Hsp90-mediated regulation of DYRK3 couples SG disassembly and growth via mTORC1 signaling (2021)
Figure 5

Figure 5: ALS cells are sensitive to Hsp90 inhibition and show decreased DYRK3 expression.

(A) WT and P525L FUS eGFP iPSCs were differentiated into MNs and were treated with sodium arsenite (500 μM) for 2 hrs. MNs were fixed and the number of SGs per cell was counted. WT FUS eGFP iPSC-MNs have no SGs. Number of P525L cells counted: 888 (arsenite); 1119 (arsenite + rec.). Number of SGs in P525L cells: 1498 (arsenite); 222 (arsenite + rec.). n = 6, ± sem (Student's t-test).

(B) Confocal microscopy images of P525L FUS eGFP MNs treated with sodium arsenite for 2 hrs and showing colocalization of FUS with TIAR-positive stress granules. Scale bar is 10 µm.

(C) P525L FUS eGFP MNs were treated with sodium arsenite for 2 hrs, followed by recovery in drug-free medium (+ rec. control) or in presence of 17AAG (30 μM; + rec. 17AAG) or of GSK (5 μM; +rec. GSK) for 6 hrs. Quantitation of the number of SGs per cell in P525L FUS eGFP MNs. Number of P525L cells counted and n/condition: 2488, n = 13 (arsenite); 1045, n = 12 (arsenite + rec. control); 646, n = 6 (arsenite + rec. 17AAG); 924, n = 3 (arsenite + rec. GSK), ± sem (One-way ANOVA).

(D) HeLa cells expressing GFP-tagged wild-type (WT) FUS or the ALS-associated mutant G156E were treated with sodium arsenite (50 µM) for 45 min to induce SGs, followed by recovery in drug-free medium (Recovery Control) or in presence of GA (5 µM). Images were taken over a time period of 4 hrs every 10 min. Dashed lines = 95% confidence intervals. Number of cells counted: GFP-FUS WT cells, 227 (Recovery Control) and 254 (GA 5 µM); GFP-FUS G156E cells, 149 (Recovery Control) and 177 (GA 5 µM).

(E) HeLa cells expressing GFP-tagged wild-type (WT) FUS or the ALS-associated mutant G156E were lipofected with non-targeting control siRNA or siRNA pools specific for Hsp90 α and β. 72 hrs post-transfection, cells were treated with sodium arsenite (50 µM) for 45 min to induce SGs, followed by recovery in drug-free medium. Images were taken over a time period of 4 hrs every 10 min. Dashed lines = 95% confidence intervals. Number of cells counted: GFP-FUS WT cells, 532 (siRNA Control) and 449 (siRNA Hsp90); GFP-FUS G156E cells, 566 (siRNA Control) and 504 (siRNA Hsp90).

(F) HeLa cells expressing GFP-tagged wild-type (WT) FUS or the ALS-associated mutant G156E were treated with sodium arsenite (500 µM) for 45 min to induce SGs, followed by recovery in drug-free medium (ars. + rec. control) or in presence of GSK (5 µM). Treatment of the cells with GSK (5 µM) for 4 hrs was included as a control. Cells were fixed and the % of SG-positive cells was counted. Number of GFP-FUS-WT cells counted: 641 (control); 726 (ars.); 846 (ars. + rec. control 4 hrs); 1010 (ars. + rec. GSK 4 hrs); 422 (GSK 4 hrs). Number of GFP-FUS-G156E cells counted: 538 (control); 602 (ars.); 737 (ars. + rec. control 4 hrs); 922 (ars. + rec. GSK 4 hrs); 424 (GSK 4 hrs). n = 4-9 independent samples, ± sem; p = 10-5 (between recovery control and recovery with GSK) (One-way ANOVA).

(G) DYRK3 and HSP90AA1, HSP90AB1 mRNA levels in three fibroblast lines from healthy individuals. Fibroblast lines were either left untreated (control) or exposed to sodium arsenite (500 μM) for 45 min (arsenite); where indicated, cells were allowed to recover in drug-free medium for 4 hrs prior to RNA extraction (arsenite + recovery 4 hrs). n = 3 independent experiments, ± sem (One-way ANOVA).

(H) Fibroblasts from one sporadic ALS (sALS) patient and from one familiar ALS (fALS) patient, carrying the p.R191Q mutation in the VCP gene were treated as described in G. Fold change of DYRK3, HSP90AA1 (AA1) and HSP90AB1 (AB1) mRNA levels compared to the average of the three fibroblast lines from healthy individuals (control) are shown. n = 3 independent experiments, ± sem (One-way ANOVA).

(I) Double immunofluorescence labeling using antibodies against DYRK3 and FUS in lumbar spinal cord α-MNs of healthy subjects (control) and fALS patients carrying the p.521C mutation in the FUS gene. Control (upper panel): α-MNs showing uniform cytoplasmic (white arrows) and speckled pattern of strong nuclear immunoreactivity (white arrowhead) of DYRK3, as well as diffuse nuclear FUS immunoreactivity. fALS (p.R521C-FUS; lower panel): surviving α-MNs harboring large FUS aggregates (yellow arrows) showed markedly reduced DYRK3 immunoreactivity both in the cytoplasm as well as in the nucleus (red arrowheads). Instead, α-MNs devoid of FUS aggregates (white arrows) showed normal DYRK3 immunoreactivity similar to the one of α-MNs from normal controls. Asterix (*) represents non-specific lipofuscin granules in one of the α-MN in FUS-ALS. Paraffin sections; scale bars is 50 µm.

(J) Quantification of α-MNs showing low or absent DYRK3 staining (upper graphic) and FUS aggregates (lower graphic) in lumbar spinal cord from five healthy subjects (control) and five fALS patients carrying the p.521C mutation in the FUS gene. Total number of α-MNs analyzed: 403 (control); 132 (fALS). n = 5, ± sem (Student's t-test).

Data information: Related to Movie EV10.