# Potential Therapeutic Targets for PPARγ after  Spinal Cord Injury

Authors: Dana M. McTigue
Journal: PPAR Research
 (2008-02-05)

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## Abstract

Traumatic injury to the spinal cord results in multiple anatomical, physiological, and functional deficits as a result of local neuronal and glial cell death as well as loss of descending and ascending axons traversing the injury site. The many different mechanisms thought to contribute to protracted secondary cell death and dysfunction after spinal cord injury (SCI) are potential therapeutic targets. Agents that bind and activate the transcription factor peroxisome proliferator-activated receptor-γ (PPAR-γ) show great promise for minimizing or preventing these deleterious cascades in other models of CNS disorders. This review will summarize the major secondary injury cascades occurring after SCI and discuss data from experimental CNS injury and disease models showing the exciting potential for PPARγ therapies after SCI.


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## Body

## 1. What is PPARγ?

“PPAR” is an acronym for peroxisome
proliferator-activated receptor, which refers to a family of nuclear hormone
receptors that function as ligand-activated transcription factors. Three PPAR isoforms
have been identified to date, including PPARα, PPARβ/δ, and PPARγ (for review, see [1–3]). Upon activation, PPAR
molecules heterodimerize with retinoid X receptors (RXRs), which are the
nuclear receptors for 9-cis retinoic
acid.  After dimerization, the PPAR/RXR
complex binds to specific promoter sequences on target genes where it can promote
or repress gene activation. Most initial research on these molecules focused on
their role in lipid metabolism and homeostasis [1, 4]. However, it is now known that PPARs function in a
much broader array of physiological functions, both under normal conditions and
following injury or disease. In particular, upregulation of PPARγ mRNA has
been detected in inflammatory cells and in experimental models of CNS injury
such as ischemic stroke [5, 6]. PPARγ agonists appear to have potent anti-inflammatory and
neuroprotective actions [7–9]; thus, this transcription factor may be involved in
coordinating cellular responses to CNS injury. This also presents the
opportunity to enhance neuroprotection by leveraging PPARγ expression
through administration of specific agonists following CNS damage. Indeed, over the
past decade, several studies have revealed beneficial actions of promoting PPARγ
activation in experimental models of CNS injury, ischemia, and disease. Less
work has examined the potential of promoting PPARγ activation following injury to the spinal cord, for
which current clinical therapies are limited. This review will summarize the
documented beneficial actions of PPARγ following CNS injury and illustrate how they may also
promote anatomical and behavioral recovery after spinal cord injury (SCI).


## 2. Spinal Cord Injury: The Facts

In the United States, a
new SCI is sustained on average every 41 minutes, which results in∼1100 new
cases each year. The majority of these injuries are caused by motor vehicle
accidents, followed by accidents such as falling from ladders or diving into
shallow water [10]. Most SCI's occur in young individuals, particularly
males—in their late
teens or twenties. Because medical care has improved dramatically during the
previous century, most individuals can expect to live many years following an
SCI. Their lives, however, are not easy and they have many issues with which to
deal on a daily basis. In the eyes of most uninjured people, the most obvious
problem affecting SCI individuals is their inability to walk. While this is clearly
a significant obstacle to overcome, a recent survey of paraplegics and
quadriplegics revealed that regaining locomotor function is actually of lesser
importance to them compared to the many other issues they face [11]. For instance, quadriplegics would prefer
restoration of hand and arm function over walking. Paraplegics’ top choice
would be regaining normal sexual function, an important issue in terms of
relationships with significant others, and the ability to have a family.  Also high on the list for all spinal-injured
people was return of bowel and bladder function. Other serious issues many face
include potentially fatal autonomic dysreflexia, pressure sores that can take several
months to heal, and untreatable intractable pain [10].Since it is impossible to prevent the occurrence of most SCIs, our best hope is to improve the level of
recovery achievable after an SCI occurs. Most SCIs result from a
contusion-type injury in which the vertebral bodies and/or intervertebral discs
are rapidly displaced into the spinal canal causing crushing and bruising of
the delicate spinal tissue [12, 13]. The initial impact leads to immediate hemorrhage and
rapid cell death at the impact site. This is followed by multiple secondary
injury cascades that cause further tissue loss and dysfunction [10, 14]. If these secondary injury processes
were minimized or
eliminated, the outcome for patients would be greatly improved. Many of these
cascades are potential targets for intervention by activation of the
transcription factor PPARγ. Indeed, two recent studies demonstrated that
treatment of SCI rodents with a PPARγ agonist results in significantly improved anatomical
sparing and locomotor abilities [15, 16]. The rest of this review will discuss specific secondary
injury processes that occur after SCI and how PPARγ
activation may be used to lessen their impact.


