Catherine Yardin

Tau protein kinases: Involvement in Alzheimer's disease

Tau phosphorylation is regulated by a balance between tau kinase and phosphatase activities. Disruption of this equilibrium was suggested to be at the origin of abnormal tau phosphorylation and thereby might contribute to tau aggregation. Thus, understanding the regulation modes of tau phosphorylation is of high interest in determining the possible causes at the origin of the formation of tau aggregates in order to elaborate protection strategies to cope with these lesions in Alzheimers disease. Among the possible and specific interventions that reverse tau phosphorylation is the inhibition of certain tau kinases. Here, we extensively reviewed tau protein kinases, their physiological roles and regulation, their involvement in tau phosphorylation and their relevance to AD. We also reviewed the most common inhibitory compounds acting on each tau kinase.

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.

Apoptosis is Induced by Radiofrequency Fields through the Caspase-Independent Mitochondrial Pathway in Cortical Neurons

Abstract Joubert, V., Bourthoumieu, S., Leveque, P. and Yardin, C. Apoptosis is Induced by Radiofrequency Fields through the Caspase-Independent Mitochondrial Pathway in Cortical Neurons. Radiat. Res. 169, 38–45 (2008). In the present study, we investigated whether continuous-wave (CW) radiofrequency (RF) fields induce neuron apoptosis in vitro. Rat primary neuronal cultures were exposed to a CW 900 MHz RF field with a specific absorption rate (SAR) of 2 W/kg for 24 h. During exposure, an increase of 2°C was measured in the medium; control experiments with neurons exposed to 39°C were then performed. Apoptosis was assessed by condensation of nuclei with 4′,6-diamino-2-phenylindole (DAPI) staining observed with an epifluorescence microscope and fragmentation of DNA with TdT-mediated dUTP nick-end labeling (TUNEL) analyzed by flow cytometry. A statistically significant difference in the rate of apoptosis was found in the RF-field-exposed neurons compared to the sham-, 37°C- and 39°C-exposed neurons either 0 or 24 h after exposure using both methods. To assess whether the observed apoptosis was caspase-dependent or -independent, assays measuring caspase 3 activity and apoptosis-inducing factor (AIF) labeling were performed. No increase in the caspase 3 activity was found, whereas the percentage of AIF-positive nuclei in RF-field-exposed neurons was increased by three- to sevenfold compared to other conditions. Our results show that, under the experimental conditions used, exposure of primary rat neurons to CW RF fields may induce a caspase-independent pathway to apoptosis that involves AIF.

Microwave exposure of neuronal cells in vitro : Study of apoptosis

Purpose:The aim of this study was to investigate microwave (MW) effects on neuronal apoptosis in vitro. Materials and methods:Human neuroblastoma cells SH-SY5Y were exposed to a 900 MHz global system for mobile communication (GSM) or continuous-wave (CW) radiofrequency fields for 24 h in a wire-patch cell. The specific absorption rates (SAR) used were 2 W/kg for CW and 0.25 W/kg average for GSM. During CW exposure, an increase of 2°C was measured, and controls with cells exposed to 39°C were then performed. Apoptosis rate was assessed immediately or 24 h after exposure using three methods: (i) 4′,6-diamino-2-phenylindole (DAPI) staining; (ii) flow cytometry using double staining with TdT-mediated dUTP nick-end labeling (TUNEL) and propidium iodide (PI); and (iii) measurement of caspase-3 activity by fluorimetry. Results:No statistically significant difference in the apoptosis rate was observed between sham and 24 h MW-exposed cells, either GSM-900 at an average SAR of 0.25 W/kg, or CW 900 MHz at a SAR of 2 W/kg, either 0 h or 24 h post-exposure. Furthermore, for CW-exposure, apoptosis rates were comparable between sham-, CW-, 37°C- and 39°C-exposed cells. All three methods used to assess apoptosis were concordant. Conclusion:These results showed that, under the conditions of the present experiment, MW-exposure (either CW or GSM-900) does not significantly increase the apoptosis rate in the human neuroblastoma cell line SH-SY5Y.

BAD and Bcl-2 regulation are early events linking neuronal endoplasmic reticulum stress to mitochondria-mediated apoptosis

The mechanism of endoplasmic reticulum (ER)-mediated apoptosis in neurons was examined. Using primary cortical neurons, we show that nordihydroguaiaretic acid (NDGA) and brefeldin A (BFA), two ER stressors, induce early ER stress as shown by Western blotting of the eukaryotic initiation factor-2alpha (eIF2alpha), an ER stress marker. This event was associated with an enhancement of neuronal apoptosis as demonstrated by the time-dependent increase in caspase-3 activity and by nuclear fragmentation. The study of the apoptotic signaling showed the translocation of cytochrome c from the mitochondrial matrix to the cytosol. Further evaluation of the apoptotic process revealed that NDGA and BFA induced a rapid dephosphorylation of BAD and decrease expression of Bcl-2. Altogether, our results indicate that neuronal ER stress is associated with an apoptotic cascade involving the mitochondria.

