# Regulation of Glial Cell Functions by PPAR-γ  Natural and Synthetic Agonists

Authors: Antonietta Bernardo; Luisa Minghetti
Journal: PPAR Research
 (2008)

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## Abstract

In the recent years, the peroxisome proliferator-activated receptor-γ (PPAR-γ), a well known target for type II diabetes treatment, has received an increasing attention for its therapeutic potential in inflammatory and degenerative brain disorders. PPAR-γ agonists, which include naturally occurring compounds (such as long chain fatty acids and the cyclopentenone prostaglandin 15-deoxy Δ12,14 prostaglandin J2), and synthetic agonists (among which the thiazolidinediones and few nonsteroidal anti-inflammatory drugs) have shown anti-inflammatory and protective effects in several experimental models of Alzheimer's and  Parkinson's diseases, amyotrophic lateral sclerosis, multiple sclerosis and stroke, as well as in few clinical studies. The pleiotropic effects of PPAR-γ agonists are likely to be mediated by several mechanisms involving anti-inflammatory activities on peripheral immune cells (macrophages and lymphocytes), as well as direct effects on neural cells including cerebral vascular endothelial cells, neurons, and glia. In the present article, we will review the recent findings supporting a major role for PPAR-γ agonists in controlling neuroinflammation and neurodegeneration through their activities on glial cells, with a particular emphasis on microglial cells as major macrophage population of the brain parenchyma and main actors in brain inflammation.


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## Body

## 1. Introduction

The peroxisome proliferator-activated receptor-γ
(PPAR-γ) belongs to the hormone nuclear receptor super family. It is a ligand-dependent
transcription factor activated by both naturally occurring compounds, such as
long chain fatty acids and the cyclopentenone prostaglandin 15-deoxy Δ12,14 prostaglandin J2 (15d-PGJ2), and synthetic agonists,
including the thiazolidinediones (TZDs), and few nonsteroidal anti-inflammatory
drugs (NSAIDs). Because of their role in the regulation of genes involved in
lipid and carbohydrate metabolism, PPAR-γ and the other two isoforms PPAR-α and δ,
deeply affect lipid homeostasis and insulin sensitivity [1–3]. The TZDs
rosiglitazone (Avandia®), and pioglitazone (Actos®), introduced on the market
in the early 1990s, are currently in clinical use to control blood glucose
levels in subjects affected by type II diabetes.In the last decade, accumulating evidence suggests
that, besides diabetes and metabolic syndrome [4], PPAR-γ agonists have significant therapeutic potential
in brain disorders. A large number of experimental studies and few clinical
observations have suggested that PPAR-γ
ligands may be successfully exploited to treat a wide range of neurological
diseases, ranging from neurodegenerative diseases, to traumatic injuries,
stroke, and demyelinating diseases, as recently reviewed by Heneka et al. [5].
In Alzheimer's disease (AD) transgenic mouse models, the TZD rosiglitazone attenuated
learning and memory deficits [6], in line with its ability to promote cognitive
preservation in patients with early AD [7, 8]. In
amyotrophic lateral sclerosis (ALS) and Parkinson's disease animal models, the
TZD pioglitazone ameliorated the disease symptoms [9, 10]. In rodent
focal ischemia models, both pioglitazone and rosiglitazone decreased the
infarct volume [11–13]. Furthermore,
the natural agonist 15d-PGJ2 was shown to decrease the neurological
deficits after experimental intracerebral hemorrhage [14] and its plasma levels
in stroke patients were found directly correlated to the neurological outcome [15].
Rosiglitazone and pioglitazone decreased secondary neuronal damage,
astrogliosis, microglial activation, myelin loss, and neuropathic pain in
animal models of spinal cord injury while improving motor function recovery [16].
In experimental autoimmune encephalomyelitis (EAE), a well known model for
autoimmune demyelinating diseases, synthetic, and natural PPAR-γ ligands—as well as some α or δ agonists—have been reported to ameliorate
clinical symptoms, to reduce expression of pro-inflammatory cytokines and chemokines,
to decrease brain inflammation, demyelination and glial activation, and to
delay the onset of disease [17–25]. More
recently, promising results obtained in experimental models of ocular diseases have
evidenced that PPAR-γ could be targeted to control inflammation and treat invalidating diseases such
as diabetic retinopathy and optic neuritis, a demyelinating disease of the
optic nerve frequently associated to multiple sclerosis (MS) (see for review [26]).
Nonetheless, in spite of the amount of data on the therapeutic activities of
PPAR agonists in EAE, clinical studies are still lacking and reports on their
clinical use in MS or optic neuritis are still anecdotal [27]. Clinical trials
are, however, in course with pioglitazone and rosiglitazone [5].The beneficial effects of PPAR-γ agonists in
degenerative, inflammatory and traumatic brain pathologies are most likely
mediated by several mechanisms, which may be disease-specific and involve both
peripheral and central anti-inflammatory activities, by affecting crucial
functions of peripheral (macrophages and/or lymphocytes) and central (microglial
cells) immune cells. Besides microglia, PPAR-γ agonists can act on other neural
cell types, including astrocytes, neurons, and oligodendrocytes (Figure 1).Figure 1
Cellular targets of PPAR-γ agonists in neurodegenerative diseases. PPAR-γ
agonists can control neuroinflammation, neurodegeneration, and demyelination by
effecting several cellular targets and by several direct and indirect
mechanisms. PPAR-γ agonists can control glial activation, preventing a number of proinflammatory
activities that can contribute to myelin/OL damage and neurotoxicity PPAR-γ
agonists may also affect OLs and neurons, by preventing release inflammatory
mediators and/or promote the synthesis of soluble factors or membrane-bound
molecules that control glial activation.Several of the beneficial effects of PPAR-γ
result from its ability, once activated by specific ligand, to control the
expression of proinflammatory genes, through the binding of specific sequences
in their promoter regions—the peroxisome proliferator response elements
(PPREs)—but also independently from its DNA-binding
activity, by a mechanism termed transrepression, which have just begun to be
elucidated [28]. In addition, some PPAR-γ ligands may exert specific activities
independently from PPAR-γ.
Among these, of great interest is the ability of a few TZDs to directly
influence mitochondrial function by binding to target sites in mitochondria
including the Complex I of the respiratory chain and the newly described
protein mitoneet [29].


