Mutant FUS proteins that cause amyotrophic lateral sclerosis incorporate into stress granules

Abstract

Mutations in the RNA-binding protein FUS (fused in sarcoma) are linked to amyotrophic lateral sclerosis (ALS), but the mechanism by which these mutants cause motor neuron degeneration is not known. We report a novel ALS truncation mutant (R495X) that leads to a relatively severe ALS clinical phenotype compared with FUS missense mutations. Expression of R495X FUS, which abrogates a putative nuclear localization signal at the C-terminus of FUS, in HEK-293 cells and in the zebrafish spinal cord caused a striking cytoplasmic accumulation of the protein to a greater extent than that observed for recessive (H517Q) and dominant (R521G) missense mutants. Furthermore, in response to oxidative stress or heat shock conditions in cultures and in vivo, the ALS-linked FUS mutants, but not wild-type FUS, assembled into perinuclear stress granules in proportion to their cytoplasmic expression levels. These findings demonstrate a potential link between FUS mutations and cellular pathways involved in stress responses that may be relevant to altered motor neuron homeostasis in ALS.

Structural and energetic basis of ALS-causing mutations in the atypical proline–tyrosine nuclear localization signal of the Fused in Sarcoma protein (FUS)

“…4 and Table 1). Loss of Kapβ2 binding in these mutants correlates with the degree of cytoplasmic mislocalization in cells (2,(6)(7)(8)(9). Fig.…”

“…Such high affinities may be consistent with the initiation of disease later in life. High affinities for Kapβ2 may allow near-normal nuclear function and localization for some FUS mutants or partial mislocalization for others, which are tolerated until the aging cells encounter additional hits such as further impairment of the nuclear transport machinery or other cellular stress that exacerbate FUS mislocalization or trigger aggregation of mislocalized FUS (2,5,7,9). Furthermore, even though ALS mutants bind Kapβ2 rather tightly, effects of decreased affinities compared with those of wild-type FUS may be amplified in cells due to competition with other high-affinity Kapβ2 cargos and with nonspecific competitors (16,17,30).…”

“…More recently, the signal was shown to direct Kapβ2-mediated nuclear import in cells (2) and to be heavily mutated in ∼5% of familial amyotrophic lateral sclerosis (ALS) (3)(4)(5), a progressive and fatal neurodegenerative disorder. ALS mutations in the PY-NLS disrupted nuclear import of FUS, causing its mislocalization and aggregation in the cytoplasm, as evidenced by cytoplasmic FUS inclusions in motor neurons of ALS patients (2,(6)(7)(8)(9). The PY-NLS of FUS is also mutated in another neurodegenerative disease, frontotemporal lobar dementia (FTLD), which also is characterized by cytoplasmic mislocalization and aggregation of FUS (10,11).…”

Astrocytes expressing ALS‐linked mutant FUS induce motor neuron death through release of tumor necrosis factor‐alpha

“…Because the redistribution of nuclear FUS has been observed in a variety of cell culture models (Bosco et al, 2010; Dormann et al, 2010; Farg et al, 2012), we examined it in our in vitro system of cultured astrocytes transduced with mutFUS. Isolated primary astrocytes were firstly tested for purity using the glutamate transporter GLAST as a marker of mature astrocytes (Supporting Information Figure S1a,c).…”

ALS mutations in TLS/FUS disrupt target gene expression

“…1, cf. wild-type FUS in the top row and mutant derivatives in the bottom three rows, aggregates are denoted by arrows, and the nuclear boundary is highlighted in blue via DAPI stain). FUS Q -which, unlike other characterized FUS ALS-associated mutations, is recessive (Bosco et al 2010)-did not show cytoplasmic localization but did display altered nuclear accumulation such that the size and intensity of FUS-containing speckle-like nuclear structures were larger and more intense in FUS Q -expressing cells compared with wild-type FUS (Fig. 1, cf.…”