Geneviève Bureau

Resveratrol and Quercetin, Two Natural Polyphenols, Reduce Apoptotic Neuronal Cell Death Induced by Neuroinflammation

Parkinsons disease (PD) is a movement disorder characterized by a progressive loss of nigrostriatal dopaminergic neurons. Microglia activation and neuroinflammation have been associated with the pathogenesis of PD. Indeed, cytokines have been proposed as candidates that mediate the apoptotic cell death of dopaminergic neurons seen in PD. In this study, we investigated the effect of two natural polyphenols, resveratrol and quercetin, on neuroinflammation. For glial cells, we observed that lipopolysaccharide (LPS)‐induced mRNA levels of two proinflammatory genes, interleukin 1‐α and tumor necrosis factor‐α, are strongly decreased by treatments with resveratrol or quercetin. We also undertook microglial‐neuronal coculture to examine the influence of resveratrol and quercetin on dopaminergic neuronal cell death evoked by LPS‐activated microglia. Cytotoxicity assays were performed to evaluate the percentage of cell death, with apoptotic cells identified by both the TdT‐mediated dUTP nick end labeling technique and the detection of cleaved caspase‐3. We report that treatment of N9 microglial cells with resveratrol or quercetin successfully reduced the inflammation‐mediated apoptotic death of neuronal cells in our coculture system. Altogether our results demonstrate that resveratrol and quercetin diminished apoptotic neuronal cell death induced by microglial activation and suggest that these two phytoestrogens may be potent antiinflammatory compounds.

Striatal inhibition of PKA prevents levodopa-induced behavioural and molecular changes in the hemiparkinsonian rat

l-3,4-dihydroxyphenylalanine methyl ester hydrochloride (l-DOPA) is the gold standard for symptomatic treatment of Parkinsons disease (PD), but long-term therapy is associated with the emergence of abnormal involuntary movements (AIMS) known as l-DOPA-induced dyskinesias (LID). The molecular changes underlying LID are not completely understood. Using the 6-hydroxydopamine-lesioned rat model of PD, we showed that l-DOPA elicits profound alterations in the activity of three LID molecular markers, namely DeltaFosB, dopamine, cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) and extracellular signal-regulated kinases 1 and 2 (ERK1/2), as well as in phosphorylation levels of the cytoskeletal-associated protein tau. These modifications are triggered by protein kinase A (PKA) activation and intermittent stimulation of dopamine receptors as they are totally prevented by intrastriatal injections of Rp-cAMPS, a PKA inhibitor, or by continuous administration of l-DOPA via subcutaneous mini-pump. Importantly, Rp-cAMPS does not modulate the positive effect of l-DOPA on locomotor deficits and significantly attenuates the emergence of AIMS in 6-hydroxydopamine hydrobromide-lesioned rats. Even if decreased PKA signalling in the striatum may represent a clinical challenge, these data provide novel evidence that PKA activation, through modification of striatal signalling and alterations of cytoskeletal constituents, plays a key role in the manifestation of LID.

Alpha and beta estradiol protect neuronal but not native PC12 cells from paraquat-induced oxidative stress

Oxidative stress is currently considered a mediator of cell death in several neurodegenerative diseases. Notably, it may play an important role in the degeneration of dopamine neurons of the substantia nigra in Parkinson’s disease. We examined the effect of a strong oxidant, the herbicide paraquat, on cell distress using native and neuronal pheochromocytoma PC12 cells. Paraquat administration for 8 hours induced a significant cellular death in both native and in neuronal PC12 cells. Since the anti-oxidant properties of estrogens may promote neuroprotectionin vitro andin vivo, we then investigated the ability of estradiol stereoisomers, 17α-estradiol and 17β-estradiol, to rescue PC12 cells submitted to paraquat-induced oxidative stress. Our results show a protective effect of both estradiol stereoisomers in neuronal PC12 cells treated with paraquat, whereas this effect could not be observed in native PC12 cells. We also demonstrate that estrogen receptor β protein expression is modulated by paraquat administration in native PC12 cells, while paraquat does not change estrogen receptor β expression in neuronal PC12 cells. Paraquat also decreases estrogen receptor α in neuronal PC12 cells, thus suggesting new routes for paraquat to collapse cellular metabolism. Besides, the oxidation of dihydrodhodamine-123 into fluorescent rhodamine in the presence of paraquat but not in presence of paraquat and 17α-estradiol or 17β-estradiol,sustain a possible direct scavenging role of both estradiol stereoisomers.

Intrastriatal inhibition of extracellular signal-regulated kinases impaired the consolidation phase of motor skill learning.