## 2. PPAR-γ: Structure, Functions, and Agonists

The PPAR-γ and the two closely related α and PPAR-δ (also known as β, NUC-1, or FAAR) share a high homology, but
differ for tissue distribution and ligand specificity [2]. α is mainly
expressed in tissues with high catabolic rates of fatty acids, such as the
liver, muscle, and heart, whereas PPAR-δ shows a much wider distribution. PPAR-γ
is highly expressed in adipose tissue and in cells of the immune system,
including lymphocytes and macrophages. In the brain, PPAR-γ is expressed in several cell types including
microglia, astrocytes, oligodendrocytes, and neurons.PPAR-γ protein shows a remarkable conservation across species. Human and the murine PPAR-γ proteins
show 95% identity at the amino acid level. The human PPAR-γ
gene is located on chromosome 3 and generates at least three mRNA transcripts,
PPAR-γ1, PPAR-γ2, and PPAR-γ3 [30–32]. PPAR-γ1 e PPAR-γ3
mRNAs encode for the same protein, while PPAR-γ2 mRNA gives rise to a protein containing 28
additional amino acids at the N-terminus.At protein level, all three PPARs show a similar organization in five different
functional domains, two of which—the DNA-binding domain (DBD) and the
ligand-binding domain (LBD)—are the highly conserved [2]. The DBD contains the two zinc finger-like motifs
that recognize the DNA target, and can be considered the hallmark of the
nuclear receptor superfamily. The LBD conserves a common three-dimensional
structure, which hosts a particularly large ligand-binding cavity, of which
only 30–40% is occupied by the ligand. The relatively free nonspecific interaction between the cavity
and the hydrophobic domains of the ligand explains the low ligand-specificity
of PPARs. Nonetheless, the LBDs of the three PPAR isotypes have sufficiently
divergent amino acid sequences to allow some ligand specificity.Several unsaturated fatty acids bind to all three PPAR isoforms, whereas saturated
fatty acids are in general poor PPAR ligands. However, given the relatively
high concentration of lipids required for PPAR activation (in the micromolar or
submicromolar concentration range), their “in vivo” role as PPAR ligands remains
a controversial issue. Some arachidonic acid metabolites are more effective
PPAR-γ
ligands than the precursor. In particular, 15d-PGJ2, characterized
by a reactive α,β-unsaturated
ketone in the cyclopentenone ring, was the first PPAR-γ endogenous ligand, described in 1995 by two
independent groups [33, 34].The implication of PPAR-γ in several important metabolic and
degenerative disorders, has strongly pushed the research of specific PPAR-γ
agonists and antagonist (for review see [35]). A
major group of synthetic PPAR-γ agonists is represented by the antidiabetic drugs TZDs, originally identified for
their ability to improve the insulin sensitivity of diabetic animals. Pioglitazone
and rosiglitazone belong to this group of high-affinity ligand. A different
series of synthetic PPAR-γ ligands are derived by L-tyrosine GI262570, GW1929, and GW7845, which were
developed on the basis of their activity on human PPAR-γ and are among the most potent PPAR-γ
agonists, being active at low nanomolar concentrations.In addition to these groups of ligands, several members of
the heterogeneous NSAID family have been described as agonists for PPARs [35] and reference therein. In most cases, the doses required for PPAR-γagonist activity are in the high micromolar range, thus largely exceeding those
required for in vivo inhibition of cyclooxygenases (COXs), the main target of
these drugs. Among NSAIDs, aspirin and acetaminophen (or paracetamol) lack of
agonistic activity for any of the PPAR subtypes, whereas indomethacin,
ibuprofen, and diclofenac are selective for the γsubtype.
Recently, we have shown that the two nitric oxide (NO)-releasing derivative of
flurbiprofen, HCT1026 and NXC 2216, were both able to activate PPAR-γ
and induce its specific binding to a PPRE sequence [36, 37]. Few antagonists are also available, but their
use is often limited by partial agonistic activity. The plasticizer biphenol A
diglycidyl ether (BADGE) and the irreversible antagonist GW9662 are among the
most widely used.


