Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Targeting STMN2 for neuroprotection and neuromuscular recovery in Spinal Muscular Atrophy: evidence from in vitro and in vivo SMA models

doi: 10.1007/s00018-024-05550-3

Figure Lengend Snippet: Human spinal SMA iPSC-derived MNs showed STMN2 dysregulation. A Experimental strategy used to assess the cellular effect of STMN2 modulation by JNK-inhibitor SP600125 (15 µM) or by lentivirus delivering STMN2 cDNA (MOI 5) in SMA MNs differentiated from iPSCs via embryoid bodies (EBs). Created with BioRender.com. B Representative immunofluorescence images of Ctrl and SMA d18 MNs stained for STMN2 (red), β-tubulin (green), and DAPI (blue) and acquired with a confocal microscope at 40X magnification. Scale bar = 50 μm. C Quantification of STMN2 level intensity ( n = 3 cell lines/group) in B reporting separately soma, axon, and the ratio axon/soma data. STMN2 levels were normalized to SMA average values. Values show means ± SEM from 3 independent experiments. Statistical significance was determined by Student’s t -test. **** P < 0.0001. All inserts are 4X magnification

Article Snippet: In particular, cells were treated for 72 hours (h) after seeding at day 15 with 15 μM SP600125 (Cell Signaling, #8177), an anthrapyrazolone ATP-competitive inhibitor of JNK, or transduced with a lentivirus- STMN2 (MOI 5; Origene RC202914L4V, accession number NM_007029) or with a null vector as the negative control.

Techniques: Derivative Assay, Immunofluorescence, Staining, Microscopy

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Targeting STMN2 for neuroprotection and neuromuscular recovery in Spinal Muscular Atrophy: evidence from in vitro and in vivo SMA models

doi: 10.1007/s00018-024-05550-3

Figure Lengend Snippet: JNK-inhibitor SP600125 protected SMA MNs from degeneration and ameliorated axon length and outgrowth defects in vitro. A Representative immunofluorescence images of untreated (SMA) and SP-treated (SMA + SP) d18 MNs, labeled for STMN2 (red), β-tubulin (green) and DAPI (blue) and acquired with a confocal microscope at 40X magnification. Scale bar = 50 μm. B Intensity quantification of STMN2 in A ( n = 3 cell lines/group) analyzing separately soma, axon, and the ratio axon/soma. STMN2 levels were normalized to untreated SMA average values. Values show means ± SEM from 3 independent experiments. Statistical significance was determined by Student’s t -test. C Representative immunofluorescence images of Ctrl, SMA, and SMA + SP d18 MNs stained for TUNEL (green) and DAPI (blue) and acquired with a confocal microscope at 40X magnification. Scale bar = 50 μm. D Representative immunofluorescence images of Ctrl, SMA and SMA + SP d18 MNs stained for β-tubulin (black) and acquired with a confocal microscope at 63X magnification. Scale bar = 50 μm. E Quantification of TUNEL-positive cells ( n = 3 cell lines/group) in C , expressed as percentage of apoptotic cells on the total amount of nuclei. Values were normalized to untreated SMA average. Values show means ± SEM from 3 independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc test. F , G Measurement of axon length ( F ) of D , expressed as normalized values to untreated SMA average, and dendritic complexity performed with Sholl analysis ( G ) of D , expressed as number of intersections at different distances from soma ( n = 3 cell lines/Ctrl; n = 4 cell lines/SMA and /SMA + SP). Values show means ± SEM from 3 independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc test. ** P < 0.01, *** P < 0.001, **** P < 0.0001. All inserts are 4X magnification

Article Snippet: In particular, cells were treated for 72 hours (h) after seeding at day 15 with 15 μM SP600125 (Cell Signaling, #8177), an anthrapyrazolone ATP-competitive inhibitor of JNK, or transduced with a lentivirus- STMN2 (MOI 5; Origene RC202914L4V, accession number NM_007029) or with a null vector as the negative control.

Techniques: In Vitro, Immunofluorescence, Labeling, Microscopy, Staining, TUNEL Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Targeting STMN2 for neuroprotection and neuromuscular recovery in Spinal Muscular Atrophy: evidence from in vitro and in vivo SMA models

