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Nodal β spectrins are required to maintain Na+ channel clustering and axon integrity

  1. Cheng-Hsin Liu
  2. Sharon R Stevens
  3. Lindsay H Teliska
  4. Michael Stankewich
  5. Peter J Mohler
  6. Thomas J Hund
  7. Matthew N Rasband  Is a corresponding author
  1. Department of Neuroscience, Baylor College of Medicine, United States
  2. Program in Developmental Biology, Baylor College of Medicine, United States
  3. Department of Pathology, Yale University, United States
  4. Department of Physiology and Cell Biology, The Ohio State University, United States
  5. Biomedical Engineering, The Ohio State University, United States
Research Article
Cite this article as: eLife 2020;9:e52378 doi: 10.7554/eLife.52378
7 figures, 1 video, 1 table and 1 additional file

Figures

Mice lacking β4 spectrin in PNS sensory neurons have normal behaviors, action potential conduction, and Nav channel clustering at nodes or Ranvier.

(A) Immunoblotting of brain homogenates from 3 month-old Sptbn4F/F and Nes-cre;Sptbn4F/F mice using antibodies against the C-terminal SD domain of β4 spectrin and actin. (B) Immunostaining of cortical brain sections from 3 month-old Sptbn4F/F and Nes-cre;Sptbn4F/F mice using antibodies against the C-terminal SD domain (green) and N-terminal domain (red) of β4 spectrin. Scale bar, 50 μm. (C) Accelerating rotarod test performed on 6 week-old mice. Sptbn4F/F, N = 7; Avil-cre;Sptbn4F/F, N = 6. Data are mean ± SEM, p=0.9708. (D) Hot plate test performed on 6 week-old mice. Sptbn4F/F, N = 6; Avil-cre;Sptbn4F/F, N = 5. Data are mean ± SEM, p=0.5003. (E) Representative compound action potentials recorded from 5 week-old Sptbn4F/F (black) and Avil-cre;Sptbn4F/F (red) dorsal roots. (F) Conduction velocities recorded from 5 week-old mice. Sptbn4F/F, N = 3 mice, 12 dorsal roots. Avil-cre;Sptbn4F/F, N = 3 mice, 11 dorsal roots. Data are mean ± SEM. p=0.9202. (G) Immunostaining of dorsal roots from 1.5 month-old Sptbn4F/F and Avil-cre;Sptbn4F/F mice using antibodies against pan-neurofascin (blue), β4 spectrin SD-domain (green), and pan-Nav channels (red). Scale bar, 10 μm. (H) Quantification of the percentage of dorsal root nodes labeled for β4 spectrin and Nav channels in the indicated tissues and genotypes. N = 3 animals per tissue per genotype, with at least 80 nodes counted per data point. Data are mean ± SEM. For β4 spectrin labeling, ***p=1.97595E-07 between Sptbn4F/F and Avil-cre;Sptbn4F/F dorsal roots, or ***p=1.07844E-05 between Avil-cre;Sptbn4F/F dorsal roots and ventral roots, respectively. For Nav labeling, *p=0.0246 between Sptbn4F/F and Avil-cre;Sptbn4F/F dorsal roots, or p=0.1783 between Avil-cre; Sptbn4F/F dorsal roots and ventral roots, respectively.

Figure 1—source data 1

Original immunoblotting image for Figure 1A and statistical summary for Figure 1C,D,F,H.

https://cdn.elifesciences.org/articles/52378/elife-52378-fig1-data1-v1.xlsx
β1 spectrin and AnkR rescue Nav channel clustering at nodes in β4 spectrin deficient dorsal roots.

