Journal: Neurobiology of disease

Article Title: Protective effects of NAMPT or MAPK inhibitors and NaR on Wallerian degeneration of mammalian axons

doi: 10.1016/j.nbd.2022.105808

Figure Lengend Snippet: Western blot and Degeneration Index analysis reveal protective effects of DLK inhibition in transected axons. A-B. Semi-quantitative Western blot analysis of distal axons at 4 and 8 h after transection and treatment with the DLK inhibitor GNE3511 or vehicle (DMSO). A shows representative immunoblot and B quantification of signals normalized to actin and with reference to uninjured/untreated axon samples. GNE3511 suppresses phosphorylation of JNK at 4 h, t 5 = 6.17, p = 0.0005. NMNAT2 is significantly degraded between 4 and 8 h post-axotomy (t 5 = 3.5, p = 0.016) in vehicle treated axons but not in the presence of GNE (t 5 = 2.8, p = 0.046). Similarly, GNE suppresses the injury associated degradation of SCG10 at both time points (t 5 = 3.06, p = 0.031; and t 5 = 4.02, p = 0.008 respectively), and the injury associated degradation of spectrin at 8 h (calculated as the index of the p150 fragment to total spectrin, p284; t 5 = 3.08, p = 0.03). Signals were analyzed by 2-way ANOVAs with Holm-šídák’s multiple comparisons. C. Axons were treated with GNE 2 h before or at the time of transection, or 2 and 4 h later, or with vehicle (DMSO) at the time of injury. Statistical analysis of DI was performed by two-way ANOVA for the effect of time (F 7,35 = 164.7, p < 0.0001), treatment (F 4,5 = 47.78, p = 0.0004) and their interactions (F 28,35 = 16.22, p < 0.0001) with Holm-šídák’s multiple comparisons. GNE treatment at any time point offers protection against fragmentation early after axotomy (12 h ) compared to vehicle treated axons (t 40 = 5.7–8.8, p < 0.0001). However, pre-treatment or treatment at the time of injury shows significantly less degeneration compared to delayed treatment at 24 h (t 40 = 2.71–5.8, p < 0.03–0.0001). D. Representative photomicrographs from C. Scale bar, 50 μm. Error bars indicate ±1 SEM.

Article Snippet: Membranes were air dried, rehydrated and then blocked with 50% Odyssey blocking solution-tris buffered saline (Li-Cor, Germany) and incubated overnight at 4 °C with the target antibodies: mouse alpha-spectrin (Millipore Cat# MAB1622, RRID:AB_11214057) mouse actin (Santa Cruz Biotechnology Cat# sc-47,778 HRP, RRID:AB_2714189), rabbit SCG10 (Proteintech Cat# 10586–1-AP, RRID:AB_2197283), rabbit p Thr183/Tyr185 -JNK (Cell Signaling Technology Cat# 9251, RRID:AB_33165).

Techniques: Western Blot, Inhibition, Phospho-proteomics

Journal: bioRxiv

Article Title: STMN2 protein depletion via translation deficits and stress granules and its compensation in ALS

doi: 10.64898/2026.01.25.701321

Figure Lengend Snippet: a Stress granule assembly and translational repression under stress and their modulation by small molecules. Schematic was prepared using BioRender. b,c eIF4A inhibition with small molecules leads to rapid and dramatic STMN2 depletion. Representative western blot (b) and quantification (c) are shown. Cells were treated with the indicated compounds for 1 hour. N=3; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with Dunnett’s post-hoc test. d eIF4A inhibitors and phospho-eF2α dependent stressors induce stress granule assembly. Representative images are shown. Scale bar, 10 μm. e STMN2 mRNA segregates into distinct foci in response to stress. Single molecule RNA-FISH with a custom probe was used. Arrowheads point to the foci with STMN2 mRNA enrichment. Scale bar, 25 μm. f STMN2 mRNA is significantly enriched in stress granules. Quantification and representative images are shown. Arrowheads point to G3BP1-positive foci – stress granules (SGs). Stress granules from 114 cells were included in the analysis. **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with Dunnett’s post-hoc test. Scale bar, 10 μm. g STMN2 translation occurs outside stress granules. Newly synthesised STMN2 protein molecules were visualised by puro-PLA and SGs – by G3BP1 immunocytochemistry. 570 stress granules were included in the analysis. Scale bar 10 µm. h ISRIB and lipoamide interfere with stress granule assembly. NaAsO 2 was used. Representative images and automated quantification data are shown. N=3 (500-1000 cells analysed in each experiment). ***/ ### p<0.001, ****/ #### p<0.0001, two-way ANOVA. Scale bar 10 µm. i ISRIB and lipoamide attenuate STMN2 depletion during the recovery from stress. Cells were pre-treated with either ISRIB or lipoamide, which was replenished before the recovery from arsenite (continuous presence of the compound in the media). Representative western blots and quantification are shown. N=3; *p<0.05, Mann-Whitney U tests. SH-SY5Y cells were used.

Article Snippet: Immunostaining was performed as described earlier ( ) using commercially available antibodies (all at 1:1000 dilution): STMN2 (rabbit polyclonal, Proteintech, 10586-1-AP or mouse monoclonal, Proteintech, 67201-1-Ig); TDP-43 (rabbit polyclonal, C-terminal, Sigma or mouse monoclonal, R&D Systems, MAB7778); G3BP1 (rabbit polyclonal, Proteintech, 13057-2-AP); GM130 (rabbit polyclonal, Proteintech 11308-1-AP); Tuj/betaIIItubulin (rabbit recombinant monoclonal Alexa488-labelled, Abcam, AB237350).

Techniques: Inhibition, Western Blot, Immunocytochemistry, MANN-WHITNEY