doi: 10.1007/s00018-024-05550-3

Figure Lengend Snippet: STMN2 modulation by lentivirus specifically restored axon length and outgrowth defects in SMA MNs. A Representative immunofluorescence images of null vector (indicated as SMA) and STMN2 -LV (indicated as SMA + STMN2 -LV) treated d21 MNs, labeled with STMN2 (red), β-tubulin (green) and DAPI (blue) and acquired with a confocal microscope at 40X magnification. Scale bar = 50 μm. B Intensity quantification of STMN2 ( n = 3 cell lines/group) in A analyzing separately soma, axon, and the ratio axon/soma. STMN2 levels were normalized to SMA MNs treated with the null vector. Values show means ± SEM from 3 independent experiments. Statistical significance was determined by Student’s t -test. C Representative immunofluorescence images of Ctrl, SMA and SMA + STMN2 -LV d21 MNs ( n = 3 cell lines/group) stained for TUNEL (green) and DAPI (blue) acquired with a confocal microscope at 40X magnification. Scale bar = 50 μm. D Representative immunofluorescence images of Ctrl, SMA and SMA + STMN2 -LV d21 MNs stained for β-tubulin (black) and acquired with a confocal microscope at 63X magnification. Scale bar = 50 μm. E Quantification of TUNEL-positive cells ( n = 3 cell lines/group) in C expressed as percentage of apoptotic cells on the total amount of nuclei. Values were normalized to the average of SMA treated with the empty vector. Values show means ± SEM from 3 independent experiments. F , G Measurement of axon length ( F ) of D , expressed as normalized on average of SMA treated with the null vector values, and dendritic complexity performed with Sholl analysis ( G ) of D , expressed as number of intersections at different distances from soma ( n = 3 cell lines/Ctrl; n = 4 cell lines/SMA and /SMA + STMN2 -LV). Values show means ± SEM from 3 independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc test. *** P < 0.001, **** P < 0.0001. All inserts are 4X magnification

Article Snippet: In particular, cells were treated for 72 hours (h) after seeding at day 15 with 15 μM SP600125 (Cell Signaling, #8177), an anthrapyrazolone ATP-competitive inhibitor of JNK, or transduced with a lentivirus- STMN2 (MOI 5; Origene RC202914L4V, accession number NM_007029) or with a null vector as the negative control.

Techniques: Immunofluorescence, Plasmid Preparation, Labeling, Microscopy, Staining, TUNEL Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Targeting STMN2 for neuroprotection and neuromuscular recovery in Spinal Muscular Atrophy: evidence from in vitro and in vivo SMA models

doi: 10.1007/s00018-024-05550-3

Figure Lengend Snippet: AAV9:: Stmn 2 treatment improved survival and neuromuscular functions in SMA mice. A Representative Western blot of STMN2 in the brain, spinal cord, and sciatic nerves of SMA mice treated with AAV9:: Stmn2 or AAV9::null at P10. B Densitometric quantification of STMN2/actin levels in A normalized to SMA + AAV9::null ( n = 4 animals/group). The values are represented as mean value ± SEM. Statistical significance was determined by Student’s t -test. C Kaplan-Meier survival curves of WT ( n = 20), SMA + AAV9::null ( n = 23) and SMA + AAV9:: Stmn2 ( n = 18). Statistical significance was determined by the Log-rank test. D Postnatal mean body weight ± SEM expressed in grams at different times of WT ( n = 20), SMA + AAV9::null ( n = 23) and SMA + AAV9:: Stmn2 ( n = 18). Statistical significance was determined by multiple unpaired t -test. E Righting response score expressed as percentage of mice that can perform the test (WT n = 20; SMA + AAV9::null n = 23, SMA + AAV9:: Stmn2 n = 18). The values are represented as mean value ± SEM. Statistical significance was determined by two-way ANOVA followed by Dunnett’s post-hoc test. F Macroscopic phenotype of WT, SMA + AAV9::null and SMA + AAV9:: Stmn2 at P8. * P < 0.05, *** P < 0.001, **** P < 0.0001

Article Snippet: In particular, cells were treated for 72 hours (h) after seeding at day 15 with 15 μM SP600125 (Cell Signaling, #8177), an anthrapyrazolone ATP-competitive inhibitor of JNK, or transduced with a lentivirus- STMN2 (MOI 5; Origene RC202914L4V, accession number NM_007029) or with a null vector as the negative control.

Techniques: Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Targeting STMN2 for neuroprotection and neuromuscular recovery in Spinal Muscular Atrophy: evidence from in vitro and in vivo SMA models

doi: 10.1007/s00018-024-05550-3

Figure Lengend Snippet: AAV9:: Stmn2 ameliorated muscle-related features in SMA mice. A Representative immunofluorescence images of gastrocnemius muscle of WT, AAV9::null and AAV9:: Stmn2 -treated SMA mice at P10 stained for α-bungarotoxin (α-BTX, red) and neurofilament-M (NF-M, green) and acquired with a confocal microscope at 63X magnification. Scale bar = 100 μm. B Representative gastrocnemius muscle fibers of WT, AAV9::null and AAV9:: Stmn2 -treated SMA mice at P10 stained with hematoxylin/eosin and acquired with an optical microscope with a magnification of 20X. Scale bar = 200 μm. C Respective quantification of the percentage of innervated NMJs on the total number ( n = 6 animals/group). Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc test. D , E Relative fiber CSA quantification expressed in µm 2 ( D ) and fibrotic/necrotic tissue quantification expressed as non-muscular area percentage ( E ) were measured ( n = 6 animals/group). The values are represented as mean value ± SEM. Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc test. **** P < 0.0001. All inserts are 4X magnification

Article Snippet: In particular, cells were treated for 72 hours (h) after seeding at day 15 with 15 μM SP600125 (Cell Signaling, #8177), an anthrapyrazolone ATP-competitive inhibitor of JNK, or transduced with a lentivirus- STMN2 (MOI 5; Origene RC202914L4V, accession number NM_007029) or with a null vector as the negative control.

Techniques: Immunofluorescence, Staining, Microscopy

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