Lithium down-regulates tau in cultured cortical neurons: A possible mechanism of neuroprotection

In tauopathies such as Alzheimers disease (AD), the moleccular mechanisms of tau protein agregation into neurofibrillary tangles (NFTs) and their contribution to neurodegeneration are not fully understood. Recent studies indirectly demonstrated that tau, regardless of its aggregation, might represent a key mediator of neurodegeneration, especially that induced by the amyloid (Abeta) pathology. Lithium is a medication for bipolar mood disorders. Its therapeutic mechanism of action remains unclear, in part because of the large number of biochemical effects attributed to lithium. Since lithium directly inhibits glycogen synthase kinase-3beta (GSK3beta), a key enzyme involved in tau phosphorylation, it was suggested that the therapeutic use of lithium could be expanded from mood disorders to neurodegenerative conditions. Lithium has been also reported to protect cultured neurons against Abeta toxicity, and to prevent NFTs accumulation and cognitive impairments in transgenic models of tauopathies. However, the exact mechanism of neuroprotection provided by lithium remains unknown. Here, we show that exposure of cultured cortical neurons to lithium decreased tau protein levels. This decrease was not linked to the activation of proteolytic processes including calpains, caspases and proteasome or to neuronal loss, but was rather associated with a reduction in tau mRNA levels. Moreover, prior exposure to lithium, at concentrations effective in reducing tau protein levels, markedly reduced pre-aggregated Abeta-induced neuronal apoptosis. Our findings raise the possibility that lithium could exert its neuroprotective effect against Abeta toxicity through the downregulation of tau proteins and that, at least, by acting at the level of tau mRNA.

Mild kainate toxicity produces selective motoneuron death with marked activation of CA2+-permeable AMPA/kainate receptors

Motoneuron death could be produced by higher sensitivity to excitoxicity during the development and pathological conditions. We report here that in ventral spinal cord cultures mild kainate exposure (12.5 microM, 20 min or 100 microM, 2.5 min) induced selective cobalt stain of motoneurons, indicating a specific Ca2+ entry through the Ca(2+)-permeable AMPA/kainate receptors. This result was associated with a selective motoneuron death as previously described. In these cultures, motoneuron immunoreactivity for the Ca2+ buffering protein, calretinin was negative. These findings suggest that the selective motoneuron death due to a mild excitotoxic insult could be linked to a marked Ca2+ influx associated with the lack of some Ca2+ buffering proteins.

N-methyl-d-aspartate receptor blockade enhances neuronal apoptosis induced by serum deprivation

Neuronal apoptosis a hallmark of brain development could also be involved in neurodegenerative diseases. Glutamate toxicity is widely proposed as an important factor in the pathogenesis of neurological disorders. We show here that, in rat primary cortical cultures, the blockade of N-methyl-D-aspartate (NMDA) glutamate receptors exacerbated neuronal apoptosis induced by serum deprivation. This effect is observed at early stage of cultures (9 days in vitro (DIV)) and mildly decreases in more mature cultures (13 and 15 DIV). At the opposite, low concentrations of NMDA (5 microM) or glutamate (5 microM) prevented the neuronal apoptosis induced by trophic support withdrawal. In primary cortical cultures, the proapoptotic effect of trophic support removal can be modulated by NMDA receptors depending upon the magnitude of these glutamate receptor activation.

Involvement of glycogen synthase kinase‐3β and tau phosphorylation in neuronal Golgi disassembly

The dissociation of the neuronal Golgi complex is a classical feature observed in neurodegenerative disorders including Alzheimers disease. The goal of this study is to determine if the phosphorylation of tau protein is involved in neuronal Golgi disassembly. Primary cortical cultures were exposed to two Golgi toxins, brefeldin A (BFA) or nordihydroguaiaretic acid (NDGA). Immunocytochemical studies using the anti58 k antibody revealed that Golgi disassembly started in exposed neurons a few minutes after treatment. BFA and NDGA induced a rapid and transient increase in tau phosphorylation in a site‐specific manner on immunoblots. In addition, the increase in tau phosphorylation directly correlated with a transient dissociation of tau from the cytoskeleton and a decrease of the acetylated tubulin. Furthermore, the activity of glycogen synthase kinase‐3β (GSK‐3β) increased transiently, as demonstrated by the kinase activity assay and by immunoblottings of serine‐9 and tyrosine‐216 phosphorylated of GSK‐3β. A decrease of the Akt phosphorylated form was also shown. The increase in tau phosphorylation was inhibited by the GSK‐3β inhibitor, lithium. Finally, morphometric studies showed that lithium partially blocked the Golgi disassembly caused by BFA or NDGA. Together these findings indicate that GSK‐3β activity and tau phosphorylation state are involved in the maintenance of the neuronal Golgi organization.

Monozygotic twins concordant for blood karyotype, but phenotypically discordant: a case of "mosaic chimerism".

We report on 23 years old discordant monozygotic (MZ) twins, one with minor anomalies and mental delay, the other one being normal. Both had 46,XX,dup(11)(p12p15)/46,XX mosaicism in blood, with a similar proportion of abnormal cells (respectively, 16% and 17%). However, interphase fluorescence in situ hybridization (FISH) analysis performed on buccal smear and urinary sediment using specific probes located at the duplicated region showed that mosaicism was only present in the abnormal twin, with 68% abnormal cells. We hypothesize that the postzygotic chromosomal rearrangement may have occurred early in one embryo after the twinning event, and the blood mosaicism observed in both twins would have resulted from blood exchanges via placental anastomoses. This hypothesis of chimerism is strongly supported by twin‐to‐twin transfusion syndrome observed during fetal life of our twins. This case and those previously reported lead us to suggest that blood is particularly unsuitable for cytogenetic investigations of twins.