How microwaves can help to study membrane ageing.

This paper studies original protocols of rapid PES/PVP membrane NaOCl degradation allowing at reaching ageing states that are representative of industrial ageing. The long term objective is to propose basis for further fundamental studies aiming at the improvement of the impact of membrane ageing on behaviour in UF (fouling and cleaning mastering). The key of several protocols is the use of ageing acceleration thanks to microwave irradiation, either continuous or pulsed ones, that can be further associated (or not) with short ageing time in UF conditions. To evaluate the representativeness of obtained aged membranes, comparisons are achieved between pristine, voluntary laboratory aged membranes and an industrial membrane at the end of its service-life. Several physico-chemical analyses were used (ATR-FTIR, SEM-EDX, contact angle, SEC-HPLC). Evaluation of UF performances were made in UF of a model protein (Lysozyme, 14,300 g.mol-1). Finally, the proof of concept is done that conditions using MW exist to reach ageing state representative of industrial ageing.

On the impact of ethanol on the rejection and transfer mechanism during ultrafiltration of a charged macromolecule in water/ethanol.

Ultrafiltration (UF) is a sustainable membrane separation technique. It could be useful for the concentration/purification of bio-sourced molecules that are extracted either by pure ethanol or by water/ethanol mixtures. Nevertheless, the process optimization requires an in-depth understanding of the transfer mechanisms of solute through membranes, especially for charged solutes, that are nowadays not sufficiently documented. Previous studies achieved in aqueous media have shown that the rejection of charged solutes by an UF membrane involves at least three mechanisms: convection, diffusion and electrostatic interactions. The present study aims at a systematic analysis of the transfer mechanisms of a model protein (lysozyme) in water/ethanol mixtures (100/0-70/30 v/v) during UF by a zirconia inorganic membrane. The influence of the pH varying in the 4-9 range and of the ionic strength (I) is also discussed. The ionic strength I can be adjusted by addition of an indifferent electrolyte (NaCl) only aiming at the screening of the electrostatic interactions or by addition of a selectively adsorbed electrolyte(KH2PO4) that is able to change the isoelectric pH of the protein and thus to modulate the electrostatic interactions in a different way when compared to NaCl. Of course, both salts have an impact on the protein rejection in UF. The results are analysed using the CDE model previously developed in our group to explain the behaviour of a single protein during UF in water and accounting for convection, diffusion and electrophoretic migration. The applicability of the CDE model in water/ethanol mixtures up to 70/30 v/v is finally shown.

Progesterone-estrogen interactions in synaptic plasticity and neuroprotection.

17ß-Estradiol and progesterone exert a number of physiological effects throughout the brain due to interactions with several types of receptors belonging to the traditional family of intracellular hormonal receptors as well as to membrane-bound receptors. In particular, both hormones elicit rapid modifications of neuronal excitability that have been postulated to underlie their effects on synaptic plasticity and learning and memory. Likewise, both hormones have been shown to be neuroprotective under certain conditions, possibly due to the activation of pro-survival pathways and the inhibition of pro-apoptotic cascades. Because of the similarities in their cellular effects, there have been a number of questions raised by numerous observations that progesterone inhibits the effects of estrogen. In this manuscript, we first review the interactions between 17ß-estradiol (E2) and progesterone (P4) in synaptic plasticity, and conclude that, while E2 exerts a clear and important role in long-term potentiation of synaptic transmission in hippocampal neurons, the role of P4 is much less clear, and could be accounted by the direct or indirect regulation of GABAA receptors. We then discuss the neuroprotective roles of both hormones, in particular against excitotoxicity. In this case, the neuroprotective effects of these hormones are very similar to those of the neurotrophic factor BDNF. Interestingly, P4 antagonizes the effects of E2, possibly through the regulation of estrogen receptors or of proteins associated with the receptors or interactions with signaling pathways activated by E2. Overall, this review emphasizes the existence of common molecules and pathways that participate in the regulation of both synaptic plasticity and neurodegeneration.

Calpain-mediated regulation of stargazin in adult rat brain.

