Journal: eLife

Article Title: Axon-specific microtubule regulation drives asymmetric regeneration of sensory neuron axons

doi: 10.7554/eLife.104069

Figure Lengend Snippet: ( A ) In vitro DRG neurons labeled with βIII-tubulin depicting different development stages. Scale bar, 10 µm. ( B ) Percentage of different DRG neuron morphologies at DIV21 (n = 3 independent experiments, 100 cells per experiment). ( C ) Imaris segmentation of a pseudo-unipolar DRG neuron transduced with AAV-CMV-eGFP. Scale bar, 7 µm. ( D ) In vitro DRG neurons transduced with AAV-CMV-eGFP depicting stem axon formation. Scale bar, 10 µm. ( E, F ) Stem axon diameter ( E ) and length ( F ) of DRG neuron axons from the formation of the stem axon (Initial) to the final stage of pseudo-unipolarization (Final) (n = 13 neurons; paired t -test, diameter ****p<0.0001, length ***p=0.0004). ( G ) Stem axon and cell-body displacement during pseudo-unipolarization (n = 13 neurons; paired t -test, **p=0.0020). ( H ) In vitro diameter of DRG axons; n = 5–8 independent experiments, 5–10 neurons/experiment; paired t -test in bipolar neurons, ***p=0.0003; repeated measures (RM) one-way ANOVA in pseudo-unipolar neurons, stem-peripheral *p=0.0196, stem-central **p=0.0069, peripheral-central **p=0.0048; for comparisons amongst peripheral and central-like axons from bipolar and pseudo-unipolar neurons, a two-way ANOVA was used (peripheral: **p=0.0039; central: p=0.9829). ( I ) In vitro pseudo-unipolar DRG neuron transduced with the lentivirus CMV-EB3-GFP depicting different axon diameter. Scale bar, 5 µm. Data are represented as mean ± SEM.

Article Snippet: Pseudo-unipolar DRG neurons were selected for segmentation using Imaris software (version 10.1.1).

Techniques: In Vitro, Labeling, Transduction

Journal: eLife

Article Title: Axon-specific microtubule regulation drives asymmetric regeneration of sensory neuron axons

doi: 10.7554/eLife.104069

Figure Lengend Snippet: ( A ) In vitro pseudo-unipolar DRG neuron transduced with a Tom20-GFP lentivirus, labeling mitochondria. Scale bar, 5 µm. ( B ) Quantification of the anterograde mitochondria flux (n = 4 independent experiments, five DRGs/experiment; paired t -test, *p=0.0143). ( C ) EB3-GFP comet density in in vitro DRG axons (n = 6–7 independent experiments, 5–10 neurons/experiment; paired t -test in bipolar axons, **p=0.0038; repeated measures [RM] one-way ANOVA in pseudo-unipolar axons, stem-central *p=0.0221, peripheral-central *p=0.0171). ( D ) Kymographs of in vitro pseudo-unipolar DRG axons. ( E ) EB3-GFP comet velocity in in vitro pseudo-unipolar DRG axons (n = 6 independent experiments, 5–10 neurons/experiment; RM one-way ANOVA, stem-central *p=0.0443, peripheral-central *p=0.0183). ( F ) Representation of naive DRG neurons connected to the peripheral nerve (containing peripheral axons) and dorsal root (containing central axons). The dashed squares indicates the imaging locations. ( G ) Live imaging of DRG axons from Thy1-EB3-eGFP mice. Scale bar, 5 µm. ( H ) EB3-GFP comet density in DRG explants from naive mice (n = 12–17 animals; 3–6 axons/animal, **p=0.0037) and mice with a peripheral conditioning lesion (CL) (n = 9–10 animals, 3–5 axons/animal, p=0.1423). Two-way ANOVA; peripheral naive-peripheral CL, *p=0.0276; central naive central CL, **p=0.0026. ( I ) High-magnification electron microscopy images within individual naive DRG axons, depicting axonal microtubules (red arrowheads). Scale bar, 100 nm. ( J ) Total density of microtubules in naive DRG axons (n = 8 animals, 5–10 axons/animal; paired t -test, p=0.2299). ( K ) EB3-GFP comet velocity in DRG explants from naive mice (n = 11–15 animals, 3–6 axons/animal, **p=0.0048) and mice with peripheral CL (n = 8–9 animals, 3–5 axons/animal, **p=0.0035). Two-way ANOVA, peripheral naive-peripheral CL, ***p=0.0003; central naive-central CL, **p=0.0038. ( L ) Representation of a sciatic nerve injury to DRG neurons (conditioning lesion). The dashed square indicates the imaging location, while the dashed line and scissor marks the lesion site. ( M ) Total density of axonal microtubules in DRG peripheral and central axons after peripheral CL (n = 8 animals, five axons/animal; paired t -test, p=0.4624). Data are represented as mean ± SEM.

Article Snippet: Pseudo-unipolar DRG neurons were selected for segmentation using Imaris software (version 10.1.1).

