This study demonstrates a role for altered GSK-3? activity resulting in enhanced DRG
neurite outgrowth following excitotoxic spinal injury. Biochemical and immunohistochemical
approaches showed that excitotoxic spinal lesions resulted in strong (early and persistent)
GSK-3? inhibition in the spinal cord dorsal horn and adjacent DRG neurons. Intrathecal
treatment with a PI3K inhibitor (LY294002), a known GSK-3? activator, blunted injury
induced neurite initiation and elongation. Short term intrathecal delivery of LY294002,
initiated at the time of injury, significantly reduced GSK-3? inhibition in the spinal
dorsal horn and adjacent DRG and prevented the development of at-level dysesthesias.
These data suggest that QUIS induced spinal injury results in GSK-3? inhibition and
peripheral afferent growth that contributes to the development of at-level neuropathic
pain following SCI.
Peripheral growth responses in the development of at-level sensory disturbances
Historically, efforts to understand the development of neuropathic pain following
SCI have focused on the injury epicenter and supraspinal regions 51]. This study along with previous results from our lab 6] has shown that QUIS spinal injury results in at-level induced dysesthesias associated
with enhanced DRG growth. These finding are supported by Bedi et al. which showed
similar at-level peripheral growth promoting effects along with spontaneous activity/hyperexcitability
in a contusion model, suggesting that the growth responses were not unique to the
SCI model 7], 8]. Consistent with these reports, we demonstrated that spinal injury induced DRG growth
was evident early (3Â days post-injury) and persisted throughout a 14Â days survival
period. Although latent DRG growth (14Â day post-injury) was evident in small and large
fibers, early injury induced growth (3Â day post-injury) was most prominent in small
DRG neurons, a finding that is supported by reports showing a temporal window of nociceptive
sprouting occurring early post-SCI 8], 14]. It is possible that these growth promoting effects in small (C and A?) and large
(A?) fibers contribute to the intraspinal sprouting and structural plasticity in dorsal
horn circuitry that may, in part, contribute to pain post-SCI 11], 14], 15], 52]–56]. These findings along with the increased GSK-3?
P
expression (and expansion into deeper layers) in the dorsal horn, provide further
evidence to support a role for maladaptive structural plasticity post-spinal injury.
Collectively, the early growth of peripheral afferent post-spinal injury suggests
that these responses may lead to maladaptive plasticity and reorganization in the
dorsal horn afferents contributing to the development of at-level sensory disturbances.
GSK-3? as a regulator of DRG outgrowth following SCI
These studies provide the first evidence to support a role for GSK-3? signaling as
a regulator of peripheral afferent plasticity following spinal injury. GSK-3? is well-established
for its role in neuronal morphogenesis via regulation of an extensive list protein
substrates, such as collapsin response proteins 29], 32], 57], APC 20], CLASP-associated proteins 58], 59], ?-catenin 28], 30], microtubule associated proteins 60]–64]. Regulation of these cytoskeletal proteins to promote neurite elongation is accomplished
through fine-tuned control of GSK-3? activity 22], 39], were partial inhibition of GSK-3? via phosphorylation on Serine-9 leads to neurite
elongation, and strong (inhibition) suppression or activation of GSK-3? leads to termination
of neurite outgrowth. Here we demonstrated that QUIS induced SCI results in GSK-3?
inhibition (spinal cord dorsal horn and adjacent DRG) and robust DRG neurite growth.
We demonstrated that opposing actions directed towards GSK-3? activation, using the
PI3K inhibitor LY294002, effectively suppressed DRG neurite initiation and elongation
following QUIS induced spinal injury. Although LY294002 is a potent inhibitor of PI3K/Akt
pathways that targets GSK-3? activation, its effects are not direct; and therefore
LY294002 may also activate Akt/mTOR pathways to restrict neurite outgrowth 20], 41], 65]. While LY294002 leads to activation of the mTOR pathway in the central nervous system,
emerging evidence suggests that mTOR signaling does not control sensory afferent growth
providing further evidence that PI3K-GSK-3? signaling is critical in mediating post
injury DRG growth 42]. Our finding that intrathecally delivered LY294002 blocked the SCI induced increases
in GSK-3?
