Michèle Bastide

PPAR: a new pharmacological target for neuroprotection in stroke and neurodegenerative diseases

PPARs (peroxisome-proliferator-activated receptors) are ligand-activated transcriptional factor receptors belonging to the so-called nuclear receptor family. The three isoforms of PPAR (alpha, beta/delta and gamma) are involved in regulation of lipid or glucose metabolism. Beyond metabolic effects, PPARalpha and PPARgamma activation also induces anti-inflammatory and antioxidant effects in different organs. These pleiotropic effects explain why PPARalpha or PPARgamma activation has been tested as a neuroprotective agent in cerebral ischaemia. Fibrates and other non-fibrate PPARalpha activators as well as thiazolidinediones and other non-thiazolidinedione PPARgamma agonists have been demonstrated to induce both preventive and acute neuroprotection. This neuroprotective effect involves both cerebral and vascular mechanisms. PPAR activation induces a decrease in neuronal death by prevention of oxidative or inflammatory mechanisms implicated in cerebral injury. PPARalpha activation induces also a vascular protection as demonstrated by prevention of post-ischaemic endothelial dysfunction. These vascular effects result from a decrease in oxidative stress and prevention of adhesion proteins, such as vascular cell adhesion molecule 1 or intercellular cell-adhesion molecule 1. Moreover, PPAR activation might be able to induce neurorepair and endothelium regeneration. Beyond neuroprotection in cerebral ischaemia, PPARs are also pertinent pharmacological targets to induce neuroprotection in chronic neurodegenerative diseases.

Increase in endogenous brain superoxide dismutase as a potential mechanism of lipopolysaccharide-induced brain ischemic tolerance.

A low dose (0.5 mg/kg) of lipopolysaccharide (LPS), administered 72 hours before 60-minute middle cerebral artery occlusion, induced a delayed neuroprotection proven by the significant decrease (–35%) of brain infarct volume in comparison with control, whereas infarct volumes remained unchanged in rats treated 12, 24, or 168 hours before ischemia. This delayed neuroprotective effect of LPS was induced only with low doses (0.25 to 1 mg/kg), whereas this effect disappeared with a higher dose (2 mg/kg). The delayed neuroprotection of LPS was induced in the cortical part of the infarcted zone, not in the subcortical part. The beneficial effect of LPS on consequences of middle cerebral artery occlusion was suppressed by dexamethasone (3 mg/kg) and indomethacin (3 mg/kg) administered 1 hour before LPS, whereas both drugs had no direct effect on infarct volume by themselves, suggesting that activation of inflammatory pathway is involved in the development of LPS-induced brain ischemic tolerance. Preadministration of cycloheximide, an inhibitor of protein synthesis, also blocked LPS-induced brain ischemic tolerance suggesting that a protein synthesis is also necessary as a mediating mechanism. Superoxide dismutase (SOD) could be one of the synthesized proteins because lipopolysaccharide increased SOD brain activity 72 hours, but not 12 hours, after its administration, which paralleled the development of brain ischemic tolerance. In contrast, catalase brain activity remained unchanged after LPS administration. The LPS-induced delayed increase in SOD brain content was suppressed by a previous administration of indomethacin. These data suggest that the delayed neuroprotective effect of low doses of LPS is mediated by an increased synthesis of brain SOD that could be triggered by activation of inflammatory pathway.

Differential role of nitric oxide pathway and heat shock protein in preconditioning and lipopolysaccharide-induced brain ischemic tolerance.

The purposes of this study were to investigate the role of nitric oxide (NO), nitric oxide synthase (NOS), and 70 kDa heat shock protein in brain ischemic tolerance induced by ischemic preconditioning and lipopolysaccharide. Focal cerebral ischemia was induced in rats by intraluminal middle cerebral artery occlusion. Infarct volume was significantly reduced (1) in rats subjected to 3 min ischemia 72 h prior to 60 min ischemia; (2) in rats administered lipopolysaccharide (0.5 mg/kg; i.p.) 72 h prior to 60 min ischemia compared with controls. The beneficial effect of ischemic preconditioning was unchanged despite prior administration of nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor. Conversely, the protective effect of lipopolysaccharide was nullified by L-NAME. Using immunohistochemical techniques, we observed that (1) ischemic preconditioning but not lipopolysaccharide induces the expression of 70 kDa heat shock protein in cerebral cortex and (2) lipopolysaccharide induces early increased expression of endothelial NOS in cerebral blood vessels. The results suggest that (1) endothelium-derived NO plays a role of a trigger in the brain tolerance induced by lipopolysaccharide, and (2) 70 kDa heat shock protein is involved in the protection afforded by ischemic preconditioning but not by lipopolysaccharide.