Techniques: In Vitro, Transduction, Labeling, Imaging, Electron Microscopy

Journal: eLife

Article Title: Axon-specific microtubule regulation drives asymmetric regeneration of sensory neuron axons

doi: 10.7554/eLife.104069

Figure Lengend Snippet: ( A ) Representative images of a peripheral DRG axon after performing RNAscope analysis to detect Spastin and Dpysl5 (CRMP5 mouse gene) mRNA particles. Scale bar, 10 µm. ( B, C ) The density of ( B ) total Spastin mRNA and ( C ) total Dpysl5 mRNA in the DRG peripheral nerve and dorsal root (n = 7 animals, three non-consecutive DRG peripheral nerve and dorsal root sections were analyzed per animal). Paired t -test, spastin: p=0.2112, CRMP5 p=0.7671. ( D ) In vitro pseudo-unipolar DRG neurons transduced with a CMV-EB3-GFP lentivirus. Scale bar, 5 µm. ( E, F ) Sequential images of EB3-GFP comets, either stopping ( E ) or crossing ( F ) the DRG T-junction. This analysis spanned a 5 µm region from the end of the DRG stem axon to the start of either the peripheral-like (red line) or central-like (blue line) axonal branch. Scale bar, 5 µm. ( G ) Number of EB3-GFP comets that either stop or cross the DRG-T junction (n = 8 independent experiments, 10 DRGs/experiment; RM two-way ANOVA, stop *p=0.0439, cross *p=0.0162, stop-cross peripheral p=0.9169, stop-cross central **p=0.0032). ( H ) Immunofluorescence of polyglutamylated tubulin and βIII-tubulin in DRG peripheral and central-like axonal branches. Scale bar, 10 µm, and close-up 5 µm. ( I ) Quantification of polyglutamylated tubulin fluorescence intensity normalized to βIII-tubulin (n = 5 independent experiments, eight pseudo-unipolar DRGs per experiment, paired t -test, p=0.2957). ( J ) Immunofluorescence of acetylated tubulin and βIII-tubulin in DRG peripheral and central-like axonal branches. Scale bar, 10 µm, and close-up 5 µm. ( K ) Quantification of acetylated tubulin fluorescence intensity normalized to βIII-tubulin (n = 4 independent experiments, eight pseudo-unipolar DRGs per experiment, paired t -test, p=0.2946). ( L ) Immunofluorescence of Δ2 tubulin and βIII-tubulin in DRG peripheral and central-like axonal branches. Scale bar, 10 µm, and close-up 5 µm. ( M ) Quantification of Δ2 tubulin fluorescence intensity (n = 4 independent experiments, eight DRGs/experiment; paired t -test, **p=0.0012). Data are represented as mean ± SEM.

Article Snippet: Pseudo-unipolar DRG neurons were selected for segmentation using Imaris software (version 10.1.1).

Techniques: RNAscope, In Vitro, Transduction, Immunofluorescence, Fluorescence

Journal: eLife

Article Title: Axon-specific microtubule regulation drives asymmetric regeneration of sensory neuron axons

doi: 10.7554/eLife.104069

Figure Lengend Snippet: ( A ) Density of EB3-GFP comets in wild-type DRG axons in vitro (n = 4 independent experiments, 10 cells/experiment; repeated measures [RM] one-way ANOVA, stem-peripheral p=0.0536, stem-central **p=0.0082, peripheral-central **p=0.0027). ( B ) Density of EB3-GFP comets in Spastin knockout DRG axons in vitro (n = 4 independent experiments, 10 cells/experiment; RM one-way ANOVA, stem-peripheral *p=0.0168, stem-central *p=0.0250, peripheral-central p=0.8762). ( C, D ) EB3-eGFP comet density in ( C ) wild-type and ( D ) Spastin knockout mice (n = 10–14 animals; three axons/animal; unpaired t -test; wild-type, *p=0.0388; knockout, p=0.9792). ( E, F ) EB3-eGFP comet velocity in ( E ) wild-type and ( F ) Spastin knockout mice (n = 5–7 animals; three axons/animal; unpaired t -test; wild-type, *p=0.0405; knockout, p=0.0823). ( G ) Representation of the conditioning lesion (CL). A dorsal column hemisection is preceded by a sciatic nerve transection 1 week before. Lesion sites are indicated with dashed red lines and DRG axons in green. ( H–J ) Longitudinal spinal cord sections of ( H ) wild-type mice with spinal cord lesion or ( I ) CL and ( J ) Spastin knockout mice with CL. Dorsal column tract axons were traced with cholera toxin-B (white). The lesion border is highlighted by a yellow line. Regenerating axons are highlighted by red arrowheads. C, caudal; R, rostral; D, dorsal; V, ventral. Scale bar, 100 µm. ( K ) Number of regenerating axons in wild-type mice with spinal cord injury (n = 5 animals) and CL (n = 6 animals), and Spastin knockout with CL (n = 7 animals); six sections per animal. One-way ANOVA; wild-type SCI-CL, ***p=0.0005; wild-type-knockout CL, **p=0.0020; wild-type SCI-knockout CL, p=0.3335. ( L ) Representative in vitro wild-type and Spastin knockout adult DRG neurons labeled with βIII-tubulin. Scale bar, 30 µm. ( M ) Quantification of the number of primary neurites in adult wild-type and Spastin knockout DRG neurons. n = 4–5 independent experiments for wild-type and Spastin knockout; unpaired t -test; *p=0.0205. Data are represented as mean ± SEM.