## 3. PPAR-γ Agonists and Oligodendrocyte  Biology

Oligodendrocytes (OLs) are the myelin-forming cells of the CNS. Their differentiation from
precursor to mature cells occurs through a series of stages that can be defined
by morphological and antigenic changes occurring in vivo as well as in culture
systems [38]. During development and repair OLs extend elongated processes,
forming multilamellar sheaths around neuronal axons. The formation, growth, and
maintenance of the myelin sheath are prominent parts of neural development and
nervous system function. As for OL maturation, myelin formation is a multistep
process, involving recruitment to germination sites, proliferation of
undifferentiated OL progenitors and their differentiation to mature OLs,
producing myelin. Damage to OLs as a result of oxidative stress is considered a
key pathogenetic pathway in several adult and infant human diseases. A
substantial number of in vitro and in vivo studies has shown a maturation-dependent
vulnerability to oxidative stress of the OL lineage [39–41], suggesting that OL progenitor is a key target for
limit white matter damage and promote myelin repair [42].
Oligodendrocytes are major lipid producing cells, as required for myelin
formation and maintenance. Given the role of PPARs in lipid metabolism it is
conceivable that this group of nuclear receptor play a major role in OL
differentiation and function. Although PPAR-β/δ
has been long considered the PPAR type mainly expressed in OLs and involved in
myelination [43, 44], recent findings support an important role for PPAR-γ
activators in OL protection and differentiation. The first evidence for a role
of PPAR-γ in OL differentiation was reported by Roth et al. [45].
By using the B12, oligodendrocyte-like cell line and primary cultures of spinal
cord OL precursors, the authors first demonstrated that these cells expressed
all three PPAR isoforms and found that natural and synthetic PPAR-γ
agonists, but not other isoform activators, enhance process extension and cell
maturation. These effects were blocked by the PPAR-γ antagonist GW9662. The maturation of pre-OLs
was accompanied by enhanced expression of alkyl-dihydroxyacetone phosphate
synthase (ADAPS), a peroxisomal enzyme required for the synthesis of
plasmalogen, an etherphospholipid essential for myelin formation. These
observations suggest that PPAR-γ mediated mechanisms may be important for OL differentiation and peroxisome
functions. An important role for these organelles in maintaining OL and white
matter integrity has been recently demonstrated in mutant mice characterized by
the selective absence of functional peroxisomes from OLs [46]. In
line with the proposed role of PPAR-γ in controlling OL differentiation and functions, we have recently confirmed the
expression of PPAR-γ in highly purified rat OL cultures (Figure 2(a)). The level of expression is
increased with the OL maturation in vitro (Bernardo et al., in preparation). In
addition, we found an increased expression of PPAR-γ in white matter of young rats (post natal day
19) exposed to perinatal global asphyxia (Figure 2(b)). This model mimics some
of the features of perinatal asphyxia, a major cause of immediate and delayed
brain damage in the newborn [47, 48], and is characterized by early oxidative stress,
delayed behavioral deficits, and alteration in myelin formation, as indicated
by the strong reduction of myelin basic protein (MBP) expression (Figure 2(b)).
Whether PPAR-γ over-expression is part of an adaptive response to the hypoxic condition aimed
at restoring myelin formation or is part of an aberrant program leading behavioral
impairment remain to be established.Figure 2
PPAR-γ expression in culture rat oligodendrocytes and in white matter (postnatal
day 19) in rat model of global perinatal asphyxia. (a) Immunocytochemistry of rat OL progenitor cultures, prepared as previously
described [40] for PPAR-γ (upper
panel) and the OL marker O4 (lower panel). (b) Western blot analysis of white
matter homogenates from rats at postnatal day 19 subjected to 20 minutes of
perinatal asphyxia (hypoxic) and from controls, prepared as described in
Piscopo et al. [48]. Inset show the decreased levels of MBP in hypoxic
rats at pnd 19.In apparent contrast with the above findings, Xiang et al. [49],
reported that the PPAR-γ
natural ligand 15d-PGJ2, but not other PGs, induced apoptosis of OL
precursor cell lines (mOP and CG4 cell lines). The toxic effect was developmental
stage-dependent, being the undifferentiated mOP cells more susceptible than
differentiated cells. In line with observations previously reported in
microglia cultures [50],
cell death was independent of the nuclear receptor
PPAR-γ. Since the toxic effect of 15d-PGJ2 was prevented by
preincubation of cell cultures with N-acetyl cysteine, a reducing agent and a
precursor molecule for glutathione (GSH) synthesis, but not with free radical
scavengers, the authors suggest that the underlying mechanism is related to
oxidative stress due to depletion of GSH.


