Journal: bioRxiv

Article Title: PTBP1 Regulates Injury Responses and Sensory Pathways in Adult Peripheral Neurons

doi: 10.1101/2020.10.22.348631

Figure Lengend Snippet: ( A ) RNA structures and schematic of RNA affinity chromatography for mass spectrometry (MS) or western blot (WB). Secondary structures for MAIL (motif for axonal importin localization), and mutated GMAIL and IMAIL motifs, were generated using Mfold with modified bases shown in red (see Methods for sequences). ( B ) MS results for MAIL vs. GMAIL pulldown from rat axoplasm. Enrichment score (cutoff in red = 0.667) vs. Peptide count (PC)/ molecular weight (MW) shown in log scale (cutoff in red = 0.0004), for details see Methods. ( C ) WB for PTBP1 from mouse sciatic nerve (SN) axoplasm after pull-down with MAIL or control RNA motifs. Note that PTBP1 is enriched in the MAIL pulldown, while depleted from the supernatant. Similar results were obtained from rat and bovine axoplasm (data not shown). ( D ) WB analysis for PTBP1 protein levels in liver, brain and dorsal root ganglia (DRG) from adult mice. PTBP1 levels were normalized to GAPDH and shown as fold change (FC) from liver. Individual datapoints with mean ± SEM, unpaired t-test for Brain vs. DRG, *** indicates p< 0.001, n = 6 mice. ( E ) Tissue sections of adult lumbar DRG ganglia, stained for DAPI, PTBP1 and TUJ1 (Tubulin Beta 3 Class III). Scale bar = 20 μm. Merged images show outlined neuronal nuclei (white line) and cytoplasm (white dashed line), glia nuclei are marked by a black cross. ( F ) Quantification of PTBP1 signal in nuclei shows PTBP1 expression in both cell types with enrichment in glia. Individual datapoints with mean ± SEM, ratio paired t-test, ** indicates p< 0.01, n = 4 mice (> 100 nuclei for each mouse). ( G ) Quantification of PTBP1 in Tuj1 positive neurons in nucleus and cytoplasm. Individual datapoints with mean ± SEM, ratio paired t-test, ** indicates p< 0.01, n = 4 mice (> 60 cells for each mouse). Grey dashed lines indicate paired datapoints. H-J , Longitudinal sections of SN stained for NFH (Neurofilament heavy chain) and PTBP1 (protein), combined with fluorescent in situ hybridization (FISH) for KPNB1 mRNA. No primary antibody was used as control for PTBP1 staining (CTRL) and scramble (SCRM) mRNA for KPNB1 FISH. ( H ) Representative images of the staining (top), FISH (middle) and axonal colocalization of PTBP1 with KPNB1 or SCRM mRNA in white (bottom). Scale bar = 10 μm. ( I ) Quantification of KPNB1 mRNA vs. SCRM and PTBP1 protein vs. CTRL in SN. Separate quantification for mRNA and protein signal, individual datapoints with mean ± SEM, t-test, * indicates p< 0.05, *** indicates p< 0.001, n = 3 mice. ( J ) Quantification of axonal colocalization by Person’s correlation. R-values as individual datapoints with mean ± SEM, t-test, **** indicates p< 0.0001, n = 10 sciatic nerve sections from 3 mice.

Article Snippet: Furthermore, anti-PTBP1 (abcam, ab133734, WB 1:1000), anti-PTBP2 (Abnova, H00058155-A01, IF 1:1000, WB 1:2000), anti-PTBP2 (abcam, ab154787, WB 1:1000). anti-ROD1 (Santa Cruz, sc-100845, WB 1:200), anti-GFP (abcam, ab6556, IHC 1:500, ICC & WB 1:5000), anti-NFH (abcam, ab72996, IHC 1:1000, ICC 1:2000), anti-GAPDH (Santa Cruz Biotechnology, sc-32233, WB 1:5000), anti-ERK1/2 (Sigma, M5670, WB 1:30000), anti-β-III tubulin TUJ1 (abcam, ab18207, IF 1:1000, WB: 1:6000), anti-CGRP (AbD SEROTEC 1720-9007, IF 1:1000), anti-Nucleolin (abcam, ab50279, IF & WB 1:1000).