It is well known that motor skill learning is characterized by rapid improvement in performances within the first training session and a slower progression in the following sessions that is correlated to the consolidation phase. Our goal was to establish the regional mapping of neural activity in relation to the motor skill learning included in the accelerating rotarod task using Zif268, c-Fos and ERK 1/2. As ERK 1/2 activity is also a marker of adaptive response to synaptic activation for newly learned events, its role was also verified. Learning the rotarod task did not affect levels of Zif268, but induced a selective upregulation of c-Fos in the cerebellum, motor cortex M1 and M2, cingulate cortex CG1 and CG2 as well as dorsal striatum. Notably, levels of phosphorylated ERK 1/2 were selectively increased in this later region during consolidation phase. To further study this effect, we injected inhibitors of ERK activation, the SL327 intraperitoneally or the PD98059 directly into the dorsal striatum, and observed that motor performances were exclusively impaired in this phase. These findings indicate that ERK 1/2 activity of the dorsal striatum is critical for the consolidation of late but not early phase of motor skill memory.

Effects of estradiol, phytoestrogens, and ginkgo biloba extracts against 1-methyl-4-phenyl-pyridine-induced oxidative stress

Oxidative stress has been recently considered as a mediator of nerve cell death in several neurodegenerative diseases. We studied the effect of the parkinsonism-inducing toxine 1-methyl-4-phenyl-pyridine (MPP+) on several parameters of cell distress using native and neuronal PC12 cells. Then, since estrogens have been reported to prevent neuronal degeneration caused by oxidative damage, we investigated the ability of 17β-estradiol (E2); two Ginkgo biloba extracts, EGb 761 and Cp 202; as well as two flavonoids, quercetin and kaempferol, to rescue PC12 cells submitted to MPP+-induced oxidative stress. Our results consistently show that both Ginkgo biloba extracts could prevent cell death in native and neuronal PC12 cells, while in neuronal PC12 cells also quercetin and E2 could reverse MPP+ neurotoxic effet. Western blot analysis demonstrated that MPP+ injuries might modulate dopamine transporter (DAT) protein expression but not estrogen receptor β (ERβ) protein expression. EGb 761 and Cp 202 also modulate DAT and ERβ protein expression in neuronal cells. From these studies, we outline the importance of testing estrogen-like plant-derived molecules as potent antioxidants and examine their effect on protein expression.

Blockade of NMDA receptors 2A subunit in the dorsal striatum impairs the learning of a complex motor skill.

Accumulating evidence proposes that the striatum, known to control voluntary movement, may also play a role in learning and memory. Striatum learning is thought to require long-lasting reorganization of striatal circuits and changes in the strength of synaptic connections during the memorization of a complex motor task. Whether the ionotropic glutamate receptor N-methyl-D-aspartate (NMDAR) contributes to the molecular mechanisms of these memory processes is still unclear. The aim of the present study was to investigate the role of striatal NMDAR and its subunit composition during the learning of the accelerating rotarod task in mice. To this end, we injected directly into the dorsal striatum of mice, via chronically implanted cannula, the NMDAR channel blocker MK-801 as well as the NR2A and NR2B subunit-selective antagonists NVP-AAM077 and Ro 25-6981, respectively, before rotarod training. There was no effect in the motor performances of mice treated with 1.0 μg/side of MK-801, 0.1 μg/side of NVP-AAM077, or 5 and 10 μg/side of Ro 25-6981. In contrast, injections of 2.5 and 5 μg/side of MK-801 or 0.5 and 1 μg/side of NVP-AAM077 impaired motor learning at Day 3 and 8. Interestingly, treatments with MK-801 and NVP-AAM077 did not alter the general motor capacities of mice as revealed by the stepping, wire suspension, and pole tests. Our study demonstrates that the NMDAR of the dorsal striatum contributes to motor learning, especially during the slow acquisition phase, and that NR2A subunits play a critical role in this process.

Partial dopamine depletion in MPTP-treated mice differentially altered motor skill learning and action control.

Recent findings suggest that the neurotransmitter dopamine (DA) system plays a role in motor control and the acquisition of habits and skills. However, isolating DA-mediated motor learning from motor performance remains challenging as most studies include often severely DA-depleted mice. Using the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we investigated the effect of various degrees of DA-depletion in mice on three tests of motor behaviors: the accelerating rotarod, wire suspension and pole tests. Three protocols were performed to decrease DA synthesis to various extents: 4 injections (i.p.) of 9 mg/kg in 1 day; 4 injections (i.p.) of 15 mg/kg in 1 day; or 5 injections (s.c.) of 30 mg/kg in 5 days. Severity of DA-depletion was assessed by the evaluation of tyrosine hydroxylase (TH) and dopamine transporter levels in the striatum using the Western blot technique. Mice were gathered into four different groups according their TH levels: mild, moderate, marked and severe. In these mice, the general motor abilities such as coordination, motion speed and muscular strength were relatively intact whereas impaired acquisition of skilled behavior occurred in mice with marked and severe reduction in TH levels. Marked and severely DA-depleted mice exhibited lower scores within the first trials of the first training day as well as a much slower progression in the following days on the accelerating rotarod. Based on these results, we conclude that the learning of a skilled behavior is more vulnerable to DA depletion than the DA-mediated control of motor activity.