Delayed Cerebrovascular Protective Effect of Lipopolysaccharide in Parallel to Brain Ischemic Tolerance

Cerebrovascular abnormalities, in endothelium and smooth muscle compartments, occur in the course of cerebral ischemia–reperfusion as evidenced by the impairment of endothelium-dependent relaxation and decrease in potassium inward rectifier density in occluded middle cerebral arteries (MCAs). The authors investigated whether a delayed vascular protection occurred in a model of brain ischemic tolerance. A low dose of lipopolysaccharide (0.3 mg/kg) administered 72 h before MCA occlusion induced a significant decrease in infarct volume. In parallel to this delayed neuroprotective effect, lipopolysaccharide prevented the ischemia–reperfusion–induced impairment of endothelium relaxation. In addition, lipopolysaccharide prevented the postischemic alteration of potassium inward rectifier–dependent smooth muscle relaxation as well as the decrease in potassium inward rectifier density measured by patch-clamp in dissociated vascular smooth muscle cells originated from the occluded MCA. These results suggest that during brain ischemic tolerance, lipopolysaccharide is able to induce both a delayed neuroprotective and vasculoprotective effect.

Ferroptosis, a newly characterized form of cell death in Parkinson's disease that is regulated by PKC.

Parkinsons disease (PD) is a complex illness characterized by progressive dopaminergic neuronal loss. Several mechanisms associated with the iron-induced death of dopaminergic cells have been described. Ferroptosis is an iron-dependent, regulated cell death process that was recently described in cancer. Our present work show that ferroptosis is an important cell death pathway for dopaminergic neurons. Ferroptosis was characterized in Lund human mesencephalic cells and then confirmed ex vivo (in organotypic slice cultures) and in vivo (in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model). Some of the observed characteristics of ferroptosis differed from those reported previously. For example, ferroptosis may be initiated by PKCα activation, which then activates MEK in a RAS-independent manner. The present study is the first to emphasize the importance of ferroptosis dysregulation in PD. In neurodegenerative diseases like PD, iron chelators, Fer-1 derivatives and PKC inhibitors may be strong drug candidates to pharmacologically modulate the ferroptotic signaling cascade.

Relationship between Inward Rectifier Potassium Current Impairment and Brain Injury after Cerebral Ischemia/Reperfusion

Functional alterations of barium-sensitive potassium inward rectifier (Kir) current, which is involved in the vasodilation of middle cerebral arteries (MCA) in rat brain, have been described during brain ischemiaireperfusion (I/R). The authors investigate the effects of I/R on Kir current recorded in isolated myocytes from MCA of control rats and from contralateral and ipsilateral MCA of ischemic rats by the whole-cell patch-clamp technique, and the relationship between its alteration and The severity of brain injury. The vascular smooth muscle cells exhibited similar morphologic features in all conditions, and the Kir was present in the three groups of myocytes, exhibiting a characteristic inward rectification and a normal external potassium dependence. The Kir density was significantly reduced in cell of MCA ipsilateral to occlusion with a maximum at −135 mV, whereas there was no difference between control and contralateral cells. This alteration in Kir density in occluded MCA was significantly correlated with severity of brain injury and brain edema. These results suggest that the alteration of Kir density in MCA myocytes after I/R and the consecutive impaired dilation of MCA may contribute to aggravation of the brain injury.

Repolarization abnormalities and their arrhythmogenic consequences in porcine tachycardia-induced cardiomyopathy

OBJECTIVES Action potential prolongation related to the alteration of several membrane currents is constantly reported in heart failure (HF) but reports about its role in arrhythmogenesis are sparse. Our aim was to determine, by analogy with long QT syndromes, whether prolonged repolarization is associated with increased dispersion or linked to bradycardia-dependent ventricular arrhythmias in pacing-induced cardiomyopathy. METHODS QT intervals, action potentials and transmural activation-to-recovery intervals (ARIs) along with whole-cell delayed rectifier (I(K)) and transient outward (I(to1)) K+ currents were recorded in left ventricle from pigs with HF and controls. HF was obtained after 14 days of rapid pacing at 250 ms. RESULTS Repolarization was delayed as indexed by corrected QT intervals (13.7% increase, P<0.01) or ARIs (252+/-4 to 340+/-7 ms, P<0.01). ARIs were uniformly prolonged with disappearance of the transmural gradient, spatial dispersion of repolarization decreased by 50% (P<0.05). I(to1) density was reduced in HF from 1.35+/-0.1 to 0.57+/-0.04 pA/pF subepicardially, from 1.05+/-0.19 to 0.55+/-0.08 pA/pF midmyocardially and from 1.04+/-0.1 to 0.48+/-0.04 pA/pF subendocardially. I(K) density was significantly decreased in HF pigs vs. controls: subepicardially from 0.46+/-0.04 to 0.22+/-0.02 pA/pF; midmyocardially from 0.46+/-0.05 to 0.25+/-0.03 pA/pF; and subendocardially from 0.49+/-0.04 to 0.20+/-0.04 pA/pF following depolarization at +50 mV. Electrocardiogram (ECG) monitoring at the time of death did not disclose any polymorphic ventricular tachyarrhythmia. CONCLUSION Despite a profound alteration in K+ currents, repolarization is uniformly prolonged in this model with no proclivity to develop bradycardia-dependent arrhythmias.

