1. Neuroscience

EphrinB2 knockdown in cervical spinal cord preserves diaphragm innervation in a mutant SOD1 mouse model of ALS

  1. Mark W Urban
  2. Brittany A Charsar
  3. Nicolette M Heinsinger
  4. Shashirekha S Markandaiah
  5. Lindsay Sprimont
  6. Wei Zhou
  7. Eric V Brown
  8. Nathan T Henderson
  9. Samantha J Thomas
  10. Biswarup Ghosh
  11. Rachel E Cain
  12. Davide Trotti
  13. Piera Pasinelli
  14. Megan C Wright
  15. Matthew B Dalva  Is a corresponding author
  16. Angelo C Lepore  Is a corresponding author
  1. Department of Neuroscience, Jefferson Synaptic Biology Center, Vickie and Jack Farber Institute for Neuroscience, Sidney Kimmel Medical College at Thomas Jefferson University, United States
  2. Jefferson Weinberg ALS Center, Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, United States
  3. Department of Biology, Arcadia University, United States
  4. Department of Cell and Molecular Biology, Tulane Brain Institute, Tulane University, United States
https://doi.org/10.7554/eLife.89298.4
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Cite this article
  1. Mark W Urban
  2. Brittany A Charsar
  3. Nicolette M Heinsinger
  4. Shashirekha S Markandaiah
  5. Lindsay Sprimont
  6. Wei Zhou
  7. Eric V Brown
  8. Nathan T Henderson
  9. Samantha J Thomas
  10. Biswarup Ghosh
  11. Rachel E Cain
  12. Davide Trotti
  13. Piera Pasinelli
  14. Megan C Wright
  15. Matthew B Dalva
  16. Angelo C Lepore
(2024)
EphrinB2 knockdown in cervical spinal cord preserves diaphragm innervation in a mutant SOD1 mouse model of ALS
eLife 12:RP89298.
https://doi.org/10.7554/eLife.89298.4
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6 figures, 1 table and 1 additional file

Figures

Figure 1
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EphrinB2 expression was increased in ventral horn astrocytes.

SOD1G93A mouse ventral horn cervical spinal cord tissue immuinostained for ephrinB2 at 60 days (b), 120 days (c), endstage (d), and WT mouse age-matched control (a), scale bar: 200 µm. Quantification of ephrinB2 expression within the ventral horn shows a progressive increase in expression over time compared to WT controls (e). Endstage ephrinB2 expression in the thoracic (f, g) and lumbar (h–k) regions; scale bar: 200 µm, 100 µm, respectively. Endstage SOD1G93A mouse cervical spinal cord tissue co-immuostained for ephrinB2 (l, n, o, q) and neuronal and astrocyte lineage-specific markers NeuN (m–n) and GFAP (p–q), respectively; scale bar: 30 µm. Analysis in panels A-K: n=3–4 mice per genotype and per time point; 1–2 females and 2 males per condition. Analysis in panels L-O: n=3 mice per genotype and per time point; 1 female and 2 males per condition.

Figure 1—source data 1

File contains the raw data for Figure 1, panel E.

https://cdn.elifesciences.org/articles/89298/elife-89298-fig1-data1-v1.xlsx
Figure 2
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Lenti-shRNA injection reduced ephrinB2 expression in cervical spinal cord astrocytes.

We injected 60-day-old SOD1G93A mice with Lenti-GFP control or Lenti-shRNA-Efnb2 into cervical ventral horn (a). Injections were made bilaterally into six sites throughout C3-C5 to target the PhMN pool (b).Thirty µm transverse tissue sections of the cervical spinal cord show robust expression of the GFP reporter bilaterally within the ventral horn (c); scale bar: 500 µm. Cell lineage of viral transduction was assessed using three markers: neuronal marker NeuN, oligodendrocyte lineage marker Olig2, and astrocyte marker GFAP. Spinal cord tissue collected from SOD1G93A injected with Lenti-GFP vector was sectioned at 30 µm and immunostained for NeuN (d-f), Olig2 (g–i) and GFAP (j–l); scale bar: 150 µm. Quantification of transduction lineage was assessed by counting the total numbers of GFP+ cells that were co-labeled with each lineage-specific marker and expressing this as a percentage of the total number of GFP+ cells (m). We assessed the amount of knockdown achieved by the Lenti-shRNA-Efnb2 vector by immunostaining endstage SOD1G93A cervical spinal cord tissue with an anti-ephrinB2 antibody in both Lenti-GFP control (o, q) and Lenti-shRNA-Efnb2 (p, r) tissue; scale bar: 100 µm. Knockdown was quantified by counting the total number of GFP+ cells expressing ephrinB2+ within the cervical ventral horn (n). Analyses in all panels: n=3 mice per condition; 1 female and 2 males per condition.

Figure 2—source data 1

File contains the raw data for Figure 2, panels M and N.

https://cdn.elifesciences.org/articles/89298/elife-89298-fig2-data1-v1.xlsx
Figure 3
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EphrinB2 knockdown protected cervical spinal cord motor neurons and preserved functional innervation of the diaphragm in SOD1G93A mice.