Article Snippet: Pseudo-unipolar DRG neurons were selected for segmentation using Imaris software (version 10.1.1).

Techniques: In Vitro, Knock-Out, Labeling

Journal: Pain

Article Title: Pharmacologically enabling the degradation of Na V 1.8 channels to reduce neuropathic pain

doi: 10.1097/j.pain.0000000000003470

Figure Lengend Snippet: PY(A) peptide decreased Na V 1.8 expression. (A) PY(A) peptide sequence. (B) Proposed mechanism of lipidated peptides partitioning in the membrane and affecting Magi-1. (C) Cultured rat DRG neurons treated with 10 µM PY(A) peptide or 10 µM scrambled peptide control. Na V 1.8 immunofluorescence progressively decreases over 6 and 24 hours after incubation with PY(A) peptide. There is also an internalization of Na V 1.8 seen after 6 hours and continuing until 24 hours. (D) Quantification of Na V 1.8 immunofluorescence in cultured DRGs. Significance was determined using a one-way ANOVA with * P < 0.05; ** P < 0.01; *** P < 0.001. (E) Cultured rat DRG neurons treated with PY(A) peptide + vehicle, PY(A) peptide + bafilomycin (100 nM), or scrambled peptide + vehicle. Na V 1.8 immunofluorescence is decreased at 6 and 24 hours; however, the addition of Bafilomycin prevents the loss of Na V 1.8 immunofluorescence. (F) Quantification of Na V 1.8 immunofluorescence in cultured DRG neurons. Significance was determined using a one-way ANOVA with * P < 0.05; ** P < 0.01; *** P < 0.001. (G) Representative action potential firing from cultured neurons after 24 incubation with scrambled (above) PY(A) peptide (below). (H) Representative rheobase determination in cultured DRG neurons. PY(A) peptide increased threshold of firing. Red trace indicates selected trace to measure rheobase. For scrambled −140 pA, and for PY(A) peptide −440 pA. (I) Threshold of firing as a function of PY(A) peptide dose. The threshold for action potentials increased as the concentration of the peptide increased. Estimated IC 50 = 928 nM. ANOVA, analysis of variance; DRG, dorsal root ganglion.

Article Snippet: Cultured human DRG neuronal suspensions were purchased from Anabios (San Diego, CA).

Techniques: Expressing, Sequencing, Membrane, Cell Culture, Control, Immunofluorescence, Incubation, Concentration Assay

Journal: Pain

Article Title: Pharmacologically enabling the degradation of Na V 1.8 channels to reduce neuropathic pain

doi: 10.1097/j.pain.0000000000003470

Figure Lengend Snippet: PY(A)-H peptide decreased Na V 1.8 expression in human DRG neurons. (A) Human PY peptide (PY(A)-H) sequence. (B) Incubation of peptide alters Na V 1.8 expression in a first human donor. A similar Na V 1.8 immunofluorescence decrease is seen within 6 hours and continues to decrease at 24 hours of 10 µM PY(A)-H incubation. The scrambled peptide had no effect on Na V 1.8 expression. (C) Quantification of fluorescence in scrambled and PY(A)-H peptide–treated neurons over time. Significance was determined using one-way ANOVA ** P < 0.01; *** P < 0.001. (D) Incubation to peptide alters Na V 1.8 expression in a second human donor. Na V 1.8 immunofluorescence decreased within 6 hours of 10 µM PY(A)-H incubation and continued to decrease at 24 hours. The scrambled peptide did not affect Na V 1.8 expression. (E) Quantification of fluorescence in scrambled and PY(A)-H peptide–treated neurons over time. Significance was determined using one-way ANOVA ** P < 0.01; *** P < 0.001. (F) Immunofluorescence images of human DRG neurons taken from the third donor 24 hours after incubation with 10 µM scrambled peptide or PY(A)-H peptide. There was a reduction of Na V 1.8 immunofluorescence in PY(A)-H peptide–treated human DRG neurons compared to scrambled peptide. A marked reduction in immunofluorescence was noted particularly in the processes (indicated by white arrows). (G) Quantification of Na V 1.8 staining fluorescence in DRGs. Significance determined using unpaired Student t test * P < 0.05. (H) Representative I Na at +20 mV of human DRG neurons after 24-hour incubation with 10 µM scrambled (top) or PY(A) peptide (bottom). (I) Quantification of I Na current density at +20 mV pooled from donors 1, 2, and 3. Significance determined using unpaired Student t test *** P < 0.005 (n = 12). ANOVA, analysis of variance; DRG, dorsal root ganglion.

Article Snippet: Cultured human DRG neuronal suspensions were purchased from Anabios (San Diego, CA).

Techniques: Expressing, Sequencing, Incubation, Immunofluorescence, Fluorescence, Staining