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: Promoting axon regeneration by enhancing the non-coding function of the injury-responsive coding gene Gpr151

doi: 10.1101/2021.02.19.431965

Figure Lengend Snippet: (a) Schematic structure of Gpr151 gene in wild type control (black) or targeted knockout (KO) mice (blue). Gpr151 gene was disrupted by the insertion of LacZ -cassette in the CDS region. (b) Schematic diagram of timeline illustrating the pre-conditioning injury and sample preparation. Sciatic nerves of wild type control or KO mice were injured by crushing the nerves with forceps and dissected at 3 days after injury. The sciatic nerves were prepared as cryosections and subjected to immunohistochemistry with anti-SCG10 and βIII tubulin antibodies. L4,5 DRGs were subjected to RT-qPCR analysis (ÄÄCt) or adult DRG neuron cultures. (c) RT- qPCR analysis of mouse L4,5 DRGs. Average of normalized levels of wild type control and KO mRNA levels at 3 days after introducing sciatic nerve crush injury. The averages of relative Gpr151 mRNA levels from uninjured KO, injured control and KO samples was compared to the average level from control uninjured samples (set to 1). (n=6 for each condition; *** p <0.001, ANOVA followed by Tukey test). (d) Representative longitudinal sections of immunohistochemistry of sciatic nerves from in vivo axon regeneration assay. The longitudinal cryosections were immunostained with anti-SCG10 (white; top and yellow converted; bottom), a marker protein specifically labelling regenerating axon. βIII tubulin antibody was used for counterstaining to visualize the nerve sections (blue; bottom). The red dotted arrow line indicates the crush site. Scale bar, 500 μm. (e) Average of normalized SCG10 intensity from immunostained sections in (d). Immunostained SCG10 fluorescence intensity was acquired by measurement windows with 100 pixel-width (equivalent to 87.7 μm) from crush sites to distal parts of nerves sections at an every 100-pixel distance from ImageJ . The acquired intensity from every measurement window was normalized to the intensity of the crush site (n=7 for control mice and n=5 for KO mice; * p <0.05 by t -test; mean ± SEM). (f) Regeneration index calculated from (e). Regeneration index was defined as a distance of an indicated SCG10 intensity. Average distance of normalized SCG10 intensity 0.5 or 0.2 was calculated from (e) and presented as regeneration index (mm). ( # p = 0.59, ## p = 0.05 by t -test). (g) Mouse L4,5 DRG tissues were dissected at 3 days with (+SNI) or without (-SNI) sciatic nerve crush injury (SNI). DRG neurons were plated and cultured to monitor pre-conditioning effect-induced neurite outgrowth. Scale bar, 100 μm. (h) Average of the longest axon length from (g) (three independent biological replicates; total 6 mice; total 304, 313, 291, 283 cells for each condition, -SNI Control, -SNI KO, +SNI Control, +SNI KO; *** p <0.001 by ANOVA followed by Tukey tests). (i) Cumulative frequency of the longest axon length from (g). (j) Percentage of neurons in three categories of the longest axon length.

Article Snippet: Anti-HA antibody (Abcam, ab9110; RRID: AB_307019), anti-GPR151 antibody (Aviva Systems Biology, OAAF06441), anti-α tubulin antibody (Santa Cruz Biotechnology, sc-53030; RRID: AB_2272440), anti-c-Jun antibody (Cell Signaling Technology, 9165; RRID: AB_2130165), anti-SCG10 antibody (Novus Biologicals, NBP1-49461; RRID: AB_10011569), anti-βIII tubulin antibody (TUJ1, Abcam, ab41489, RRID: AB_727049), anti-GAPDH antibody (Santa Cruz Biotechnology, sc-32233; RRID: AB_627679), anti-FLAG antibody (Cell Signaling Technology, 14793; RRID: AB_2572291).

Techniques: Control, Knock-Out, Sample Prep, Immunohistochemistry, Quantitative RT-PCR, In Vivo, Marker, Fluorescence, Cell Culture

Journal: bioRxiv

Article Title: Promoting axon regeneration by enhancing the non-coding function of the injury-responsive coding gene Gpr151