(A) Immunostaining of dorsal roots from 1.5 month-old Sptbn4F/F and Avil-cre;Sptbn4F/F mice using antibodies against pan-neurofascin (blue), β4 spectrin SD-domain (green), and β1 spectrin (red). Scale bar, 10 μm. (B) Immunostaining of dorsal roots from 1.5 month-old Sptbn4F/F and Avil-cre;Sptbn4F/F mice using antibodies against pan-neurofascin (blue), AnkG (green), and AnkR (red). Scale bar, 10 μm. (C) Quantification of the percentage of dorsal root nodes in Sptbn4F/F and Avil-cre;Sptbn4F/F mice labeled for β1 spectrin, β4 spectrin, AnkG and AnkR. N = 3 mice per genotype with more than 80 nodes counted per data point. Data are mean ± SEM. β1 spectrin, ***p=4.62544E-05; β4 spectrin, ***p=1.97595E-07; AnkG, **p=0.0014; AnkR, ***p=0.0002.

Mice lacking β1 spectrin in PNS sensory neurons have normal behaviors, action potential transmission, and Nav channel clustering at nodes.

(A) Immunoblotting of brain homogenates from 3 month-old SptbF/F and Nes-cre;SptbF/F mice using antibodies against β1 spectrin and actin. (B) Immunostaining of cortex, hippocampal CA1 region and cerebellum from 3 month-old SptbF/F and Nes-cre;SptbF/F mice using antibodies against β1 spectrin (green). Scale bar, 50 μm. (C) Accelerating rotarod test performed on 6 week-old mice. SptbF/F, N = 8; Avil-cre;SptbF/F, N = 11. Data are mean ± SEM, p=0.2706. (D) Hot plate test performed on 6 week-old mice. SptbF/F, N = 6; Avil-cre;SptbF/F, N = 8. Data are mean ± SEM, p=0.4403. (E) Representative CAPs recorded from 5 week-old SptbF/F (black) and Avil-cre;SptbF/F (red) dorsal roots. (F) Conduction velocities recorded from 5 week-old mice. SptbF/F, N = 3 mice, 12 dorsal roots. Avil-cre;SptbF/F, N = 3 mice, 12 dorsal roots. Data are mean ± SEM. p=0.7562. (G) Immunostaining of dorsal roots from 6-week-old SptbF/F and Avil-cre;SptbF/F mice using antibodies against pan-neurofascin (blue), β4 spectrin SD-domain (green), and pan-Nav channels (red). Scale bar, 10 μm. (H) Quantification of the percentage of dorsal root nodes labeled for β4 spectrin and Nav channels in SptbF/F and Avil-cre;SptbF/F mice. N = 3 animals per genotype, with at least 80 nodes counted per animal. Data are mean ± SEM.

Figure 3—source data 1

Original immunoblotting image for Figure 3A and statistical summary for Figure 3C,D,F,H.

https://cdn.elifesciences.org/articles/52378/elife-52378-fig3-data1-v1.xlsx
Mice lacking both β1 and β4 spectrin in PNS sensory neurons have motor dysfunction and impaired action potential conduction.

(A) Avil-cre;SptbF/F;Sptbn4F/F mice have a hindlimb clasping reflex. (B) Footprint assay in 6 week-old SptbF/F;Sptbn4F/F and Avil-cre;SptbF/F;Sptbn4F/F mice. (C) Accelerating rotarod test performed on 6 week-old mice. SptbF/F;Sptbn4F/F, N = 8; Avil-cre; SptbF/F;Sptbn4F/F, N = 10. Data are mean ± SEM, ***p=7.17995E-09. (D) Tail immersion test performed on 6 week-old mice. SptbF/F, N = 8; Avil-cre;SptbF/F;Sptbn4F/F, N = 10. Data are mean ± SEM, p=0.2011. (E) Representative CAPs recorded from 5 week-old SptbF/F;Sptbn4F/F (black) and Avil-cre;SptbF/F;Sptbn4F/F (red) dorsal roots. (F) Conduction velocities recorded from 5 week-old mice. SptbF/F;Sptbn4F/F, N = 3 mice, 12 dorsal roots. Avil-cre;SptbF/F;Sptbn4F/F, N = 3 mice, 11 dorsal roots. Data are mean ± SEM. ***p=2.76334E-14.