P
expression also supports that these effects are, in part, mediated through PI3K-GSK-3?
signaling. This is in agreement with studies showing potent effects of LY294002 to
activate GSK-3? and prevent neurite outgrowth 20], 41], 66]. Collectively, these data provide morphological evidence for the role of PI3K-GSK-3?
signaling in SCI induced primary afferent growth.
GSK-3? is a convergent signaling target to pro-nociceptive factors released in response
to spinal injury
In support of GSK-3? as a mediator of aberrant afferent growth and sensory disturbances,
GSK-3? is a convergent signaling effector of established pro-nociceptive signals including
nerve growth factor (NGF), PI3K, and Wnts 26], 34], 35], 43], 45], 46], 67], 68]. Numerous studies have shown that peripheral and central nervous system injury results
in upregulation of neurotrophins and Wnts that may inhibit GSK-3? and contribute the
anatomical remodeling in spinal dorsal horn and DRG to induce neuropathic pain 34], 67], 69]–71]. Furthermore, efforts to block the effects of NGF, Wnts, and PI3K are effective at
preventing the development of neuropathic pain and autonomic dysreflexia 11], 34], 36], 45], 46], 69], 72]. Substantial evidence suggests that these extracellular signals released following
injury converge to phosphorylate/inactivate GSK-3? to cause neurite elongation 20], 26], 73]–77]. Interestingly, studies have demonstrated that activation of PI3K is required for
NGF-induced neurite elongation, and therefore our finding that demonstrated alterations
PI3K-GSK-3? signaling following QUIS spinal cord injury provides an intracellular
link that underlies these structural responses following injury 20], 78], 79].
Altered GSK-3? activity in the spinal dorsal horn and DRG in neuropathic pain
Recent studies have provided evidence for the role of altered GSK-3? activity in development
of neuropathic pain, showing that GSK-3?
P
(inactive) is distributed in the spinal cord dorsal horn axons where it is positioned
to influence sensory growth and synaptic plasticity 66], 80]. Although the temporal relationship between GSK-3? activities and pain following
nervous system injury are largely unexplored, Weng et al. recently showed that peripheral
nerve injury resulted in early increases in GSK-3?
P
in the spinal cord dorsal horn 80]. This finding is consistent with our data (Figure 1, 3) showing early and persistent inhibition of GSK-3? in the DRG and spinal cord dorsal
horn following excitotoxic SCI (evident at 3 and 14Â days post-injury). In support
of these findings, peripheral nerve injury induces early activation PI3K-Akt in spinal
dorsal horn and DRG neurons, an established upstream signal to inhibit GSK-3? activity
36]. Furthermore, activation of PI3K/Akt following peripheral nervous system injury are
strongly expressed in small, nociceptive neurons that have an established role in
early central sensitization which contributes to the development of neuropathic pain
36], 43], 81]. Interestingly, reports have shown that activation of PI3K-GSK-3? following injury
are transient, occurring within 3Â days post-injury 36], 80]. Our reported inhibition of GSK-3? that extended throughout 14Â days post-injury may
be attributed to differences between peripheral vs. central injury, as well as the
progressive nature created by the QUIS-excitotoxic spinal injury 48]. Additional evidence for the role of GSK-3? in SCI pain is supported by the finding
that NGF, an inhibitor of GSK-3? with a pivotal role in primary afferent sprouting,
is increased in the spinal cord and DRG following SCI 20], 25], 35], 67]. Further studies are needed to examine how different SCI models impact GSK-3?, activity
as well as the time course for these changes. Nonetheless, our studies provide the
first link by which spinal injury can directly impact GSK-3? activity in a manner
that would lead to sensory afferent outgrowth/sprouting and sensory dysesthesias.
