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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 8, 2001 as doi:10.1096/fj.00-0857fje. |
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* Department of Anesthesiology and
Pathology, University of California, San Diego, School of Medicine, and VA Medical Center, San Diego, La Jolla, California 92093-0629, USA
2Correspondence: Department of Anesthesiology, School of Medicine, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0629, USA. E-mail: wcampana{at}ucsd.edu
SPECIFIC AIMS
Using two experimental models of painful neuropathy and axonal degeneration, including one that caused enhanced TUNEL staining (apoptosis), we compared Epo/EpoR events in the peripheral nerve fibers undergoing Wallerian degeneration and in their corresponding DRG with and without apoptosis. We hypothesized that DRG neurons and Schwann cells express Epo/EpoR constitutively and that their expression is altered during painful nerve injury and includes activation of JAK2 signaling in Schwann cells.
PRINCIPAL FINDINGS
1. Identification of Epo/EpoR in normal and injured sciatic nerves
To identify the presence of Epo/EpoR in peripheral nerve, we
performed immunohistochemistry of rat sciatic nerve sections with and
without nerve injury. Nerve injury was caused by either gently crushing
the rat sciatic nerve 2 mm distal to the DRG or by applying four loose,
chromic gut ligatures to the distal sciatic nerve, this latter method
being known as chronic constriction injury (CCI). Both forms produce
Wallerian degeneration of many, but not all, nerve fibers distal to the
lesion. Using an anti-Epo polyclonal antibody, immunoreactive material
was present in the axoplasm and Schwann cell cytoplasm. After 5 days of
Wallerian degeneration caused by CCI, less Epo immunoreactivity was
noted in axoplasm, yet Schwann cells demonstrated enhanced
immunoreactivity for Epo. EpoR was expressed in normal axons and
Schwann cells and was reduced only in the axoplasm during CCI
neuropathy.
2. Identification of Epo/EpoR in normal and injured DRG neurons
Epo was present in 
one-half of normal DRG neurons and stained
nuclear and cytoplasmic spaces in both small- and large-diameter
neurons. The distribution was not appreciably changed by CCI
neuropathy. Immunostaining of EpoR in DRGs of normal rats revealed the
presence of EpoR on many cell bodies. Generally, a large proportion of
EpoR was present in the cytoplasmic portion of the DRG cell bodies.
However, with higher concentrations of anti-EpoR (1:500), distinct
punctate staining at the plasma membrane was observed. After two
different injuries—CCI and crush—the proportion of EpoR
immunostaining in DRG cell bodies changed only in the crush model,
which was created closer to the DRG. There was a significant reduction
in EpoR after 7 days of adjacent crush injury compared with D0 controls
(P<0.01).
EpoR was identified at a molecular mass of 70 and 90 kDa.
Immunoprecipitates from adjacent crush-injured rats contained less EpoR
(both the 70 and 90 kDa protein) than uninjured rats (Fig. 1
). We also probed the blot for antiphosphotyrosine and observed that
after adjacent crush injury, phosphorylation of the 90 kDa band was
greater than the 70 kDa band (Fig. 1)
.
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3. Activation of EpoR in primary Schwann cells
Because of the abundant immunolocalization of EpoR in Schwann
cells, we investigated the direct effects of Epo on JAK2
phosphorylation known to be associated with EpoR. Upon Epo binding to
EpoR, there is an increased affinity for JAK2, a member of the Janus
family of cytoplasmic tyrosine kinases. JAK2 is activated by
transphosphorylation of its active site and phosphorylates tyrosine
residues on the intracellular domain of the Epo receptor.
Immunoprecipitation of primary Schwann cells stimulated with or without
Epo revealed prominent phosphorylated bands at 130 kDa and 70 kDa
(Fig. 2
). The smaller tyrosine-phosphorylated protein at 70 kDa is likely to
be EpoR. There was also an increase in JAK2 phosphorylation by Epo in a
rat Schwannoma cell line, RN22 (Fig. 2)
.
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CONCLUSIONS
We report localized Epo and EpoR in the sciatic nerve and DRG of adult rats. Our data indicate that Epo is produced in the cell bodies and axons in normal DRG and is up-regulated in Schwann cells after painful chronic constriction injury. The distribution of EpoR was different from Epo, indicating that ligand binding was not the sole reason for Epo immunoreactivity in tissues. We identified EpoR in some axons and neuronal cell bodies in the DRG, endothelial cells, and Schwann cells of normal nerve. However, the distribution and intensity of immunostaining for EpoR in Schwann cells were similar for both injured and uninjured sciatic nerve. These findings are consistent with changes in Epo and EpoR mRNA expression in astrocytes and neurons under hypoxic conditions in culture: Epo is up-regulated and EpoR is not.
