fusidic acid  (MedChemExpress)

Journal: Acta Neuropathologica

Article Title: PP2A and GSK3 act as modifiers of FUS-ALS by modulating mitochondrial transport

doi: 10.1007/s00401-024-02689-y

Figure Lengend Snippet: PP2A and GSK3 pharmacological inhibition rescue FUS cytoplasmic mislocalization in patient iPSC-derived motor neurons. a . Representative confocal images showing FUS distribution in patient iPSC-derived motor neurons with the P525L mutation, as well as in P525P isogenic controls after treatment with lithium chloride (LiCl, 1 mM 48 h), tideglusib (TD, 15 μM 48 h) or okadaic acid (OA, 1 nM 72 h). Scale bars 10 μm. b Quantification of nuclear/cytoplasmic ratios (N/C ratio) fluorescent intensity of FUS in motor neurons shows a rescue of FUS mislocalization after treatments. Each dot represents one analyzed cell. Three different colors indicate data combined from three independent differentiations (70–80 cells/condition). Data are shown as Grand mean; Kruskal–Wallis with Dunn’s multiple comparisons test. **** p < 0.0001, ns not significant

Article Snippet: HEK293T cells were transfected with FLAG-tagged WT and P525L FUS-encoding plasmids and treated with 10 nM calicheamicin γ1 (MedChemExpress, HY-19609) for 2 h. Next, the cells were treated with 10 nM OA (dissolved in H 2 O) for different time periods.

Techniques: Inhibition, Derivative Assay, Mutagenesis

Journal: Acta Neuropathologica

Article Title: PP2A and GSK3 act as modifiers of FUS-ALS by modulating mitochondrial transport

doi: 10.1007/s00401-024-02689-y

Figure Lengend Snippet: PP2A and GSK3 inhibition improves ALS-associated NMJ impairments. a Representative confocal micrographs of NMJs formed by FUS-P525L cells with LiCl, TD, and OA treatments. NMJs are impaired in FUS-P525L ALS, and inhibition of PP2A or GSK3 improves the phenotype (see Fig. S4 for additional images). Scale bars 10 μm. b Quantification of NMJ-like structures for all conditions, based on the neurite/presynaptic marker morphology and/or based on Btx ( red )-SYP/NEFH ( green ) co-localization per myotube. Each dot represents one analyzed myotube. Three different colors indicate data combined from three independent differentiations (70–80 myotubes/condition). Data are shown as Grand mean; N = 3 biological replicates; Kruskal–Wallis test with Dunn’s multiple comparisons test. * p < 0.05; **** p < 0.0001, ns not significant

Article Snippet: HEK293T cells were transfected with FLAG-tagged WT and P525L FUS-encoding plasmids and treated with 10 nM calicheamicin γ1 (MedChemExpress, HY-19609) for 2 h. Next, the cells were treated with 10 nM OA (dissolved in H 2 O) for different time periods.

Techniques: Inhibition, Marker

Journal: Acta Neuropathologica

Article Title: PP2A and GSK3 act as modifiers of FUS-ALS by modulating mitochondrial transport

doi: 10.1007/s00401-024-02689-y

Figure Lengend Snippet: Pharmacological inhibition of PP2A and GSK3 rescue mitochondrial transport deficits. a – c Example kymographs (time-distance plots) of mitochondria (MitoTracker Green) after treatment of 30-day-old P525P isogenic control motor neurons and P525L mutant motor neurons with 1 mM lithium chloride (LiCl) for 48 h ( a ), 15 μM tideglusib (TD) for 48 h ( b ), and 1 nM okadaic acid (OA) for 72 h ( c ). Mock conditions were untreated. Scale bars 30 μm d – f . Percentage of mitochondria that are motile in the motor neurons (day 30) comparing isogenic control and mutant with or without LiCl ( d ), TD ( e ), and OA ( f ). Each dot represents one analyzed neurite. Three different colors indicate data combined from three independent differentiations (70–80 neurites/condition). OA and TD treatments were performed at the same time, so the mock data are the same for both conditions. Data shown as Grand Mean; N = 3 differentiations; Kruskal–Wallis with Dunn’s multiple comparisons test. **** p < 0.0001, ns not significant

Article Snippet: HEK293T cells were transfected with FLAG-tagged WT and P525L FUS-encoding plasmids and treated with 10 nM calicheamicin γ1 (MedChemExpress, HY-19609) for 2 h. Next, the cells were treated with 10 nM OA (dissolved in H 2 O) for different time periods.