Figure 5 with 1 supplement
Mice lacking both β1 and β4 spectrin in PNS sensory neurons progressively lose their nodal Nav channels.

(A) Immunostaining of dorsal roots from P8, 1 month-old and 6 month-old SptbF/F;Sptbn4F/F and Avil-cre;SptbF/F;Sptbn4F/F mice using antibodies against pan-neurofascin (blue) and panNav channels (green). Arrowheads point to intact nodal Nav clusters, whereas arrows point to nodes devoid of Nav channels. Scale bar, 10 μm. (B) Quantification of the percentage of dorsal root nodes labeled for Nav channels in SptbF/F;Sptbn4F/F and Avil-cre;SptbF/F;Sptbn4F/F mice at the indicated ages. N = 3 animals per genotype per time point, except N = 4 in 6 month-old Avil-cre;SptbF/F;Sptbn4F/F. Data are mean ± SEM. P8: p=0.3508; 1M: *p=0.0249; 6M:***p=2.8444E-05. (C) The percentage of nodes labeled for AnkG or AnkR at 1 month or 6 months of age in Avil-cre;SptbF/F;Sptbn4F/F mice. N = 3 or four animals per genotype per timepoint, with a minimum of 80 nodes analyzed per animal. Data are mean ± SEM, *p=0.0296. (D) Immunostaining of dorsal roots from 6 month-old SptbF/F;Sptbn4F/F and Avil-cre;SptbF/F;Sptbn4F/F mice using antibodies against pan-neurofascin (blue), caspr (green), and Kv1.2 (red). Scale bar, 10 μm. (E) Immunostaining of teased dorsal roots from 9 month-old SptbF/F;Sptbn4F/F and Avil-cre;SptbF/F;Sptbn4F/F mice using antibodies against pan-neurofascin (blue), β2 spectrin (green), and caspr (red). Scale bar, 10 μm. (F) Immunostaining of teased dorsal roots from 9 month-old SptbF/F;Sptbn4F/F and Avil-cre;SptbF/F;Sptbn4F/F mice using antibodies against pan-neurofascin (blue), β3 spectrin (green), and panNav channels (red). Scale bar, 10 μm.

Figure 5—figure supplement 1
Quantification of nodes per field of view (FOV) by paranodal neurofascin immunostaining in SptbF/F;Sptbn4F/F and Avil-cre;SptbF/F;Sptbn4F/F mice dorsal root at the indicated ages.

N = 3 animals per genotype per time point, except N = 4 in 6 month-old Avil-cre; SptbF/F;Sptbn4F/F. Data are represented as mean ± SEM. 1M: p=0.897; 6M: p=0.7976.

PNS sensory axons lacking nodal spectrins have an axon injury response, deformation, and hypermyelination.