Techniques: Affinity Chromatography, Mass Spectrometry, Western Blot, Generated, Modification, Molecular Weight, Control, Staining, Expressing, In Situ Hybridization

Journal: bioRxiv

Article Title: PTBP1 Regulates Injury Responses and Sensory Pathways in Adult Peripheral Neurons

doi: 10.1101/2020.10.22.348631

Figure Lengend Snippet: ( A ) In vitro binding assay using recombinant PTBP1 or Nucleolin (NCL) with MAIL (motif for axonal importin localization), IMAIL (inactive MAIL) or zipcode motif (ZIP) of β-Actin. Detection by western blot (WB). This assay proves a direct association of PTBP1 with the RNA localization motif of KPNB1. ( B ) WB for different PTBP paralogues (PTBP1, PTBP2, PTBP3) in several tissues. GAPDH as housekeeping gene, n = 6 mice for PTBP1 in liver, brain and dorsal root ganglia (DRG), quantified in . ( C ) Whole tissue staining (iDISCO) on lumbar DRG: co-localization of PTBP1 and PTBP2 in the nucleus of DRG neurons. PTBP2 strictly neuronal, PTBP1 expression in neurons and glia cells. n = 3 mice, scale bar = 50 μm, NFH = Neurofilament heavy chain. ( D ) PTBP1 expression in DRG neurons of all sizes. Simple linear regression for PTBP1 intensity in the cell body (CB) versus cell body area (μm 2 ), n = 330 neurons (from 4 mice), regression line ± 95% CI in magenta, R 2 = 0.008524. e , PTBP1 levels in axons of the sciatic nerve. Simple linear regression for axonal PTBP1 versus axon area (μm 2 ), n = 24,736 axons (from 3 mice), regression line ± 95% CI in magenta, R 2 = 0.0009398. Objects >30μm 2 have been discarded for the analysis, for illustration purposes 6 datapoints with PTBP1 intensity >800 are not shown (but included in liner regression analysis). D & E show that in DRG neurons PTBP1 is expressed in neurons of different CB sizes and axons of different diameters. Images in D & E are not exposure matched (independent experiments, different tissues). They show PTBP1 in Magenta HOT scale (0-255), outline of TUJ1 indicated with white dashed line, scale bar D = 20 μm, scale bar E = 5 μm. ( F ) Quantification of PTBP1 in glial nuclei vs. TUJ1 positive axons within the same image. Individual datapoints with mean ± SEM, ratio paired t-test, p-value * < 0.05, n = 3 mice (> 1000 axons for each mouse). Grey dashed line indicates paired datapoints. ( G ) Images corresponding to F , with panels for TL (transmitted light), DAPI, TUJ1 and PTBP1. Nuclear and axonal signal for PTBP1 is shown in magenta hot scale (exposure matched, same image, 0-255), with the mask for DAPI or TUJ1 as a white outline. Scale bar = 5 μm.

Article Snippet: Furthermore, anti-PTBP1 (abcam, ab133734, WB 1:1000), anti-PTBP2 (Abnova, H00058155-A01, IF 1:1000, WB 1:2000), anti-PTBP2 (abcam, ab154787, WB 1:1000). anti-ROD1 (Santa Cruz, sc-100845, WB 1:200), anti-GFP (abcam, ab6556, IHC 1:500, ICC & WB 1:5000), anti-NFH (abcam, ab72996, IHC 1:1000, ICC 1:2000), anti-GAPDH (Santa Cruz Biotechnology, sc-32233, WB 1:5000), anti-ERK1/2 (Sigma, M5670, WB 1:30000), anti-β-III tubulin TUJ1 (abcam, ab18207, IF 1:1000, WB: 1:6000), anti-CGRP (AbD SEROTEC 1720-9007, IF 1:1000), anti-Nucleolin (abcam, ab50279, IF & WB 1:1000).