Risperidone prolongs cardiac action potential through reduction of K+ currents in rabbit myocytes

Prolongation of QT interval by antipsychotic drugs is an unwanted side effect that may lead to ventricular arrhythmias. The antipsychotic agent risperidone has been shown to cause QT prolongation, especially in case of overdosage. We investigated risperidone effects on action potentials recorded from rabbit Purkinje fibers and ventricular myocardium and on potassium currents recorded from atrial and ventricular rabbit isolated myocytes. The results showed that (1) risperidone (0.1-3 microM) exerted potent lengthening effects on action potential duration in both tissues with higher potency in Purkinje fibers and caused the development of early afterdepolarizations at low stimulation rate; (2) risperidone (0.03-0.3 microM) reduced significantly the current density of the delayed rectifier current and at 30 microM decreased the transient outward and the inward rectifier currents. This study might explain QT prolongation observed in some patients treated with risperidone and gives enlightenment on the risk of cardiac adverse events.

Repercussions of pharmacologic reduction in ionic currents on action potential configuration in rabbit Purkinje fibers: Are they indicative of proarrhythmic potential?

The evaluation of the cardiac safety of newly developed drugs by electrophysiological studies are today mandatory in several countries. A number of experimental models performed either on multicellular preparations or on native or cloned ionic channels have been used but the predictivity of the results remains a matter of debate, particularly if a drug exerts mixed ionic channel‐blocking effects. The present study was designed to scrutinize the repercussions of selectively decreasing the main ionic currents which play a role in the repolarization process. Using conventional microelectrodes, action potentials were recorded from rabbit Purkinje fibers stimulated at 1 Hz and 0.2 Hz and exposed to a broad range of cumulative and increasing concentrations. The compounds used were 1) potassium channel blockers (4‐aminopyridine, dofetilide, terikalant, and indapamide), 2) chloride current blocker (4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid), and 3) calcium channel antagonists (nifedipine, verapamil, and cadmium). It appears from our results that the modifications observed in the repolarization phase and in the spike‐and‐dome aspect showed that the antagonists evaluated were not as specific as one would expected and that they affected action potential profiles as a result of additional nonspecific effects which may influence the incidence of proarrhythmic events. In conclusion, we propose the use of rabbit Purkinje fibers as an in vitro model adequate for the evaluation of the cardiac safety of newly developed drugs. Drug Dev. Res. 47:63–76, 1999.

Withdrawal of fenofibrate treatment partially abrogates preventive neuroprotection in stroke via loss of vascular protection.

To explore the mechanisms of action of preventive neuroprotection induced by PPAR-alpha activation, we have evaluated the neuronal, vascular effects of preventive treatment with fenofibrate up until the induction of experimental brain ischaemia and fenofibrate treatment withdrawn 3days before ischaemia induction. Fenofibrate (200mg/kg/day) was administered in rats for 14days or withdrawn 3days before induction of cerebral ischaemia. Animals underwent a 1-hour middle cerebral artery occlusion (MCAo), followed by reperfusion for 24h. The MCAs vasoreactivity was analyzed and brain sections were used to assess infarct size, inflammatory and oxidative stress markers. Fenofibrate administration significantly decreases the cerebral infarct volume. This effect was associated with partial prevention of post-ischaemic endothelial dysfunction. However, withdrawal of the fenofibrate treatment 3days before the induction of ischaemia reduced the neuroprotection and was less beneficial in preventing endothelial dysfunction as well as superoxide anion production. In contrast, fenofibrate significantly reduced microglial activation and neutrophil infiltration into the ischaemic zone to a similar extent in both treatment modes. Our results show that the fenofibrate-induced cerebral protective effect may be related to both an acute effect and a preconditioning-like mechanism. The vascular protective effect appears rather to translate the acute effects of fenofibrate administration.