## 4. PPAR-γ Agonists and Astrocytes

Astrocytes are most abundant glial cells in the CNS and crucial players in brain
homeostasis. Among other functions, they provide metabolic support for neurons,
uptake neurotransmitters such as glutamate, synthesize neurotrophic factors,
and contribute to ion homeostasis (i.e., potassium uptake) and blood-brain
barrier induction and maintenance [51]. In addition, astrocytes exert important
roles also in brain inflammation and immunity, as they express several—though
fewer than microglia—pattern-recognition receptors (PRRs) such as for example
the Toll-like receptors, and release cytokines and chemokines that can trigger
or amplify the local inflammatory response [52]. Similar to
microglia, astrocytes rapidly react to a wide array of insults or damaging
events. Reactive astrocytes, which are characterized by increased expression of
glial fibrillary acidic protein (GFAP), a constituent of the intermediate
filaments, are typical of most brain pathologies. Thus astrocytes represent an
important target for anti-inflammatory and neuroprotective therapeutic
strategies.Astrocytes express PPAR-γ [53, 54], and accumulating evidence over the last ten years indicates
that PPAR-γ agonists modulate astrocyte functions.In rat cortical slices and cultured astrocytes, the TZD pioglitazone was found to significantly
increase glucose consumption in time- and dose-dependent manners, through a
mechanism independent of PPAR-γ and involving cAMP/PKA signaling [55]. Pioglitazone did not modify the expression of the glucose
transporter GLUT-1, which is mainly expressed in glial and endothelial cells,
but rather it increased glucose flux through pre-existing GLUT-1 protein. In addition,
pioglitazone increased lactate production and release, induced mitochondrial
membrane hyperpolarization, and protected astrocytes against
hypoglycemia-induced cell death. On the basis of their studies, the authors suggest that
TZDs modulate enzyme activities present within the mitochondrial membrane causing increased cytosolic pyruvate,
resulting in greater lactate production. The inhibitory effect on mitochondrial function is
compensated by an increase in anaerobic glycolysis allowing for continued ATP production. Eventually,
the reduced intracellular glucose levels are replenished by glucose transport through the GLUT-1. At
later times, mitochondrial respiration recovers, and accumulated ATP utilized to maintain and
increase the membrane potential. Because hyperpolarization of the mitochondrial membrane is postulated
to be protective, the net result of TZD treatment, at least in astrocytes, is protective and
allows cells to withstand subsequent noxious stimuli [55]. Altogether,
these results suggest that TZD-induced alteration of astrocyte metabolism and mitochondrial
function could be beneficial in neurological conditions, in which glucose
availability is reduced.Another important mechanism by which PPAR-γ agonists could exert neuroprotection by influencing astrocyte functions is the
enhancement of glutamate uptake. Romera et al. [56] reported that the PPAR-γ antagonists
T0070907 prevented the ischemic preconditioning-induced (IPC) neuroprotection
in neuronal-astrocytic cocultures subjected to oxygen-glucose deprivation (ODG)
and reversed the inhibitory effect of IPC on OGD-induced glutamate release. In
addition, rosiglitazone and the non-TZD agonist L-796,449 induced a
concentration-dependent increase in glutamate transporter GLT-1 expression and [3H] glutamate
uptake in rat astrocytes. In addition the authors identified six putative PPREs
in the promoter region of GLT1/EAAT2 gene, suggesting GLT1/EAAT2 glutamate
transporter is a novel PPAR-γ target gene [56]. Finally, 15d-PGJ2 remarkably increase the
synthesis and release of neurotrophic factor nerve growth factor (NGF) in mouse
primary astrocytes, which could further contribute to neuroprotection [57].As mentioned above, activated astrocytes produce cytokines and other molecules involved
in inflammatory response, which are thought to significantly contribute to
