Microporous Hydroxyapatite-Based Ceramics Alter the Physiology of Endothelial Cells through Physical and Chemical Cues.

Incorporation of silicate ions in calcium phosphate ceramics (CPC) and modification of their multiscale architecture are two strategies for improving the vascularization of scaffolds for bone regenerative medicine. The response of endothelial cells, actors for vascularization, to the chemical and physical cues of biomaterial surfaces is little documented, although essential. We aimed to characterize in vitro the response of an endothelial cell line, C166, cultivated on the surface CPCs varying either in terms of their chemistry (pure versus silicon-doped HA) or their microstructure (dense versus microporous). Adhesion, metabolic activity, and proliferation were significantly altered on microporous ceramics, but the secretion of the pro-angiogenic VEGF-A increased from 262 to 386 pg/mL on porous compared to dense silicon-doped HA ceramics after 168 h. A tubulogenesis assay was set up directly on the ceramics. Two configurations were designed for discriminating the influence of the chemistry from that of the surface physical properties. The formation of tubule-like structures was qualitatively more frequent on dense ceramics. Microporous ceramics induced calcium depletion in the culture medium (from 2 down to 0.5 mmol/L), which is deleterious for C166. Importantly, this effect might be associated with the in vitro static cell culture. No influence of silicon doping of HA on C166 behavior was detected.

Synthesis by solid route and physicochemical characterizations of blends of calcium orthophosphate powders and mesoporous silicon particles.

The purpose of the study was to investigate the synthesis of economic calcium phosphate powders from recycled oyster shells, using a ball milling method. The oyster shell powder and a calcium pyrophosphate powder were used as starting materials and ball milled, then heat treated at 1,050°C for 5 h to produce calcium phosphate powders through a solid-state reaction. Electrochemically synthesized mesoporous silicon microparticles were then added to the prepared phosphate powders by mechanical mixer. The final powders were characterized using X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy to analyze their chemical composition and determine the most suitable process conditions. The biocompatibility of the produced powders was also tested in vitro using murine cells and the results showed good biocompatibility.

Coherent anti-Stokes Raman scattering cell imaging and segmentation with unsupervised data analysis.

Coherent Raman imaging has been extensively applied to live-cell imaging in the last 2 decades, allowing to probe the intracellular lipid, protein, nucleic acid, and water content with a high-acquisition rate and sensitivity. In this context, multiplex coherent anti-Stokes Raman scattering (MCARS) microspectroscopy using sub-nanosecond laser pulses is now recognized as a mature and straightforward technology for label-free bioimaging, offering the high spectral resolution of conventional Raman spectroscopy with reduced acquisition time. Here, we introduce the combination of the MCARS imaging technique with unsupervised data analysis based on multivariate curve resolution (MCR). The MCR process is implemented under the classical signal non-negativity constraint and, even more originally, under a new spatial constraint based on cell segmentation. We thus introduce a new methodology for hyperspectral cell imaging and segmentation, based on a simple, unsupervised workflow without any spectrum-to-spectrum phase retrieval computation. We first assess the robustness of our approach by considering cells of different types, namely, from the human HEK293 and murine C2C12 lines. To evaluate its applicability over a broader range, we then study HEK293 cells in different physiological states and experimental situations. Specifically, we compare an interphasic cell with a mitotic (prophase) one. We also present a comparison between a fixed cell and a living cell, in order to visualize the potential changes induced by the fixation protocol in cellular architecture. Next, with the aim of assessing more precisely the sensitivity of our approach, we study HEK293 living cells overexpressing tropomyosin-related kinase B (TrkB), a cancer-related membrane receptor, depending on the presence of its ligand, brain-derived neurotrophic factor (BDNF). Finally, the segmentation capability of the approach is evaluated in the case of a single cell and also by considering cell clusters of various sizes.

Advanced protocol to functionalize CaP bioceramic surface with peptide sequences and effect on murine pre-osteoblast cells proliferation.

