Fig 1: Phospho-mimetic substitution at Ser505 variably enhances the effects of ALS-linked mutations. (A) Intracellular localization of TLS and its mutants with or without S505D substitution in N2a (top) or COS-7 (bottom) cells. Bars represent average NCI values (mean ± SD, n = 3). For each mutant, open and red bars indicate non-substituted S505 and S505D, respectively. *P < 0.05 in comparison with S505 in two-tailed t-test. (B) Intracellular localization of S505D/H509Q (left panels) and S505D/H509P (right panels) in N2a cells. Scale bar indicates 10 µm. (C) S505D substitution modulates splicing regulation of E1A/mut12s in N2a cells. Bars represent the ratio of the 9s spliced product as in Figure 5 (mean ± SD, n = 3). TLS, S505D and S505D/H509Q showed significantly higher ratio of 9s compared to EGFP (P < 0.05). On the other hand, S505D and S505D/H509Q was not significantly different from TLS. In addition, S505D/H509P and S505D/R510K showed significantly decreased activity compared to TLS, with a greater extent for S505D/H509P (**P < 0.01 and *P < 0.05, ANOVA and Tukey's test).
Fig 2: Psychiatric history of donors: proportion of pathological classes among (A) donors differentially diagnosed with a primary psychiatric disorder after the clinical onset of dementia; (B) donors with a medical history of psychiatric disorder; and (C) donors with a family history of primary psychiatric disorder. AD = Alzheimer's disease; FTLD = frontotemporal lobar degeneration; FUS = fused in sarcoma; LBD = Lewy body disease; TDP = TAR DNA‐binding protein.
Fig 3: Neostriatum of FTLD-FUS cases. a and b Semi-macro photograph of the rostal neostriatum including the caudate nucleus head (rectangle), nucleus accumbens (asterisk), and putamen. Severe gliosis (Holzer stain) (a) and atrophy (hematoxylin and eosin stain) (b). c Marked neuronal loss and astrocytosis in the magnified area of the nucleus accumbens (shown by asterisk in b). d Severe neuronal loss with astrocytosis in the magnified area of the caudate nucleus (shown by the rectangle in b). In FTLD-FUS cases, the ventral putamen (e) is more involved than the dorsal region (f)
Fig 4: Impaired endplate maturation in iPSC-derived myotubes from FUS-ALS patients. a, Structure of AChR clusters on hiPSC-derived myotubes was visualized with BTX (green) and assorted based on their morphology in three categories with increasing maturity (diffuse puncta, aligned puncta, dense/cluster). Myotubes were immunolabeled with actinin (red) and nuclei with DAPI (blue). Scale bar: 10µm. b, Quantification of maturation state revealed less mature endplate structures in both patient-derived cell lines compared to CNTL2 and CNTL3. Two-sided Fisher’s Exact Test with Bonferroni correction for multiple testing; ***p<0.0005; N (number of endplates)= 127 (CNTL1), 116 (CNTL2), 71 (CNTL3), 88 (FUS1), 94 (FUS2). N= 3 independent cultures per condition. c, Structure of AChR clusters in hiPSC-derived motor neuron-myotube co-cultures was visualized with BTX (green) and assorted based on their morphology in four categories with increasing maturity (diffuse puncta, aligned puncta, dense/cluster or complex). Myotubes were immunolabeled with actinin (red) and nuclei with DAPI (blue). Scale bar: 10µm. d, Quantification of maturation state is shown in the bar graph, revealing less mature endplate-like structures in co-cultures in which myotubes and/or motor neurons were derived from FUS-ALS patients. Two-sided Fisher’s Exact Test with Bonferroni correction for multiple testing; ***p<0.00005; N (number of endplates)= 99 (CNTL1 MT + CNTL 1 MN), 74 (CNTL1 MT + FUS1 MN), 226 (FUS1 MT + CNTL1 MN), 52 (FUS1 MT + FUS1 MN), 35 (CNTL1 MT + FUS2 MN), 19 (FUS2 MT + CNTL1 MN), 93 (FUS2 MT + FUS2 MT). N= 3 independent co-cultures per condition. e, Quantification of maturation state in motor-neuron myotube co-cultures derived from FUS1 and its isogenic CNTL3. Two-sided Fisher’s Exact Test with Bonferroni correction for multiple testing; ***p<5x10-8; N (number of endplates)= 273 (CNTL3 MT + CNTL3 MN), 73 (CNTL3 MT + FUS1 MN), 381 (FUS1 MN + CNTL3 MT), 285 (FUS1 MT + FUS1 MN). N= 3 independent co-cultures per condition.
Fig 5: Characterization of HeLa cells expressing SG markers and mutant misfolding‐prone proteins HeLa stable cell lines expressing BAC‐encoded FUS‐mCherry or FUS‐GFP were analyzed by Western blotting using anti‐FUS antibody. Positions of endogenous FUS and the transgenes are indicated.HeLa cells expressing BAC‐encoded SG markers (FUS‐mCherry, G3BP1‐mCherry, FUS‐GFP, or G3BP2‐GFP) were transfected with plasmids expressing SOD1(A4V)‐GFP or Ubc9TS‐mCherry. Live cells were imaged under normal conditions (37°C, no treatment). FUS is localized in the nucleus, and G3BP is diffusely distributed in the cytoplasm. Misfolding‐prone proteins Ubc9TS and SOD1(A4V) are also diffusely distributed. Scale bar = 10 μm.HeLa cells expressing SOD1(A4V)‐GFP, SOD1(WT)‐GFP, Ubc9TS‐mCherry, or Ubc9WT‐mCherry were treated with MG132 (10 μM) for 14 h. Cells were fixed and stained for p62. p62 localized to large perinuclear inclusions (arrows) together with misfolded proteins but not WT variants of SOD1 or Ubc9. Scale bar = 10 μm.HeLa cells expressing G3BP2‐GFP and Ubc9TS‐mCherry were imaged during the treatment with MG132 (10 μM). G3BP2 assembled into SGs (arrowheads), Ubc9TS accumulated over time and localized to aggresome (arrows). Scale bar = 10 μm. Images are from Movie EV1.
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