Techniques: In Vitro, Binding Assay, Recombinant, Western Blot, Staining, Expressing

Journal: bioRxiv

Article Title: PTBP1 Regulates Injury Responses and Sensory Pathways in Adult Peripheral Neurons

doi: 10.1101/2020.10.22.348631

Figure Lengend Snippet: (A) PTBP1 protein expression in sciatic nerve (SN) axoplasm under naive (na) and injury (inj) conditions. Detection by western blot (WB). PTBP1 levels were normalized to Extracellular Signal-Regulated Kinase 1/2 (ERK) and the data is shown as fold change (FC) to naive (red dashed line). Individual datapoints with mean ± SEM, one-way ANOVA with Tukey’s multiple comparison test, ** indicates p< 0.01, n = 3 mice for each timepoint. (B) Representative images of sciatic nerve cross sections from naive nerve and 7d after crush injury. Axonal PTBP1 is shown in magenta hot scale (0-255), outline of TUJ1 (Tubulin Beta 3 Class III) indicated with a white line, TL (transmission light), scale bar = 5 μm. (C) Quantification of axonal PTBP1 signal in TUJ1 positive axons of different sizes (Binning of axon area: 0-1, 1-2, 2-5, 5-10 μm 2 ). PTBP1 intensity is shown as FC to naive (red dashed line), individual datapoints with mean ± SEM, two-way repeated measure (RM) ANOVA, **** indicates p< 0.0001, Sidak’s multiple comparison test, ** indicates p< 0.01, * indicates p< 0.05, n = 3 (>700 axons/mouse for each size category). (D) PTBP1 protein expression in cell bodies of dorsal root ganglia (DRG). WB of PTBP1 with normalization to GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase), shown as FC to naive (red dashed line). Individual datapoints with mean ± SEM, one-way ANOVA with Tukey’s multiple comparison test, not significant, n = 3-4 mice for each timepoint. (E) Representative images of DRG tissue sections from naive, 3d and 7d after SN crush. Scale bar = 20 μm. (F) Quantification of average PTBP1 signal in DRG neurons (summary of all cell sizes, NHF+ & – cells). Individual datapoints with mean ± SEM, one-way ANOVA with Tukey’s multiple comparison test, not significant, n = 3-7 mice (> 95 cells/mouse).

Article Snippet: Furthermore, anti-PTBP1 (abcam, ab133734, WB 1:1000), anti-PTBP2 (Abnova, H00058155-A01, IF 1:1000, WB 1:2000), anti-PTBP2 (abcam, ab154787, WB 1:1000). anti-ROD1 (Santa Cruz, sc-100845, WB 1:200), anti-GFP (abcam, ab6556, IHC 1:500, ICC & WB 1:5000), anti-NFH (abcam, ab72996, IHC 1:1000, ICC 1:2000), anti-GAPDH (Santa Cruz Biotechnology, sc-32233, WB 1:5000), anti-ERK1/2 (Sigma, M5670, WB 1:30000), anti-β-III tubulin TUJ1 (abcam, ab18207, IF 1:1000, WB: 1:6000), anti-CGRP (AbD SEROTEC 1720-9007, IF 1:1000), anti-Nucleolin (abcam, ab50279, IF & WB 1:1000).

Techniques: Expressing, Western Blot, Comparison, Transmission Assay

Journal: bioRxiv

Article Title: PTBP1 Regulates Injury Responses and Sensory Pathways in Adult Peripheral Neurons

doi: 10.1101/2020.10.22.348631

Figure Lengend Snippet: ( A ) Quantification of PTBP1 levels in sciatic nerve (SN) axoplasm under naive (na) and injury (inj) conditions, combining several experiments. Detection by western blot (WB). PTBP1 levels were normalized to a housekeeping gene and the data is shown as fold change (FC) to naive, which was used in all experiments as a reference (red dashed line). The data is shown as mean ± SEM (black curve) with an overlay of the individual data points in grey. One-way ANOVA with Tukey’s multiple comparison test. Time points 5-21 d are significantly different compared to na, 1d, 2d, 3d, 28d and 56d. p-value * -**** < 0.05-0.0001, n = 4-11 mice for each timepoint. Most relevant timepoints for this study are shown also in (Experimental set with 3 replicates, performed with the same blotting conditions). B-E , Quantification of PTBP1 intensity in different categories of axons and glia nuclei. Individual datapoints with mean ± SEM, ratio paired t-test, p-value * < 0.05, n = 3 mice (>1000 positive objects/mouse). ( B ) Clusters of small axons (TUJ1, Tubulin beta class III); ( C) NFH (Neurofilament heavy chain) positive axons; ( D ) CHAT (Choline O-Acetyltransferase) positive axons; ( E ) Glia cell nuclei (DAPI positive objects). Images below show PTBP1 in magenta HOT scale within the outlines of the corresponding mask and immunofluorescent images for the corresponding markers, scale bar = 5 μm. ( F ) PTBP1 protein expression in purified neurons of DRG tissue (cells after percoll gradient). WB of PTBP1 with normalization to TUJ1, shown as FC to naive (red dashed line). Individual datapoints with mean ± SEM, t-test, not significant, n = 3 mice. ( G ) Quantification of PTBP1 signal in DRG neurons according to different cell body sizes (Binning of cell body (CB) area: 0-200, 200-300, 300-500, >500 μm 2 ). PTBP1 intensity is shown as FC to naive (red dashed line), individual datapoints with mean ± SEM, two-way RM ANOVA with Tukey’s multiple comparison test, not significant, n = 3-7 mice. ( H ) Quantification of PTBP1 signal in NFH positive neurons. FC to na (red dashed line), individual datapoints with mean ± SEM, one-way ANOVA with Tukey’s multiple comparison test, not significant, n = 3-7 mice.

