A cell-permeable peptide inhibitor TAT-JBD reduces the MPP+-induced caspase-9 activation but does not prevent the dopaminergic degeneration in substantia nigra of rats.

Many studies showed that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) which was widely used to produce Parkinson's disease (PD)-like models in animals can elicit apoptosis with increase of caspase activity via its neurotoxic metabolite 1-methyl-4-phenylpyridinium ion (MPP(+)). Another pathway shown in MPTP-mediated nigrostriatal dopaminergic cell death involved the c-Jun-N-terminal kinases (JNKs) which are stress-activated protein kinases (SAPKs). Activation of the JNKs leads to the activation of transcription factors such as c-Jun that regulates its own expression. However, it is not known whether the activation of c-Jun is crucial in the stimulation of caspases leading to apoptosis observed in PD-like models. The aim of this study was to investigate the cellular expression and phosphorylation of c-Jun and the caspase-9 activity in rat injured with an intranigral injection of MPP(+). Furthermore, we determined the effects of a cell-permeable peptide TAT-JBD, inhibiting selectively JNKs, on apoptosis markers and on the expression of tyrosine hydroxylase (TH). Our results showed that MPP(+) induced not only an activation of c-Jun but also an early and robust stimulation of caspase-9 in midbrain of rats. Furthermore, a preliminary intravenous injection of TAT-JBD reduced the caspase-9 activation specifically induced by MPP(+) suggesting a control of the JNKs pathway on the intrinsic way of apoptosis in MPP(+)-toxicity. However, the inhibition of the JNK pathway did not prevent TH inhibition, DNA fragmentation and Bad expression in MPP(+)-lesioned substantia nigra of rats. Therefore, the possibility of intervention on the JNK pathway as a therapeutic strategy in Parkinson's disease is questionable.

Regulation of initiation factors controlling protein synthesis on cultured astrocytes in lactic acid-induced stress.

The goals of this work were first to assess whether the lactic acidosis observed in vivo in ischemia may by itself explain the inhibition of protein synthesis described in the literature and second to study the factors controlling the initiation of protein synthesis under lactic acid stress. Primary rat astrocyte cultures exposed to pH 5.25 underwent cell death and a strong inhibition of protein synthesis assessed by [3H]methionine incorporation, which was solely due to acidity of the extracellular medium and was not related to lactate concentrations. This result was associated with a weak phosphorylation of eukaryotic initiation factor (eIF)4E and a rapid phosphorylation of eIF2alpha via the kinases PKR and PKR-like endoplasmic reticulum kinase. The inhibition of PKR by PRI led first to a significant but not complete dephosphorylation of eIF2alpha that probably contributed to maintain the inhibition of the protein synthesis and second to surprising phosphorylations of extracellular signal-regulated protein kinase, p70S6K and eIF4E, suggesting a possible cross-link between the two pathways. Conversely, cell death was weak at pH 5.5. Protein synthesis was decreased to a lesser extent, the phosphorylation of eIF2alpha was limited, extracellular signal-regulated protein kinase 1/2 was activated and its downstream targets, p70S6K and eIF4E, were phosphorylated. However, the strong phosphorylation of eIF4E was not associated with an activation of the eIF4F complex. This last result may explain why protein synthesis was not stimulated at pH 5.5. However, when astrocytes were exposed at pH 6.2, corresponding to the lower pH observed in hyperglycemic ischemia, no modification in protein synthesis was observed. Consequently, lactic acidosis cannot, by itself, provide an explanation for the decrease in protein synthesis previously reported in vivo in ischemia.

Group I metabotropic glutamate receptors activate the p70S6 kinase via both mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK 1/2) signaling pathways in rat striatal and hippocampal synaptoneurosomes.