SOD1G93A mice injected with Lenti-GFP control or Lenti-shRNA-Efnb2 were cresyl violet stained and MN counts were performed at 117 days of age. Thirty µm transverse cervical spinal cord tissue sections were stained with cresyl violet (a); scale bar: 250 µm. The dotted box outlines the ventral horn and area of the image shown in (c). MN populations within the ventral horn were quantified (b). Representative images show a greater loss of MNs in Lenti-Control (c) compared to the Lenti-shRNA-Efnb2 (d) group; scale bar: 100 µm. SOD1G93A mice injected with Lenti-GFP control or Lenti-shRNA-Efnb2 were assessed in vivo for PhMN-diaphragm innervation by electrophysiological analysis at 117 days of age. CMAP amplitudes were recorded from each hemi-diaphragm following ipsilateral phrenic nerve stimulation. Representative traces of Lenti-GFP (e) and Lenti-shRNA-Efnb2 (f) recordings show a larger CMAP amplitude in the Lenti-shRNA-Efnb2 group. Quantification of maximal CMAP amplitude shows significant preservation in the Lenti-shRNA-Efnb2-treated group compared to control (g). Analysis in panels A-D: n=4 mice per condition; 2 females and 2 males per condition. Analysis in panels E-G: n=4 mice per genotype and per time point; 2 females and 2 males per condition.

Figure 3—source data 1

File contains the raw data for Figure 3, panels B and G.

https://cdn.elifesciences.org/articles/89298/elife-89298-fig3-data1-v1.xlsx
Figure 4
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Knockdown of ephrinB2 in the cervical spinal cord ventral horn did not extend survival or delay onset of disease in SOD1G93A mice.

Biweekly weights of Lenti-shRNA-Efnb2 and Lenti-Control mice were recorded until endpoint sacrifice (a), and disease onset was measured for each animal when there was a 10% drop in body weight (b). Biweekly individual hindlimb grip strengths were assessed for each animal (c), and disease onset was recorded when the animal had a 10% decline in hindlimb grip strength (d). Each animal was also tested for forelimb grip strength (e), and disease onset was recorded when the animal had a 10% decline in forelimb grip strength (f). Weights, forelimb grip strength and hindlimb grip strength were taken biweekly starting one week prior to injection of Lenti-shRNA-Efnb2 or Lenti-GFP control, and all force measurements plotted were the average force (lb) of all animals combined in each group. Disease duration was determined by time from weight onset to endstage (g), hindlimb disease onset to endstage (h), and forelimb diseaseonset to endstage for each animal (i). Disease duration was also measured from the time of forelimb disease onset to time of hindlimb disease onset (j). Survival was measured as the day each animal reached endstage, which was determined by the righting reflex (k). Analyses in all panels: n=8–10 mice per genotype and per time point; 4–5 females and 4–5 males per condition.

Figure 4—source data 1

File contains the raw data for Figure 4, panels A-K.

https://cdn.elifesciences.org/articles/89298/elife-89298-fig4-data1-v1.xlsx
Figure 5
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EphrinB2 knockdown preserved morphological innervation of the diaphragm in SOD1G93A mice.

Diaphragm muscles were labeled with SMI-312 (green), SV2, (green) and alpha-Bungarotoxin (blue). Representative images of fully-innervated (a), partially-denervated (b) and completely-denervated ((c): arrowheads denote completely-denervated NMJs) NMJs are shown; all scale bars: 30 µm. Compared to SOD1G93A mice treated with Lenti-GFP (d), animals injected with Lenti-shRNA-Efnb2 (e) showed greater preservation of PhMN innervation of the diaphragm NMJ. Quantification revealed a significant increase in the percentage of fully-innervated NMJs (f) and a decrease in the percentage of completely-denervated NMJs (g) in the Lenti-shRNA-Efnb2 group compared to Lenti-GFP controls. The percentage of partially-denervated NMJs was not statistically different between the two groups (h). Analyses in all panels: n=4 mice per condition; 2 females and 2 males per condition.

Figure 5—source data 1

File contains the raw data for Figure 5, panels F-H.

https://cdn.elifesciences.org/articles/89298/elife-89298-fig5-data1-v1.xlsx
Figure 6
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EphrinB2 upregulation in human spinal cord from ALS donors with an SOD1 mutation.

Immunoblotting analysis on postmortem samples from human ALS donors with an SOD1 mutation and also non-diseased human samples. In the lumbar enlargement, there was a large increase in ephrinB2 protein expression in the SOD1 mutation ALS samples compared to the non-diseased controls (top blot). GFAP immunoblotting on the same lumbar spinal cord samples shows a robust increase in GFAP protein levels in the two samples with increased ephrinB2 expresison (middle blot). There was not increased ephrinB2 expression in a disease unaffected region in these same three ALS donor samples, as ephrinB2 protein levels were not elevated in the frontal cortex (top blot). Immunoblot for total protein (bottom blot). Demographic information: Donor 1 – death at 67 years; male; non-ALS; Donor 2 – death at 70 years; male; non-ALS; Donor 3 – death at 70 years; female; non-ALS; Donor 4 – death at 42 years; female; SOD1-D102H mutation; absence of C9orf72 repeat expansion; Donor 5 – death at 55 years; male; SOD1-A4V mutation; absence of C9orf72 repeat expansion; Donor 6 – death at 58 years; male; SOD1-V87A mutation; absence of C9orf72 repeat expansion.

