Guylène Page

mTOR/p70S6k signalling alteration by Aβ exposure as well as in APP‐PS1 transgenic models and in patients with Alzheimer's disease

In Alzheimers disease, neuropathological hallmarks include the accumulation of β‐amyloid peptides (Aβ) in senile plaques, phosphorylated tau in neurofibrillary tangles and neuronal death. Aβ is the major aetiological agent according to the amyloid cascade hypothesis. Translational control includes phosphorylation of the kinases mammalian target of rapamycin (mTOR) and p70S6k which modulate cell growth, proliferation and autophagy. It is mainly part of an anti‐apoptotic cellular signalling. In this study, we analysed modifications of mTOR/p70S6k signalling in cellular and transgenic models of Alzheimers disease, as well as in lymphocytes of patients and control individuals. Aβ 1–42 produced a rapid and persistent down‐regulation of mTOR/p70S6k phosphorylation in murine neuroblastoma cells associated with caspase 3 activation. Using western blottings, we found that phosphorylated forms of mTOR and p70S6k are decreased in the cortex but not in the cerebellum (devoid of plaques) of double APP/PS1 transgenic mice compared with control mice. These results were confirmed by immunohistochemical methods. Finally, the expression of phosphorylated p70S6k was significantly reduced in lymphocytes of Alzheimers patients, and levels of phosphorylated p70S6k were statistically correlated with Mini Mental Status Examination (MMSE) scores. Taken together, these findings demonstrate that the mainly anti‐apoptotic mTOR/p70S6k signalling is altered in cellular and transgenic models of Alzheimers disease and in peripheral cells of patients, and could contribute to the pathogenesis of the disease.

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.

Tyk2/STAT3 Signaling Mediates β-Amyloid-Induced Neuronal Cell Death: Implications in Alzheimer's Disease

One of the pathological hallmarks of Alzheimers disease (AD) is deposition of extracellular amyloid-β (Aβ) peptide, which is generated from the cleavage of amyloid precursor protein (APP). Accumulation of Aβ is thought to associate with the progressive neuronal death observed in AD. However, the precise signaling mechanisms underlying the action of Aβ in AD pathophysiology are not completely understood. Here, we report the involvement of the transcription factor signal transducer and activator of transcription 3 (STAT3) in mediating Aβ-induced neuronal death. We find that tyrosine phosphorylation of STAT3 is elevated in the cortex and hippocampus of APP/PS1 transgenic mice. Treatment of cultured rat neurons with Aβ or intrahippocampal injection of mice with Aβ both induces tyrosine phosphorylation of STAT3 in neurons. Importantly, reduction of either the expression or activation of STAT3 markedly attenuates Aβ-induced neuronal apoptosis, suggesting that STAT3 activation contributes to neuronal death after Aβ exposure. We further identify Tyk2 as the tyrosine kinase that acts upstream of STAT3, as Aβ-induced activation of STAT3 and caspase-3-dependent neuronal death can be inhibited in tyk2−/− neurons. Finally, increased tyrosine phosphorylation of STAT3 is also observed in postmortem brains of AD patients. Our observations collectively reveal a novel role of STAT3 in Aβ-induced neuronal death and suggest the potential involvement of Tyk2/STAT3 signaling in AD pathophysiology.

Fluoro-Jade B staining as useful tool to identify activated microglia and astrocytes in a mouse transgenic model of Alzheimer's disease.

Fluoro-Jade B is known as a high affinity fluorescent marker for the localization of neuronal degeneration during acute neuronal distress. However, one study suggested that fluoro-Jade B stains reactive astroglia in the primate cerebral cortex. In this study, we analyzed the staining of fluoro-Jade B alone or combined with specific markers for detection of glial fibrillary acidic protein (GFAP) or activated CD68 microglia in the double APP(SL)/PS1 KI transgenic mice of Alzheimers disease (AD), which display a massive neuronal loss in the CA1 region of the hippocampus. Our results showed that fluoro-Jade B did not stain normal and degenerating neurons in this double mouse transgenic model. Fluoro-Jade B was co-localized with Abeta in the core of amyloid deposits and in glia-like cells expressing Abeta. Furthermore, fluoro-Jade B was co-localized with CD68/macrosialin, a specific marker of activated microglia, and with GFAP for astrocytes in APP(SL)/PS1 KI transgenic mice of AD. Taken together, these findings showed that fluoro-Jade B can be used to label activated microglia and astrocytes which are abundant in the brain of these AD transgenic mice. It could stain degenerating neurons as a result of acute insult while it could label activated microglia and astrocytes during a chronic neuronal degenerative process such as AD for example.

