Stéphanie Pain

Activated double-stranded RNA-dependent protein kinase and neuronal death in models of Alzheimer’s disease

Neuronal death is a pathological hallmark of Alzheimers disease. We have shown previously that phosphorylated double-stranded RNA-dependent protein kinase is present in degenerating hippocampal neurons and in senile plaques of Alzheimers disease brains and that genetically down-regulating double-stranded RNA-dependent protein kinase activity protects against in vitro beta-amyloid peptide neurotoxicity. In this report, we showed that two double-stranded RNA-dependent protein kinase blockers attenuate, in human neuroblastoma cells, beta-amyloid peptide toxicity evaluated by caspase 3 assessment. In addition, we have used the newly engineered APP(SL)/presenilin 1 knock-in transgenic mice, which display a severe neuronal loss in hippocampal regions, to analyze the activation of double-stranded RNA-dependent protein kinase. Western blots revealed the increased levels of activated double-stranded RNA-dependent protein kinase and the inhibition of eukaryotic initiation factor 2 alpha activity in the brains of these double transgenic mice. Phosphorylated RNA-dependent protein kinase-like endoplasmic reticulum-resident kinase was also increased in the brains of these mice. The levels of activated double-stranded RNA-dependent protein kinase were also increased in the brains of patients with Alzheimers disease. At 3, 6 and 12 months, hippocampal neurons display double stranded RNA-dependent protein kinase labelings in both the nucleus and the cytoplasm. Confocal microscopy showed that almost constantly activated double-stranded RNA-dependent protein kinase co-localized with DNA strand breaks in apoptotic nuclei of CA1 hippocampal neurons. Taken together these results demonstrate that double-stranded RNA-dependent protein kinase is associated with neurodegeneration in APP(SL)/presenilin 1 knock-in mice and could represent a new therapeutic target for neuroprotection.

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.

Neuroprotective Effect of PACAP on Translational Control Alteration and Cognitive Decline in MPTP Parkinsonian Mice

Parkinson’s disease (PD) is characterized by a triade of motor symptoms due to the degeneration of nigrostriatal pathway. In addition to these motor impairments, cognitive disturbances have been reported to occur in PD patients in the early stage of the disease. The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin widely used to produce experimental models of PD. In a previous work, we showed that MPTP altered the expression of proteins involved in mTOR antiapoptotic and PKR apoptotic pathways of translational control (TC) in neuroblastoma cells. In the present study, the results indicated that a subchronic MPTP intoxication in mice decreased the dopaminergic neuron number, produced an activation of PKR way and an inhibition of mTOR way of TC especially in striatum and frontal cortex associated with a great activation of PKR in hippocampus. Moreover, in parallel to biochemical analysis, the mnesic disturbances induced by MPTP were characterized in C57Bl/6 mice, by testing their performance in three versions of the Morris Water Maze task. Behavioral results showed that the MPTP lesion altered mice learning of a spatial working memory, of a cued version and of a spatial reference memory task in the water maze. Furthermore, we previously demonstrated that the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) could counteract the MPTP toxicity on TC factors in neuroblastoma cells. Thus, the second objective of our study was to assess the PACAP effect on MPTP-induced TC impairment and cognitive deficit in mice. The pretreatment with PACAP27 by intravenous injections partially protected TH-positive neuron loss induced by MPTP, prevented the MPTP-induced protein synthesis control dysregulation and mnesic impairment of mice. Therefore, our results could indicate that PACAP may be a promising therapeutic agent in Parkinson’s disease.