Changes in AMPA receptors have been proposed to underlie changes in synaptic efficacy in hippocampus and other brain structures. Calpain activation has also been discussed as a potential mechanism to produce lasting modifications of synaptic structure and function. Stargazin is a member of the family of transmembrane AMPA receptor associated proteins (TARPs), which participates in trafficking of AMPA receptors and regulates their kinetic properties. We report here that preincubation of thin (20 µm) frozen rat brain sections with calcium changes the immunological properties of stargazin, an effect totally blocked by a calpain inhibitor. Immunocytochemistry indicates that in situ calpain activation produces a decreased immunoreactivity for stargazin in the neuropil throughout the brain, and Western blots confirmed that a similar treatment decreased stargazin levels. Interestingly, the same treatment did not modify the immunoreactivity for another TARP member, γ-8, although it increased immunoreactivity in cell bodies in hippocampus, an effect that was not blocked by calpain inhibition. These results strongly suggest the involvement of calpain in the regulation of AMPA receptor targeting and function through truncation of stargazin.

A methodology for monitoring globular milk protein changes induced by ultrafiltration: a dual structural and functional approach.

Understanding filtration mechanisms at a molecular level is important for predicting structural and functional properties of globular milk proteins after membrane operations. This stage is thus highly decisive for the further development of membrane separations as an efficient alternative to chromatographic processes for the fractionation of milk proteins. In this study, we proposed an original and complete analytical package for the examination of the putative effect of filtration at both macroscopic and molecular levels. We then investigated the pertinence of this analytical package during ultrafiltration (UF) of globular milk proteins in both dead-end and crossflow modes. Reverse-phase HPLC combined with statistical computing was shown to be relevant for the assessment of even slight physically induced modifications. Adaptations of circular dichroism and solubility measurements, regarding their respective dependence on temperature and pH, were also useful for an accurate evaluation of functional modifications. At last, immunochemistry was proven to be a pertinent tool for the specific detection of modifications affecting a targeted protein, even in mixed solutions. Moreover, results obtained by such methods were shown to be coherent with data obtained from classical techniques such as fluorescence. For beta-lactoglobulin, some physically induced modifications were noticed in the permeate because of shear stress inside membrane pores. In the case of alpha-lactalbumin dead-end UF, permeation was shown to affect protein characteristics because of an increase in the relative calcium content responsible for a conformational transition from the apo-form to the holo-form of the protein. Finally, during crossflow UF with limited transmission of BSA, observations were coherent with a partial aggregation because of the circulation of proteins in the filtration pilot. Such a hypothesis corroborates results previously mentioned in the literature.

BDNF mediates the neuroprotective effects of positive AMPA receptor modulators against MPP+-induced toxicity in cultured hippocampal and mesencephalic slices.

Neurotoxicity is involved in various neurodegenerative diseases including Parkinson's disease (PD), which affects mesencephalic dopaminergic neurons of the substantia nigra (SN). Positive alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor modulators (PARMs, a.k.a. Ampakines, such as CX614) increase brain-derived neurotrophic factor (BDNF) protein levels in vivo and in cultured hippocampal slices. BDNF is a survival factor for various neuronal cell types including mesencephalic dopaminergic neurons. Using cultured mesencephalic and hippocampal slices, we investigated whether preincubation with CX614 could provide neuroprotection against MPP(+) toxicity and whether such neuroprotection was mediated by BDNF. Various treatment protocols were tested to demonstrate CX614-induced neuroprotection against MPP(+). Pretreatment with CX614 significantly reduced MPP(+)-induced toxicity and increased BDNF levels in both hippocampal and mesencephalic cultured slices; CX614 pretreatment for 6 h in hippocampal slices and 24 h in mesencephalic slices was sufficient to produce significant neuroprotection as assessed with lactate dehydrogenase release in slice medium and propidium iodide uptake in slices. Both a BDNF scavenger and an inhibitor of the BDNF receptor TrkB, abrogated CX614-mediated reduction of MPP(+)-induced toxicity. Inhibition of Ca(2+)-activated proteases, calpains, was also protective against MPP(+)-induced toxicity. However, co-application of calpain inhibitor with CX614 abolished CX614-mediated protection, suggesting a dual action of calpains in this model. We conclude that CX614 is neuroprotective against MPP(+)-induced toxicity, an effect mediated by increased BDNF expression and activation of BDNF-dependent signaling pathways. Our results provide support for using PARMs as a new therapy for neurodegenerative disorders, including PD.