(A) Immunostaining of DRG from 1 and 9 month-old Avil-cre;SptbF/F;Sptbn4F/F mice using antibodies against ATF3 (green) and the axonal marker Tuj1 (red). Scale bar, 200 μm. (B) Quantification of the percentage of Tuj1-positive DRG neurons labeled for ATF3 in SptbF/F;Sptbn4F/F, Avil-cre;SptbF/F, Avil-cre;Sptbn4F/F and Avil-cre;SptbF/F;Sptbn4F/F mice at the indicated ages. N = 3 mice per genotype per age, except N = 2 for 1 month-old Avil-cre;SptbF/F, and N = 2 for 6 month-old Avil-cre;Sptbn4F/F mice. For 1 month-old: SptbF/F;Sptbn4F/F vs. Avil-cre;SptbF/F;Sptbn4F/F, p=0.6321; Avil-cre;Sptbn4F/F vs. Avil-cre;SptbF/F;Sptbn4F/F, p=0.2712. For 6 month-old: SptbF/F;Sptbn4F/F vs. Avil-cre;SptbF/F;Sptbn4F/F, **p=0.0044; Avil-cre;SptbF/F vs. Avil-cre;SptbF/F;Sptbn4F/F, **p=0.0041; For 9+ months old: SptbF/F;Sptbn4F/F vs. Avil-cre;SptbF/F;Sptbn4F/F, **p=0.0085; Avil-cre;SptbF/F vs. Avil-cre;SptbF/F;Sptbn4F/F, **p=0.0094; Avil-cre;Sptbn4F/F vs. Avil-cre;SptbF/F;Sptbn4F/F, **p=0.0099. Data are mean ± SEM. (C) Quantification of total and ATF3+ DRG cell body diameters in Avil-cre; SptbF/F;Sptbn4F/F mice at the indicated ages. N = 3 animals per time point. Data are shown in a box-and-whisker plot (median: a line across 25th and 75th percentiles: lower and upper box edges, respectively; minimum and maximum: the values below and above the box, respectively). Total number of DRG neurons counted are 1078 at 6M, and 1005 at 9M. At 6M: ***p=6.61E-07; 9M: ***p=8.06E-10. (D) TEM images of 9 month-old dorsal root cross sections from SptbF/F;Sptbn4F/F and Avil-cre;SptbF/F;Sptbn4F/F mice. Scale bar, 2 μm. (E) Quantification of axon circularity in 9 month-old SptbF/F;Sptbn4F/F and Avil-cre;SptbF/F;Sptbn4F/F mice. N = 3 mice per genotype. SptbF/F;Sptbn4F/F, n = 533 axons; Avil-cre;SptbF/F;Sptbn4F/F, n = 873 axons. **p=0.0056. Data are mean ± SEM. (F) Scatter plot of g-ratio versus axon diameter from dorsal roots of 9 month-old SptbF/F;Sptbn4F/F and Avil-cre; SptbF/F;Sptbn4F/F mice. SptbF/FSptbn4F/F, n = 266 axons; Avil-cre; SptbF/F;Sptbn4F/F, n = 303 axons pooled from 3 mice of each genotype. (G) Quantification of g-ratio of dorsal roots from 9 month-old SptbF/F;Sptbn4F/F and Avil-cre;SptbF/F;Sptbn4F/F mice. N = 3 mice per genotype. **p=0.0045. Data are mean ± SEM. (H) Percentage of dorsal root axon diameter in 9 month-old SptbF/F;Sptbn4F/F and Avil-cre; SptbF/F;Sptbn4F/F mice. Axons are shown in 1 μm bins. (I) Quantification of axon diameter of dorsal root from 9-month-old SptbF/F;Sptbn4F/F and Avil-cre; SptbF/F;Sptbn4F/F mice. Data are shown in a box-and-whisker plot (median: a line across the box; 25th and 75th percentiles: lower and upper box edges, respectively; minimum and maximum: the values below and above the box, respectively). **p=0.0023. In (H) and (I), SptbF/F;Sptbn4F/F, n = 266 axons; Avil-cre; SptbF/F;Sptbn4F/F, n = 303 axons pooled from 3 mice of each genotype.

Cartoon illustrating the phenotypes of the mice analyzed here.

Mice lacking β1 spectrin have normal nodes of Ranvier. Mice lacking β4 spectrin have normal nodes of Ranvier due to compensation by a β1 spectrin/AnkR protein complex. Mice lacking all nodal β spectrins cluster Nav channels during early development. However, with increasing age these mice lose nodal Nav channels, Ankyrins, and NF186, and show an axon injury response including altered membrane organization.

Videos

Video 1
9 month-old WT and Avil-cre;SptbF/F;Sptbn4F/F mice.

Mice lacking both β1 and β4 spectrin in sensory neurons are ataxic.

