Journal: bioRxiv

Article Title: Cdc48/VCP and endocytosis regulate TDP-43 and FUS toxicity and turnover

doi: 10.1101/668798

Figure Lengend Snippet: (A) Cdc48-GFP strain alone (left), and Cdc48-GFP strain transformed with a TDP-43-mRuby2 plasmid (right) were imaged in both mid log (M-L) and stationary phase (S-P). Arrowhead and percentage values indicate Cdc48 and TDP-43 co-localization. Scale bar: 2 µm. (B) Cdc48-TAP strain was transformed with an empty vector or TDP-43-YFP plasmid, and then TAP immunoprecipitation was performed. (C) As in (B), but with GFP immunoprecipitation i.e. reciprocal pull down. (D) Cdc48-TAP strain with TDP-43 was transformed with vector or Pab1-GFP and the interaction was determined. (E) WT and cdc48-3 strains were treated with 0.2 mg/ml Cycloheximide (CHX) for the indicated time at either 25°C or 35°C. TDP-43 protein levels detected and normalized relative to a PGK1 loading control. (F) Serial dilution growth assay of WT and cdc48-3 transformed with empty vector or GAL1 -regulated TDP-43-YFP plasmid at different temperatures.

Article Snippet: Primary antibodies were as follows: α-GFP (902602; Biolegend), α-TDP-43 (60019-2-Ig; ProteinTech), α-Vinculin (V4505; Sigma-Aldrich), α-Pgk1 (ab113687; Abcam), α-VCP (ab11433; Abcam), α-TAP (Thermofisher CAB1001) and α-GAPDH (MA5-15738, Invitrogen).

Techniques: Transformation Assay, Plasmid Preparation, Immunoprecipitation, Serial Dilution, Growth Assay

Journal: bioRxiv

Article Title: Cdc48/VCP and endocytosis regulate TDP-43 and FUS toxicity and turnover

doi: 10.1101/668798

Figure Lengend Snippet: (A) Growth assay of WT and indicated isogenic null strains transformed with an empty vector or GAL1 -regulated TDP-43-GFP plasmid. (B) Ubx3-GFP strain was transformed with a TDP-43-mRuby2 plasmid and images were taken at mid-log phase. Arrowhead indicates Ubx3 and TDP-43 co-localization. Scale bar: 2 µm. (C) Serial dilution growth assay of indicated strains transformed with GAL1 -regulated TDP-YFP plasmid (D) Cdc48-GFP strain was transformed with an untagged TDP-43 plasmid and either a Vps34-or Vps38-mRuby2 plasmid; Arrowhead indicates Cdc48 co-localization with both Vps34 and Vps38. Scale bar: 2 µm.

Article Snippet: Primary antibodies were as follows: α-GFP (902602; Biolegend), α-TDP-43 (60019-2-Ig; ProteinTech), α-Vinculin (V4505; Sigma-Aldrich), α-Pgk1 (ab113687; Abcam), α-VCP (ab11433; Abcam), α-TAP (Thermofisher CAB1001) and α-GAPDH (MA5-15738, Invitrogen).

Techniques: Growth Assay, Transformation Assay, Plasmid Preparation, Serial Dilution

Journal: bioRxiv

Article Title: Cdc48/VCP and endocytosis regulate TDP-43 and FUS toxicity and turnover

doi: 10.1101/668798

Figure Lengend Snippet: (A) WT and cdc48-3 cells were cultured at 35°C for 2 hours and then stained with FM4-64 dye (8 uM) for time periods indicated and examined. Arrowhead indicates vacuolar staining. Persistence of plasma membrane staining in cdc48-3 cells and reduced vacuolar membrane staining relative to WT indicates endocytic defect. (B-C) As in (A) except with additional expression of TDP-43-GFP (B) or FUS-GFP (C). Scale bar = 2 µm. (D) Analysis of vacuolar staining intensity of (A-C). **P < 0.01, ***P < 0.001 by Student’s unpaired two-tailed t test. Data was shown as mean ± s.e.m.