The immunosuppressant rapamycin exacerbates neurotoxicity of Aβ peptide

Alzheimers disease (AD) is a neurodegenerative disease of the central nervous system characterized by two major lesions: extracellular senile plaques and intraneuronal neurofibrillary tangles. β‐Amyloid (Aβ) is known to play a major role in the pathogenesis of AD. Protein synthesis and especially translation initiation are modulated by different factors, including the PKR/eIF2 and the mTOR/p70S6K pathways. mRNA translation is altered in the brain of AD patients. Very little is known about the translation control mediated by mTOR in AD, although mTOR is a central regulator of translation initiation and also ribosome biogenesis and cell growth and proliferation. In this study, by using Western blotting, we show that mTOR pathway is down‐regulated by Aβ treatment in human neuroblastoma cells, and the underlying mechanism explaining a transient activation of p70S6K is linked to cross‐talk between mTOR and ERK1/2 at this kinase level. This phenomenon is associated with caspase‐3 activation, and inhibition of mTOR by the inhibitor rapamycin enhances Aβ‐induced cell death. Moreover, in our cell model, insulin‐like growth factor‐1 is able to increase markedly the p70S6K phosphorylation controlled by mTOR and reduces the caspase‐3 activity, but its protective effect on Aβ cell death is mediated via an mTOR‐independent pathway. These results demonstrate that mTOR plays an important role as a cellular survival pathway in Aβ toxicity and could represent a possible target for modulating Aβ toxicity.

Neuroprotection by neuropeptide Y in cell and animal models of Parkinson's disease

This study was aimed to investigate the potential neuroprotective effect of neuropeptide Y (NPY) on the survival of dopaminergic cells in both in vitro and in animal models of Parkinsons disease (PD). NPY protected human SH-SY5Y dopaminergic neuroblastoma cells from 6-hydroxydopamine-induced toxicity. In rat and mice models of PD, striatal injection of NPY preserved the nigrostriatal dopamine pathway from degeneration as evidenced by quantification of (1) tyrosine hydroxylase (TH)-positive cells in the substantia nigra pars compacta, levels of (2) striatal tyrosine hydroxylase and dopamine transporter, (3) dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) as well as (4) rotational behavior. NPY had no neuroprotective effects in mice treated with Y(2) receptor antagonist or in transgenic mice deficient for Y(2) receptor suggesting that NPY effects are mediated through this receptor. Stimulation of Y(2) receptor by NPY triggered the activation of both the ERK1/2 and Akt pathways but did not modify levels of brain derived neurotrophic factor (BDNF) or glial cell line-derived neurotrophic factor. These results open new perspectives in neuroprotective therapies using NPY and suggest potential beneficial effects in PD.

Neuroprotective effects of pituitary adenylate cyclase-activating polypeptide (PACAP) in MPP+-induced alteration of translational control in Neuro-2a neuroblastoma cells.

Parkinsons disease (PD) and 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) toxicity are both associated with dopaminergic neuron death in the substantia nigra. Although a variety of evidence has shown that degenerative cells have apoptotic features, the role of apoptosis in disease pathology remains controversial. The 1‐methyl‐4‐phenylpyridinium ion (MPP+), a metabolite of MPTP, was recently shown to alter the expression of proteins involved in translational control. The initiation step of translational control is regulated by a cascade of phosphorylation affecting proteins of the antiapoptotic way controlled by mammalian target of rapamycin (mTOR) and of the proapoptotic way controlled by double‐stranded RNA protein–dependent kinase (PKR). A study showed that MPP+ induced an increase in eIF2α phosphorylation, leading to inhibition of protein synthesis. The aims of our study were: (1) to assess the effects of MPP+ toxicity on molecular factors of PKR and mTOR signaling pathways in murine neuroblastoma cells, and (2) to examine the ability of VIP and PACAP peptides to counteract the MPP+ toxicity. Our findings showed that MPP+ induced phosphorylation of eIF2α and significantly reduced the expression of phosphorylated mTOR, p70S6K, eIF4E, and 4E‐BP1, suggesting its toxicity in controlling protein synthesis. Furthermore, the VIP peptide had no effect on either the PKR or the mTOR signaling pathway. On the contrary, the PACAP 27 neuropeptide prevented MPP+‐induced eIF2α phosphorylation and blocked MPP+ toxicity in molecular factors of the mTOR pathway. And last, PACAP 27 seemed to protect Neuro‐2a cells from the apoptotic process as assessed by the decreased nuclear condensation after DAPI staining. These results could open new paths of research of PACAP in PD.

