Design and functional comparison of TDP-43 loss-of-function reporter systems.

Comparison of stable polyclonal HEK cells expressing UNC13A-TS, CFTR-TS, or CUTS following treatment with siRNA control (siCTRL) (20nM) or TDP-43 (siTDP-43) (0.6nM - 20nM). Cells were reverse-transfected with siRNA treatment in complete media supplemented with doxycycline (1000 ng/mL). As a negative control, we use ND: No doxycycline. After 72 h, cells were analyzed by live imaging and protein lysate was harvested for western blot analysis. (A) Schematic of the TDP-43 loss-of-function Sensor (TS) system design. (B) Overview of the UNC13A-TS, CFTR-TS, and CUTS cryptic exon cassette design. (C) Representative live imaging of TS comparison (10X) nuclear GFP indicates TDP-43 LOF. (D) Mean intensity quantification of GFP signal intensity as shown in (C). (E) Western blot analysis of CUTS immunoblotting for TDP-43 and GFP proteins. (F-G) Relative pixel quantification of TDP-43 and GFP band normalized to total protein (Ponceau S) from (E). Statistical significance was determined by one-way ANOVA and Tukey’s multiple comparison test (*, p < 0.05; **, p< 0.01; ***, p < 0.001; ****, p < 0.0001). Green = GFP signal; red = mCherry signal. Scale bar = 100 µm. N=3 biological replicates.

Differential reporter sensitivity to TDP-43 depletion.

Comparison of stable polyclonal HEK cells expressing UNC13A-TS, CFTR-TS, or CUTS following treatment with siRNA control (siCTRL) (20nM) or TDP-43 (siTDP-43) (0.6nM - 20nM). Cells were reverse-transfected with siRNA treatment in complete media supplemented with doxycycline (1000 ng/mL). After 72 h, cells were analyzed by live imaging and protein lysate was harvested for western blot analysis. (A) Mean intensity quantification of GFP signal intensity from live imaging, presented as fold change from mock. (B) Relative pixel quantification of GFP normalized to total protein (Ponceau S), presented as fold change from mock. (C-E) WB analysis of UNC13A-TS, developing again TDP-43 and GFP proteins, and pixel quantification of TDP-43 and GFP intensity from bands show in (C) normalized to total protein (Ponceau S). (F-H) WB analysis of CFTR-TS, developing again TDP-43 and GFP proteins, and pixel quantification of TDP-43 and GFP intensity from bands show in (F) normalized to total protein (Ponceau S). Statistical significance was determined by two-way ANOVA and Tukey’s multiple comparison test (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). N=3 biological replicates.

CUTS exhibits linear, dose-responsive activation following low-level TDP-43 knockdown.

Low-dose siRNA TDP-43 (siTDP-43) treatment was performed in stable polyclonal HEK cells expressing CUTS. CUTS-expressing cells were reverse transfected with siRNA control (siCTRL) or siTDP-43 in a dose-response curve (38 to 1200pM) in doxycycline supplemented media (1000ng/ml) for 72hr. (A) Representative immunofluorescence images of CUTS-expressing HEK cells under low doses of siRNA TDP-43 treatment. (60X). (B) Percentage of GFP-positive cells normalized to the number of CUTS-expressing mCherry-positive cells. (C) Western blot of TDP-43 and GFP proteins from HEK cell lysate expressing CUTS under low doses of siRNA TDP-43. Ponceau S is shown as a loading control. (D) Pixel intensity quantification of the TDP-43 and (E) GFP bands shown in (C), presented as fold-change from the mock-treated sample. (F) Schematic showing the position of qPCR primers, developed to detect CUTS cryptic exon inclusion (referred to as ‘CUTS-CE’ and ‘CUTS-J’). (G) Representative agarose gel showing qPCR product from melting curve detecting CUTS cryptic exon inclusion using the primers shown in (F). (H-I) qPCR quantification of TDP-43 and CUTS cryptic exon J from the siTDP-43 dose curve presented as fold-change from the mock-treated sample. Linear regression analysis shown in (D), (E), (H) and (I) was performed on Log values. Fitting method = least squares regression. (****, p < 0.0001). N=3 biological replicates. Green = GFP; red = mCherry. Scale bar = 50 µm. N=3 biological replicates.