doi: 10.1101/2021.02.19.431965

Figure Lengend Snippet: (a) Schematic diagram of AAV-control and AAV-5’UTRm adeno-associated virus vector and experimental timeline of in vivo axon regeneration assay in sciatic nerves (wk, weeks). U6, U6 promoter; Scrambled shRNA sequence 48-mer; T, transcription termination sequence; CMV, human cytomegalovirus immediate early enhancer/promoter; GFP, enhanced green fluorescence protein CDS. (b) Representative image of adult DRG neurons. L4,5 DRGs were dissected from AAV-injected mice and cultured for 12 hours and immunostained with βIII tubulin antibody. GFP fluorescence signal indicated AAV-mediated in vivo gene delivery and expression of target genes. Scale bar, 100 μm. (c) In vivo axon regeneration assay from crushed sciatic nerves. Representative longitudinal sections of sciatic nerves from AAV-control or AAV-5’UTRm- injected mice. Red dotted arrows indicate the injury site. Scale bar, 500μm. (d) Normalized SCG10 intensity measured from SCG10 immunostained sections in (c). Measurement window (100-pixel width, equivalent to 87.7 μm) was placed at the crush site and intensities were acquired at every 100-pixel to the distal part of the sections. (e) Regeneration index calculated from (d). (n=8 for AAV-control, 10 for AAV-5’UTRm; ** p <0.01, **** p <0.0001 by t -test; mean±SEM). (f) Experimental timeline of in vivo axon regeneration assay in optic nerves (wk, weeks). (g) Representative longitudinal sections of optic nerves from AAV-control or AAV-5’UTRm-injected mice. Regenerating axons were labeled by Alexa594-conjugated CTB injection. Red dotted arrows indicate the injury site. Red bars indicated the locations analyzed the estimation of regenerating axon numbers in (h). Scale bar, 200μm. (h) Estimated numbers of regenerating axons (n=5 for AAV-control, 6 for AAV-5’UTRm; * p <0.05, ** p <0.01, *** p <0.001 by one-way AVONA with Bonferroni test; mean±SEM). (i) Relative expression level of Gpr151 and Atf3 after injury in mouse retina tissues (injured / uninjured). RT-qPCR analysis of Gpr151 and Atf3 from mouse retina tissues dissected at 3 days with optic nerve crush injury (injured, purple) or without injury (uninjured, black) (n=3 for each condition; *** p <0.001, ns, not significant by t-test; mean±SEM).

Article Snippet: Anti-HA antibody (Abcam, ab9110; RRID: AB_307019), anti-GPR151 antibody (Aviva Systems Biology, OAAF06441), anti-α tubulin antibody (Santa Cruz Biotechnology, sc-53030; RRID: AB_2272440), anti-c-Jun antibody (Cell Signaling Technology, 9165; RRID: AB_2130165), anti-SCG10 antibody (Novus Biologicals, NBP1-49461; RRID: AB_10011569), anti-βIII tubulin antibody (TUJ1, Abcam, ab41489, RRID: AB_727049), anti-GAPDH antibody (Santa Cruz Biotechnology, sc-32233; RRID: AB_627679), anti-FLAG antibody (Cell Signaling Technology, 14793; RRID: AB_2572291).

Techniques: Control, Virus, Plasmid Preparation, In Vivo, shRNA, Sequencing, Fluorescence, Injection, Cell Culture, Expressing, Labeling, Quantitative RT-PCR

Journal: eLife

Article Title: Translatome analysis reveals cellular network in DLK-dependent hippocampal glutamatergic neuron degeneration

doi: 10.7554/eLife.101173

Figure Lengend Snippet:

Article Snippet: STMN2 antibodies were a mouse monoclonal anti-STMN2 (R&D Systems, MAB6930) and a rabbit polyclonal anti-STMN2 (Proteintech, 10586–1-AP).

Techniques: Knock-Out, Over Expression, Sequencing, RNAscope, TUNEL Assay, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Multiplex Assay, Software

Journal: Scientific Reports

Article Title: Enhanced axonal regeneration of ALS patient iPSC-derived motor neurons harboring SOD1 A4V mutation

doi: 10.1038/s41598-023-31720-7

Figure Lengend Snippet: Antibodies/Reagent used for immunofluorescence staining.

Article Snippet: Stathmin-2 (STMN2) , Early regeneration marker , Novus , 1:2000.

Techniques: Immunofluorescence, Staining, Marker

Journal: Scientific Reports

Article Title: Enhanced axonal regeneration of ALS patient iPSC-derived motor neurons harboring SOD1 A4V mutation

doi: 10.1038/s41598-023-31720-7

Figure Lengend Snippet: SOD1 +/+ and SOD1 +/A4V axons regenerate similarly 24 h following axotomy. ( A ) Mean axon length 24 h post-axotomy (SOD1 +/+ n = 13; SOD1 +/A4V n = 12). Each data point represents 50–100 traced axons from 1 individual axonal compartment. ( B ) Frequency distribution of axon length 24 h post-axotomy. ( C ) SOD1 +/+ (left) and SOD1 +/A4V (right) regenerating axons 24 h post axotomy stained for GAP-43. Scale bar = 50 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upmu$$\end{document} μ m. ( D-E ) Growth cones 24 h post axotomy stained for smi-31 (green), F-actin (white), stathmin-2 (red). Scale bar = 2 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upmu$$\end{document} μ m. ( D ) SOD1 +/+ (top panels) ( E ) SOD1 +/A4V (bottom panels). ( F ) Average growth cone area 24 h post-axotomy. (G) Average STMN2 fluorescence intensity in growth cone area 24 h post-axotomy. Bars represent mean ± SEM. ns indicates p > 0.05.

Article Snippet: Stathmin-2 (STMN2) , Early regeneration marker , Novus , 1:2000.

Techniques: Staining, Fluorescence

Journal: Cell reports

Article Title: Efficient generation of lower induced motor neurons by coupling Ngn2 expression with developmental cues

doi: 10.1016/j.celrep.2022.111896

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-SCG10/STMN2 , Novus Biologicals , NBP49461.

Techniques: Virus, Recombinant, SYBR Green Assay, Software