Group I metabotropic glutamate receptors (mGluRs) have been demonstrated to play a role in synaptic plasticity via a rapamycin-sensitive mRNA translation signaling pathway. Various growth factors can stimulate this pathway, leading to the phosphorylation and activation of mammalian target of rapamycin (mTOR), a serine/threonine protein kinase that modulates the activity of several translation regulatory factors, such as p70S6 kinase. However, little is known about the cellular and molecular mechanisms that bring the plastic changes of synaptic transmission after stimulation of group I mGluRs. Here, we investigated the role of the mTOR-p70S6K and the ERK1/2-p70S6K pathways in rat striatal and hippocampal synaptoneurosomes after group I mGluR stimulation. Our findings show that (S)-3,5-dihydroxyphenylglycine (DHPG) increases significantly the activation of mTOR and p70S6K (Thr389, controlled by mTOR) in both brain areas. The mTOR activation is dose-dependent and requires the stimulation of mGluR1 subtype receptors as for the p70S6K activation observed in striatum and hippocampus. In addition, the p70S6K (Thr421/Ser424) activation via the ERK1/2 activation is increased and involved also mGluR1 receptors. These results demonstrate that group I mGluRs are coupled to mTOR-p70S6K and ERK1/2-p70S6K pathways in striatal and hippocampal synaptoneurosomes. The translational factor p70S6K could be involved in the group I mGluRs-modulated synaptic efficacy.

Lactic acidosis stimulates ganglioside and ceramide generation without sphingomyelin hydrolysis in rat cortical astrocytes.

Acidosis is a ubiquitous feature of cerebral ischemia, and triggers a cascade of biochemical events that results in neuronal injury. The purpose of this study was to evaluate the effects of lactic acidosis on the ganglioside composition, the ceramide and sphingomyelin (SM) levels in rat cortical astrocytes. Primary astrocyte cultures were exposed to lactic acid (pH 5.5) for 2, 5 and 17 h, and cell death was evaluated at each time point. Gangliosides, ceramides and SM were analyzed by high-performance thin layer chromatography. Lactic acidosis caused a progressive increase of both GM3 and GD3 gangliosides up to 5 h of treatment. However, at 17 h of acidosis, GM3 tented to return to the normal level whereas GD3 accumulated. Additionally, ceramides were gradually generated, whereas no significant decrease of SM occured for 17 h of acidosis. These results suggest that ceramides were not produced by the breakdown of SM and may be served as metabolic precursor for the biosynthesis of GM3 and GD3. Since these lipids are important messengers of the adaptative responses to stress, accumulation of sphingolipids triggered by lactic acid exposure of astrocytes might play an important role in determining the outcomes of injurious processes.

The up-regulation of the striatal dopamine transporter's activity by cAMP is PKA-, CaMK II- and phosphatase-dependent.

The neuronal dopamine transporter (DAT) is a presynaptic plasma membrane protein mediating the re-uptake of dopamine released from synaptic cleft into the nerve terminals. While the regulation of its activity by protein kinase C signalling is well-characterized, there is controversial debate about its regulation by protein kinase A (PKA) signalling. In rat striatal synaptosomes, we showed that a cell-permeable cyclic adenosine 3',5'-monophosphate analogue up-regulated the DAT capacity without modification of its efficiency. This acute effect was PKA-, calcium calmodulin dependent kinase II- and phosphatase-dependent. Together, these results suggest that the activity of DAT may depend on a state of the transporter with both specific phosphorylated and dephosphorylated sites.

Effect of ischemia on TBARS and lactate production in several cerebral regions of anaesthetised and awake rats.

The premise of neuroprotective therapy for acute ischemic stroke is based upon the possibility to interfere with the cellular ischemic cascade, so the understanding of the mechanisms and consequences of cerebral ischemia is necessary. The relationship between lipid peroxidation and acidosis was investigated in several regions of rat brain following ischemia without reperfusion. Male Wistar rats (280-300 g) were anaesthetised (Ketalar 33 mg/kg and Rompun 6.66 mg/kg) or not and underwent a four-vessel occlusion for 5 minutes. Then, thiobarbituric acid-reactive substances (TBARS) and lactate levels were measured in different brain regions (cerebellum, bulb, striatum, hippocampus, cortex). Induction of ischemia by ligation of two common carotid arteries and two vertebral arteries resulted in a production of TBARS (40-120%, p < 0.05) and lactate (20-60%, p < 0.05) in all cerebral regions of awake rats, especially in striatum, suggesting a potential link between lipid peroxidation and acidosis. When ischemia was realised on anaesthetised animals, an increase of lactate levels (30-50%, p < 0.05) was shown in all brain regions but TBARS were produced only in striatum (82%, p < 0.05). These data showed the particular vulnerability of striatum to ischemia and the possible opposite effects of an anaesthesia.