Article Snippet: Furthermore, anti-PTBP1 (abcam, ab133734, WB 1:1000), anti-PTBP2 (Abnova, H00058155-A01, IF 1:1000, WB 1:2000), anti-PTBP2 (abcam, ab154787, WB 1:1000). anti-ROD1 (Santa Cruz, sc-100845, WB 1:200), anti-GFP (abcam, ab6556, IHC 1:500, ICC & WB 1:5000), anti-NFH (abcam, ab72996, IHC 1:1000, ICC 1:2000), anti-GAPDH (Santa Cruz Biotechnology, sc-32233, WB 1:5000), anti-ERK1/2 (Sigma, M5670, WB 1:30000), anti-β-III tubulin TUJ1 (abcam, ab18207, IF 1:1000, WB: 1:6000), anti-CGRP (AbD SEROTEC 1720-9007, IF 1:1000), anti-Nucleolin (abcam, ab50279, IF & WB 1:1000).

Techniques: Western Blot, Comparison, Expressing, Purification

Journal: bioRxiv

Article Title: PTBP1 Regulates Injury Responses and Sensory Pathways in Adult Peripheral Neurons

doi: 10.1101/2020.10.22.348631

Figure Lengend Snippet: ( A ) Viral constructs used for knockdown experiments. ITR (inverted terminal repeat); U6 (type III RNA polymerase III promoter), shRNA (small hairpin RNA), EF1α (elongation factor 1 alpha) promoter, EYFP (enhanced yellow fluorescent protein), WPRE (Woodchuck hepatitis virus posttranscriptional regulatory element), polyA (polyadenylation signal). ( B ) Schematic of in vivo knockdown experiments. shRNA constructs were delivered via AAV9 (Adeno associated virus serotype 9) through intrathecal (IT) injection, followed by sciatic nerve (SN) crush 3 weeks later. At day 7 after crush DRG cells were collected for culture and SN for immunohistochemistry. ( C ) Representative images for DRG neurons in culture (20 h after plating) for naive and injury conditioned cells from shCtrl and shPTBP1 treated mice. TUJ1 (Tubulin Beta 3 Class III), scale bar = 100 μm. D & E , Quantification of growing cells in (c) . Individual datapoints with mean ± SEM, two-way RM (repeated measure) ANOVA with Sidak’s multiple comparison test, ** indicates p< 0.01, *** indicates p< 0.001, n = 5 mice per condition. ( D ) Percent of growing cells. ( E ) Total outgrowth in μm. ( F ) SN longitudinal sections 7 days after crush for shCtrl and shPTBP1. Staining for CGRP (Calcitonin Gene-Related Peptide 1), injury site indicated by the white dashed line, scale bar = 500 μm. ( G ) Quantification of the number of positive axons (average of several regions up to 4 mm) proximal and distal to the crush site, shown as ratio distal/proximal for NFH (Neurofilament heavy chain, images in ) and CGRP. Individual datapoints with mean ± SEM, twoway RM ANOVA with Sidak’s multiple comparison test, p-value **** < 0.001, n = 3-4 mice.