The presence of Epo and EpoR in axons of normal nerve suggests they are integral components of neuronal function and that changes after nerve injury may contribute to aberrant cellular activity. The up-regulation of Epo in Schwann cells after nerve injury is not surprising, as it has been shown that hypoxic conditions increased Epo mRNA in central nervous system (CNS) glia. Enhanced production of Epo at the injury site may regulate several processes. Increased Epo may facilitate retrograde transport of Epo to the sensory neurons of the DRG. Transported Epo/EpoR complexes to specific subpopulations of neurons could potentially regulate signaling cascades involved in aberrant neuronal activity. In addition, increased levels of Epo at the injury site may potentiate Schwann cell proliferation through an autocrine mechanism involving JAK2 activation. After nerve injury, Schwann cells proliferate in the absence of axons producing their own growth factors. Thus, similar to Epo’s role in hematopoiesis (a potent inducer of erythroid precursor cells), Epo may induce dedifferentiated Schwann cells to proliferate. These studies are ongoing.
The role of Epo/EpoR after injury may also be linked to antiapoptotic activity. In the CNS, Epo is a potent survival factor for several types of neurons including rat septal cholinergic neurons in vivo, hippocampal, and cerebral cortical neurons. Epo has also been demonstrated to protect hippocampal neurons from lethal ischemic damage and to prevent ischemia-induced learning disability. We demonstrated in the CCI model that there was very little induction of DRG neuronal TUNEL staining (an indicator of apoptosis), a finding that is consistent with the expectation that adult sensory neurons typically are insensitive to peripheral nerve injury at the sciatic notch and growth factor withdrawal. However, in our model of nerve crush 2 mm distal to the DRG, cell bodies stained positively by TUNEL labeling, which suggests the presence of apoptosis. This finding was also expected since it had been demonstrated earlier that nerve injury 7 mm distal to the DRG induced a 15% absolute loss of L5 DRG neurons. We observed reduced neuronal staining and a generalized loss of EpoR intensity within 4 days, with the greatest losses observed in small ‘B’-type neurons. At similar time points corresponding to TUNEL labeling in adjacent crush neurons, cell bodies and axons of the DRG demonstrated a significant loss of EpoR without loss of NF200-positive neurons. The loss of EpoR correlated consistently with TUNEL labeling of DRG cell bodies, which would suggest that the loss of EpoR was not due simply to a loss of neurons.
The type of neurons in the DRG that expressed EpoR included a subpopulation of large, light-staining neurons that corresponded to typically trkA-independent neurons with myelinated axons, which represented 40% of the lumbar DRG neurons. These DRG neurons are identified by NF200, a marker of neurofilaments. These myelinated axons are connected in the periphery to mechanosensitive endings such as Pacinian corpuscles, hair follicle afferents, and muscle spindles. These findings suggest that EpoR may also play a unique role in the maintenance of touch sensation. However, EpoR was also clearly expressed on other DRG cell bodies, including IB4-positive neurons (data not shown), suggesting that EpoR effects are not limited to myelinated axons.
We observed the presence of EpoR in the DRG and identified its
molecular mass to be 70 and 90 kDa. Epo receptor size has been shown
to be tissue-specific and to possess different affinities for Epo.
Cross-linking experiments with PC12 cells demonstrated an Epo receptor
of 105 kDa with a Kd of 16 nM. Erythroid cells have
two forms of Epo receptor, including a 140 kDa and 120 kDa form with a
Kd of 90 pM. Our findings are most closely related
to the size observed for endothelial cells: 79 kDa with an affinity of
2–8 nM. We also showed changes in phosphorylation state of the 90 kDa
protein after adjacent crush injury suggesting that post-translational
processing of different sized Epo receptors is also relevant to EpoR
activity. In Schwann cells, where EpoR was abundantly expressed, Epo
appears to activate signaling molecules similar to those observed in
hematopoietic cell types. JAK2 was phosphorylated in response to Epo
and further confirms the essentiality of JAK2 in Epo signaling outside
of erythropoiesis.
We conclude that Epo and EpoR are produced locally in cells of the peripheral nervous system. Epo is up-regulated in the sciatic nerve, particularly in Schwann cells after painful CCI neuropathy. EpoR is located on Schwann cells and stimulates JAK2 phosphorylation. In a neuropathy model having many TUNEL-labeled DRG cell bodies (crush) and mechanical hyperalgesia, EpoR was down-regulated in small and large NF200-positive, mechanosensitive neurons. In a painful neuropathy model where minimal TUNEL labeling (CCI) was observed, EpoR did not appear to be down-regulated.
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FOOTNOTES
1 To read the full text of this article, go
to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0857fje ; to
cite this article, use FASEB J. (June 8, 2001)
10.1096/fj.00-0857fje 
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