A chemical genetic screen identifies TDP-43 phase modulators

(A) The workflow of the chemical genetic screen. (B) Dose dependent reduction of anisosome number by identified chemicals. DLD1 TDP-43 2KQ-Clover cells were treated with doxycycline to induce anisosome for 24 h and then treated with the drugs as indicated for 15 h. Cells were imaged for anisosome count.

Two distinct types of TDP-43 phase modulators

(A) DLD1 TDP-43 2KQ-Clover cells treated with doxycycline for 24 h were further treated with the indicated compounds for 6 h and imaged (SPA, Spautin-1, 5 μM; KPT, KPT-276, 15 μM; CP, CP-673451, 30 μM; TRP, Tripterin, 1 μM; BTZ, Bortezomib, 10 nM). Scale bar, 5 μm. (B) FRAP analyses of anisosomes after treatment with the indicated drugs for 3-5 h. Circles indicate bleached areas. Scale bars, 1 μm. (C, D) Quantification of the experiments represented in B. FL, Fluorescence. (E) Reverse FRAP analyses of anisosome dynamics. The areas indicated by the dashed lines were bleached. Scale bar, 5 μm. (F) Quantification of the fluorescence loss over time in unbleached anisosomes as shown in E. N=4-6. (G) Quantification of the initial rate of fluorescence loss in unbleached anisosomes. ****, p<0.0001; ***, p<0.001 by unpaired Student’s t-test.

A genome-wide siRNA screen identifies modifiers of TDP43 phase behavior

(A) The workflow of the siRNA genetic screen. GW, genome-wide; AS, anisosome; HT, high-throughput. (B) Pathway analysis of genes whose knockdown reduces anisosome number in cells. The relative anisosome count are indicated by both color and size of the nodes. (C) GO molecular function analysis of TDP-43 phase modifiers. (D) A list of identified genes linked to ALS in C.

Anisosome phase behavior is modulated by RNA splicing and protein translation

(A) The splicing inhibitor Pladienolide-B (PlaB) reduces anisosome number in a dose dependent manner. Representative images of anisosome-induced cells treated with DMSO (control), 5 nM, or 20 nM PlaB for 16 h. Scale bar: 10 µm. (B) Quantification showing the number of anisosome (AS) per cell in TDP-43 2KQ-Clover cells treated with PlaB as indicated. * p <0.05, ** p <0.01, **** p < 0.0001 by One-way ANOVA. n=3 biological repeats. (C) Representative FRAP images of anisosomes in cells treated for 16 h with DMSO or PlaB (20 nM). BB, before photobleaching, right after photobleaching (0 s), or 4 and 30 seconds after photobleaching (4 s and 30 s). Scale bar, 1 µm. (D) The graph shows the quantification of the remaining TDP-43 fluorescence (FL) in C. Error bars indicate mean ± SD, N = 28 for control and 23 for PlaB-treated cells. (E) Live cell imaging of anisosome fusion in TDP-43 2KQ-Clover cells treated with 20 nM PlaB for 5 h before tracking the fusion. Representative images from Movie S1 showing fusion events indicated by arrows. Scale bar, 1 µm. (F) Representative images of anisosome-induced (24 h) cells treated with DMSO (control) or ANS (200 nM) for 16 h. Scale bar, 1 µm. (G) Quantification of the number of anisosomes per cell in randomly selected images of DMSO-, Cycloheximide (CHX)-, or ANS-treated cells. **** p < 0.0001 by one-way ANOVA; each dot represents an image with at least 20 cells counted. n=3 biological repeats. (H) Quantification of anisosome size in control or cells treated with CHX or ANS. ****, p < 0.0001 by One-way ANOVA. n=3 biological repeats. AU, arbitrary unit. (I) As in C except that anisosome-induced cells were treated with CHX or ANS before photobleaching. Scale bar 1 µm. (J) Quantification of fluorescence recovery in CHX- or ANS-treated cells vs the DMSO control.