## 3. Glutamate Excitotoxicity: A Good Transmitter Gone Bad

Within minutes of SCI,
extracellular glutamate levels rise within and around the injury site [17]. This potent neurotransmitter can then diffuse to
surviving cells, bind to surface receptors, and lead to what is known as
excitotoxic cell death [18]. Especially vulnerable to excitotoxicity are neurons
and oligodendrocytes, which express a full complement of glutamate receptors.
Loss of neurons at the injury site will lead to direct denervation and
paralysis of muscle fibers innervated by those neurons, thereby contributing to
motor deficits. Because a significant amount of sensory processing occurs
within the spinal cord, especially that involved in pain and temperature
sensation, loss of neurons can also lead to hypersensitivity, paresthesia,
enhanced and prolonged pain, and/or total loss of pain and temperature
sensation. Excitotoxic injury to oligodendrocytes can result in demyelination
of axons around the injury site. This, in turn, will lead to a drastic
reduction or complete halt of axonal transmission, thereby enhancing the disconnection between the
brain and spinal segments below the level of injury. Thus, excitotoxicity has
the potential to markedly exacerbate the functional problems encountered after
SCI. Indeed, the involvement of excess glutamate in cell death after SCI was
demonstrated by studies in which early treatment with glutamate antagonists
significantly enhanced tissue preservation and functional recovery following
SCI in rats [19, 20].A major mechanism responsible
for maintaining low extracellular glutamate levels is astrocytic uptake via
glutamate transporters, including GLT1/EAAT2
which is responsible for removal of up to 90% of extracellular glutamate. While
glutamate transporters are effective under basal conditions, they become
saturated when glutamate levels rise substantially above normal. Thus, a
mechanism for increasing expression of GLT1/EAAT2
and other glutamate transporters could be highly beneficial after SCI. Recent
work reveals that PPARγ activation may do just that. Using a cell culture model
of ischemic preconditioning, Romera et al. [21] showed that preconditioning upregulates PPARγ
expression in neurons and astrocytes, and that treatment of the cultures with a
PPARγ agonist significantly increased astrocytic expression of GLT1/EAAT2
mRNA and protein. They also showed that this increased expression translated
into enhanced glutamate uptake and reduced cell death. The proposed mechanism
was a direct increase in EAAT2 promoter activity induced by activated PPARγ. A
direct neuroprotective action by PPARγ activation under
excitotoxic conditions has also been demonstrated using
cultures of pure cortical neurons [22]. In vivo evidence supports the notion that PPARγ activation is protective
against glutamate excitotoxicity. For instance, treatment with a PPARγ agonist decreased neuron
loss caused by intracortical injection of a glutamate receptor agonist [22]. While changes in glutamate
levels in SCI models treated with PPARγ agonists have not yet been measured, protection
against glutamate excitotoxicity is a plausible mechanism by which PPARγ could
improve outcome after SCI.


