Journal: Neuron

Article Title: RNA binding antagonizes neurotoxic phase transitions of TDP-43.

doi: 10.1016/j.neuron.2019.01.048

Figure Lengend Snippet: (A) Purified TDP43-MBP WT or 5FL (5μM) were incubated for 2 hours (10% dextran buffer, 150 mM NaCl) in the absence or presence of 1,6 hexanediol (10% w/v). Scale bar = 50 μm. (B) Representative DIC images of purified TDP43-MBP WT or 5FL following incubation with increasing concentrations of yeast total RNA. Scale bar = 50 μm. (C) Representative electron micrographs of TDP-43 WT and 5FL following TEV cleavage in the absence (left) or presence (right) of yeast total RNA (15 μg). Scale bars = 4 μm. (D) Turbidity changes (normalized OD395 readings) of TDP-43 WT (blue) and 5FL (red) following TEV cleavage in the absence (open circles) or presence (solid circles) of yeast total RNA (15 μg). (E) Turbidity changes (normalized OD395 readings) of TDP-43 WT proteins following TEV cleavage in the presence of yeast total RNA-only (25 μg) (green) or yeast total RNA (25μg) followed by RNase A addition (2.5μg) at 90 min post-TEV cleavage (red prior to/purple following RNase A). (F) Construct designs for non-optogenetic TDP-43 vectors containing functional (WT) or RNA-binding deficient (5FL) RRMs. TDP-43cyto constructs contain point mutations in the nuclear localization sequence to mimic cytoplasmic mislocalization. (G-H) Immunofluorescence analysis of cells expressing EGFP-TDP-43 constructs (G) or EGFP-TDP43cyto constructs (H) with/without functional RRMs (WT/5FL) for hyperphosphorylated TDP-43 (red) and p62 (purple). Scale bars = 10 μm. (I-J) FRAP analysis of EGFP-TDP43 5FL (G) and EGFP-TDP43cyto 5FL (H) inclusions, n = 14-23 (I) and 20-24 cells (J). Data shown are mean +/− S.E.M. Scale bars = 10 μm.

Article Snippet: All wild-type optoTDP43 vectors were constructed using the same TDP-43 insert. optoTDP43 constructs containing the TDP43cyto, 5FL and/or M337V point mutations were generated from mutant TDP43 plasmids (Plasmids 84912, 84914, 98674, Addgene).

Techniques: Purification, Incubation, Construct, Functional Assay, RNA Binding Assay, Sequencing, Immunofluorescence, Expressing

Journal: Neuron

Article Title: RNA binding antagonizes neurotoxic phase transitions of TDP-43.

doi: 10.1016/j.neuron.2019.01.048

Figure Lengend Snippet: (A) Cytoplasmic EGFP-TDP43 constructs were expressed with functional (WT, top) or RNA-binding deficient (5FL, bottom) RRMs in HEK293 cells prior to heat shock or sodium arsenite treatment and immunostaining for G3BP1 (red) and eIF4G (not shown). (B-C) Percentage of EGFP-TDP43 granules that co-localized with G3BP1/eIF4G were calculated (SG+/−). n = 99-316 granules. (D) RNA FISH targeting Poly-A tails was performed to assess mRNA presence in SG+/− TDP-43 granules following sodium arsenite treatment. Scale bar = 10 μm. (E) Mean granule area for SG+ and SG− TDP-43 granules following heat shock and sodium arsenite treatment was determined by immunofluorescence, n = 24-68 granules per condition. (F-G) HEK293 cells were co-transfected with cytoplasmic EGFP-TDP43cyto and G3BP1-mCh and exposed to sodium arsenite treatment to induce stress granule formation. FRAP analysis of TDP-43 granules that co-localized (SG+) or did not co-localize (SG−) with G3BP-mCh was performed to assess material state of SG+/− granules, n = 17-27 cells. (H) Immunohistochemical analysis of ALS/FTLD spinal cord tissue was performed to determine whether patient TDP-43 inclusions contain SG proteins. Arrows or insets indicate a lack of co-localization between TDP-43 inclusions (red) and the SG components G3BP1 (top) and ATXN2 (bottom) (green). Scale bar = 20 μm. Data shown are mean +/− S.E.M. ****, p < 0.0001.

Article Snippet: All wild-type optoTDP43 vectors were constructed using the same TDP-43 insert. optoTDP43 constructs containing the TDP43cyto, 5FL and/or M337V point mutations were generated from mutant TDP43 plasmids (Plasmids 84912, 84914, 98674, Addgene).

Techniques: Construct, Functional Assay, RNA Binding Assay, Immunostaining, Immunofluorescence, Transfection, Immunohistochemical staining