Jacques Hugon

CNTF promotes survival of retinal ganglion cells after induction of ocular hypertension in rats: the possible involvement of STAT3 pathway.

We examined the neuroprotective effect of ciliary neurotrophic factor (CNTF) on retinal ganglion cells (RGCs) in a rat glaucoma model with increased intraocular pressure (IOP) and studied the CNTF‐mediated activation of Janus kinase/signal transducer and activator of transcription (JAK‐STAT) pathway. Elevated IOP was induced by laser photocoagulation of the episcleral and limbal veins. The survival of RGCs was studied using Fluoro‐Gold labelled in ocular hypertensive eyes with or without CNTF intravitreal injection. Immunochemical staining and immunoblot analysis for CNTF and phosphorylated STAT3 (pSTAT3) were performed. There was a significant and progressive loss of RGCs in the retinas following the induction of elevated IOP. A single intravitreal injection of 2 µg in 2 µL CNTF significantly protected RGCs up to 4 weeks. pSTAT3 was only transiently expressed in ocular hypertensive eyes. However, in eyes treated with CNTF, pSTAT3 was observed up to 2 weeks after the induction of elevated IOP. In ocular hypertensive eyes, CNTF‐positive cells were found in the inner nuclear layer (INL), and there was a transient increase in the pSTAT3 cells in the ganglion cell layer and INL. Immunoblots showed that STAT3 was transiently phosphorylated after IOP increase, but with an injection of CNTF, pSTAT3 protein was observed up to 2 weeks after hypertensive glaucoma induction. Laser‐induced chronic ocular hypertension in rats resulted in the death of RGCs and a transient activation of STAT3 in the retina. Intravitreal injection of CNTF showed a significant protection of RGCs, and the JAK‐STAT signalling could be one of the important pathways that underlie the mechanism of CNTF neuroprotection in this rat glaucoma model.

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.

Involvement of double‐stranded RNA‐dependent protein kinase and phosphorylation of eukaryotic initiation factor‐2α in neuronal degeneration

Inhibition of protein translation plays an important role in apoptosis. While double‐stranded RNA‐dependent protein kinase (PKR) is named as it is activated by double‐stranded RNA produced by virus, its activation induces an inhibition of protein translation and apoptosis via the phosphorylation of the eukaryotic initiation factor 2α (eIF2α). PKR is also a stress kinase and its levels increase during ageing. Here we show that PKR activation and eIF2α phosphorylation play a significant role in apoptosis of neuroblastoma cells and primary neuronal cultures induced by the β‐amyloid (Aβ) peptides, the calcium ionophore A23187 and flavonoids. The phosphorylation of eIF2α and the number of apoptotic cells were enhanced in over‐expressed wild‐type PKR neuroblastoma cells exposed to Aβ peptide, while dominant‐negative PKR reduced eIF2α phosphorylation and apoptosis induced by Aβ peptide. Primary cultured neurons from PKR knockout mice were also less sensitive to Aβ peptide toxicity. Activation of PKR and eIF2α pathway by Aβ peptide are triggered by an increase in intracellular calcium because the intracellular calcium chelator BAPTA‐AM significantly reduced PKR phosphorylation. Taken together, these results reveal that PKR and eIF2α phosphorylation could be involved in the molecular signalling events leading to neuronal apoptosis and death and could be a new target in neuroprotection.

mTOR‐dependent signalling in Alzheimer's disease

•  Introduction •  Neurodegeneration and neurofibrillary degeneration in Alzheimers disease brains •  Dysfunction of protein synthesis mediated by mTOR‐dependent signalling in Alzheimers disease brains •  Neurodegeneration mediated by mTOR‐dependent signalling •  Tau phosphorylation mediated by mTOR‐dependent signalling •  mTOR‐dependent signalling in lymphocytes of AD: potential biomarkers for AD diagnosis •  mTOR‐dependent signalling and other neurodegenerative diseases •  Perspectives •  Conclusions

Upstream Signaling Pathways Leading to the Activation of Double-stranded RNA-dependent Serine/Threonine Protein Kinase in β-Amyloid Peptide Neurotoxicity

One of the hallmarks of Alzheimers disease is extracellular accumulation of senile plaques composed primarily of aggregated β-amyloid (Aβ) peptide. Treatment of cultured neurons with Aβ peptide induces neuronal death in which apoptosis is suggested to be one of the mechanisms. We have demonstrated previously that Aβ peptide induces activation of double-stranded RNA-dependent serine/threonine protein kinase (PKR) and phosphorylation of eukaryotic initiation factor 2α (eIF2α) in neurons in vitro. Degenerating neurons in brain tissues from Alzheimers disease patients also displayed high immunoreactivity for phosphorylated PKR and eIF2α. Our previous data have also indicated that PKR plays a significant role in mediating Aβ peptide-induced neuronal death, because neurons from PKR knockout mice and neuroblastoma SH-SY5Y cells stably transfected with dominant negative mutant of PKR are less susceptible to Aβ peptide toxicity. Therefore, it is important to understand how PKR is activated by Aβ peptide. We report here that inhibition of caspase-3 activity reduces phosphorylation of PKR and to a certain extent, cleavage of PKR and eIF2α in neurons exposed to Aβ peptide. Calcium release from the endoplasmic reticulum and activation of caspase-8 are the upstream signals modulating the caspase-3-mediated activation of PKR by Aβ peptide. Although in other systems HSP90 serves as a repressor for PKR, it is unlikely the candidate for caspase-3 to affect PKR activation in neurons after Aβ peptide exposure. Elucidation of the upstream pathways for PKR activation can help us to understand how this kinase participates in Aβ peptide neurotoxicity and to develop effective neuroprotective strategy.

