Journal: Neuron

Article Title: Cytoplasmic TDP-43 de-mixing independent of stress granules drives inhibition of nuclear import, loss of nuclear TDP-43, and cell death

doi: 10.1016/j.neuron.2019.02.038

Figure Lengend Snippet: (A) Schematic of experimental design to assess the properties of de-mixed cytoplasmic TDP-43 particles in human iPSC-derived motor neurons with or without sodium arsenite treatment. Human iPSC-derived motor neuron precursor cells were differentiated for 7 days and then the differentiated motor neurons were infected with a lentivirus driving expression of Ubi::TDP-43∆NLS-GFP. After 1–2 weeks of expression the cells were analyzed. (B-C) Immunostaining of G3BP1 of mature motor neurons expressing cytoplasmic TDP-43 in absence of sodium arsenite treatment (B) or with 50 µM sodium arsenite (C). MAP2 was stained for neuron marker. (D-E) Representative images of cytoplasmic TDP-43∆NLS-GFP particles formed in absence of stress. FRAP example of TDP-43∆NLS-GFP particles in a neuron after a complete bleaching. (E) Mean fluorescence intensity of the fully bleached TDP-43∆NLS-GFP particles over time. Data are normalized to the average intensity of a particle before photobleaching and are represented as mean ± SEM from the recovery curves of 8 particles in a total of four independent experiments. (F-G) Representative images of cytoplasmic TDP-43∆NLS-GFP particles after 3 hours of 50 µM sodium arsenite treatment. FRAP example of TDP-43∆NLS-GFP particles in a motor neuron after a complete bleaching. (G) Mean fluorescence intensity of the fully bleached TDP-43∆NLS-GFP particles over time. Data are normalized to the average intensity of a particle before photobleaching and are represented as mean ± SEM from the recovery curves of nine particles in a total of four independent experiments.

Article Snippet: Cells Lines iPSC-derived motor neurons Human iPSC cells, derived from peripheral blood mononuclear cells donated by a 58-year-old healthy Caucasian male, were generated by iXCells Biotechnologies and selected for normal karyotype with normal self-renewal and differentiation ability ( Melamed et al., 2019 ). iPS cells were first differentiated into motor neuron precursors in 21 days and then further differentiated into motor neurons for another 7 days using a differentiation protocol patented by iXCells Biotechnologies (Provisional Application no. 14359–001-888) ( Melamed et al., 2019 ).

Techniques: Derivative Assay, Infection, Expressing, Immunostaining, Staining, Marker, Fluorescence

Journal: Neuron

Article Title: Cytoplasmic TDP-43 de-mixing independent of stress granules drives inhibition of nuclear import, loss of nuclear TDP-43, and cell death

doi: 10.1016/j.neuron.2019.02.038

Figure Lengend Snippet: (A) Schematic of experimental design to assess the properties of de-mixed cytoplasmic TDP-43 particles in human iPSC-derived motor neurons with or without sodium arsenite treatment. Human iPSC-derived motor neuron precursor cells were differentiated for 7 days and then the differentiated motor neurons were infected with a lentivirus driving expression of Ubi::TDP-43∆NLS-GFP. After 1–2 weeks of expression the cells were analyzed. (B-C) Immunostaining of G3BP1 of mature motor neurons expressing cytoplasmic TDP-43 in absence of sodium arsenite treatment (B) or with 50 µM sodium arsenite (C). MAP2 was stained for neuron marker. (D-E) Representative images of cytoplasmic TDP-43∆NLS-GFP particles formed in absence of stress. FRAP example of TDP-43∆NLS-GFP particles in a neuron after a complete bleaching. (E) Mean fluorescence intensity of the fully bleached TDP-43∆NLS-GFP particles over time. Data are normalized to the average intensity of a particle before photobleaching and are represented as mean ± SEM from the recovery curves of 8 particles in a total of four independent experiments. (F-G) Representative images of cytoplasmic TDP-43∆NLS-GFP particles after 3 hours of 50 µM sodium arsenite treatment. FRAP example of TDP-43∆NLS-GFP particles in a motor neuron after a complete bleaching. (G) Mean fluorescence intensity of the fully bleached TDP-43∆NLS-GFP particles over time. Data are normalized to the average intensity of a particle before photobleaching and are represented as mean ± SEM from the recovery curves of nine particles in a total of four independent experiments.

Article Snippet: We thank iXCells Biotechnologies for providing human iPSC-derived motor neuron precursor cells.

Techniques: Derivative Assay, Infection, Expressing, Immunostaining, Staining, Marker, Fluorescence