Toxicity of MPTP on neurotransmission in three mouse models of Parkinson's disease.

Parkinsons disease (PD) is recognized as the second most common neurodegenerative disorder after Alzheimers disease. PD is mainly characterized by a selective degeneration of the dopaminergic neurons in the substantia nigra. Also, it is observed imbalances in some nondopaminergic systems, including the serotonergic system. Serotonergic dysfunction appears to play a role in some parkinsonian symptoms, including motor function, L-dopa-induced dyskinesia, mood, psychosis, and constipation. The fact that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes a parkinsonian syndrome was discovered in 1982 and has been used extensively and successfully in various mammalian species, including monkeys and mice, to produce an experimental model of PD. Three common dosing regimens of the MPTP-induced mice model of PD were compared on dopaminergic neurotransmission and serotonin levels in various brain regions. Results showed that tyrosine hydroxylase activity and dopaminergic transporter density were reduced in striatum and substantia nigra of mice and that this reduction was dependent on the cumulative dose of MPTP injected. Furthermore, for the three protocols, a decrease of dopamine (DA) level was observed in striatum, associated with a significant diminution of DA concentration in frontal cortex only for the chronic treatment. Moreover, a decrease of serotonin level was observed in midbrain and hippocampus of acute and sub-acute intoxicated-mice. In all, the results suggested that dosing regimen should be carefully pre-considered. Furthermore, the acute and sub-acute MPTP protocols represent good models of early, subclinical stages of PD, ideal in the development of neuroprotective strategies.

Activation of the protein p7OS6K via ERK phosphorylation by cholinergic muscarinic receptors stimulation in human neuroblastoma cells and in mice brain.

Stimulation of cholinergic muscarinic receptors has been shown to provide substantial protection from DNA damage, oxidative stress and mitochondrial impairment, insults that may be encountered by neurons in development, aging, or neurodegenerative diseases. A study recently indicated that the activation of muscarinic receptors in astrocytoma cells modified the expression of the kinase p70S6K involved in the translational control. The translational control is in part regulated by a cascade of phosphorylation affecting proteins of the anti-apoptotic way controlled by mTOR (mammalian target of rapamycin) and the pro-apoptotic way controlled by PKR. The aim of our study was to investigate the effect of cholinergic muscarinic stimulation by an agonist oxotremorine on the anti-apoptotic way of translational control, in human neuroblastoma cells and in mice brain. Our results showed that muscarinic receptor activation significantly increased the expression of phosphorylated p70S6K, eIF4E and ERK without modification of mTOR activity in neuroblastoma cells and in cerebral cortex and hippocampus of mice, suggesting a stimulation of protein synthesis. Our findings support the notion that synaptic activity, through activation of neurotransmitter receptors, can provide substantial support of cellular survival mechanisms and suggest that loss of such synaptic input increases vulnerability to insult-induced programmed cell death.

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 eIF2α 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 eIF2α 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 eIF2α 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.

Time course of MPTP toxicity on translational control protein expression in mice brain

The present study investigated in mice brain, the time course of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity on the expression of translational control proteins. Mice received intraperitoneal injections of MPTP (30 mg/kg/day) for 5 days and were sacrificed 1, 2, 3, 4 and 7 days after the last injection. The results, obtained by western blot, indicated that MPTP produced an alteration of the expression of proteins involved in the mTOR anti-apoptotic way and the PKR pro-apoptotic pathway of translational control especially in striatum and frontal cortex of mice. These disturbances were associated with a great activation of PKR in hippocampus at D9, the time point corresponding to maximal translational control alterations. Furthermore, whereas no modification of translational control protein expression was observed in mice substantia nigra after western blot procedure, immunofluorescent labeling revealed, in this target region of the toxin MPTP, a decrease of the expression of phospho-mTOR and a great activation of the phosphorylated form of PKR, marker of pathogenesis.