XPO1 regulates anisosome liquid-to-solid transition

(A) Pharmacological inhibition of XPO-1 with Leptomycin B (LMB) reduces the number of anisosome. The graph on top indicates the experimental design. Arrows show cells with enlarged anisosomes. Scale bar 10 µm (B) Quantification of anisosome numbers per cell in experiments represented by A. Error bars indicate mean ± SD; **** p <0.0001 by one-way ANOVA. n=3 biological repeats. (C) LMB dose dependently reduces anisosome number. Anisosome were induced in TDP-43 2KQ-Clover cells followed by treatment with LMB at the indicated concentrations for 16 h. The histogram shows the distribution of cells (50-90 cells/condition) by anisosome count. (D) FRAP experiments demonstrate that anisosomes remain in a liquid phase following LMB treatment (200 nM, 16 h). Anisosome-induced (24 h) cells were treated with DMSO or LMB for 16 h and then photobleached at the indicated areas. Scale bar 1 µm. (E) Quantification of the FRAP experiment in D. (F) Time-lapse confocal microscopy detects anisosome fusion after LMB (200 nM, 5 h) treatment in TDP-43 2KQ-Clover cells. Arrows indicate fusion events. Scale bar, 1 µm (G) Overexpression of mCherry-tagged XPO-1 in TDP-43 2KQ-Clover cells induces cytoplasmic TDP-43 puncta. TDP-43 2KQ-Clover cells (green) transfected with mCherry-XPO1 (red) were stained with DAPI (blue) to label nuclei (dashed lines). Cells were imaged 48 h post-transfection. Panels 1, 2 show a representative confocal section, while panels 3-6 show reconstructed 3-D views. The position and volume of anisosomes were also presented in magenta in a surface-rendered view (SRV) in panel 5. The arrow in panel 3 indicates an example of cytoplasmic TDP-43 aggregate. Scale bar, 10 µm. (H) Quantification of the percentage of cells showing cytosolic TDP-43 puncta in XPO-1 positive (pos) and negative (neg) cells in randomly selected fields from 3 independent experiments. **** p <0.0001 by unpaired Student’s t-test. (I) FRAP-based confocal imaging reveals the transition of anisosomes into a gel-like state upon XPO-1 overexpression. Scale bar, 1 µm. (J) Anisosome formation changes endogenous XPO-1 localization. TDP-43 2KQ-Clover before or after anisosome induction were stained with anti-XPO-1 antibodies (red) and DAPI (blue). The bleached areas were indicated by dashed lines. Scale bar, 10 µm. (K) Quantification of nuclear endogenous XPO-1 levels in individual cells (indicated by dots) before or after anisosome induction. **** p < 0.001 by unpaired Student’s t-test. n=2 independent repeats. (L) A schematic model depicting how the nuclear XPO-1 activity influences TDP-43 liquid-to-phase transition. AS, anisosome.

Inhibition of XPO-1 reduces TDP-43 hyperphosphorylation but only partially restore neuronal gene expression in K181E organoids

(A) Confocal fluorescence imaging reveals significant reduction in phosphorylated TDP-43 (p-TDP43) in K181E/K181E organoids following KPT-276 treatment. Organoids of the indicated genotypes at day 87 were treated with DMSO as a control, or with KPT-276 (20 nM) for 35 days. Organoids were fixed, sectioned, and stained with antibodies against TUJ1, a neuronal marker (Magenta), TDP-43 (green) and p-TDP-43 (red). n= 3-5 organoids from two individual batches. (B) Quantification of p-TDP-43 Mean Spot Volume (top) and percentage of cells lacking nuclear TDP-43 (bottom) in experiments represented by A. Error bars indicate mean ± s.e.m., * p < 0.05, **** p < 0.0001 by one-way ANOVA, n=3 organoids. (C) Gene Ontology (GO) analysis of genes whose expression was disrupted in K181E/K181E organoids but restored by KPT-267 as shown in Supplemental Fig. S3. BP, Biological Pathway; CC, Cellular Component; MF, Molecular Function.

HSP90 inhibitor Geldanamycin converts TDP-43 anisosome to a gel-like state

(A) DLD1 cells were treated with doxycycline to induce anisosome formation and then treated with Geldanamycin (GA) at 10 μM for 4 h. Cells were stained with Hoechst (Blue) and imaged by a confocal microscope. Scale bar, 5 μm. (B) A TDP-43-bearing anisosome (dashed circle) in a GA-treated cell was photobleached and then imaged. Scale bar, 1 μm. The graph shows the quantification. Error bars, s.e.m. (N>5 anisosomes). FL, fluorescence.

The impact of splicing inhibitor and translation inhibitor on anisosomes

(A) A violin plot showing the relative size distribution of anisosomes (AS) in DLD1 cells treated with Pladienolide-B at the indicated concentration for 16 h. ****, p<0.0001 by one-way ANOVA. n=3 biological repeats. (B) DLD1 cells were treated with doxycycline to induce anisosome formation and then treated with Cycloheximide (CHX) at 20 μg/mL or DMSO as a control for 16 h. Cells were stained with Hoechst (Blue) and imaged by a confocal microscope. Scale bar, 5 μm.

KPT-276 modestly reverses gene expression defects in K181E mutant organoids

Heatmap showing genes whose expression was altered in K181E/K181E organoids and restored after KPT-276 treatment.