## 4. Lipid Peroxidation

A well-documented pathological
process occurring early after SCI is the formation of reactive oxygen and
nitrogen species (ROS and RNS,
resp.); this results from increased intracellular calcium levels,
mitochondrial dysfunction, arachidonic acid breakdown, and activation of inducible
nitric oxide synthase (iNOS) [23–25]. Initially thought to be a problem only in acute SCI
tissue, newer studies have revealed that indices of free radical damage are
present throughout the first week after injury [26, 27]. ROS and RNS cause lipid peroxidation as well as oxidative and nitrative damage to
proteins and nucleic acids [27, 28]. In addition, oxidative damage exacerbates
mitochondrial dysfunction [29] and contributes to intracellular calcium overload
which activates proteases resulting in breakdown of cytoskeletal proteins [27, 30]. Thus, the collective damage induced by ROS and RNS is far-reaching and likely contributes
to cellular death and functional loss after SCI.PPARγ activation
after SCI could dampen the damage induced by ROS and RNS in multiple ways. First, PPARγ activation may reduce the
overall level of free radicals present in the injured tissue since PPARγ activation leads to decreased
nitric oxide, cyclooxygenase-2 (COX-2),
iNOS, and nitrotyrosine levels in animal models of amyotrophic lateral
sclerosis (ALS), multiple
sclerosis (MS), ischemia, and neuroinflammation [31–37]. In addition, PPARγ agonists may increase the levels of antioxidants
in or around the injured tissue. For instance, catalase levels were elevated by
PPARγ agonist treatment following
intracerebral hemorrhage [38]; with increased antioxidant levels, the surviving tissue will be
better equipped to fend off assault by free radicals. Thus, agonists that
stimulate PPARγ may reduce the levels of free radicals and at the
same time, elevate enzymes essential for combating free radicals that remain.
This in turn would reduce the number of neurons and glial cells that die in the
subacute phase of SCI. Studies by our group and others have shown that
treatment with the PPARγ agonist pioglitazone resulted
in an increase in the number of motor neurons spared after SCI, which might
have been due, at least in part, to a reduction in post-SCI oxidative damage [15, 16]. By promoting motor neuron survival in human SCI, significant
preservation of segmental function may be possible. Although complete recovery
of “normal” function may not be feasible, even partial recovery of hand
function, for instance, could drastically improve the quality of life for an
individual with SCI.


## 5. Inflammatory-Mediated Cell Death

A well-characterized event
after spinal trauma is local microglial activation, inflammatory cell
infiltration, and upregulation of proinflammatory mediators. Indeed, several
studies have shown the rapid rise in proinflammatory cytokines and chemokines
which stimulate inflammatory cell infiltration into the injured spinal cord [14]. Once present within the damaged and surrounding
parenchyma, inflammatory cells such as neutrophils, macrophages, and lymphocytes
can exacerbate tissue damage. For instance, activated macrophages and microglia
produce cytotoxic molecules such as iNOS, TNFα, IL-1β, and IL-6. Interestingly, PPARγ levels
are upregulated in activated microglia and macrophages [35, 39, 40] and activation of PPARγ in these cells can
decrease production of proinflammatory mediators [40–42]. The mechanisms contributing to these effects include antagonism of
AP-1, STAT, and NFκB
levels as well as concomitant increases in IκBα levels. Collectively, these actions will reduce
the inflammatory potential of the treated cells [37, 38, 40, 43]. Accordingly, PPAR-induced inhibition of microglia/macrophage
accumulation and release of proinflammatory cytokines has been detected in
animal models of Alzheimer’s disease, Parkinson’s disease, and MS [33, 34, 44–46]. PPARγ activation can also reduce the differentiation of
monocytes into macrophages [45], promote macrophage apoptosis [47], and decrease T-cell proliferation, which collectively would result in
reduced numbers of infiltrating inflammatory cells into the injured spinal
cord [48]. Indeed, PPARγ-induced reductions in neutrophil, T-cell, and macrophage infiltration have been shown
in animal studies of experimental allergic encephalomyelitis (EAE, an animal
model of MS) and intracerebral hemorrhage [33, 38, 48]. This may be due, in part to, a PPARγ-mediated reduction in chemokines, which elicit inflammatory
cell recruitment to the CNS. For instance, mice with EAE given oral PPARγ agonists expressed lower
levels of MIP1α and RANTES within the
brain compared to control mice [33].Collectively,
these data suggest that PPARγ activation provides a
potent means for reducing proinflammatory mediators after CNS injury, including
trauma to the spinal cord. This is further suggested by recent SCI studies
which revealed a reduction in gliosis, cytokines, and adhesion molecules [16]. Many SCI studies have demonstrated that postinjury treatment with anti-inflammatory
agents results in significantly improved anatomical and functional recovery [49–54]. Thus, the anti-inflammatory actions of activating the PPARγ pathway could provide
another mechanism for reducing the deleterious proinflammatory cascades
initiated after SCI.