CUTS signal scales with dose-dependent TDP-43 knockdown.

Low-dose siTDP-43 treatment was performed in stable polyclonal HEK cells expressing CUTS. CUTS-expressing cells were reverse transfected with siCTRL or siTDP43 in a dose-response curve (38 - 1200 pM) in doxycycline supplemented media (1000ng/ml) for 72 h. (A) Pixel intensity quantification of the GFP and TDP-43 bands shown in (C), presented as fold-change from the mock-treated sample. (B) qPCR quantification of the siTDP-43 dose curve presented as fold-change from the mock-treated sample. Purple text above indicates the GFP fold change from the mock-treated sample. Red text above indicates the percentage of total detectable TDP-43 knockdown. Linear regression analysis shown in (D) and (G) was performed on Log values. Fitting method = least squares regression. (C) GFP pixel intensity from the Western blot shown in Figure 2C plotted against the percentage of TDP-43 knockdown detected under siTDP-43 treatment (38-1200 pM). (D) CUTS CE transcript quantification plotted against the percentage of TDP-43 knockdown detected under siTDP-43 treatment (38 - 1200 pM). (E) RT-PCR of endogenous cryptic exons and the CUTS biosensor following increasing doses of siTDP43 treatment.

Mislocalization and aberrant phase transitions induce TDP-43 LOF.

Stable HEK cells expressing CUTS were induced with doxycycline (1000 ng/mL) for 24 hours before transfection with the following plasmids: pCMV backbone, TDP-43WT, TDP-43ΔNLS, TDP-435FL, TDP-43ΔNLS 5FL, or non-transfected. Following transfection, plasmids were expressed for 72 h, followed by live imaging and protein analysis. (A) Live-imaging of CUTS HEK cells expressing WT or mutant TDP-43 gene cassettes. (B) Representative western blot of exogenous and endogenous GFP and TDP-43. Ponceau S is shown as a loading control. (C) Relative GFP pixel intensity quantification of the band is shown in (B). (D–E) Immunofluorescence of stable HEK cells expressing CUTS with siCTRL (20 nM), siTDP-43 (20 nM), mock, and NaAsO₂ (250 µM). (E) Immunofluorescence GFP signal quantification of (D) with 250 µM NaAsO₂ treatment relative to siTDP-43 (20 nM). (F-G) RT-qPCR from CUTS-expressing HEK cells treated with 250 µM NaAsO2 of TDP-43, CUTS-J transcript, and endogenous cryptic exons (ATGB4, GPSM2, and KCNQ2) relative to siRNA TDP-43 20 nM (dotted line) and normalized to 18S. (I) RT-PCR from Figure F-H. Expression values were normalized to 18S rRNA, and fold changes were calculated relative to the siTDP-43 (20 nM) condition. Black squares= Mock: Red circle= NaAsO₂. Statistical significance was determined by one-way ANOVA and Tukey’s multiple comparison test (*, p < 0.05; **. p < 0.01; ***. p < 0.001; ****, p < 0.0001). Green = GFP; red = mCherry. Scale bar = 100 µm. N = 3 biological replicates.

TDP-43wt expression rescues loss-of-function in a HeLa TARDBP Knockout Cell Line.

Transient CUTS expression in HeLa TDP-43 KO we induced with doxocycline (1000n/ml) for 24 hours before transfection of pCM backbone or TDP-43WT plasmids. Following transfection, plasmids were expressed for 72 h, followed by live imaging and protein analysis. (A) Live-imaging of HeLa TDP-43 KO expressing CUTS in combination with TDP-43WT or pCMV backbone control. (B) Representative WB of exogenous and endogenous GFP and TDP-43. Ponceau S is shown as a loading control. (C) Relative GFP pixel intensity quantification of the protein bands shown in (B). (D)RT-PCR product from CUTS cryptic exon inclusion from wildtype or TDP-43 KO HeLa cell lines using the CUTS-CE primers shown in Figure 2E. Statistical significance was determined by one-way ANOVA and Tukey’s multiple comparison test (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). Green = GFP; red = mCherry. Scale bar = 100 µm. N = 3 biological replicates.

