ARTICLE
Auteur(s) : Catherine Heurteaux
Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097,
660 route des Lucioles, 06560 VALBONNE
Introduction
Cerebral ischemia and temporal lobe epilepsy carry a high risk of
permanent brain damage mainly due to excitotoxic cell death. These
both pathologies have therapeutic and economic considerations,
because they affect almost 2% of the intellectual deficits.
Cerebral ischemia is the third cause of mortality and the first
cause of long term disability. Temporal lobe epilepsy (TLE),
characterized by recurrent complex partial seizures (SRS) is one of
the most prevalent forms of epilepsy and is frequently associated
with pharmacoresistance. During an ischemia or an epilepsy, neurons
at risk die as a result of a neurotoxic biochemical cascade
initiated by reduced energy stores, membrane depolarisation,
excessive neurotransmitter release, accumulation of free fatty
acids and lysophospholipids, elevated intracellular calcium,
increased oxygen free radicals and neuronal hyperexcitability [1].
The evolution of the major pathophysiological entities of tissue
destruction in stroke and epilepsy follows a temporal profile going
from minutes-hours to weeks, which corresponds to the acute
mechanisms of excitotoxicity and the delayed mechanisms of
apoptosis and inflammation leading to the neuronal damage. Since
classical therapeutic strategies, that consist in blocking the
death pathways were unsuccessful to pass from the bench to the
bedside, it can be interesting to test an alternative approach that
consists to increase the neuronal resistance by using the
activation of potassium channels (K+ channels) to
prevent the neuronal hyperexcitability.
K+ channels are known to be involved in the
endogenous regulation of the nervous cell excitability. Opening of
K+ channels may reduce the depolarization triggered by
ischemia or epileptic seizure and consequently may reduce brain
damage. At a presynaptic level, the K+ channel
activation under physiological conditions will lead to efflux of
K+ resulting in hyperpolarization of the membrane and
decrease of the synaptic glutamate release. At a postsynaptic
level, activation of the same K+ channels will prevent
some of the postsynaptic effects of glutamate at N-methyl-D
aspartate (NMDA) receptors by hyperpolarizing cells and thus
favoring blockade by magnesium of NMDA receptor-associated ion
channels. Today, more than 77 genes encoding K+ channels
have been identified in the human genome. Recently, a newly
discovered family of K+ channels, called tandem pore
domain K+ channels (K2P), with four
transmembrane-spanning domains and two pore-regions for each
protein subunit has been identified [2]. These K2P
channels (also called background K+ channels) and their
regulation by membrane-receptor-coupled second messengers, as well
as pharmacological agents are therefore important in tuning
neuronal resting membrane potential, action potential duration,
membrane input resistance and, consequently regulating transmitter
release [3]. The class of mammalian K2P channel subunits
now includes 15 members. One of them is the TREK-1 channel, which
is the most extensively studied [4-6]. The specificity of its
regulation is particularly interesting in relation with neuronal
disease states. Mechano-gated and arachidonic acid-activated
TWIK-related K+1 (TREK-1), highly expressed in the brain
[7] is a signal integrator responding to a wide range of
physiological and pathological inputs. It can be activated by
physical stimuli such as stretch, depolarization, intracellular
acidosis and warm temperature. In relation with neuroprotection,
TREK-1 is upmodulated by volatile anaesthetics, riluzole [8] (a
well-known neuroprotective agent) [9-12] and also with
lysophospholipids and polyunsaturated fatty acids (PUFA) including
arachidonic acid (AA), docosahexaenoic acid (DHA) and
alpha-linolenic acid (ALA) [6, 13].
PUFA and neuronal protection
Using in vivo models of ischemia, our laboratory has shown that
PUFAs are able to induce a strong neuronal protection against the
deleterious effects of cerebral [14, 15] and spinal [16] ischemia.
In the model of global ischemia [14], induced in rats by
cauterization of vertebral arteries and transient (20 min)
clamping of both carotids, an intravenous injection of ALA at a
dose of 500 nmoles/kg 30 min up to 2 hours post-ischemia
strongly reduces the neuronal loss of CA1 pyramidal cell layer
induced by severe ischemia and blocks apoptosis revealed by TUNEL
assay (figure
1). The transient (60 min) occlusion of middle
cerebral artery (focal ischemia) induces in mice focal cortical and
subcortical lesions and reproduces human clinical observations of a
stroke. The quantitation of infarct volume at 24-hour postischemia
shows that an injection of ALA, but not palmitic acid, a saturated
fatty acid reduces the infarction volume with a therapeutic window
from 30 min to 6 hours postischemia [15]. Interestingly, at
one month after reperfusion, the best protection is obtained with a
three-week therapy of ALA (250 nmoles/kg) with a 70% survival rate
(figure 1).