brain damage. Such neurotoxic activities have been shown to be reduced by PPAR-γ
agonists in several experimental paradigms. The two TZD compounds NP00111 and
NP01138 were reported to inhibit the production of nitric oxide (NO), IL-6, and
TNF-α as well as expression of the inducible enzymes iNOS and COX2 induced in LPS-stimulated
astrocyte and microglial cultures [58]. Consistently with
the described anti-inflammatory activities, the two compounds were
neuroprotective in an animal model in which of brain damage is induced by kainic
acid administration [59]. Both in vitro and in vivo effects were substantially
attenuated by cotreatment with the PPAR-γ antagonist GW9662, supporting the involvement
of PPAR-γ activation.In contrast to the above described TZDs, the natural ligand 15d-PGJ2 prevented
the IL-1β-induced COX-2 mRNA accumulation in human astrocytes, through a PPARγ-independent
mechanism [60]. Similarly, Lennon and colleagues [61]
showed that ciglitazone and 15d-PGJ2 activated the MAP kinase
cascades (Erk, Jnk, and p38 MAP kinase) in astrocytes by a PPAR-γ independent mechanism, which required the
presence of ROS. Again, independently of PPAR-γ, 15d-PGJ2 and rosiglitazone reduced
the phosphorylation of signal transducers and activators of transcription
(STAT) 1 and 3 as well as Janus kinase 1 (JAK1) and JAK2 in activated
astrocytes and microglia [62].Recently, Xu and Drew [63] extended the analysis of the anti-inflammatory
activity of PPAR-ligands to other inflammatory mediators belonging to the IL-12
family of cytokines. They found that in primary astrocytes, LPS induced the
production of IL-12p40, IL-23, and IL-27p28 proteins, which was significantly reduced
in the presence of 15d-PGJ2. Since these cytokines play critical
roles in the differentiation of T helper (Th) 1 and Th17 cells and are likely
to contribute to the development of multiple sclerosis, this observation
further support the potential role of PPAR-γ agonists in MS treatment [5, 64].In line with the beneficial effect of PPAR-γ agonists in experimental models of inflammatory diseases, PPAR-γ has also been involved in anti-inflammatory
functions of IL-4, a Th2 type cytokine, which plays an important role in controlling Th1 cell
responses and resolution of inflammation. Paintlia et al. [65]
demonstrated that the inhibition of iNOS expression and the increase of
survival of differentiating OPs induced by IL-4 in inflammatory
cytokine-stimulated mixed cultures are mediated by PPAR-γ activation. In support of their conclusions, the
authors describe a coordinate increase in the expression of both PPAR-γ  and its natural ligand-producing enzyme
12/15-lipoxygenase (12/15-LOX) in IL-4-treated glial cells and show that
IL-4-induced PPAR-γ activation antagonizes NF-κB transactivation in inflammatory
cytokine-stimulated astrocytes. A similar upregulation of PPAR-γ by IL-4 was demonstrated in cultured microglial
cells [66]. To link between IL-4 and PPAR-γ is completed by the observation that the
anti-inflammatory activity of the TZD troglitazone was mediated by its ability
to increase IL-4 expression in glial cultures [67].Astrocytes recognize and react to several pathogens through their repertoire of PPRs [52].
In a recent study, 15d-PGJ2 and ciglitazone suppress the production
of IL-1β and NO in Staphylococcus aureus-stimulated astrocytes [68].
Interestingly, 15d-PGJ2 attenuated TLR2 expression, the PPR
recognizing Staphylococcus aureus. Importantly, 15d-PGJ2 and
ciglitazone were still capable of inhibiting the release of proinflammatory
mediators induced by Staphylococcus aureus in PPAR-γ-deficient astrocytes,
supporting PPAR-γ-independent effects of these compounds. In another study, 15d-PGJ2 significantly attenuated astrocyte reaction to mycotoxin ochratoxin A (OTA), a
widespread food contaminant that accumulates in the brain. At noncytotoxic concentrations,
OTA down-regulated GFAP expression while it upregulated vimentin. Interestingly,
OTA increased PPAR-γ expression, possibly increasing the susceptibility of OTA-exposed cells to PPAR-γ
agonist treatment [69].


