Amandine Magnaudeix

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.

Tau protein phosphatases in Alzheimer's disease: The leading role of PP2A

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 that might contributes to tau aggregation. Thus, understanding the regulation modes of tau dephosphorylation is of high interest in determining the possible causes at the origin of the formation of tau aggregates and to elaborate protection strategies to cope with these lesions in AD. Among the possible and relatively specific interventions that reverse tau phosphorylation is the stimulation of certain tau phosphatases. Here, we reviewed tau protein phosphatases, their physiological roles and regulation, their involvement in tau phosphorylation and the relevance to AD. We also reviewed the most common compounds acting on each tau phosphatase including PP2A.

The new indirubin derivative inhibitors of glycogen synthase kinase-3, 6-BIDECO and 6-BIMYEO, prevent tau phosphorylation and apoptosis induced by the inhibition of protein phosphatase-2A by okadaic acid in cultured neurons

Alterations in glycogen synthase kinase‐3β (GSK3β) and protein phosphatase‐2A (PP2A) have been proposed to be involved in the abnormal tau phosphorylation and aggregation linked to Alzheimers disease (AD). Interconnections between GSK3β and PP2A signaling pathways are well established. Targeting tau kinases was proposed to represent a therapeutic strategy for AD. However, which tau kinases should be blocked and to what extent, keeping in mind that kinases have physiological roles? Because most kinase inhibitors are relatively specific and many of them interfere with the cell cycle, it is necessary to develop more specific tau kinase inhibitors devoid of cell toxicity. Here, we used the PP2A inhibition by okadaic acid (OKA) in primary cultured cortical neurons as an in vitro model of increased tau phosphorylation and apoptosis. We tested the effects of two newly characterized indirubin derivative inhibitors of GSK3, 6‐BIDECO (6‐bromoindirubin‐3′‐[O‐(N,N‐diethylcarbamyl)‐oxime] and 6‐BIMYEO (6‐bromoindirubin‐3′‐[O‐(2‐morpholin‐1‐ylethyl)‐oxime] hydrochloride) on OKA‐induced tau phosphorylation and neuronal apoptosis. Both compounds exhibit higher selectivity toward GSK3 compared with other tau kinases (for 6‐BIDECO, IC50 is 0.03 μM for GSK3, >10 μM for CDK1, and 10 μM for CDK5; for 6‐BIMYEO, IC50 is 0.11 μM for GSK3, 1.8 μM for CDK1, and 0.9 μM for CDK5). We show that 6‐BIDECO and 6‐BIMYEO used at micromolar concentrations are not neurotoxic and potently reversed tau phosphorylation and apoptosis induced by OKA. The neuroprotection by these compounds should be further validated in animal models of AD.

Study of p53 expression and post-transcriptional modifications after GSM-900 radiofrequency exposure of human amniotic cells†

The potential effects of radiofrequency (RF) exposure on the genetic material of cells are very important to determine since genome instability of somatic cells may be linked to cancer development. In response to genetic damage, the p53 protein is activated and can induce cell cycle arrest allowing more time for DNA repair or elimination of damaged cells through apoptosis. The objective of this study was to investigate whether the exposure to RF electromagnetic fields, similar to those emitted by mobile phones of the second generation standard, Global System for Mobile Communications (GSM), may induce expression of the p53 protein and its activation by post-translational modifications in cultured human cells. The potential induction of p53 expression and activation by GSM-900 was investigated after in vitro exposure of human amniotic cells for 24 h to average specific absorption rates (SARs) of 0.25, 1, 2, and 4 W/kg in the temperature range of 36.3-39.7 °C. The exposures were carried out using a wire-patch cell (WPC) under strictly controlled conditions of temperature. Expression and activation of p53 by phosphorylation at serine 15 and 37 were studied using Western blot assay immediately after three independent exposures of cell cultures provided from three different donors. Bleomycin-exposed cells were used as a positive control. According to our results, no significant changes in the expression and activation of the p53 protein by phosphorylation at serine 15 and 37 were found following exposure to GSM-900 for 24 h at average SARs up to 4 W/kg in human embryonic cells.

GSM-900MHz at low dose temperature-dependently downregulates α-synuclein in cultured cerebral cells independently of chaperone-mediated-autophagy.

The expanding use of GSM devices has resulted in public concern. Chaperone-mediated autophagy (CMA) is a way for protein degradation in the lysosomes and increases under stress conditions as a cell defense response. α-synuclein, a CMA substrate, is a component of Parkinson disease. Since GSM might constitute a stress signal, we raised the possibility that GSM could alter the CMA process. Here, we analyzed the effects of chronic exposure to a low GSM-900MHz dose on apoptosis and CMA. Cultured cerebral cortical cells were sham-exposed or exposed to GSM-900MHz at specific absorption rate (SAR): 0.25W/kg for 24 h using a wire-patch cell. Apoptosis was analyzed by DAPI stain of the nuclei and western blot of cleaved caspase-3. The expression of proteins involved in CMA (HSC70, HSP40, HSP90 and LAMP-2A) and α-synuclein were analyzed by western blot. CMA was also quantified in situ by analyzing the cell localization of active lysosomes. 24 h exposure to GSM-900MHz resulted in ∼0.5°C temperature rise. It did not induce apoptosis but increased HSC70 by 26% and slightly decreased HSP90 (<10%). It also decreased α-synuclein by 24% independently of CMA, since the localization of active lysosomes was not altered. Comparable effects were observed in cells incubated at 37.5°C, a condition that mimics the GSM-generated temperature rise. The GSM-induced changes in HSC70, HSP90 and α-synuclein are most likely linked to temperature rise. We did not observe any immediate effect on cell viability. However, the delayed and long term consequences (protective or deleterious) of these changes on cell fate should be examined.