Spinal ischemia is a devastating complication of thoracic and
thoracoabdominal aortic surgery, which induces a severe and often
definitive paraplegia. In the model of spinal ischemia [16], rats
are submitted to cross-clamping of the aortic arch and left
subclavian artery for 15 min. The rats treated with 200
nmol/kg of ALA at the onset of reperfusion have a better neurologic
function (figure
2). PUFA are also able to induce a brain tolerance [17].
Cerebral preconditioning is a powerful endogenous protective
mechanism in which moderate ischemic or epileptic insult provide a
neuroprotective adaptation of the brain against subsequent severe
ischemic or epileptic insult, normally lethal for neurons [18].
Because ATP-sensitive potassium channels opening through adenosine
A1 receptor activation are a central early step in
cerebral preconditioning, it is possible to mimic preconditioning
pharmacologically with adenosine agonists and KATP
openers [19, 20]. Similarly, an intravenous injection of ALA at 500
nanomoles/kg induce a potent brain tolerance when it is
administered as early as 3 days before severe ischemic or epileptic
injury [21] (figure
2). Palmitic acid, which does not activate TREK-1 channels
fails to protect the brain in both pathologies. The potent delayed
neuroprotection induced by ischemic, epileptic or pharmacological
preconditioning requires de novo synthesis of proteins including
manganese superoxide dismutase, Bcl2 and heat shock
protein 70 in the time window of protection (1 to 3 days).
ALA-induced preconditioning induces a strong HSP70 expression in
the cerebral structures including the CA1 region of hippocampus,
normally damaged by severe ischemia [17] (figure 2).
TREK-1 channel, lipids and neuronal protection
The TREK-1 channels have no specific blockers. In order to study
the physiopathological role of TREK-1 in vivo, its gene has been
disrupted by homologous recombination in the mouse. Using this
knockout (KO) mouse model, recent studies indicate a central role
for TREK-1 in general anaesthesia, pain perception, depression and
neuroprotection [13, 22-24]. To test the resistance of KO mice to
global ischemia, a 30 min transient bilateral occlusion of
common carotid arteries (CCA) is associated with a systemic
hypotension (Mean Arterial Blood Pressure, MABP 30 ± 3 mmHg).
During the recovery period, most of the knockout mice developed
seizures of progressive severity leading to a 40% increase in the
number of deaths for the Trek1–/– mice compared to the
Trek1+/+ mice [22] (figure 3). Using two
models of epilepsy (seizures induced by epileptogenic doses of
kainate (a glutamate agonist, 22 mg/kg) or pentylenetetrazole
(a GABA antagonist, 40 to 55 mg/kg), results show that
Trek1–/– mice were much more sensitive to epilepsy. More
than 75% of the mutant mice died within 3 days of kainate
administration, compared with 3% of Trek1+/+ mice, and
the average maximum intensity of seizures observed in
Trek1–/– mice increased by 33%. Trek1–/– mice
developed generalized convulsive seizures with the appearance of
bilateral spike-wave discharges with spike frequencies and
amplitudes higher than in Trek1+/+ mice. Activation of
c-fos, routinely used as a biochemical marker of neuronal
excitability is drastically enhanced in Trek1-/- mice
compared to Trek1+/+ mice, particularly in CA3 subfield
at 120 min after kainate injection [22]. While ALA treatment
is neuroprotective against global ischemia and seizures, such
neuroprotection is lost in Trek1–/– mice, which
indicates that protection by PUFA is mediated by TREK-1 opening
[13, 22] (figure
3).
Conclusion
It has been well established that PUFA and particularly ALA and DHA
administered in acute treatment are potent neuroprotectors against
ischemia and epilepsy. In term of prevention, PUFA induce a strong
ischemic and epileptic tolerance. Trek1 knockout mice provide
evidence for the important functional role of this K+
channel in PUFA-neuroprotection. At the pharmacological level,
future studies will be needed to identify high-affinity openers of
the TREK-1 channel that might prove useful for the treatment of a
range of neuronal disease states.
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