Improving bioassay sensitivity for neurotoxins detection using volterra based third order nonlinear analysis.

Based on a novel analytical method for analyzing short-term plasticity (STP) of the CA1 hippocampal region in vitro, a screening tool for the detection and classification of unknown chemical compounds affecting the nervous system was recently introduced [1], [2]. The recorded signal consisted of evoked population spike in response to Poisson distributed random train impulse stimuli. The developed analytical approach used the first order Volterra kernel and the Laguerre coefficients of the second order Volterra model as classification features [3]. The biosensor showed encouraging results, and was able to classify out of sample compounds correctly [2]. We have taken an exploratory step to investigate the advantage of introducing a third order model [4]. DAP5, an NMDA channel blocker, did not show major changes in the second order kernel and in its corresponding Laguerre coefficients. Data were reanalyzed using a third order model. DAP5 showed discernable changes in the third order kernel as well as in the some of the corresponding Laguerre coefficients. Hence, the third order Volterra based model has the potential to improve the sensitivity and the discriminatory power of the proposed bioassay.

Role of polyamine metabolism in kainic acid excitotoxicity in organotypic hippocampal slice cultures.

Polyamines are ubiquitous cations that are essential for cell growth, regeneration and differentiation. Increases in polyamine metabolism have been implicated in several neuropathological conditions, including excitotoxicity. However, the precise role of polyamines in neuronal degeneration is still unclear. To investigate mechanisms by which polyamines could contribute to excitotoxic neuronal death, the present study examined the role of the polyamine interconversion pathway in kainic acid (KA) neurotoxicity using organotypic hippocampal slice cultures. Treatment of cultures with N1,N(2)-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527), an irreversible inhibitor of polyamine oxidase, resulted in a partial but significant neuronal protection, especially in CA1 region. In addition, this pre-treatment also attenuated KA-induced increase in levels of lipid peroxidation, cytosolic cytochrome C release and glial cell activation. Furthermore, pre-treatment with a combination of cyclosporin A (an inhibitor of the mitochondrial permeability transition pore) and MDL 72527 resulted in an additive and almost total neuronal protection against KA toxicity, while the combination of MDL 72527 and EUK-134 (a synthetic catalase/superoxide dismutase mimetic) did not provide additive protection. These data strongly suggest that the polyamine interconversion pathway partially contributes to KA-induced neurodegeneration via the production of reactive oxygen species.

Cyclic changes in estradiol regulate synaptic plasticity through the MAP kinase pathway.

Hippocampal synaptic structure and function exhibit marked variations during the estrus cycle of female rats. Estradiol activates the mitogen-activated protein (MAP) kinase pathway in numerous cell types, and MAP kinase has been shown to play a critical role in the mechanisms underlying synaptic plasticity. Here, we report that endogenous estrogen produces a tonic phosphorylation/activation of extracellular signal-regulated kinase 2 (ERK2)/MAP kinase throughout the female rat brain and an increase in tyrosine phosphorylation of NR2 subunits of N-methyl-D-aspartate (NMDA) receptors. Moreover, cyclic changes in estrogen levels during the estrus cycle of female rats are associated with corresponding changes in the levels of activation of ERK2, the state of tyrosine phosphorylation of NR2 subunits of NMDA receptors, and the magnitude of long-term potentiation in hippocampus. Thus, cyclic changes in female sexual hormones result in marked variations in the state of activation of a major cellular signaling pathway critical for learning and memory and in a cellular model of learning and memory.

Kainate excitotoxicity in organotypic hippocampal slice cultures: evidence for multiple apoptotic pathways.