## 5. PPAR-γ Agonists and Microglial Cells

Microglia derive from myeloid precursors that enter the developing CNS to become the
major population of brain resident macrophages. Under physiological conditions,
microglia show a ramified morphology and the absence of cell-surface and
cytoplasmic molecules typically associated with other tissue macrophages. In
this quiescent or “resting” state microglia are able to “sense” subtle
environmental changes to which they rapidly react [70].
Although our knowledge on microglial in physiological conditions is still
limited, using transgenic mice showing specific expression of enhanced green
fluorescent protein in microglia and in vivo two-photon microscopy, it was
shown that “resting” microglia constantly survey the surrounding
microenvironment with extremely motile processes and protrusions [71].
Once activated, microglia rapidly undergo morphological changes, characterized
by cell body enlargement, loss of ramified processes, and upregulation of
cell-surface and/or cytoplasmic antigens. In addition, activated microglia can
synthesize a range of different molecules, including free radicals,
inflammatory cytokines, chemokines, lipid mediators, and neurotrophic factors,
whose typical profile will determine the outcome of microglial activation in
term of repair or injury [70]. Although in the past activated microglia have
been regarded mainly as detrimental for the surrounding cells and as major
players in neurodegenerative processes, it is now clear that activated
microglia play  complex and multifaceted
roles, which need to be defined within each disease. Importantly, the different
states of activation can be switched between one state and another during the
course of disease or in response to further stimuli or signals from the
periphery [72].A deeper knowledge of microglial biology and of the molecular mechanisms
underlying the acquisition of protective versus detrimental functions is
crucial for finding new molecular targets and developing effective treatments
for a wide range of neurological disorders.In this view, PPAR-γ agonists have been extensively studied in the last decade for their therapeutic
potential as key molecules in preventing the undesired toxic effects of microglial
activation [35, 73].One of the first finding supporting a role for 15d-PGJ2 as endogenous
regulator of microglial activation—15d-PGJ2 derives from PGD2,
a major PG synthesized within the brain by most neural cells—was provided by
Petrova et al. [74], who demonstrated that this PPAR-γ natural ligand attenuates iNOS expression, and
the subsequent NO accumulation, in the murine BV-2 microglial cell line
stimulated by LPS. Since the TZD troglitazone did not affect the NO pathway, it
was suggested that 15d-PGJ2 inhibits iNOS expression by a PPAR-γ
independent mechanism. The same authors then demonstrated that 15d-PGJ2 decreases the production of TNF-α,
IL-1β and the expression of COX-2 in the same cell system while increasing the expression of the antioxidant enzyme
hemeoxygenase-1 and the intracellular levels of glutathione [75].Bernardo et al. [76] showed for the first time that primary microglial
cells, unlike BV-2 cells, express PPAR-γ
and that such basal expression is increased by its specific agonists, while it
is reduced in the presence of microglial activators such as LPS and IFN-γ.
Microglial PPAR-γ
was subsequently shown to be functionally active, being able to bind specific
PPRE sequences upon activation by natural and synthetic agonists [50].
Similar to BV-2 cell line, in primary microglial cultures 15d-PGJ2 prevented LPS-induced iNOS expression and TNF-α production as well as IFN-γ-induced expression of major histocompatibility
complex (MHC) class II antigens, by mechanisms involving PPAR-γ activation and reduced activation of STAT-1
and NF-κB, which are known to mediate IFN-γ and LPS signaling [76].