Figure 6—source data 1

File contains the raw data for Figure 6.

https://cdn.elifesciences.org/articles/89298/elife-89298-fig6-data1-v1.xlsx

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Mus musculus)Transgenic SOD1G93A mice:
C57BL/6 J congenic line B6.Cg-Tg(SOD1*G93A)1Gur/J		The Jackson LaboratoryRRID: IMSR_JAX:004435Both female and male mice used
Genetic reagent (Mus musculus)Transgenic SOD1G93A mice:
B6SJL-Tg(SOD1*G93A)1Gur/J		The Jackson LaboratoryRRID:IMSR_JAX:002726Both female and male mice used
Biological sample (Homo sapiens)Spinal cord and cortex
samples from ALS donors		Biorepository of the Jefferson Weinberg ALS Center (1 of 3 ALS samples)
Project ALS (2 of 3 ALS samples)		All three ALS donors had an SOD1 mutation (donor 1: D102H mutation; donor 2: A4V; donor 2: V87A), and all three donors did not have a C9orf72 repeat expansion.
These three donors succumbed to ALS at 42 (female), 55 (male), or 58 (male) years of age.
Biological sample (Homo sapiens)Spinal cord and cortex
from non-ALS donors		NIH NeuroBioBankAge of death for these three non-ALS donors was 67, 70, and 70 years.
AntibodyMouse monoclonal
Anti-SV2
(Used for IHC)		Developmental Studies Hybridoma Bank, Iowa City, IARRID: AB_23153871:10
AntibodyMouse monoclonal
Anti-SMI312
(Used for IHC)		Covance, Greenfield, INRRID: AB_23149061:1000
AntibodyMouse monoclonal
Anti-NeuN
(Used for IHC)		EMD-Millipore, Temecula, CARRID: AB_22987721:200
AntibodyRabbit polyclonal
Anti-GFAP
(Used for IHC)		Dako, Carpinteria, CARRID: AB_100134821:400
AntibodyMouse monoclonal
Anti-GFAP
(Used for Westerns)		BD Bioscience, Franklin Lakes, NJCat. #6105661:2000
AntibodyRabbit polyclonal
Anti-Olig2
(Used for IHC)		EMD-Millipore, Temecula, CARRID: AB_22990351:200
AntibodyGoat polyclonal
Anti-ephrinB2
(Used for IHC)		R&D Systems, Minneapolis, MNRRID: AB_22619671:50
AntibodyRabbit polyclonal
Anti-ephrinB2 antibody
(Used for Westerns)		Abcam, Cambridge, MARRID: AB_111568961:500
AntibodyGoat polyclonal
Anti-EphA4
(Used for IHC)		R&D Systems, Minneapolis, MNRRID: AB_20993711:100
AntibodyRabbit polyclonal
Anti-GFP
(Used for IHC)		Aves Labs, Davis, CARRID: AB_100002401:500
Recombinant DNA reagentLenti-shRNA-Efnb2;
VSVG.HIV.SIN.cPPT.U6.
SbRmEphrinB2.4.CMV.EGFP		This studyVector generated by the Dalva lab
Recombinant DNA reagentLenti-Control;
VSVG.HIV.SIN.cPPT.
U6.Empty.CMV.EGFP		This studyVector generated by the Dalva lab
SoftwareScope 3.5.6 softwareADInstruments, Colorado Springs, CORRID: SCR_001620
SoftwareImageJ/Fiji softwareRRID: SCR_003070
SoftwareBio-Rad Image Lab softwareBio-Rad, Hercules, CARRID:SCR_014210
SoftwareGraphpad Prism 6Graphpad Software Inc, LaJolla, CARRID: SCR_002798
Other (Microscope)Olympus FV1000 confocal microscopeOlympus, Center Valley, PARRID: SCR_014215‘NMJ analysis’ subsection of the Materials and methods section
Other
(Microscope)		Zeiss Axio M2 Imager confocal microscopeZeiss, Hebron, KYRRID:SCR_020922‘Motor neuron counts’ subsection of the Materials and methods section

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  1. Mark W Urban
  2. Brittany A Charsar
  3. Nicolette M Heinsinger
  4. Shashirekha S Markandaiah
  5. Lindsay Sprimont
  6. Wei Zhou
  7. Eric V Brown
  8. Nathan T Henderson
  9. Samantha J Thomas
  10. Biswarup Ghosh
  11. Rachel E Cain
  12. Davide Trotti
  13. Piera Pasinelli
  14. Megan C Wright
  15. Matthew B Dalva
  16. Angelo C Lepore
(2024)
EphrinB2 knockdown in cervical spinal cord preserves diaphragm innervation in a mutant SOD1 mouse model of ALS
eLife 12:RP89298.
https://doi.org/10.7554/eLife.89298.4