Article Snippet: Primary antibodies were as follows: α-GFP (902602; Biolegend), α-TDP-43 (60019-2-Ig; ProteinTech), α-Vinculin (V4505; Sigma-Aldrich), α-Pgk1 (ab113687; Abcam), α-VCP (ab11433; Abcam), α-TAP (Thermofisher CAB1001) and α-GAPDH (MA5-15738, Invitrogen).

Techniques: Cell Culture, Staining, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Cdc48/VCP and endocytosis regulate TDP-43 and FUS toxicity and turnover

doi: 10.1101/668798

Figure Lengend Snippet: (A) HEK 293A cells were either treated with 40µM dynasore for 24 hours or subject to VCP knock down, followed by determination of TDP-43 and FUS protein levels and analyzation. (B) HEK293A cells expressing stably integrated TDP-43-GFP or TDP-35 YFP were examined under normal growth or following DBeQ treatment (10µM, 1hr). Scale bar: 5µm (C-D) Fluorescence immunohistochemistry of control (CTL) and ALS patients (P) frontal cortex tissue with TDP-43, VCP and DAPI and quantification. Arrowhead indicates VCP co-localization with TDP-43. Scale bar: 5 µm. *P < 0.05 by Student’s unpaired to-tailed t test. Data is shown as mean ± s.e.m.

Article Snippet: Primary antibodies were as follows: α-GFP (902602; Biolegend), α-TDP-43 (60019-2-Ig; ProteinTech), α-Vinculin (V4505; Sigma-Aldrich), α-Pgk1 (ab113687; Abcam), α-VCP (ab11433; Abcam), α-TAP (Thermofisher CAB1001) and α-GAPDH (MA5-15738, Invitrogen).

Techniques: Expressing, Stable Transfection, Fluorescence, Immunohistochemistry

Journal: bioRxiv

Article Title: Cdc48/VCP and endocytosis regulate TDP-43 and FUS toxicity and turnover

doi: 10.1101/668798

Figure Lengend Snippet: In yeast, the toxicity, turnover and aggregation of both TDP-43 and FUS depend on endocytosis function (this study and ). We suggest that large microscopically visible aggregates of TDP-43 and FUS are most likely cleared from the cytoplasm by autophagy, with oligomeric or soluble forms of TDP-43 and FUS being subject to clearance by endocytic or proteasomal means. If true, given our yeast toxicity data, in which endocytic but not autophagic mutants enhance TDP-43 and FUS toxicity, this would suggest that oligomeric, rather than microscopically visible forms of TDP-43 and FUS are the “toxic” cellular species. Cdc48/VCP, which localizes in microscopically visible TDP-43 or FUS aggregates, may facilitate TDP-43 and FUS conversion into oligomeric and soluble forms. Red arrows broadly represent antagonistic aggregation-promoting processes which may include cellular stress, impaired proteostasis, RNA metabolism defects and perturbation of nuclear-cytoplasmic trafficking, all of which are implicated in ALS pathology. Cdc48/VCP may facilitate entry of TDP-43 and FUS into the endocytic pathway. Finally, sequestration of Cdc48/VCP (and various endocytic proteins) within TDP-43 or FUS aggregates may contribute to the observed inhibition of endocytosis rates caused by TDP-43 or FUS expression, owing to various roles described for Cdc48/VCP in the endocytic pathway. The means by which TDP-43 and FUS enter the endocytic pathway remains mechanistically undefined.

Article Snippet: Primary antibodies were as follows: α-GFP (902602; Biolegend), α-TDP-43 (60019-2-Ig; ProteinTech), α-Vinculin (V4505; Sigma-Aldrich), α-Pgk1 (ab113687; Abcam), α-VCP (ab11433; Abcam), α-TAP (Thermofisher CAB1001) and α-GAPDH (MA5-15738, Invitrogen).