Sulfatide and GM1 ganglioside modulate the high-affinity dopamine uptake in rat striatal synaptosomes: evidence for the involvement of their ionic charges.

The present study was undertaken to examine the effects of the anionic glycolipids GM1 ganglioside and sulfatide on the high-affinity dopamine (DA) uptake in rat striatal synaptosomes. After 1h of incubation, GM1 stably bound to synaptosomes and modified the activity of the neuronal dopamine transporter (DAT). With 1.2 and 12 microM GM1, V(max) decreased by 13 and 23%, respectively, reflecting a slight reduction of the number of functional uptake sites and K(m) was lowered by 21 and 33%, thus showing an increase of the affinity. Treatment of synaptosomes with 1.2 microM of sulfatide, which possesses an anionic sulfated group, led to a similar decrease of V(max) (19%) than GM1, but to a significantly higher reduction of K(m) (35%). In fact, sulfatide associated to synaptosomes in a 3.5-fold higher extent than GM1. Conversely, when GM1 and sulfatide were replaced by GM1 alcohol and galactosylceramide, respectively, no modification of the DA uptake occurred, although these neutral glycolipids incorporated into the synaptosomes to the same extent as the related anionic compounds.Altogether, these results demonstrate the key role of negative charges linked to the oligosaccharide chains of glycolipids in the modulation of DA transport across the synaptosomal membrane.

Induction of heat shock proteins (HSPs) by sodium arsenite in cultured astrocytes and reduction of hydrogen peroxide-induced cell death.

Induction of heat shock proteins (HSPs) protects cells from oxidative injury. Here Hsp72, Hsp27 and heme oxygenase-1 (HO-1) were induced in cultured rat astrocytes, and protection against oxidative stress was investigated. Astrocytes were treated with sodium arsenite (20-50 micro m) for 1 h, which was non-toxic to cells, 24 h later they were exposed to 400 micro m H2O2 for 1 h, and cell death was evaluated at different time points. Arsenite triggered strong induction of HSPs, which was prevented by 1 micro g/mL cycloheximide (CXH). H2O2 caused cell loss and increased cell death with features of apoptosis, i.e. TdT-mediated dUTP nick-end labelling (TUNEL) reaction and caspase-3 activation. These features were abrogated by pre-treatment with arsenite, which prevented cell loss and significantly reduced the number of dead cells. The protective effect of arsenite was not detected in the presence of CHX. Pre-treatment with arsenite increased protein kinase B (Akt) and extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation after H2O2. However, while Akt phosphorylation was prevented by CHX, Erk1/2 phosphorylation was further enhanced by CHX. The results show that transient arsenite pre-treatment induces Hsp72, HO-1 and, to a lesser extent, Hsp27; it reduces H2O2-induced astrocyte death; and it causes selective activation of Akt following H2O2. It is suggested that HSP expression at the time of H2O2 exposure protects astrocytes from oxidative injury and apoptotic cell death by means of pro-survival Akt.

Aluminum L-glutamate complex in rat brain cortex: in vivo prevention of aluminum deposit by magnesium D-aspartate.

Our previous experiments in the rat showed that aluminum L-glutamate complex (Al L-Glu) crosses the blood-brain barrier and accumulates in selective brain areas and that Al salts may increase D-aspartic acid forms in living brain proteins, probably by inducing more thermodynamically stable structures than L isomers. As magnesium blocks NMDA receptors, D-aspartic acid was used in the present study in the form of magnesium salt to prevent the excitotoxicity of dicarboxylic amino acids. Effects on brain amino acids and Al cortex levels in mature rats were studied after chronic treatment with Al L-Glu or Na L-Glu alone or in association with magnesium D-aspartate (Mg D-Asp). Results demonstrate that treatment with Mg D-Asp induces a decrease in the Al concentration in brain cortex of Al L-Glu-treated rats. In aluminum-free treated controls, treatment with Mg D-Asp in association with Na L-Glu also induces a decrease in Al concentration in brain cortex. These data indicate that Mg D-Asp administration protects rat brain cortex from Al accumulation and suggest that this treatment may be useful in preventing brain Al intoxication.