The Ins and Outs of Nanoparticle Technology in Neurodegenerative Diseases and Cancer.

As we enter the twenty-first century, several therapies based on using nanoparticles (NPs) ranging in size 1 - 1000 nm have been successfully brought to the clinic to treat cancer, pain and infectious diseases. These therapies bring together the ability of NPs to target the delivery of drugs more precisely, to improve solubility, to prevent degradation, to improve their therapeutic index and to reduce the immune response. NPs come in all shapes and sizes, designed specifically for biomedical applications such as solid lipid polymers, liposomes, dendrimers, nanogels, and quantum dots. These NPs offer many attractive characteristics such as biological stability and biocompatibility, thus incorporating different biological or drug molecules. Among the major therapeutic challenges from neurological diseases through to cancer is the development of nanomaterials that are able to be effective against the disease. In the case of neurodegeneration, one of the most difficult areas to penetrate for drug discovery in the body is the central nervous system, protected by the blood-brain-barrier. Whilst in the case of cancer, the biggest problem is how to specifically target a tumor with sufficient drug without causing side effects or inducing resistance. A new generation of intelligent NPs are emerging for the treatment of human disease such as neurological disorders and cancer. The use of natural alternative therapy is an encouraging idea in drug discovery. To this end as we gain more knowledge into the biological function of exosomes, this will allow us to harness their potential as natural NPs in future therapeutics.

DC2 and Keratinocyte-associated Protein 2 (KCP2), Subunits of the Oligosaccharyltransferase Complex, Are Regulators of the γ-Secretase-directed Processing of Amyloid Precursor Protein (APP)

The oligosaccharyltransferase complex catalyzes the transfer of oligosaccharide from a dolichol pyrophosphate donor en bloc onto a free asparagine residue of a newly synthesized nascent chain during the translocation in the endoplasmic reticulum lumen. The role of the less known oligosaccharyltransferase (OST) subunits, DC2 and KCP2, recently identified still remains to be determined. Here, we have studied DC2 and KCP2, and we have established that DC2 and KCP2 are substrate-specific, affecting amyloid precursor protein (APP), indicating that they are not core components required for N-glycosylation and OST activity per se. We show for the first time that DC2 and KCP2 depletion affects APP processing, leading to an accumulation of C-terminal fragments, both C99 and C83, and a reduction in full-length mature APP. This reduction in mature APP levels was not due to a block in secretion because the levels of sAPPα secreted into the media were unaffected. We discover that DC2 and KCP2 depletion affects only the γ-secretase complex, resulting in a reduction of the PS1 active fragment blocking Aβ production. Conversely, we show that the overexpression of DC2 and KCP2 causes an increase in the active γ-secretase complex, particularly the N-terminal fragment of PS1 that is generated by endoproteolysis, leading to a stimulation of Aβ production upon overexpression of DC2 and KCP2. Our findings reveal that components of the OST complex for the first time can interact with the γ-secretase and affect the APP processing pathway.

Sortilin limits EGFR signaling by promoting its internalization in lung cancer

Tyrosine kinase receptors such as the epidermal growth factor receptor (EGFR) transduce information from the microenvironment into the cell and activate homeostatic signaling pathways. Internalization and degradation of EGFR after ligand binding limits the intensity of proliferative signaling, thereby helping to maintain cell integrity. In cancer cells, deregulation of EGFR trafficking has a variety of effects on tumor progression. Here we report that sortilin is a key regulator of EGFR internalization. Loss of sortilin in tumor cells promoted cell proliferation by sustaining EGFR signaling at the cell surface, ultimately accelerating tumor growth. In lung cancer patients, sortilin expression decreased with increased pathologic grade, and expression of sortilin was strongly correlated with survival, especially in patients with high EGFR expression. Sortilin is therefore a regulator of EGFR intracellular trafficking that promotes receptor internalization and limits signaling, which in turn impacts tumor growth.Disruptions in epidermal growth factor receptor (EGFR) trafficking has been linked to tumor progression. Here the authors show that sortilin limits cell proliferation and tumor growth by promoting EGFR internalization.

Direct evidences that macroautophagy basal activity is low in cultured neurons

neuroblastoma cell line, SH-SY5Y cells, the neurite out growth was examined. Results: Using the combining promoter array, we screened an alpha-synuclein targeted promoter of cdc42 gene. As a member of the Rho family GTPase, Cdc42 controls cell migration and morphogenesis. Overexpression and mutant forms of alpha-synuclein down-regulate the expression of cdc42 and affect cytoskeleton architecture for neurite outgrowth. Conclusions: This study suggests a possible function of alphasynuclein as a transcriptional regulator by direct binding to a promoter of target gene. This novel functional mechanism of alpha-synuclein provides us a new possible clue to develop a new therapeutic strategy for the prevention of neurodegeneration through the regulation of downstream pathway of cdc42.

Advanced processing techniques for customized ceramic medical devices

Abstract Additive manufacturing methods (i.e., selective laser sintering, three-dimensional printing, slurry extrusion, and stereolithography) or freeze-templating techniques have been widely investigated to shape customized ceramic implants or scaffolds for bone repair. The principle and main advantages and limitations of these techniques are discussed in this chapter. Examples of applications are presented. A focus on advanced techniques used to characterize the in vitro biological behavior of scaffolds is also reported. These techniques include three-dimensional cell culture and the use of bioreactors which have been set up either as a tool for scaffold characterization or for production of tissue-engineered scaffolds. A literature review of the main approaches was performed. Commonly used protocols are discussed and illustrated by examples. Decisional chart and parameters to standardize are proposed.