In human microglial cells, 15d-PGJ2 did not affect NF-κB binding activity although it decreased STAT-1
expression and enhanced expression and binding activity of the AP-1 proteins
J-Jun and c-Fos [60]. It was then reported that 15d-PGJ2 inhibits IL-12 synthesis in rat primary microglia and mouse cell line N9,
activated either by LPS alone or in combination with IFN-γ or TNF-α [63, 77]. 15d-PGJ2 attenuated microglial activation also when elicited by Gram-positive bacteria
Staphylococcus aureus, by inhibiting the expression of proinflammatory cytokines
and the chemokine monocyte chemoattractant protein-1 (MCP-1) [73, 78].In cortical mixed neuron-glial cultures 15d-PGJ2, ciglitazone and
troglitazone prevented LPS-induced neuronal death, suggesting a PPAR-γ
mediated mechanism of neuroprotection [79]. Similarly, 15d-PGJ2,
ciglitazone, troglitazone and two NSAIDs indomethacin and ibuprofen reduced
the neurotoxicity of microglial cells exposed to β-amyloid fibrils [80]. In this cell system,
COX-2-specific inhibitors failed to promote neuronal survival, suggesting protective
mechanisms independent of COX-2 metabolism.In addition to indomethacin and ibuprofen, we have reported that two NO-releasing derivative of flurbiprofen,
HCT1026 and NXC 2216, were able to prevent microglial activation by activating
PPAR-γ [36, 37]. Interestingly, NCX 2216 after an initial activation induced
PPAR-γ nitration and inactivation. These effects were paralleled by a transient reduction of
TNF-α and NO production and a protracted inhibition of IL-1β and PGE2 synthesis, suggesting a
dynamic regulation of the functional state of activated microglia by NCX 2216. Long
treatment with NCX 2216 could therefore lead, after an initial activation of
PPAR-γ,
to a protracted suppression of its control over microglial activation. Our
results could help explaining why among the few NSAIDs with Aβ-lowering activity (reviewed by [81]), only in the case of protracted administration
of NCX 2216 in an AD animal model, the reduction of cerebral amyloid load accompanied by a
sustained microglial activation [82].The contribution of PPAR-γ-dependent or independent mechanisms to the inhibition of microglial activation by 15d-PGJ2 seems dependent on the cell type (primary versus transformed cell lines; fetal
versus neonatal), or on concentration of the ligand. In rat primary microglial
cultures, we have shown [50]
that 15d-PGJ2 at concentrations several
fold lower than those required for PPAR-γ activation, effectively reduced the
LPS-stimulated production of PGE2 by directly preventing the
enzymatic activity of COX-2 rather than its expression, as previously described
in activated monocytic cell lines [80, 83] and in
BV-2 cells [75]. The reduction of COX-2 enzymatic activity could be
achieved through the modifications of key cysteine residues [84],
as suggested by the ability of 15d-PGJ2 electrophilic α,β-unsaturated ketones to modify several cellular proteins [85, 86]. At concentration 10 times higher than those required
to activate PPAR-γ, 15d-PGJ2 induced microglial cell impairment and death by apoptosis [50].
The effects were stronger in activated microglia than in unstimulated cells, suggesting
that this agent may prevent excessive microglial activation by promoting their elimination
by apoptosis thus contributing to the resolution of inflammation as previously
suggested in peripheral tissues [87, 88].Although apoptosis by 15d-PGJ2 has been shown in several cells, the link
between the proapoptotic effect of 15d-PGJ2 and PPAR-γ activation is still controversial. As before
this may be linked to cell types and their degree of differentiation or
transformation. For example, as opposed to the observations reported in primary
microglia, the induction of apoptosis in T-cells and human and rat glioma cell
lines appears mediated by PPAR-γ-dependent
mechanisms [61, 87, 89–91].


