This study has identified a critical role of the C-terminus of FUS in nuclear localization and its perturbation by a subset of FUS mutants that cause fALS. Furthermore, our results revealed a mutant-specific response to cellular stress that leads to the incorporation of cytoplasmic fALS-linked FUS protein into stress granules.
Although the C-terminus of FUS differs from classical NLSs that are recognized by karyopherin α (importin α) (41
), an analysis of the evolutionarily related EWS protein (40
) suggested that the C-terminus of FUS may nonetheless function to mediate nuclear localization. Moreover, the C-terminal sequence of FUS is consistent with a ‘PY-NLS’ type of nuclear targeting signal that binds to the human karyopherin β2/transportin (Kapβ2) receptor (39
). Our results showed that truncation of this putative FUS NLS in the R495X and G515X mutants (Fig. A) caused a dramatic increase in cytoplasmic accumulation of FUS compared with the missense (H517Q and R521G) mutants, which have only a single amino acid substitution within the NLS (Fig. C and D). Thus, these studies provide compelling evidence that the C-terminus of FUS is required for normal nuclear–cytoplasmic partitioning of the protein and that fALS-linked mutations disrupt this equilibrium.
Although cytoplasmic FUS accumulation was observed previously for some overexpressed fALS-linked missense mutants (13
), the downstream consequence(s) of this mislocalization on cellular pathways, including those involved with stress responses, remains largely unknown. Here, we showed that GFP-FUS mutants retained in the cytoplasm assemble into stress granules in response to acute insults, including oxidative stress (Figs and ; Supplementary Material, Fig. S4
), ER stress (Supplementary Material, Fig. S4
) and heat shock (Figs and ). Although inhibition of transcription causes a redistribution of FUS WT from the nucleus to the cytoplasm (24
), we found here that inhibition of translation induced by other cellular stresses does not prominently alter the subcellular localization of FUS WT. However, an enhanced cytoplasmic accumulation of the autosomal recessive mutant (H517Q) was detected upon heat shock in vivo
(Fig. ), which may have implications for ALS (discussed below).
Why does cytoplasmically localized mutant FUS assemble into stress granules? One possibility is that FUS associates directly with specific mRNAs or other proteins that mediate the integration of FUS into stress granules. FUS has been shown to bind mRNAs encoding actin-related proteins, such as actin-stabilizing protein Nd1-L, which may be important for the transport of this mRNA to dendrites for local translation and maintenance of spine morphology (52
); however, comprehensive data regarding mRNAs bound by FUS are lacking. The fALS-linked R521G mutant retains the ability to complex with GGUG-containing RNAs (13
), indicating that FUS mutations do not necessarily abrogate RNA binding. Moreover, our data show that interactions involving the C-terminal NLS of FUS are not required to initiate the targeting of FUS to stress granules, as evidenced by the robust incorporation of the truncation mutants (R495X and G515X) into these structures.
The association of FUS and other RNA-binding proteins with stress granules exhibits specificity, as cytoplasmic free GFP does not localize to stress granules (Supplementary Material, Fig. S5
). Although some RNA-binding proteins such as Nova1 do not incorporate into stress granules (36
), others such as TDP-43 show this property under conditions of translational arrest induced by oxidative stress (36
) or upon the expression of a pathogenic, fragmented form of this protein (55
). One study reported the localization of acutely transfected GFP-FUS(WT) into stress granules in HT-1080 cells (22
); however, the association of GFP-FUS(WT) with stress granules was not observed in acutely transfected HEK-293 cells (56
). In addition, we did not observe stress granules in the absence of an applied stress for our stable HEK-293 cell line expressing the GFP-FUS(R521G) mutant or in HEK-293 cells acutely transfected (16 h) with FUS(R521G) (Supplementary Material, Fig. S5
), although stress granules have been detected for this mutant under acute transfection conditions in other laboratories (56
). These apparent discrepancies may result from a difference in cell lines, transfection procedures or exogenous FUS expression levels. Consistent with this hypothesis was our observation of increased cytoplasmic aggregation for FUS mutants when expressed at higher levels in transiently transfected (40 h) cells compared with stable cell lines (Supplementary Material, Fig. S3