Although GSK-3? plays a pivotal role in regulating structural neuronal plasticity
20], 22], 27], 79], GSK-3? also regulates other cellular functions including cell survival pathways,
and more recent reports demonstrating a role in pro-inflammatory processes 16], 19], 80], 82]. As such, activation of GSK-3? may contribute to enhanced secondary damage and neuronal
apoptosis associated with SCI 83]. To ensure that our observed differences with LY294002 treatment were not related
to enhanced neuronal death, cell quantification of the proportion of different neuronal
types and cell densities showed that the overall number and distribution of cell size
of our neuronal cultures from LY294002 treated were comparable to those from sham-operated
and QUIS-injected animals (data not shown) 6], 8], suggesting that our reported differences were not related to enhanced neuronal death
from drug treatment. Furthermore, there are emerging reports showing that application
of GSK-3? inhibitors may prevent the development of pain or reduce secondary damage
following peripheral or central nervous system injury potentially through reduced
inflammatory responses 83]–85]. Several of these studies employ preemptive treatments 80], 84], 85], strategies that may have limited efficacy for the treatment of neuropathic pain
following SCI resulting from unexpected trauma or ischemia. Other studies use systemic
(intraperitoneal) drug delivery which lacks targeted delivery and may, therefore,
have off target consequences that contribute to the differences observed with activation
vs. inhibitory action of GSK-3? to prevent neuropathic pain 83], 86]. It is plausible that the reported protective effects of both GSK-3? inhibition and
activation may be related to different mechanism of action based on mode of delivery
(intraperitoneal vs. intrathecal), pain model (acute vs. neuropathic), and the timing
of drug administration (preemptive vs. acute short term). Our study provides evidence
into the temporal and spatial alterations of GSK-3? activity following spinal injury;
to establish that early intratehecal delivery of LY294002 (a known GSK-3? activator)
was successful at preventing the development of at-level dysesthesias.
PI3K-GSK-3? signaling in sensory and motor neurite outgrowth
Molecular signaling pathways, such as PI3K-GSK-3? that control axonal growth are shared
between sensory and motor fibers 20], 25], making therapeutics that regulate structural plasticity challenging. The growth
promoting effects of GSK-3? has made inhibition of this enzyme an appealing target
to promote motor fiber regeneration following SCI. This has resulted in clinical and
experimental studies using GSK-3? inhibitors to promote axonal regeneration following
SCI 25], 87], 88]. Recent studies now raise the possibility that GSK-3? inhibition will not effectively
promote long-distance axonal growth and may further restrict spinal cord regeneration
32], 87], 89]. Here we also demonstrated that excitotoxic SCI resulted in GSK-3? inhibition, and
further suppression with GSK-3? inhibitors may prevent sensory outgrowth and possibly
the desired axonal motor fiber regeneration.
Understanding of the temporal and spatial nature of the growth promoting effects are
of particular importance following SCI, where efforts to control structural neuroplasticity
and regeneration are opposing, to promote motor fiber regeneration and yet limit sensory
sprouting and growth that contributes to pain syndromes post-injury 52], 53]. Our finding that DRG growth responses contribute to at-level pain syndromes is significant
as therapeutics to prevent maladaptive growth responses work towards targeted approaches
to improve clinical efficacy 52], 53], 90], 91]. Our short term intrathecal delivery of LY294002 was effective at blocking peripheral
outgrowth and at-level dysesthesias, however future studies investigating targeted
delivery to the periphery (DRG) are needed to prevent pain and allow for motor fiber
recovery post-SCI. Furthermore, our previous report and other studies have demonstrated
that SCI results in primary afferent growth and sensitization in segments remote from
the site of injury 6], 8], 14], 15], 92]. Studies are underway to investigate the effect of injury induced growth on distant
segments to determine if activation of GSK-3? can prevent and reverse the development
of below-level hyperalgesia and allodynia following SCI 48]. This study identified a therapeutic window specific to target inhibition of at-level
sensory growth. Future studies using traumatic SCI models to incorporate the motor
complications and determine the appropriate therapeutic timeline to maximize motor
recovery while minimizing the risk for the development of pain are needed.