CUTS functions as an autoregulatory controller of TDP-43 expression.

(A) Schematic of CUTS as an autoregulatory controller of TDP-43 expression (CUTS-TDP43). (B-C) TDP-43 siRNA (siTDP-43) dose-response curve in stable polyclonal HEK cells expressing CUTS, CUTS-TDP43, or CUTS-TDP-43CO (codon optimized). The codon-optimized variation allows for continued expression during siTDP-43 treatment. HEK cells expressing the CUTS, CUTS-TDP-43 and CUTS-TDP-43CO were reverse transfected with control siRNA (siCTRL) or siTDP-43 in a dose-response curve (0.6nM-20nM) in a doxycycline (1000ng/ml) supplement media for 72hr. Cells were then used for live imaging or protein analysis. (B) Live imaging of the CUTS variants. (C) Immunoblot assay of GFP and TDP-43. Ponceau S is shown as a loading control. (D-E) CFTR minigene assay in stable CUTS or CUTS-TDP-43CO expressing HEK cells. Cells were induced with doxycycline (1000 ng/mL) for 24 h before transfection with the CFTR minigene. Following an additional 24h of expression, cells were transfected with 20nM siCTRL or siTDP-43. Cells were harvested 48 h following siRNA transfection for RNA extraction and RT-PCR analysis. (D) PCR agarose gel of CFTR minigene. (E) PCR analysis of the ratio between the CFTR cryptic exon inclusion and the correctly spliced product from CFTR as shown in (D). Statistical significance was determined by One-Way ANOVA and Tukey Post-hoc (**, p < 0.01; ****, p < 0.0001). Green = GFP; red = mCherry. Scale bar = 100 µm. N=3 biological replicates.

CUTS is activated in human neuronal models in response to TDP-43 LOF.

(A) Representative immunofluorescence images of MAP-2 (magenta), mScarlet, and GFP from CUTS-expressing BE2C neuron-differentiated cells treated with siCtrl or siTDP-43 (50 nM). Scale bar = 20 μM. (B) Mean GFP/mScarlet pixel intensity ratios from (A). (C) Representative immunofluorescence images of TDP-43 (greyscale) and mScarlet-GFP signal from CUTS-expressing BE(2)-C neuron-differentiated cells show reduced TDP-43 expression following siRNA treatment. Fluorescence intensity quantification shows a 61.9 +/-9.6 percent knockdown. Scalebar = 20 μM. (D) Representative immunofluorescence images of MAP-2 (magenta), TDP-43 (grey), mScarlet, and GFP from CUTS-expressing ReN differentiated neurons with 100 nM siRNA TDP-43. Nuclear GFP signals are indicated with a white dotted circle. Scalebar = 50 μM. Non-parametric Kolmogorov-Smirnov T-test (****, p < 0.0001).

CUTS activity in differentiated BE2C and ReN Cells.

(A) Representative bright-field images of BE2C differentiation from day 0 to day 6, after 10 days of differentiation, immunofluorescent staining was performed again MAP-2. Nuclei are indicated with a white dotted circle. Scalebar = 20 mM. (B) Representative mScarlet and GFP signal from live imagining of differentiated ReN cells expressing CUTS 2 days before siTDP-43 knockdown and after 96 h of siRNA treatment at different siTDP-43 doses. Scalebar = 100 mM. (C) Representative immunofluorescent imaging of CUTS system in differentiated ReN VM cells stained for GFAP (Greyscale). Nuclei are indicated with a white dotted circle. Scalebar = 50 mM.

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