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 presented in this study provide strong evidence for a pathogenic role of hFUS and its ALS-associated mutants in neurodegeneration. 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 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 progression 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 functional 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 roles of both transcriptional and 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 comparable 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 aggregates 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 needs to be determined in future studies.
Taken together, our transgenic Drosophila model recapitulates key clinical and pathological features of FUS proteinopathy, 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 diseases.