## 6. Demyelination of Surviving Axons after SCI

Another pathological feature of
acute and chronic SCI tissue is demyelination of axons that survive the initial
traumatic event [55–57]. Loss of myelin will lead to conduction delays and/or
frank conduction block. Because axons traversing the injury site are the sole
remaining connection between the brain and caudal spinal neurons, inefficient
communication through these axons is a significant clinical issue.
Demyelination is due to loss of oligodendrocytes, which are killed at the
injury epicenter within hours of the injury and continue to undergo apoptosis
in rostral and caudal white matter for many weeks after SCI [58–60]. The potential mechanisms responsible for acute and
delayed oligodendrocyte death are numerous. For instance, oligodendrocytes are
known to be susceptible to glutamate excitotoxicity, which could contribute to the
early loss of these cells. Oligodendrocytes and their myelin membranes are also
vulnerable to lipid peroxidation, which, as stated above, is prevalent
throughout the first week after injury. Lastly, proinflammatory mediators such
as TNFα and IL-1β can lead to oligodendrocyte death.Since PPARγ activation can counteract many of these deleterious
processes, this pathway may thereby promote oligodendrocyte survival and myelin
preservation following CNS damage. Indeed, treatment with a PPARγ agonist markedly improved
myelination and decreased lesion area in the CNS of animals with EAE [33, 44, 48, 61]. In addition, a PPARγ agonist was able to reduce myelin
damage in an in vitro model of inflammatory demyelination [62]. Therefore, treatment with PPARγ agonists after SCI could potentially lead to
improved oligodendrocyte survival and better myelin preservation. Indeed, we
have noted that when the PPARγ agonist pioglitazone was given to rats after SCI, a
significant increase in sparing of white matter distal to the lesions was detected
[15]. This likely contributed to the improved locomotor function detected
in our study and others [15, 16]. Thus, acute treatment of SCI patients with a PPARγ agonist could potentially
improve tissue sparing and thereby allow for a greater level of locomotor
abilities as well as other important functional outcomes. For example, neurons
controlling bowel and bladder function are located within the lower spinal
cord, including the lumbar and sacral segments. Because most SCI’s occur in
more rostral segments, neuronal circuits that directly control bowel and
bladder are often intact after SCI. However, significant and permanent bowel
and bladder dysfunction occurs due to loss of descending signals carried by
axons that are lost or demyelinated at the impact site. Therefore, enhanced
preservation of myelin or promotion of remyelination at the injury site could
lead to functionally significant improvements in the quality of life for SCI
patients.