Article Snippet: Furthermore, anti-PTBP1 (abcam, ab133734, WB 1:1000), anti-PTBP2 (Abnova, H00058155-A01, IF 1:1000, WB 1:2000), anti-PTBP2 (abcam, ab154787, WB 1:1000). anti-ROD1 (Santa Cruz, sc-100845, WB 1:200), anti-GFP (abcam, ab6556, IHC 1:500, ICC & WB 1:5000), anti-NFH (abcam, ab72996, IHC 1:1000, ICC 1:2000), anti-GAPDH (Santa Cruz Biotechnology, sc-32233, WB 1:5000), anti-ERK1/2 (Sigma, M5670, WB 1:30000), anti-β-III tubulin TUJ1 (abcam, ab18207, IF 1:1000, WB: 1:6000), anti-CGRP (AbD SEROTEC 1720-9007, IF 1:1000), anti-Nucleolin (abcam, ab50279, IF & WB 1:1000).

Techniques: Construct, Knockdown, shRNA, Virus, In Vivo, Injection, Immunohistochemistry, Comparison, Staining

Journal: bioRxiv

Article Title: PTBP1 Regulates Injury Responses and Sensory Pathways in Adult Peripheral Neurons

doi: 10.1101/2020.10.22.348631

Figure Lengend Snippet: ( A ) Validation of shRNA in DRG culture. Transduction in vitro and culture for 10 days, positive cells (cells transduced with shPTBP1) were compared to negative control cells (Ctrl). Quantification of PTBP1 protein levels by immunofluorescence (IF) in EYFP positive cells vs. EYFP negative cells from the same mouse. Data is shown as FC (fold change) to Ctrl with individual datapoints and mean ± SEM, Mann-Whitney test, p-value **** < 0.0001, n > 30 cells. ( B ) Validation of transduction efficiency in vivo . % of positive neurons was determined by IF EYFP expression) 4 weeks after intrathecal injection of AAV9-shRNA (3 weeks +7 days crush). DRG have been dissociated for culture, fixed 20 h after plating, and stained for TUJ1 and EYFP. Transduction ~ 37% with no difference between shRNA constructs or left (naive) and right (injury) site. Individual datapoints with mean ± SEM, two-way repeated measure (RM) ANOVA with Sidak’s multiple comparison test, n = 5 mice. ( C ) SN longitudinal sections 7d after crush for shCtrl and shPTBP1. Staining for NFH (Neurofilament heavy chain), injury site indicated by the white dashed line, scale bar = 500 μm. D & E, Quantification of the number of positive axons (average of several regions up to 4 mm) proximal and distal to the crush site. Individual datapoints with mean ± SEM, two-way RM ANOVA with Sidak’s multiple comparison test, n = 3-4 mice. ( D ) NFH positive axons, not significant (ns). ( E ) CGRP (Calcitonin Gene-Related Peptide 1) positive axons, p-value * < 0.05.

Article Snippet: Furthermore, anti-PTBP1 (abcam, ab133734, WB 1:1000), anti-PTBP2 (Abnova, H00058155-A01, IF 1:1000, WB 1:2000), anti-PTBP2 (abcam, ab154787, WB 1:1000). anti-ROD1 (Santa Cruz, sc-100845, WB 1:200), anti-GFP (abcam, ab6556, IHC 1:500, ICC & WB 1:5000), anti-NFH (abcam, ab72996, IHC 1:1000, ICC 1:2000), anti-GAPDH (Santa Cruz Biotechnology, sc-32233, WB 1:5000), anti-ERK1/2 (Sigma, M5670, WB 1:30000), anti-β-III tubulin TUJ1 (abcam, ab18207, IF 1:1000, WB: 1:6000), anti-CGRP (AbD SEROTEC 1720-9007, IF 1:1000), anti-Nucleolin (abcam, ab50279, IF & WB 1:1000).

Techniques: Biomarker Discovery, shRNA, Transduction, In Vitro, Negative Control, Immunofluorescence, MANN-WHITNEY, In Vivo, Expressing, Injection, Staining, Construct, Comparison