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.

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.

The oxindole/imidazole derivative C16 reduces in vivo brain PKR activation

Inhibition of double‐stranded RNA‐dependent protein kinase (PKR) represents an interesting strategy for neuroprotection. However, inhibiting this kinase which triggers the apoptotic process could favour in counterpart cell proliferation and tumorigenesis. Here, we use an in vivo model of 7‐day‐old rat displaying a high activation of brain PKR to investigate the effects of a new PKR inhibitor identified as an oxindole/imidazole derivative (C16). We show for the first time that acute systemic injection of C16 specifically inhibits the apoptotic PKR/eIF2α signaling pathway without stimulating the proliferative mTOR/p70S6K signaling mechanism.

Inhibitory effects of ascorbic acid on dopamine uptake by rat striatal synaptosomes: relationship to lipid peroxidation and oxidation of protein sulfhydryl groups

Ascorbic acid is frequently added in the incubation medium to prevent oxidation of dopamine (DA) during uptake assays. However, a preliminary study showed that the presence of ascorbic acid induced a decrease of DA uptake after prolonged incubation. The purpose of this study was to determine the mechanism underlying ascorbic acid-induced alterations of DA uptake in rat striatal synaptosomes. In this context, the effects of physiological concentrations of ascorbic acid (100-500 microM) on DA uptake and Na+/K+ ATPase activity (which is essential for DA transporter function) were assessed in synaptosomes before and after incubation at 37 degrees C. The capacity of synaptosomes to take up DA was significantly decreased after incubation owing to a reduction in DA transporters (but with no modification of their affinity for DA). This partial inhibition was associated with a decrease of Na+/K+ ATPase activity, a production of thiobarbituric acid reactive substances (TBARS) and malonaldehyde (MDA), and a loss of sulfhydryl group content. Addition of Trolox C to the medium prevented the reduction of DA uptake, the inhibition of Na+/K+ ATPase activity, the decrease in sulfhydryl group content and the production of TBARS and MDA. These results suggest that ascorbic acid in the presence of contaminant ferrous ions induced a decrease in functional DA transporters, probably through a lipid peroxidation process involving oxidation of sulfhydryl groups and at least in part through a decrease of Na+/K+ ATPase activity.

Interaction of double-stranded RNA-dependent protein kinase (PKR) with the death receptor signaling pathway in amyloid beta (Abeta)-treated cells and in APPSLPS1 knock-in mice.

For 10 years, research has focused on signaling pathways controlling translation to explain neuronal death in Alzheimer Disease (AD). Previous studies demonstrated in different cellular and animal models and AD patients that translation is down-regulated by the activation of double-stranded RNA-dependent protein kinase (PKR). Among downstream factors of PKR, the Fas-associated protein with a death domain (FADD) and subsequent activated caspase-8 are responsible for PKR-induced apoptosis in recombinant virus-infected cells. However, no studies have reported the role of PKR in death receptor signaling in AD. The aim of this project is to determine physical and functional interactions of PKR with FADD in amyloid-β peptide (Aβ) neurotoxicity and in APPSLPS1 KI transgenic mice. In SH-SY5Y cells, results showed that Aβ42 induced a large increase in phosphorylated PKR and FADD levels and a physical interaction between PKR and FADD in the nucleus, also observed in the cortex of APPSLPS1 KI mice. However, PKR gene silencing or treatment with a specific PKR inhibitor significantly prevented the increase in pT451-PKR and pS194-FADD levels in SH-SY5Y nuclei and completely inhibited activities of caspase-3 and -8. The contribution of PKR in neurodegeneration through the death receptor signaling pathway may support the development of therapeutics targeting PKR to limit neuronal death in AD.

Neither the density nor function of striatal dopamine transporters were influenced by chronic n-3 polyunsaturated fatty acid deficiency in rodents.

We hypothesized that the chronic dietary deficiency of n-3 polyunsaturated fatty acids (n-3 PUFAs) might affect the density and/or function of dopamine transporters (DAT), which have a major role in regulating the synaptic level of dopamine. This hypothesis was tested by investigating DAT in the striatum using three complementary methods in control and deficient rats. The density of DAT was determined by quantitative autoradiography using [(125)I]PE2I, a specific ligand of this transporter. Functional investigations were performed (i) in vitro by measuring [(3)H]dopamine uptake on synaptosomes, and (ii) in vivo using intracerebral microdialysis. The results demonstrated that neither the density nor the function of DAT were influenced by n-3 PUFA deficiency in the striatum. This suggests lower sensitivity to n-3 PUFA deficiency in the striatum than that previously observed in the frontal cortex.