ALS-Related Mutant FUS Protein Is Mislocalized to Cytoplasm and Is Recruited into Stress Granules of Fibroblasts from Asymptomatic <b><i>FUS </i></b>P525L Mutation Carriers

Abstract

Background: Amyotrophic lateral sclerosis (ALS) shows a strong genetic basis, with SOD1, FUS, TARDBP, and C9ORF72 being the genes most frequently involved. This has allowed identification of asymptomatic mutation carriers, which may be of help in understanding the molecular changes preceding disease onset. Objectives: We studied the cellular expression of FUS protein and the effect of heat-shock- and dithiothreitol-induced stress in fibroblasts from FUS P525L mutation carriers, healthy controls, and patients with sporadic ALS. Methods: Western blots and immunocytochemistry were performed to study the subcellular localization of FUS protein. Control and stressed cells were double stained with FUS and the stress marker TIA-R. Results: Fibroblasts from healthy controls and sporadic ALS cases showed a prominent nuclear FUS expression. In the 2 FUS P525L mutation carriers, instead, most cells showed a protein localization in both nucleus and cytoplasm, or exclusively in the cytoplasm. Stress prompted the formation of cytoplasmic granules in all subjects and in sporadic ALS FUS mislocalization to the cytoplasm. Cytoplasmic FUS was recruited into stress granules, which showed a time-dependent decrease in all subjects. However, in the FUS P525L fibroblasts, the granules persisted longer, and they were more numerous than those detected in the cells from controls and sporadic ALS patients. Conclusions: We show that in fibroblasts of FUS P525L mutation carriers, FUS mislocalized to the cytoplasm where it redistributed into stress granules with likely a dose effect, i.e. a higher number of cells with granules, which persist longer, than in controls and ALS cases. These data represent an early molecular change occurring before ALS onset, suggesting a transient preaggregative state.

Differential subcellular expression of P525L FUS as a putative biomarker for ALS phenoconversion

“…This study confirms that in P525L FUS fibroblasts, taken from asymptomatic mutation carriers, the protein is mislocalized to the cytoplasm. 3,4 However, it also shows that after phenoconversion, in the large majority of mutant cells the protein disappear from the nucleus. This occurred to DC fibroblasts, whose number of cells expressing FUS solely in the cytoplasm doubled from some 35% in the asymptomatic phase (DC-A) to over 70% after ALS onset (DC-S).…”

“…1 This harmful point mutation is located in the nuclear localization signal (NLS) domain at the protein C-terminal. 2 Although wild-type FUS protein is expressed almost exclusively in the nucleus, the P525L FUS mutation leads to a protein mislocalization into the cytoplasm 3,4 because of its loss of capacity to bind its transporter karyopherin-2 and to be transferred back to the nucleus. 3 Here, we compare FUS expression and localization in skin fibroblasts of 2 sisters, both carriers of a P525L FUS mutation, belonging to a Sicilian family with this mutation.…”

“…3 Here, we compare FUS expression and localization in skin fibroblasts of 2 sisters, both carriers of a P525L FUS mutation, belonging to a Sicilian family with this mutation. 4,5 The first sister (DC) was seen at age 21 years when she was asymptomatic. After almost 2-year follow-up, she developed a bulbar form of ALS and died 13 months after disease onset.…”

“…The analysis of subcellular FUS expression was made through a Zeiss LSM5 confocal microscope. Cell counting for subcellular FUS expression was performed as described by Lo Bello et al 4 As expected, FUS mislocalized to the cytoplasm in almost all fibroblasts carrying the P525L FUS mutation. 4 Conversely, control fibroblasts (S-ALS and HC) expressed FUS only in the nucleus (figure, A).…”

Antiviral Immune Response as a Trigger of FUS Proteinopathy in Amyotrophic Lateral Sclerosis

“…Human fibroblasts acquisition was approved by the University of Palermo Review Board (prot.07/2017). Characterization of fibroblasts from a female patient with FUS P525L mutation was described earlier (Chiò et al, 2009;Lo Bello et al, 2017). SH-SY5Y cells and human fibroblasts were grown in high-glucose 1:1 mixture of DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin and 2 mM L-glutamine (all Invitrogen).…”