## 7. Neuron Loss after SCI

Contusion-type injuries are
the most commonly sustained trauma to the spinal cord. Because of the high degree
of vascularization and the dynamic forces encountered within the gray matter during
contusive injuries [63], the lesions evolve as centralized fluid-filled
cavities originating within gray matter regions at the lesion site that extend
into rostral and caudal segments. Thus, even mild contusions can result in significant
neuron loss over multiple segments of spinal cord. As stated above, neurons not
killed by the initial impact can fall victim to secondary cascades, including ischemia,
excitotoxicity, lipid peroxidation, and proinflammatory mediators. This neuron death
will lead directly to loss of function in the muscles innervated by motor
neurons at the segment of injury. Since the majority of injuries occur in the
cervical spinal cord, SCI often means loss of function in the arms and hands.
In addition, motor neurons driving respiration are found within C3–C5, so
injuries that directly damage these segments frequently result in respirator
dependence.Clearly, therapies that
protect neurons from secondary injury cascades after SCI are of great
importance. Given the potential beneficial actions of PPARγ
activation discussed above, neuroprotection after SCI is an important potential
therapeutic target for PPARγ agonists. Indeed, improved
neuronal survival following PPARγ agonist treatment has already been noted in
several models of CNS disorders. For instance, treatment with the PPARγ agonist pioglitazone promoted
motor neuron survival and increased muscle fiber diameter in a transgenic model
of ALS [32]. PPARγ agonists also increased neuron survival and
decreased lesion sizes in models of Parkinson’s disease [43, 46], central inflammation [34], intracerebral hemorrhage [38], and cerebral ischemia [5, 6, 31, 35]. These beneficial effects were likely mediated through a reduction in
the indirect actions noted above, including lipid peroxidation, proinflammatory
signals, and extracellular glutamate levels. However, PPARγ activation may also have a
direct effect on neurons. Neuronal expression of PPARγ has been detected in the
intact CNS and an upregulation of PPARγ was observed in neurons in the ischemic penumbra
following focal cerebral ischemia [5, 6, 31, 35]. Furthermore, cultured neurons treated with PPARγ agonists were protected
from glutamate-induced death demonstrating a direct action of PPARγ activation in neurons [22]. Thus, if a PPARγ agonist was delivered soon
after SCI, significant neuronal sparing may be achieved which would likely
translate into better functional preservation and improved quality of life for SCI
patients.


## 8. Summary

The PPAR pathway appears to
play an important role in recovery from CNS disorders (Table1). Indeed,
several studies suggest that endogenous ligands present in the damaged CNS can activate
the PPARγ pathway
and contribute to anatomical preservation. This is illustrated by studies demonstrating
that PPARγ antagonists potentiate tissue pathology after
cerebral ischemia [5, 6]. Exacerbation of neuropathology also occurs when EAE
is induced in the presence of a PPARγ antagonist or when disease is induced in PPARγ knockout
mice [61, 67]. Thus, endogenous PPARγ activation may be an essential component of promoting
spontaneous reparative mechanisms that are initiated in the injured brain and
spinal cord. This endogenous response appears submaximal, however, as the numerous
studies discussed above suggest that pharmacological activation of the PPARγ pathway
subsequent to damage may significantly improve recovery.  In the realm of SCI research, treatments are
severely lacking. Therefore, manipulating the PPARγ pathway
appears to hold great potential as a therapy for treating human SCI.Table 1
Summary of effects mediated by PPARγ  activation in CNS injury or disease models.
Disease/injury modelKnown PPARγ  effectsReferencesSpinal cord injury↑ locomotor recovery[15, 16]↑ myelin sparing↑ motor
neuron sparing↓ glial
activation↓ proinflammatory
cytokinesExperimental allergic encephalomyelitis(model of multiple sclerosis)↑ myelin sparing[33, 44, 48, 61]↓ lesion size↓ inflammatory cell infiltrate↓ proinflammatory
cytokines & chemokines↓ clinical score (better recovery; lower no. of relapses)Amyotrophic lateral
sclerosis↑ motor
neuron survival[32]↓ glial
activation↓ COX-2↓ iNOS↑ longevity, delayed disease onsetParkinson’s disease↑ dopaminergic
neuron survival[43, 46]↓ gliosis↓ iNOS↓ NFκB translocation to the nucleus↑ IκBαCerebral ischemia↑ neuron survival in penumbra[6, 31, 35, 37, 64]↓ lesion size↓ COX-2↓ proinflammatory cytokines↑ antioxidantsCerebral hemorrhage↑ catalase[38]↓ NFκB↓ neutrophil infiltration↓ apoptosis↓ behavioral dysfunctionAlzheimer's disease↓ gliosis[7, 45, 65, 66]↓ COX-2 & iNOS↓ proinflammatory
cytokines↓ Aβ1-42+
amyloid plaques↓β-secretase
mRNA↓ monocyte differentiation into macrophages



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