To bring osteoinductive properties to calcium phosphate (CaP) bioceramics, a silicon-substituted hydroxyapatite was functionalized by integrin-adhesive cyclic-pentapeptides (c-(DfKRG)). A new two-step protocol was set up to immobilize peptides at low and controlled density on the ceramic surface and limit contamination by adsorbed molecules. To this aim, a spacer bearing c-(DfKRG)-S-PEG6-NHS molecule was synthesized and bonded to an organosilane previously covalently bonded to the ceramic surface. The functionalized ceramic was tested in vitro for MC3T3-E1 murine pre-osteoblasts. CaP ceramic surface retained good biological properties thanks to low density of bonded molecules preserving part of the bioactive CaP surface free of bioorganic molecules. The final SiHA-T-PEG6-S-c-(DfKRG) was shown to increase cell density and to improve proliferation. Furthermore, the use of a strong and stable covalent bond between inorganic and organic parts prevented early burst release of the peptide and increased the persistence of its bioactivity over time. So, this CaP ceramic associating c-(DfKRG) by covalent grafting could be considered as promising new biomaterials for bone tissue engineering.

Pre-osteoblast cell colonization of porous silicon substituted hydroxyapatite bioceramics: Influence of microporosity and macropore design.

Silicate-substituted hydroxyapatite scaffolds containing multiscale porosity are manufactured. Model parts containing macropores of five cross-sectional geometries (circle, square, rhombus, star and triangle) and two sizes are shaped by microstereolithography. Three open microporosity contents (0.5, 23 or 37 vol%) are introduced in the ceramic. MC3T3-E1 pre-osteoblasts are seeded onto these scaffolds. Analysis of cell colonization inside the macropores after 7 and 14 days of cultivation shows that the cellular filling is proportional to the macropore size and strongly influenced by macropore shape. Straight edges and convex surfaces are detrimental. High aspect ratios, the absence of reentrant angles and the presence of acute angles, by creating concavities and minimizing flat surfaces, facilitate cell colonization. Rhombus and triangle cross-sections are thus particularly favorable, while square and star geometries are the least favored. An increase in the microporosity content strongly impairs cell growth in the macropores. The data are statistically analyzed using a principal components analysis that shows that macro- and microtopographical parameters of scaffolds must be collectively considered with correlated interactions to understand cell behavior. The results indicate the important cell sensing of topography during the initial step of cell adhesion and proliferation and evidence the need for an optimized scaffold design.

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.

Osteoblast and osteoclast responses to A/B type carbonate-substituted hydroxyapatite ceramics for bone regeneration.

The influence of carbonate substitution (4.4 wt%, mixed A/B type) in hydroxyapatite ceramics for bone remodeling scaffolds was investigated by separately analyzing the response of pre-osteoblasts and osteoclast-like cells. Carbonated hydroxyapatite (CHA) (Ca9.5(PO4)5.5(CO3)0.5(OH)(CO3)0.25-CHA), mimicking the chemical composition of natural bone mineral, and pure hydroxyapatite (HA) (Ca10(PO4)6(OH)2-HA) porous ceramics were processed to obtain a similar microstructure and surface physico-chemical properties (grain size, porosity ratio and pore size, surface roughness and zeta potential). The biological behavior was studied using MC3T3-E1 pre-osteoblastic and RAW 264.7 monocyte/macrophage cell lines. Chemical dissolution in the culture media and resorption lacunae produced by osteoclasts occur with both HA and CHA ceramics, but CHA exhibits much higher dissolution and greater bioresorption ability. CHA ceramics promoted a significantly higher level of pre-osteoblast proliferation. Osteoblastic differentiation, assessed by qRT-PCR of RUNX2 and COLIA2, and pre-osteoclastic proliferation and differentiation were not significantly different on CHA or HA ceramics but cell viability and metabolism were significantly greater on CHA ceramics. Thus, the activity of both osteoclast-like and osteoblastic cells was influenced by the carbonate substitution in the apatite structure. Furthermore, CHA showed a particularly interesting balance between biodegradation, by osteoclasts and chemical dissolution, and osteogenesis through osteoblasts' activity, to stimulate bone regeneration. It is hypothesized that this amount of 4.4 wt% carbonate substitution leads to an adapted concentration of calcium in the fluid surrounding the ceramic to stimulate the activity of cells. These results highlight the superior biological behavior of microporous 4.4 wt% A/B CHA ceramics that could beneficially replace the commonly used HA of biphasic calcium phosphates for future applications in bone tissue engineering.

Hydroxyapatite microporous bioceramics as vancomycin reservoir: Antibacterial efficiency and biocompatibility investigation.