Techniques: Inhibition, Expressing

Journal: Molecular Neurodegeneration

Article Title: Mutations in α-synuclein, TDP-43 and tau prolong protein half-life through diminished degradation by lysosomal proteases

doi: 10.1186/s13024-023-00621-8

Figure Lengend Snippet: Pathogenic mutations prolong α-syn, TDP-43 and tau half-life. a-f Recombinant, full-length WT or A53T α-syn ( a , b ), WT or Q331K TDP-43 (c, d), and WT or N279K tau ( e , f ) was incubated with 50 μg of human lysosome extract for 30 min. The reaction was subjected to Western blot and results quantified. g-l , Differentiated SH-SY5Y cells expressing inducible FLAG-tagged WT or mutant α-syn ( g , h ), TDP-43 ( i , j ) or tau ( k , l ) were treated with doxycycline for 24 h to induce protein expression. Doxycycline was removed (t = 0 days), MG132 (100 nm) was added to inhibit proteasomal degradation and lysates were collected at each time point as indicated. Samples then underwent SDS-PAGE and anti-FLAG Western blotting ( n = 3 for all cell-lines tested). Samples were normalized for quantification using GAPDH as a loading control. Representative images showing the gradual clearance of of WT or mutant protein over time are shown in g , I and k with quantification of three independent replicates shown in ( h , j ) and ( i ). m-q Control or mutant iNeurons were generated as indicated, and then exposed to MG132 treatment (100 nm) for 5 days. Lysates were collected at day 0 and day 5 and underwent SDS-PAGE and anti- α-syn, anti-TDP-43, or anti-tau Western blotting ( n = 3 for all cell-lines tested). Samples were normalized for quantification using GAPDH as a loading control. Representative images demonstrating WT or mutant protein over time are shown in k with quantification of three independent replicates shown in ( l ). r Proposed model for how mutations in α-syn, TDP-43 and tau can gradually increase steady state levels of protein over decades to predispose to impaired proteostasis, protein aggregation and neurodegeneration. * p < 0.05, ** p < 0.01, or *** p < 0.001

Article Snippet: All iPSC lines (A53T/A53T α-syn, Q331K/Q331K TDP-43, N279K/N279K tau, and WT/WT TDP-43 [KOLF2.1] control) were obtained from the NIH’s iPSC Neurodegenerative Disease Initiative (iNDI) in association with the Jackson Laboratory [ ].

Techniques: Recombinant, Incubation, Western Blot, Expressing, Mutagenesis, SDS Page, Generated

Journal: The EMBO Journal

Article Title: A single N‐terminal phosphomimic disrupts TDP‐43 polymerization, phase separation, and RNA splicing

doi: 10.15252/embj.201797452

Figure Lengend Snippet: A Turbidity of 2.5 μM wild‐type (WT) and S48E TDP‐43‐MBP after 60 min (top) and 120 min (bottom) of incubation with TEV protease is consistent with phase separation at low salt concentration for the wild‐type but phase separation is absent for S48E. B Differential interference contrast micrographs of 2.5 μM full‐length TDP‐43 MBP in 150 mM NaCl (top panel) after 60 and 120 min of incubation with TEV protease. WT shows phase separation, but S48E does not until the concentration is raised (HC). C Wild‐type (WT) and variant (S48E) TDP‐43 RRM‐GFP reporters form spherical, micron‐sized nuclear droplets after overnight expression in 293T cells. Nuclei are outlined in white in representative raw images, and heat map representations of the signal intensities measured with standard and sensitive detector settings are provided below to highlight the differences in the nuclear TDP‐43 RRM‐GFP reporter signal. D Immunoblot showing total expression levels of WT and S48E reporters in 293T cells. See Appendix Fig S2C for full immunoblots with molecular weight markers. E Representative time‐dependent fluorescence recovery after half‐droplet bleaching shows that S48E enhances intra‐phase diffusion dynamics (decreases viscosity) of TDP‐43 reporter particles. F The phosphomimetic S48E mutation has a dominant, fluidizing effect on the liquid dynamics of composite droplets, as revealed by half‐bleach experiments of composite TDP‐43 droplets formed by co‐expression of wild‐type TDP‐43 RRM‐mCherry (red fluorescent, mCh) and the WT (left, blue type) or S48E (right, red type) TDP43 RRM‐GFP variants (green fluorescent, GFP) in 293T cells. G, H Quantification of fluorescence recovery after half‐droplet bleaching of (G) GFP wild‐type (black curve) and GFP S48E (red curve) or (H) mixtures of mCherry WT (black squares) plus GFP wild‐type (blue circles) or mixtures of mCherry wild‐type (inverted triangles) plus GFP S48E (red triangles). Error bars indicate s.d. of 20 measured particles from two biological replicates. I Relative splicing activity of CFTR exon 9 minigene reporter in control (N), TDP‐43 siRNA knock‐down (T) HeLa cells, RNA‐binding‐deficient mutant of F147/149L and TDP‐43 NTD variants was calculated as the ratio of percent exon inclusion relative to WT. Levels of exon inclusion using the CFTR exon 9 minigene reporter were quantified as percent of exon inclusion from Appendix Fig S2G . RNAi‐resistant wild‐type TDP‐43 (WT) and mutants were expressed in siRNA‐treated cells. Error bars indicate s.d., n ≥ 4.

