Journal: Acta Neuropathologica

Article Title: Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling

doi: 10.1007/s00401-016-1564-y

Figure Lengend Snippet: Analysis of intermediate filament (IF) levels in axons of pmn mutant mouse motoneurons. a Electron micrograph of cross sections of distal phrenic nerve from 34-day-old mice showed increased intermediate filaments in pmn axons compared to wild-type axons, scale bar 500 nm ( top ). Bottom lane shows the higher magnification of the respective image in the top lane ( scale bar 200 nm). Arrows point to IF. b Electron micrograph showing axonal compartments of motoneurons cultured for 7 days. Scale bar 500 nm. c Quantification of number of IF in pmn axons showed an increase in proximal ( P value = 0.0304), intermediate ( P value = 0.0249) and distal ( P value = 0.0336) compartments as compared to wild-type axons (Mann–Whitney one tailed test, n = 16 wild-type motoneurons and 19 pmn motoneurons from 3 independent experiments). d Western blot analysis of sciatic nerve lysate from 34-day-old mice. e , f Pmn mutant mouse nerves show increased levels of e NFL ( t = 3.210, P = 0.0326) and f NFH ( t = 2.781, P = 0.0498) proteins as compared to the wild type. Bars represent mean ± SEM, ( n = 5 wild-type and pmn and n = 3 Nefl +/−; pmn mice analyzed, * P < 0.05; one sample t test). g Expression levels of Nefl mRNA in sciatic nerve extracts of 28- to 30-day-old pmn is not changed compared to wild type. Quantification was performed by normalizing with HPRT1 as housekeeping gene. Bars represent mean ± SEM ( n = 3)

Article Snippet: Motoneurons were cultured for 3 days in vitro and stained with antibodies against NFL (red, DA2 clone, EnCor Biotechnology) and stathmin (green, rabbit monoclonal, Abcam).

Techniques: Mutagenesis, Cell Culture, MANN-WHITNEY, One-tailed Test, Western Blot, Expressing

Journal: Acta Neuropathologica

Article Title: Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling

doi: 10.1007/s00401-016-1564-y

Figure Lengend Snippet: NFL depletion increases microtubule (MT) density and axon length in pmn mutant motoneurons. a Electron micrographs of distal phrenic nerve cross-sections from 34-day-old mice ( top ), scale bar 500 nm. Bottom lane shows the higher magnification of the respective image in the top lane , scale bar 200 nm. Arrows point to MTs. b Number of MTs per axon in pmn ( P < 0.05; t = 3.640) decreased, whereas Nefl − / −; pmn mice ( P > 0.05; t = 2.701) showed MT number comparable to wild-type mice. c Density of MTs per axon in Nefl − / − ( P < 0.001; t = 10.05) and Nefl − / −; pmn ( P < 0.001; t = 9.649) mice increased as compared to wild-type axons. d Cross-sectional area of Nefl − / − ( P < 0.05; t = 3.416) and Nefl − / −; pmn ( P < 0.01; t = 4.488) reduced as compared to wild-type and pmn axons ( n = 3 pmn and Nefl − / − mice and n = 4 wild-type and Nefl − / −; pmn mice analyzed). e Representative images of motoneurons cultured for 7 days in vitro. Scale bar 100 µm. f Pmn motoneurons showed reduced axon length ( P < 0.01; t = 6.845). Nefl − / −; pmn motoneurons grew significantly longer than pmn motoneurons ( P < 0.01; t = 5.830) ( n = 3 independent experiments). Numbers in the bars represents total number of motoneurons analyzed. Bars represent mean ± SEM (one-way ANOVA and Bonferroni’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). g Bar graph showing no change in percentage survival of motoneurons after 7 days in vitro culture. h Electron micrographs showing longitudinal section of axons of wild type and Nefl − / − motoneurons cultured for 7 days in vitro. i Bar graph showing quantitative analysis of axonal diameter of cultured embryonic motoneurons from wild-type ( n = 7) and Nefl − / − ( n = 9) mice, showing no change in width after 7 days in vitro. 2–3 sections of each proximal, intermediate and distal axon were analyzed for each motoneuron and the average of all sections from the three compartments is shown in the figure

Article Snippet: Motoneurons were cultured for 3 days in vitro and stained with antibodies against NFL (red, DA2 clone, EnCor Biotechnology) and stathmin (green, rabbit monoclonal, Abcam).