Journal: bioRxiv

Article Title: PTBP1 Regulates Injury Responses and Sensory Pathways in Adult Peripheral Neurons

doi: 10.1101/2020.10.22.348631

Figure Lengend Snippet: A-C, Behavior, after knockdown of PTBP1, following sciatic nerve (SN) crush injury. Baseline (BL) testing was performed 3 weeks after AAV injection of shRNA, just prior to crush. Mice have been tested in CatWalk gait analysis (A) , heat probe (B) and von Frey (C) paradigms. PWT (paw withdrawal threshold). Individual datapoints with mean ± SEM. BL to day 7: n = 11-13 mice; day 8 onwards: n = 3-4 mice. Mixed-effects analysis with repeated measures (RM) followed by Sidak’s multiple comparison test, p-value ** < 0.01 for heat probe and von Frey, CatWalk gait analysis is not significant. Analysis for both hindlimbs left (naive), right (SN crush) at day 7 is shown in . ( D ) Schematic showing the mechanism of the decoy RNA oligo . The RNA decoy oligo for PTBP1 (PTBP1i, cyan) consists of the repetitive CU-binding motif and prevents PTBP1 protein (magenta) from binding to its endogenous mRNA targets (green). For in vivo experiments, decoy RNA oligos were labeled with a TYE665 fluorophore on the 5’ end for imaging and were modified with a phosphorothioate backbone and 2’-O-methyl modifications. e , Images of sciatic nerve (SN) cross sections after oligo injection with SN crush. Oligo (TYE665 label) was detected with high and low laser intensities to show colocalization with TUJ1 or DAPI, respectively. Big images below show oligo intensity in Magenta HOT scale (0-255) within TUJ1 or DAPI positive objects (white outline). Images are overexposed for illustration purposes, scale bar = 5 μm. ( F ) Images of dorsal root ganglia (DRG) cross sections after oligo injection into the SN. No signal for the oligo was detected in the cell bodies of DRG neurons. Scale bar = 20 μm. G-I, Behavior, after inhibition of PTBP1 via decoy RNA oligo, following sciatic nerve (SN) crush injury. BL testing was performed just prior to injections and SN crush. Mice have been tested in CatWalk gait analysis (G) , heat probe (H) and von Frey (I) paradigms. Individual datapoints with mean ± SEM, n = 9 mice. Two-way RM ANOVA with Sidak’s multiple comparison test, p-value * < 0.05, **** < 0.0001 for heat probe and von Frey, CatWalk gait analysis is not significant. Analysis for both hindlimbs left (naive), right (SN crush) at day 7 is shown in .

Article Snippet: Furthermore, anti-PTBP1 (abcam, ab133734, WB 1:1000), anti-PTBP2 (Abnova, H00058155-A01, IF 1:1000, WB 1:2000), anti-PTBP2 (abcam, ab154787, WB 1:1000). anti-ROD1 (Santa Cruz, sc-100845, WB 1:200), anti-GFP (abcam, ab6556, IHC 1:500, ICC & WB 1:5000), anti-NFH (abcam, ab72996, IHC 1:1000, ICC 1:2000), anti-GAPDH (Santa Cruz Biotechnology, sc-32233, WB 1:5000), anti-ERK1/2 (Sigma, M5670, WB 1:30000), anti-β-III tubulin TUJ1 (abcam, ab18207, IF 1:1000, WB: 1:6000), anti-CGRP (AbD SEROTEC 1720-9007, IF 1:1000), anti-Nucleolin (abcam, ab50279, IF & WB 1:1000).

Techniques: Knockdown, Injection, shRNA, Comparison, Binding Assay, In Vivo, Labeling, Imaging, Modification, Inhibition

Journal: Plants

Article Title: Effects of Chilling Treatment on Baicalin, Baicalein, and Wogonin Biosynthesis in Scutellaria baicalensis Plantlets

doi: 10.3390/plants11212958

Figure Lengend Snippet: HPLC analysis of flavones in roots of S. baicalensis plantlets after chilling treatment.

Article Snippet: HPLC analysis (model NS-6000; Futecs, Daejeon, Korea) of baicalein, baicalin, and wogonin was performed, as reported previously [ ].

Techniques: Control

Journal: Plants

Article Title: Effects of Chilling Treatment on Baicalin, Baicalein, and Wogonin Biosynthesis in Scutellaria baicalensis Plantlets

doi: 10.3390/plants11212958

Figure Lengend Snippet: HPLC analysis of flavones in shoots of S. baicalensis plantlets after chilling treatment.

Article Snippet: HPLC analysis (model NS-6000; Futecs, Daejeon, Korea) of baicalein, baicalin, and wogonin was performed, as reported previously [ ].

Techniques: Control