). These data indicate that the aggregation propensity of FUS, as well as its cellular localization and binding interactions, may be sensitive to the expression level of FUS, the translational state of individual cells and factors related to cell type; this sensitivity to altered cellular homeostasis may be expected based on the regulatory functions of proteins within the hnRNP family (57
). Although mutant FUS incorporates into stress granules, it does not significantly associate with P-bodies (Fig. ). A specific association with stress granules over P-bodies has also been reported for the ALS-associated TDP-43 protein (36
The observation that both FUS and TDP-43 can associate with stress granules raises the possibility that ALS-related mutants might perturb the normal response to stress in maladaptive ways. Although we cannot exclude the possibility that under some conditions FUS WT may also associate with stress granules, the fALS-linked mutations significantly shift the subcellular equilibrium of FUS towards the cytoplasm and enhance its association with stress granules. In fact, the R495X mutation that truncates the NLS results in the highest levels of cytoplasmic FUS compared with the other fALS-linked mutants studied here (Fig. ). Furthermore, cytoplasmic expression of R495X correlated with its incorporation into stress granules. That this mutation is also associated with a severe, early-onset clinical phenotype (Table ), which is similar to the de novo
FUS G466VfsX14 mutation (16
), raises the possibility that the vulnerability of motor neurons to mutant FUS is proportional to FUS expression in the cytoplasm. In contrast to FUS R495X, the autosomal recessive FUS H517Q mutant exhibits weak cytoplasmic expression under homeostatic cellular conditions (Figs and ). That this mutant protein could more readily translocate to the cytoplasm upon induction of heat shock in vivo
(Fig. ) suggests an enhanced susceptibility to thermal stress compared with FUS WT. Although it is not clear whether motor neuron vulnerability stems from a loss of normal FUS nuclear function or an altered cytoplasmic function, or both, the notion that cytoplasmic expression levels of RNA-binding proteins may correlate with ALS pathogenesis has also been supported for TDP-43 based on cell culture and animal model studies with mutant forms of that protein (58
We observed that mutant FUS recruitment to stress granules is reversible following an acute insult (Fig. ). In addition, we found no evidence of acute toxicity associated with the incorporation of mutant FUS into stress granules, as measured by the MTT cell proliferation assay (Supplementary Material, Fig. S4
). In light of these observations, how might this mutant-specific property, i.e. incorporation of mutant FUS into stress granules, play a role in ALS pathogenesis? It is plausible that more chronic changes in cellular homeostasis as a consequence of mutant FUS expression could produce a maladaptive outcome. For example, the inappropriate sequestration of mutant FUS and its biological binding partners into stress granules may impair the cellular response to chronic oxidative stress, protein misfolding, heat shock or other insults that cause translational arrest. Further, chronic stress may culminate in the conversion of stress granules into larger pathological aggregates, a possibility that is supported by the detection of mRNA-associated proteins within the inclusions of adult onset motor neuron disease (60
). A similar mechanism has been proposed for the accumulation of aggresomes into end-stage Lewy bodies, the pathological hallmark of Parkinson's disease and other synucleinopathies (61
). However, the detection of mRNA and its associated proteins in ALS aggregates is not a consistent finding in all reports (36
). Thus, it remains to be determined whether FUS-associated stress granules are linked to the formation of end-stage ALS aggregates, and whether FUS-containing aggregates detected in post-mortem CNS tissues of affected individuals are neurotoxic themselves or simply markers of altered cellular homeostasis.
Although we did not observe acute toxicity as a consequence of mutant FUS expression at near-endogenous levels in either the HEK-293 cells or the zebrafish embryos, further studies using model systems that more closely recapitulate the relevant neuronal environment, such as primary motor neurons and/or transgenic models, may reveal mutant-specific phenotypes related to FUS toxicity. We note that, to date, there has been no evidence published that directly links mutant FUS expression or its localization to cellular dysfunction. Nevertheless, our findings suggest that candidate pathways related to the regulation of stress defenses in motor neurons may be appropriate for further investigation of the pathogenic role of FUS in ALS. Dysregulation of specific stress responses may also contribute to the preferential motor neuron vulnerability observed in other forms of ALS caused by mutant SOD1, mutant TDP-43 and, perhaps, in sALS.