AbstarctInfections after bone reconstructive surgery are a real therapeutic and economic issue for the modern health care system. As the pathogen (most often Staphylococcus aureus) is able to develop a biofilm inside the bone, local delivery of antibiotics is of interest since high drug concentrations would be delivered directly at the target place. In this context, this study evaluated a porous hydroxyapatite implant as biocompatible bone substitute and vancomycin-delivery system to prevent post-operative infections. A simple method of impregnation with optimised conditions insured a high antibiotic loading (up to 2.3 ± 0.3 mg/m2), with a complete in vitro release obtained within 1-5 days. Additionally, the bacteriostatic and bactericidal effects of vancomycin were retained after loading on hydroxyapatite, as demonstrated after challenge with a Staphylococcus aureus strain. Regarding the biocompatibility, a wound healing assay of pre-osteoblastic MC3T3-E1 cells exposed to various concentrations of vancomycin revealed a dose-dependent reduction in cell migration for antibiotic concentrations higher than 1 mg/mL. Meanwhile, cells were able to proliferate normally on vancomycin-loaded scaffolds, although cell initial adhesion was seriously impaired for scaffolds loaded with 2.3 mg/m2 Loaded scaffolds could be stored up to three months at room temperature without any degradation of the antibiotic. Together, these results demonstrate the efficacy of these hydroxyapatite bone substitutes for local delivery of vancomycin in the context of bone infection.

Quantitative analysis of vascular colonisation and angio-conduction in porous silicon-substituted hydroxyapatite with various pore shapes in a chick chorioallantoic membrane (CAM) model.

UNLABELLED: The development of scaffolds for bone filling of large defects requires an understanding of angiogenesis and vascular guidance, which are crucial processes for bone formation and healing. There are few investigations on the ability of a scaffold to support blood vessel guidance and it this is of great importance because it relates to the quality and dispersion of the blood vessel network. This work reports an analysis of vascularisation of porous silicon-substituted hydroxyapatite (SiHA) bioceramics and the effects of pore shape on vascular guidance using an expedient ex ovo model, the chick embryo chorioallantoic membrane (CAM) assay. Image analysis of vascularised implants assessed the vascular density, fractal dimension and diameter of blood vessels at two different scales (the whole ceramic and pores alone) and was performed on model SiHA ceramics harbouring pores of various cross-sectional geometries (circles, square, rhombus, triangles and stars). SiHA is a biocompatible material which allows the conduction of blood vessels on its surface. The presence of pores did not influence angiogenesis related-parameters (arborisation, fractal dimension) but pore geometry affected the blood vessel guidance and angio-conductive potential (diameter and number of the blood vessels converging toward the pores). The measured angles of pore cross-section modulated the number and diameter of blood vessels converging to pores, with triangular pores appearing of particular interest. This result will be used for shaping ceramic scaffolds with specific porous architecture to promote vascular colonisation and osteointegration. STATEMENT OF SIGNIFICANCE: An expedient and efficient method, using chick embryo chorioallantoic membrane (CAM) assays, has been set up to characterise quantitatively the angiogenesis and the vascular conduction in scaffolds. This approach complements the usual cell culture assays and could replace to a certain extent in vivo experiments. It was applied to silicon-substituted hydroxyapatite porous bioceramics with various pore shapes. The material was found to be biocompatible, allowing the conduction of blood vessels on its surface. The presence of pores does not influence the angiogenesis but the pore shape affects the blood vessel guidance and angio-conductive potential. Pores with triangular cross-section appear particularly attractive for the further design of scaffolds in order to promote their vascular colonisation and osteointegration and improve their performances.

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.

Autophagy dysfunction and its link to Alzheimer's disease and type II diabetes mellitus.

Epidemiological data testifies the increasing incidence of Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM). Some associations were made between occidental lifestyle and development of these pathologies, moreover AD and T2DM are linked since each pathology is a causative risk factor for the other. Interestingly, autophagy, a catabolic pathway whose efficiency declines with age is importantly impaired in the affected tissues. Autophagy regulation is dependent of cell metabolic status and consequently on the 5'AMP-activated protein kinase (AMPK) and mammalian target of rapamycin signaling pathways. These pathways are altered with aging and molecular, pharmacological and physiological interventions increase lifespan in various organismal models and favours healthy aging diminishing the occurrence of age-related diseases such as diabetes, cancer, cardiovascular and neurodegenerative pathologies. Decreasing calorie intake has been known for a long time to have a beneficial effect on longevity and health. Some drug agonists of AMPK are known to mimic these effects such as metformin or resveratrol, a polyphenol extracted from plants and present in red wine, a component of the French paradox related diet. In this review, we present the epidemiological and pathogenesis links existing between AD and T2DM with an insight into the perturbations of the autophagic process highlighting the crucial role of the AMPK in development of age and metabolic related diseases. Hence, in a last part we will discuss the possible interventions susceptible to combat both T2DM and AD.