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.

In vivo activation and nuclear translocation of phosphorylated glycogen synthase kinase-3β in neuronal apoptosis: links to tau phosphorylation

The roles of glycogen synthase kinase‐3β (GSK‐3β) and tau phosphorylation were examined in seven‐day‐old rats injected with the NMDA receptor antagonist (MK801) that is known to induce neuronal apoptosis. Immunoblot and immunohistochemical analysis of brain samples demonstrated a site‐specific increase in tau phosphorylation associated with the relocalization of the protein to the nuclear/perinuclear region of apoptotic neurons. In addition, a tau 32‐kDa fragment was detected, suggesting that tau was a target of intracellular proteolysis in MK801‐treated brains. The proteolytically modified form of tau has reduced ability to bind to microtubules. GSK‐3β kinase assay and immunoblottings of active (tyrosine‐216) and inactive (serine‐9) forms of GSK‐3β revealed a rapid and transient increase in the kinase activity. Lithium chloride, a GSK‐3β inhibitor, prevented tau phosphorylation suggesting that tau phosphorylation is mediated by the activation of GSK‐3β. Confocal microscopy using double labelling of tau and GSK‐‐3β revealed that the activation of GSK‐3β in neurons was associated with early (2 h) nuclear translocation of tyrosine‐216 GSK‐3β. The execution phase of neuronal apoptosis was accompanied by a selective phosphorylation of serine‐9 and dephosphorylation of tyrosine‐216 GSK‐3β. These findings demonstrate that in vivo, GSK‐3β kinase activation and nuclear translocation are early stress signals of neuronal apoptosis.

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.

Activated mTOR and PKR Kinases in Lymphocytes Correlate with Memory and Cognitive Decline in Alzheimer’s Disease

Background: The control of translation, involving the kinases mTOR (mammalian target of rapamycin) and PKR (double-stranded RNA-dependent protein kinase), modulates cell survival and death and is altered in the brains of patients with Alzheimer’s disease (AD). In AD increased susceptibility of lymphocytes to apoptosis has been reported. Methods: We investigated the level of the kinases mTOR and PKR and the eukaryotic initiation factor 2α (eIF2α) in lymphocytes of patients with AD in comparison with controls. In AD patients we also looked for a correlation between activated proteins and cognitive and memory tests. Results: We report significant alterations of the levels of these kinases and eIF2α in lymphocytes of AD patients that were also significantly correlated with cognitive and memory test scores. Conclusion: These results suggest that the levels of mTOR, PKR and eIF2α in lymphocytes could follow the cognitive decline in AD.

Neurons overexpressing mutant presenilin-1 are more sensitive to apoptosis induced by endoplasmic reticulum-Golgi stress

Most early‐onset cases of familial Alzheimers disease (FAD) are linked to mutations in two related genes, ps1 and ps2. FAD‐linked mutant PS1 alters proteolytic processing of the amyloid precursor protein and increases vulnerability to apoptosis induced by various cell stresses. In transfected cell lines, mutations in ps1 decrease the unfolded‐protein response (UPR), which is the response to the increased amounts of unfolded proteins that accumulate in the endoplamic reticulum (ER), indicating that these mutations may increase vulnerability to ER stress by altering the UPR signalling pathway. Here we report that, in primary cultured neurons from cortices of transgenic mice, overexpression of mutated PS1 (M146L mutation) but not PS1 wild‐type (wt) enhanced spontaneous neuronal apoptosis that involved oxidative stress and caspase activation. In PS1M146L cultures, neurons displaying immunoreactivity for human PS1 were threefold more vulnerable to spontaneous apoptosis than the overall neuronal population. In addition, PS1M146L transgenic neurons were more sensitive to apoptosis induced by various stresses, including two ER‐Golgi toxins, nordihydroguaiatric acid and brefeldin A (also known to induce UPR), as well as staurosporine. In contrast, PS1 wt transgenic neurons were resistant to apoptosis induced by Golgi‐ER toxins but displayed a comparable vulnerability to staurosporine. Our study demonstrates that, as previously reported, overexpression of FAD‐linked mutant PS1 enhances neuronal vulnerability to spontaneous and induced apoptosis. In addition, we show that this vulnerability was correlated with mutant PS1 protein expression and that PS1 wt overexpression selectively prevented ER‐Golgi stress‐induced apoptosis. These data indicate that PS1 interferes with a specific apoptotic pathway that results from a dysfunction of the ER‐Golgi compartment.