Article Snippet: Immunoblotting with custom and commercial α‐TDP‐43 antibodies (CST 3449) and detection with the LI‐COR imaging system were performed as described above.

Techniques: Incubation, Concentration Assay, Variant Assay, Expressing, Western Blot, Molecular Weight, Fluorescence, Diffusion-based Assay, Viscosity, Mutagenesis, Activity Assay, Control, Knockdown, RNA Binding Assay

Journal: The EMBO Journal

Article Title: A single N‐terminal phosphomimic disrupts TDP‐43 polymerization, phase separation, and RNA splicing

doi: 10.15252/embj.201797452

Figure Lengend Snippet: A Peptides composed of TDP43 (40–53), with and without phosphorylated Ser48, were serially diluted and spotted to nitrocellulose membranes. Polyclonal antibody (α‐TDP‐43 pSer48) specific to the phosphorylated peptide was used in the top panel showing specificity for pS48, and α‐TDP‐43 “pan antibody” recognizing the same peptide irrespective of phosphorylation was used in the bottom panel. B The α‐TDP‐43 pSer48 antibody and commercial TDP‐43 antibody used in Western blots of HEK293T cell lysates both show reactivity at ˜43 kDa, consistent with TDP‐43 SDS–PAGE migration. C Standard Western blotting was performed on HEK293T cell lysates that had been transferred onto nitrocellulose membranes, except calf intestinal phosphatase (CIP, bottom) or a mock treatment (top) was used to treat the membranes prior to immunoprobing with α‐TDP‐43 (pSer48). Whole HEK293T cell lysates were used in the left panel. In the right panel, TDP‐43 was first immunoprecipitated using commercial α‐TDP‐43 antibody prior to Western blotting. D Gel filtration chromatogram of 200 μM wild‐type (black) and S48E (red) TDP‐43 NTD. The shorter retention time and skewed profile of wild‐type NTD is consistent with self‐assembly. The single‐point variant S48E results in a symmetric peak at longer retention time, consistent with predominantly monomer. E CG‐MALS derived mass average as a function of increasing TDP‐43 NTD concentration data are fit to an isodesmic self‐association model (bold black line) with K D ˜ 95 μM. Fits for dimer, trimer, tetramer, and pentamer models are poor (dashed lines), shown for comparison. F CG‐MALS data for wild‐type are effectively the same at 150 mM (black, repeated from E for clarity) and 300 mM (gray) NaCl. S48E at 150 mM NaCl (red) shows dramatically disrupted assembly with K D ˜ 2,000 μM. G, H The concentration‐dependent chemical shift deviations of 1 H‐ 15 N HSQC are large for wild‐type and small for S48E TDP‐43 NTD, consistent with disrupted binding. The CSDs are measured for 200 μM (cyan), 100 μM (green), 40 μM (yellow), and 20 μM (orange) WT compared to a monomeric control: 5 μM. For S48E, only 200 and 100 μM are shown. I The chemical shift deviations (at 100 μM with a cutoff of 0.02 ppm, shown in green) map to two different sides of TDP‐43 NTD (PDB 2N4P), supporting a view that TDP‐43 can assemble into linear chains via multiple interfaces. S48 is highlighted with red spheres.

Article Snippet: Immunoblotting with custom and commercial α‐TDP‐43 antibodies (CST 3449) and detection with the LI‐COR imaging system were performed as described above.

Techniques: Phospho-proteomics, Western Blot, SDS Page, Migration, Immunoprecipitation, Filtration, Variant Assay, Composite Gradient Multi-angle Light Scattering, Derivative Assay, Concentration Assay, Comparison, Binding Assay, Control