clinical outcome of these
MATERIALS AND METHODS
Transgenic constructs and fly strains
The openreading frames encodinghumanFUS (the wild type, R524S
or P525Lmutants)wereclonedinthe pUASTvector,whichcontainsa
UAS sequence in the promoter region (Fig. 1) (Brand et al., 1993).
FUS proteins, including the Wt or ALS mutants, contain the full-length
FUS protein fused at its carboxyl terminus to RFP with a
hemagglutinin (HA) tag. The sequences of the full length FUS
cDNAs were verified. Transgenic flies expressing either RFP vector
control or FUS-RFP protein were generated using a commercial fly
injection service (Rainbow Transgenic Flies, Inc) in a W1118
background. The Cabeza-RNAi stock was obtained from Vienna
Drosophila RNAi Center (VDRC). The Gal4 lines were obtained from
Bloomington Stock Center.
Fluorescent microscopy and scanning electron microscopy
The third instar fly larvae or adult fly heads were dissected, and the
brains were fixed with 4% paraformaldehyde (PFA) for 20min at room
temperature. After washing with PBS containing 0.1% Triton, the
brains were counterstained with DAPI, and then mounted onto
coverslips using mounting gel. Confocal images were taken under an
Olympus FV1000 confocal microscope. Volumes of MBs were
measured using Image-Pro Plus 7.0 (Media Cybernetics, Inc.).
Images of NMJs were taken from the dorsal side of larvae under a
Nikon Eclipse Ti fluorescent microscope.
For SEM, intact flies were progressively dehydrated in ethanol
(70%, 85%, 95%, 100%; 60 min each step) and then in 100% ethanol
overnight. Dehydrated flies were applied to a CPD 030 critical point
dryer, with CO2as the drying agent. SEM images were acquired
under the FEI Quanta 200 FEG electron microscope in the low
Larval movement assay
The assay was done as described (Li et al., 2010). Briefly, the larval
mobility index was measured as the number of peristaltic waves
during the period of 2min in the late third instar larvae expressing
control RFP or FUS protein under the OK371-Gal4 driver in a
controlled environment (25°C, humidity 50%±5%, illumination 2800±
All statistical analyses were performed using either one-way ANOVA,
followed by Bonferroni multiple comparison for comparing individual
groups. The bar graphs with error bars represent mean±standard
error of the mean (SEM). Significance is indicated by asterisks:
?: p<0.05;??: p<0.01;???: p<0.001.
We thank D. Kuo and other members of Wu group for critical reading
of the manuscript. We thank members of the Wu lab for stimulating
discussions and helpful suggestions. We are grateful for generous
help from Drs. C. Wang, D. Han and J. Xie at National Center for
Nanoscience and Technology in our SEM work. We thank D. Zhang
for technical assistance in the early stage of the work. JL is supported
by the National Basic Research Program (973 Program) (Grant No.
2009CB825402).YC, HYand QX are supportedby the NationalBasic
Research Program (973 Program) (Grant No. 2010CB529603).
Authors declare no competing financial interests.
Author contributions: EJR conceived the study. JYW and EJR
designed the research experiments. YC, MY, JD, YY, XC, YL, XZ, KF,
EJR and JYW performed the research and analyzed the data. HY, LZ,
JL, YS, KF, QX and JYW supervised the experiments, discussed and
analyzed the data. EHB and MM provided critical tissue samples and
revised the manuscript. YC, MY, EJR and JYW wrote the paper.
It came to our attention that a study has just been published on line
that reported similar neurodegeneration phenotypes in flies expres-
sing FUS with other three ALS-mutations (Lanson et al., 2011).
ALS, amyotrophic lateral sclerosis; FUS, Fused in sarcoma/Trans-
lated in liposarcoma gene; FTLD-FUS, frontotemporal lobar degen-
eration associated with FUS; FTLD-U, ubiquitin-positive
frontotemporal lobar degeneration; Hfus, ALS-mutant human FUS
protein; MBs, mushroom bodies; MNs, motor neurons; NMJ,
Supplementary material is available in the online version of this
article at http://dx.doi.org/10.1007/s13238-011-1065-7 and is acces-
sible for authorized uers.
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