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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  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. …

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 …

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 …

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). …

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)…

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 …

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

Additional files

MDAR checklist
https://cdn.elifesciences.org/articles/89298/elife-89298-mdarchecklist1-v1.docx

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