PP2A blockade inhibits autophagy and causes intraneuronal accumulation of ubiquitinated proteins.

Using cultured cortical neurons, we show that the blockade of protein phosphatase 2A (PP2A), either pharmacologically by okadaic acid or by short hairpin RNA (shRNA)-mediated silencing of PP2A catalytic subunit, inhibited basal autophagy and autophagy induced in several experimental settings (including serum deprivation, endoplasmic reticulum stress, rapamycin, and proteasome inhibition) at early stages before autophagosome maturation. Conversely, PP2A upregulation by PP2A catalytic subunit overexpression stimulates neuronal autophagy. In addition, PP2A blockade resulted in the activation of the negative regulator of autophagy mammalian target of rapamycin complex 1 and 5' adenosine monophosphate (AMP)-activated protein kinase (AMPK) and led to intraneuronal accumulation of p62- and ubiquitin-positive protein inclusions, likely due to autophagy downregulation. These data are consistent with previous findings showing that specific invalidation of the autophagy process in the nervous system of mouse resulted in the accumulation of p62- and ubiquitin-positive protein inclusion bodies. Furthermore, we showed that PP2A inhibition alters the distribution of the microtubule-associated protein 1 light chain(LC) 3-I (MAP LC3-I), a key component of the autophagy molecular machinery. Whether MAP LC3-I distribution in the cell accounts for autophagy regulation remains to be determined. These data are important to human neurodegenerative diseases, especially Alzheimer's disease, because they provide links for the first time between the pathological features of Alzheimer's disease:PP2A downregulation, autophagy disruption, and protein aggregation.

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 Alzheimer's 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.

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.

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.

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 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-3ß (GSK3ß) and protein phosphatase-2A (PP2A) have been proposed to be involved in the abnormal tau phosphorylation and aggregation linked to Alzheimer's disease (AD). Interconnections between GSK3ß 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.

DC2 and keratinocyte-associated protein 2 (KCP2), subunits of the oligosaccharyltransferase complex, are regulators of the gamma-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 Aß 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 Aß 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.

Inhibition of glycogen synthase kinase-3beta downregulates total tau proteins in cultured neurons and its reversal by the blockade of protein phosphatase-2A.

In tauopathies such as Alzheimer's disease (AD), the molecular mechanisms of tau protein aggregation into neurofibrillary tangles (NFTs) and their contribution to neurodegeneration remain not understood. It was recently demonstrated that tau, regardless of its aggregation, might represent a key mediator of neurodegeneration. Therefore, reduction of tau levels might represent a mechanism of neuroprotection. Glycogen synthase kinase-3beta (GSK3beta) and protein phosphatase-2A (PP2A) are key enzymes involved in the regulation of tau phosphorylation, and have been suggested to be involved in the abnormal tau phosphorylation and aggregation in AD. Connections between PP2A and GSK3beta signaling have been reported. We have previously demonstrated that exposure of cultured cortical neurons to lithium decreased tau protein expression and provided neuroprotection against Abeta. Since lithium is not a specific inhibitor of GSK3beta (ID50=2.0 mM), whether or not the lithium-induced tau decrease involves GSK3beta remained to be determined. For that purpose, cultured cortical neurons were exposed to 6-bromo-indirubin-3'-oxime (6-BIO), a more selective and potent GSK3beta inhibitor (ID50=1.5 microM) or to lithium. Analysis of tau levels and phosphorylation by western-blot assays showed that lithium and 6-BIO dose-dependently decreased both tau protein levels and tau phosphorylation. Conversely, inhibition of cyclin-dependent kinase-5 (CDK5) by roscovitine decreased phosphorylated tau but failed to alter tau protein levels. These data indicate that GSK3beta might be selectively involved in the regulation of tau protein levels. Moreover, inhibition of PP2A by okadaic acid, but not that of PP2B (protein phosphatase-2B)/calcineurin by FK506, dose-dependently reversed lithium-induced tau decrease. These data indicate that GSK3beta regulates both tau phosphorylation and total tau levels through PP2A.