## 6. Conclusions

In the last decade, there has been an increasing number of experimental studies
supporting the use of  PPAR-γ ligands to treat major disabling brain diseases, with a high social burden and
impact on health case system. The compelling evidence obtained in experimental
studies is complemented by sparse, but very encouraging clinical studies. The
positive outcomes in animal models of AD, due to the ability of PPAR-γ
agonists to reduced inflammation and the amyloid burden by various mechanisms,
have found some validation in a pilot clinical trial in which AD patients
treated for 6 months with rosiglitazone showed reduced attention and memory
deficits [7]. In a second recent trial, the improvement in cognition after 6
months of rosiglitazone treatment was significant only in AD patients who did
not have the ε4 allele of the apolipoprotein E [92], a genotype associate with a higher risk to
develop AD. Similarly, the better neurological outcome reported after
administration of PPAR-γ ligands in experimental stroke models are consistent with the result of a small
clinical trial reporting that patients with diabetes receiving pioglitazone or
rosiglitazone had an improved functional recovery after stroke compare to
patients, who have not used any TZD [93]. Furthermore, a large clinical trial
has demonstrated that pioglitazone reduced the combined risk of heart attack,
stroke, and death in high risk type 2 diabetes patients [94].The clinical use of PPAR-γ agonists in MS and ASL remains poorly investigated. Nonetheless, in a case
report, pioglitazone treatment of an MS patient resulted in increased body
weight and improved motor strength and coordination [27]. A first clinical
trial for the use of pioglitazone in ALS started in Germany  in late 2007.Although PPAR-γ synthetic ligands such as TZDs and NSAIDs appear to be very promising drugs to
treat severe human diseases, from cancer to metabolic diseases to brain
diseases, several open issues still need to be examined. Among these, the toxic
effect associated with some PPAR-γ agonists and their blood-brain-barrier permeability, which are at present still matter
of controversies. A deep knowledge of the molecular mechanisms evoked by PPAR-γ
ligands either dependent or independent of the receptor activation and of the
dependence of such effects on the specific cell type is mandatory for the
development of PPAR-γ drugs with increasing efficacy and safety.



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