Differential gene expression profiling in the mouse brain during motor skill learning : focus on the striatum structure.

Much research has implicated the striatum in motor learning, but the underlying mechanism is still under extensive investigation. In this study, genome-wide analysis of gene expression was conducted in mice that have learned a complex motor task. It is well recognized that successful learning requires repetitive training and is learned slowly over several training sessions. We therefore used mice that have fully learned the accelerating rotarod task that discriminates the faster and slower phases of motor learning. As important modulators of movement behavior, the striatum was the target of this analysis along with the cerebellum and anterior cortex. To identify potential genes implicated in long memorization process, we compared the lists of genes modulated in the striatum to those modulated in the cerebellum and cortex. As a second approach, we also determined which gene ontology categories were enriched in modulated striatal genes and identified genes with the highest numbers of annotation throughout categories. Although only some of these changes were further confirmed by RT-PCR, these two complementary analyses allowed the identification of highly relevant genes like calcium/calmodulin-dependent protein kinase 2, protein kinase C zeta and N-methyl-D-aspartate receptors. Notably, these genes are all associated with synaptic plasticity, suggesting that stabilized neuronal connections in the striatum are the foundation of durable motor memory. Our study provides the first report of a whole genome analysis of gene expression in mice that have memorized a new complex motor task, and expands our knowledge on striatal gene expression changes associated with motor skill learning.

Regulation of Tyrosine Phosphatase STEP61 by Protein Kinase A during Motor Skill Learning in Mice

Recently, striatal-enriched protein tyrosine phosphatase (STEP) and its upstream regulator protein kinase A (PKA) have been suspected to play a role in the intracellular mechanisms of fear conditioning and spatial memory. However, whether they contribute to the learning and memory of motor skills is totally unknown. In this study, we have investigated the role of STEP and PKA activities during motor skill learning associated with the accelerating rotarod task. We observed that learning the rotarod task differentially modulated the levels of phosphorylated STEP61 at serine 221, a site directly regulated by PKA, in the hippocampus, motor cortex and striatum. In a second set of experiments, we have pharmacologically inhibited PKA by the injection of Rp-cAMPS directly into the dorsal striatum of mice before rotarod trainings. PKA phosphorylation of STEP prevents the dephosphorylation of STEP substrates, whereas inhibition of PKA promotes STEP activity. Striatal PKA inhibitions dose-dependently impaired mice performances on the accelerating rotarod task. General motor abilities testing revealed an intact motor control in mice treated with 5 and 20 µg of Rp-cAMPS, but not at the highest dose of 40 µg. This suggested that motor learning was selectively affected by PKA inhibition at lower doses. Most notably, striatal inhibition of PKA reduced the levels of phosphorylated STEP61 at serine 221. Our data support that inactivation of STEP61 by the PKA activity is part of the molecular process associated with motor skill learning.

Genetic Deletion of Akt3 Induces an Endophenotype Reminiscent of Psychiatric Manifestations in Mice

The protein kinase B (PKB/Akt), found in three distinctive isoforms (PKBα/Akt1, PKBβ/Akt2, PKBγ/Akt3), is implicated in a variety of cellular processes such as cell development, growth and survival. Although Akt3 is the most expressed isoform in the brain, its role in cerebral functions is still unclear. In the present study, we investigated the behavioral, electrophysiological and biochemical consequences of Akt3 deletion in mice. Motor abilities, spatial navigation, recognition memory and LTP are intact in the Akt3 knockout (KO) mice. However, the prepulse inhibition, three-chamber social, forced swim, tail suspension, open field, elevated plus maze and light-dark transition tests revealed an endophenotype reminiscent of psychiatric manifestations such as schizophrenia, anxiety and depression. Biochemical investigations revealed that Akt3 deletion was associated with reduced levels of phosphorylated GSK3α/β at serine 21/9 in several brain regions, although Akt1 and Akt2 levels were unaffected. Notably, chronic administration of lithium, a mood stabilizer, restored the decreased phosphorylated GSK3α/β levels and rescued the depressive and anxiety-like behaviors in the Akt3 KO mice. Collectively, our data suggest that Akt3 might be a critical molecule underlying psychiatric-related behaviors in mice.