Techniques: Inhibition, Control, Mutagenesis

Journal: Acta Neuropathologica

Article Title: PP2A and GSK3 act as modifiers of FUS-ALS by modulating mitochondrial transport

doi: 10.1007/s00401-024-02689-y

Figure Lengend Snippet: GSK3 is hyperactive in FUS-ALS due to reduced inhibitory phosphorylation. a Western blot showing phospho-sgg and total-sgg protein in control vs FUS WT and R521G flies. Control was w1118 crossed to Gal80-ts; nSyb-Gal4. In Drosophila , the two splice isoforms of GSK3-β, SGG39 ( upper band ) and SGG10 ( lower band ), are seen. Beta-actin serves as a loading control. b Quantification of panel a shows that phosphorylation of SGG is reduced in FUS WT and R521G Drosophila, for the total-SGG protein and for the individual SGG39 and SGG10 isoforms, while the levels of SGG protein remain unaltered overall ( N = 3, mean ± SEM, one-way ANOVA with Sidak’s multiple comparisons test). c Western blot for GSK3α/β pSer21/9 in iPSC-derived motor neurons from FUS-P525L patients and in their CRISPR-corrected P525P isogenic control. Ser21/9 inhibitory phosphorylation of GSK3α/β appears reduced in FUS P525L patient motor neurons compared to isogenic controls, while the total levels of GSK3α/β remain unaltered among the conditions. The numbers 1, 2, and 3 represent three independent differentiations. Alpha-tubulin serves as a loading control. d Quantification of the western blot of panel c shows reduced Ser21 and Ser9 inhibitory phosphorylation for GSK3 α and β, respectively ( N = 3, mean ± SEM, two-tailed paired t test) * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: HEK293T cells were transfected with FLAG-tagged WT and P525L FUS-encoding plasmids and treated with 10 nM calicheamicin γ1 (MedChemExpress, HY-19609) for 2 h. Next, the cells were treated with 10 nM OA (dissolved in H 2 O) for different time periods.

Techniques: Phospho-proteomics, Western Blot, Control, Derivative Assay, CRISPR, Two Tailed Test

Journal: Acta Neuropathologica

Article Title: PP2A and GSK3 act as modifiers of FUS-ALS by modulating mitochondrial transport

doi: 10.1007/s00401-024-02689-y

Figure Lengend Snippet: Kinesin appears dysfunctional in FUS-ALS, perhaps due to hyperphosphorylation by GSK3. a Overexpression of Khc using the p[Khc+] stock rescues the FUS-induced fly eclosion phenotype. w1118 crossed to D42Gal4-driven FUS serves as control. ( N = 10 crosses/condition). b Overexpression of Khc strongly extends the shortened lifespan of FUS WT and R521G Drosophila at 25 ℃. w1118 crossed to FUS serves as control. (see TableS4 for statistical information). c Western blot for Khc and Klc in FUS flies vs FUS flies with p[Khc+]. Beta-actin serves as loading control. d Quantification of the western blots of panel c show a significant increase in Khc and Klc levels ( N = 6). e Western blot of Klc S433 phosphorylation in control w1118 VS FUS-expressing flies. f Quantification of the western blot of panel e shows increased levels of Klc S433p in FUS flies. g Western blot of Klc S433p in FUS flies with sgg heterozygous knockout ( N = 3). h Quantification of panel g shows that Klc S433p levels decrease after sgg inhibition. i Western blotting assessing Klc S433p in FUS flies with mts heterozygous knockout ( N = 3). j Quantification of panel i shows that Klc S433p levels decrease after mts inhibition. k Western blotting in FUS P525P and P525L MNs, assessing KLC1 S460 phosphorylation. l Quantification of panel k reveals increased KLC1 S460p in P525L patient motor neurons compared to isogenic controls ( N = 3 differentiations). m Quantification from panel k shows a trend for reduced KLC1 levels in FUS P525L MNs. Values in a , d , h , j , and l are represented as the mean ± SEM and statistical comparisons were determined using unpaired t test. One-way ANOVA with Sidak’s multiple comparison test was used for panel f . * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, ns not significant

Article Snippet: HEK293T cells were transfected with FLAG-tagged WT and P525L FUS-encoding plasmids and treated with 10 nM calicheamicin γ1 (MedChemExpress, HY-19609) for 2 h. Next, the cells were treated with 10 nM OA (dissolved in H 2 O) for different time periods.

Techniques: Over Expression, Control, Western Blot, Phospho-proteomics, Expressing, Knock-Out, Inhibition, Comparison