Techniques: Mutagenesis, Cell Culture, In Vitro

Journal: Acta Neuropathologica

Article Title: Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling

doi: 10.1007/s00401-016-1564-y

Figure Lengend Snippet: Nefl deletion increases Stat3–stathmin interaction. a Stathmin was immunoprecipitated from sciatic nerve extracts of 34-day-old mice. Western blot shows from left to right, input (Ip) and eluate (E) from wild-type and Nefl − / − mice nerve extracts after anti-IgG control and anti-stathmin pulldown. b Quantification of band intensities in the eluate showed an increased interaction of Stat3 and stathmin in Nefl − / − mice ( t = 17.08; P = 0.0034). Bars represent mean ± SEM (one sample t test, n = 3 independent experiments). c Nefl deletion increases Stat3–stathmin interaction in sciatic nerves from pmn mutant mice. d Western blot analyses show similar levels of stathmin in the sciatic nerve extracts of 34-day-old wild-type, pmn and Nefl − / − mice. Histone levels were determined to ensure equal loading of proteins. e Quantification of Stat3 per stathmin levels in the sciatic nerve extracts (input before immunoprecipitation) from 34-day-old wild-type and Nefl − / − mice ( n = 3 independent experiments). f Stathmin was immunoprecipitated from NSC-34 cell extracts. Western blot shows from left to right input for IgG, mock, and NFL overexpressing NSC-34 cells. Right blot shows eluate after IgG control pulldown and stathmin pulldown from NSC-34 cells after transfection with mock (only lipofectamine) or, NFL overexpression vector, respectively. g Western blot analysis reveals increased levels of phosphorylated Stat3 (at Y705) in the sciatic nerve extracts of 34-day-old pmn and Nefl − / − in comparison to wild-type mice. Quantification of pStat3 normalized by total Stat3 levels in h pmn mouse compared to wild type ( t = 4.928; P = 0.0044; n = 6 independent experiments) and i in Nefl − / − mouse compared to the wild-type mouse ( t = 2.865; P = 0.0242; n = 8 independent experiments). Bars represent mean ± SEM

Article Snippet: Motoneurons were cultured for 3 days in vitro and stained with antibodies against NFL (red, DA2 clone, EnCor Biotechnology) and stathmin (green, rabbit monoclonal, Abcam).

Techniques: Immunoprecipitation, Western Blot, Control, Mutagenesis, Transfection, Over Expression, Plasmid Preparation, Comparison

Journal: Acta Neuropathologica

Article Title: Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling

doi: 10.1007/s00401-016-1564-y

Figure Lengend Snippet: Levels of tyrosinated and acetylated tubulin in NFL-depleted motoneurons a Representative images of motoneurons showing increased acetylated microtubules in Nefl − / − motoneurons after 3 days in vitro culture. Neurons were stained with antibodies against tyrosinated α-tubulin ( green ), stathmin ( blue ) and acetylated α-tubulin ( red ). Scale bar 20 µm ( first and third lane ). White square boxes indicate the regions enlarged in the second and fourth lane , scale bar 2 µm. b Quantification of fluorescence intensities showed increased acetylated microtubules in Nefl − / − ( t = 3.011) as compared to wild-type motoneurons, whereas tyrosinated microtubules remained unchanged. Intensity of stathmin is used as control. Bars represent mean ± SEM (one-way ANOVA, n = 10 motoneurons from three independent experiments, * P < 0.05, *** P < 0.001)

Article Snippet: Motoneurons were cultured for 3 days in vitro and stained with antibodies against NFL (red, DA2 clone, EnCor Biotechnology) and stathmin (green, rabbit monoclonal, Abcam).

Techniques: In Vitro, Staining, Fluorescence, Control