Spatiotemporal and cell-type specific changes in ephrin-B2 in ALS
D Scura, M Urban, K Li, W Zhou, J Rothstein, M Dalva, A Lepore
Department of Neuroscience, Faber Institute for Neurosciences, Sidney Kimmel Medical College, Thomas Jefferson University, 900 Walnut Street
EphrinB2 is upregulated in end stage spinal cord Ephrin-B2/EphA4 interaction in SOD1 tissue
Introduction
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder
characterized by motor neuron (MN) loss, ultimately resulting in
patient death as a result of respiratory compromise. The exact
causative mechanism of this disease is unknown; however, studies
allude to a multitude of pathological events - one of which is altered
Eph/ephrin signaling. We evaluated the role of this pathway by
WT
SOD1G93A
examining the levels of ephrin-B2 and its receptor EphA4 in both the
Fig 1: Wild type vs SOD1G93A mice, cervical region. 5x. GFAP, green. EphrinSOD1G93A mouse, an established rodent model of disease, and
B2, blue. EphA4, red. DAPI, yellow.
human ALS post-mortem spinal cord tissue.
Additionally, we have shown in preliminary studies that knockdown
of ephrin-B2 via intraspinal shRNA injection prolongs disease
duration and extends survival of SOD1G93A mice, implicating a
potential therapeutic target for ALS patients.
Conclusion
GFAP
A
Ephrin-B2
B
Ephrin-B2
B
EphA4
C
D
GFAP/EphrinB2/EphA4/DAPI
WT
EphA4
E
F
Ephrin-B2
G
EphA4/Ephrin-B2/DAPI
GFAP/Ephrin-B2
C
SOD1G93A
EphA4
H
GFAP
Ephrin-B2
B
I
GFAP
J
EphA4/EphrinB2/DAPI
GFAP/Ephrin-B2
C
IHC analysis indicate that ephrin-B2 is upregulated in end-stage
SOD1G93A mice, in comparison to wild type controls. Our findings
also indicate the possibility that overexpression of Eph-A4 may
also contribute to disease progression.
Tissue was collected from SOD1G93A mice at 60 days (presymptomatic), 120 days (disease onset), and ~150 days (endstage). Spinal cord samples from the cervical, thoracic, and lumbar
regions was collected used for IHC analysis and mRNA
quantification. mRNA quantification was done using Qiagen RNeasy
Mini Kit. TaqMan Assays was used for qRT-PCR.
GFAP
A
DAPI ALS
Upregulation is specific to the ventral horn
A
Materials and Methods
DAPI WT
SOD1G93A
Fig 2: Comparison of ventral (top) and dorsal (bottom) horns of SOD1G93A wild
type 120 day mice . GFAP, A; Ephrin-B2, B; Merge, D. Consistent with ALS
disease pathology, the spinal cord samples show an increased number of
reactive astrocytes in the ventral horn in addition to an up-regulation of ephrinB2. Astrocytes, white arrow. Reactive astrocytes expressing ephrin-B2, red
arrow.
Thoracic region of SOD1G93A end stage mice. Top panel shows staining for wild
type end stage mice. GFAP: A, I; Ephrin-B2: B,F; EphA4: C,E,H; Merge: D,G,J.
Middle panel shows staining for only EphA4 and Ephrin-B2, while the bottom
panel shows staining for EphA4 and GFAP.
Ephrin-B2 mRNA Quantification
SOD1 End Stage GFAP/Ephrin-B2
GFAP
A
Ephrin-B2
B
C
GFAP/Ephrin-B2
C
GFAP/Ephrin-B2
Eph/ephrin Signaling
WT
GFAP
A
Left. Bidirectional
modulation of synaptic
functions by Eph/ephrin
signaling
Rüdiger Klein
Nature Neuroscience 12,
15 - 20 (2009) Published
online: 23 November
2008
Ephrin-B2
B
SOD1G93A
Fig 3: Lumbar region of SOD1G93A end stage mice. 20x. Top panel, wild type.
Bottom panel, disease pathology. GFAP,A; Ephrin-B2, B; Merge, C. Astrocytes,
white arrow. Ephrin-B2, yellow arrow. Reactive astrocytes expressing ephrinB2, red arrow.
Ephrin-B2 mRNA
expression for SOD1 and
wild-type control mice for
various regions of spinal
cord. Amplification plots
indicate consistent
amplification of cDNA. Bar
graph (right) indicates a
significant difference in
ephrin-B2 mRNA levels
between the same and
different regions of spinal
cord (p<.05).
SOD1 End Stage GFAP/Ephrin-B2/NeuN
A
GFAP
B
Ephrin-B2
C
NeuN
D
GFAP/EphrinB2/NeuN
Future Directions
Right. Cross-reactivity of
Eph/ephrin signaling. Mosch,
Birgit et al. “Eph Receptors and
Ephrin Ligands: Important Players
in Angiogenesis and Tumor
Angiogenesis.” Journal of
Oncology 2010 (2010): e135285.
Web. 6 Apr. 2015.
•
Further
analysis
of
various
time-points
of
disease
progression.
WT
• Quantification of ephrin-B2 expression and co-expression with
GFAP
Ephrin-B2
NeuN
GFAP/EphrinB2/NeuN
B
A
D
C
GFAP on astrocytes.
• Analyze EphA4 expression, cell-type specificity, and localization
of EphA4 expression across disease timepoints.
• Investigate localization of ephrin-B2/ephA4 for a potential
G93A
SOD1
interaction and subsequent signaling pathway leading to disease
progression .
Fig 4: Thoracic region of SOD1G93A end stage mice. 20X. Top panel, wild type.
Bottom panel, disease pathology. GFAP,A; Ephrin-B2, B; NeuN, C; Merge,D.
Astrocytes, white arrow. Ephrin-B2, yellow arrow. Reactive astrocytes
expressing ephrin-B2, red arrow.
Acknowledgments
This work was supported by the Margaret Q. Landenberger Research Foundation (to A.C.L.).