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Gene (Mus musculus)Sptbhttps://www.ncbi.nlm.nih.gov/gene/20741Gene ID: 20741
Gene (M. musculus)Sptbn4https://www.ncbi.nlm.nih.gov/gene/80297Gene ID: 80297
Genetic reagent
(M. musculus)
Advillin-creF. Wang (Duke University, Durham, NC)The Jackson Laboratory (Stock No:032536)
Genetic reagent (M. musculus)Sptbflox/floxThis paperSee Materials and methods, Section Animals
Genetic reagent (M. musculus)Sptbn4flox/floxPMID: 30226828
AntibodyAnti-Ankyrin G (Mouse monoclonal)NeuromabClone: N106/36; RRID: AB_10673030IF (1:500)
AntibodyAnti-PanNav (Mouse monoclonal)NeuromabClone: N419/78; RRID: AB_2493099IF (1:300)
AntibodyAnti-PanNav (Mouse monoclonal)Sigma-AldrichClone: K58/35; RRID: AB_477552IF (1:300)
AntibodyAnti-Kv1.2 (Mouse monoclonal)NeuromabClone: K14_16; RRID: AB_2296313IF (1:500)
AntibodyAnti-β1 spectrin (Mouse monoclonal)NeuromabClone: N385/21; RRID: AB_2315815IF (1:500)
WB (1:1000)
AntibodyAnti-actin (Mouse monoclonal)MilliporeCat.#: MAB1501; RRID: AB_2223041WB (1:4000)
AntibodyAnti-β2 spectrin (Mouse monoclonal)BD BiosciencesCat.#: 612563; RRID: AB_399854IF (1:1000)
AntibodyAnti-Tuj1 (Mouse monoclonal)BioLegendCat.#: 801201; RRID: AB_2313773IF (1:800)
AntibodyAnti-β3 spectrin (Rabbit polyclonal)NovusCat.#: NB110-58346; RRID: AB_877723IF (1:1000)
AntibodyAnti-ATF3 (Rabbit polyclonal)Santa CruzCat.#: SC-188, RRID: AB_2258513IF (1:1000)
AntibodyAnti-β4 spectrin NT antibody (Rabbit polyclonal)PMID: 28123356RRID: AB_2315634IF (1:50)
AntibodyAnti-β4 spectrin SD antibody (chicken polyclonal)PMID: 28123356IF (1:200)
AntibodyAnti-β4 spectrin SD antibody (Rabbit polyclonal)PMID: 28123356IF (1:500)
WB (1:1000)
AntibodyAnti-Ankyrin R (Rabbit polyclonal)PMID: 25362473IF (1:500)
AntibodyAnti-Caspr (Rabbit polyclonal)PMID: 10460258RRID: AB_2572297IF (1:500)
AntibodyAnti-Pan Neurofascin (Chicken polyclonal)R and D SystemsCat.#: AF3235; RRID: AB_10890736IF (1:500)
Sequence-based reagentGenotyping primer for Sptbn4flox/flox mouse (sense)PMID: 302268285’-GAGCTGCATAAGTTCTTCAGCGATGC-3’
Sequence-based reagentGenotyping primer for Sptbn4flox/flox mouse (anti-sense)PMID: 302268285’-ACCCCATCTCAACTGGCTTTCTTGG-3’
Sequence-based reagentGenotyping primer for Sptbflox/flox mouse (sense)This paper5’- ACAGAGACAGATGGCCGAAC-3‘
Sequence-based reagentGenotyping primer for Sptbflox/flox mouse (anti-sense)This paper5’-CTCTGGTTCCCAGGAGAGC-3’
Sequence-based reagentGenotyping primer for Avil-cre mouse (primer 1)PMID: 290382435’-CCCTGTTCACTGTGAGTAGG-3’
Sequence-based reagentGenotyping primer for Avil-cre mouse (primer 2)PMID: 290382435’- AGTATCTGGTAGGTGCTTCCAG-3’
Sequence-based reagentGenotyping primer for Avil-cre mouse (primer 3)PMID: 290382435’-GCGATCCCTGAACATGTCCATC-3’
Software, algorithmFijiNational Institutes of HealthRRID:SCR_002285
Software, algorithmQupathPMID: 29203879
Software, algorithmPrismGraph PadRRID:SCR_002798Version 6
Software, algorithmpClampMolecular DevicesRRID:SCR_011323
Software, algorithmMultiClampMolecular Devices
Software, algorithmClampfitMolecular Devices

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