The mechanisms underlying kainate (KA) neurotoxicity are still not well understood. We previously reported that KA-mediated neuronal damage in organotypic cultures of hippocampal slices was associated with p53 induction. Recently, both bax and caspase-3 have been demonstrated to be key components of the p53-dependent neuronal death pathway. Caspase activation has also been causally related to the release of mitochondrial cytochrome c (Cyto C) in the cytoplasm as a result of the collapse of the mitochondrial membrane potential (Deltapsi(M)) and the opening of mitochondrial permeability transition pores (mPTP). In the present study, we observed a rapid induction of bax in hippocampal slice cultures after KA treatment. In addition, the levels of Cyto C and caspase-3 were increased in the cytosol while the level of the caspase-9 precursor was decreased. There was also a complete reduction of Rhodamine 123 fluorescence after KA treatment, an indication of Deltapsi(M) dissipation. Furthermore, inhibition of mPTP opening by cyclosporin A partially prevented Cyto C release, caspase activation and neuronal death. These data suggest the involvement of bax, several caspases, as well as Cyto C release in KA-elicited neuronal death. Finally, inhibition of caspase-3 activity by z-VAD-fmk only partially protected neurons from KA toxicity, implying that multiple mechanisms may be involved in KA excitotoxicity.

Tyrosine phosphorylation of ionotropic glutamate receptors by Fyn or Src differentially modulates their susceptibility to calpain and enhances their binding to spectrin and PSD-95.

Both tyrosine phosphorylation and calpain-mediated truncation of ionotropic glutamate receptors are important mechanisms for synaptic plasticity. Previous work from our laboratory has shown that calpain activation results in truncation of the C-terminal domains of several glutamate receptor subunits. To test whether and how tyrosine phosphorylation of glutamate ionotropic receptor subunits modulates calpain susceptibility, synaptic membranes were phosphorylated by Fyn or Src, two members of the Src family tyrosine kinases. Tyrosine phosphorylation of synaptic membranes by Src significantly reduced calpain-mediated truncation of both NR2A and NR2B subunits of NMDA receptors, but not of GluR1 subunits of AMPA receptors. In contrast, phosphorylation with Fyn significantly protected calpain-mediated truncation of GluR1 subunits of AMPA receptors, but enhanced calpain-mediated truncation of NR2A subunits of NMDA receptors. Similar results were observed with NR2A and NR2B C-terminal domain fusion proteins phosphorylated by Fyn or Src before incubation with calpain and calcium. In addition, phosphorylation of NR2A and NR2B C-terminal fusion proteins by Fyn or Src enhanced their binding to spectrin and PSD-95. Thus, tyrosine phosphorylation impairs or facilitates calpain-mediated truncation of glutamate receptor subunits, depending on which tyrosine kinase is activated. Such mechanisms could serve to regulate receptor integrity and location, in addition to modulating channel properties.

A biosensor for detecting changes in cognitive processing based on nonlinear systems analysis.

A new type of biosensor, based on hippocampal slices cultured on multielectrode arrays, and using nonlinear systems analysis for the detection and classification of agents interfering with cognitive function is described. A new method for calculating first and second order kernel was applied for impulse input-spike output datasets and results are presented to show the reliability of the estimations of this parameter. We further decomposed second order kernels as a sum of nine exponentially decaying Laguerre base functions. The data indicate that the method also reliably estimates these nine parameters. Thus, the state of the system can now be described with a set of ten parameters (first order kernel plus nine coefficients of Laguerre base functions) that can be used for detection and classification purposes.

Attenuation of staurosporine-induced apoptosis, oxidative stress, and mitochondrial dysfunction by synthetic superoxide dismutase and catalase mimetics, in cultured cortical neurons.

Neuronal apoptosis induced by staurosporine (STS) involves multiple cellular and molecular events, such as the production of reactive oxygen species (ROS). In this study, we tested the efficacy of two synthetic superoxide dismutase/catalase mimetics (EUK-134 and EUK-189) on neuronal apoptosis, oxidative stress, and mitochondrial dysfunction produced by STS in primary cortical neuronal cultures. Exposure of cultures to STS for 24 h increased lactate dehydrogenase (LDH) release, the number of apoptotic cells, and decreased trypan blue exclusion. Pretreatment with 20 microM EUK-134 or 0.5 microM EUK-189 significantly attenuated STS-induced neurotoxicity, as did pretreatment with the caspase-1 inhibitor, Ac-YVAD-CHO, but not the caspase-3 inhibitor, Ac-DEVD-CHO. Posttreatment (1-3 h following STS exposure) with 20 microM EUK-134 or 0.5 microM EUK-189 significantly reduced STS-induced LDH release, in a time-dependent manner. Exposure of cultures to STS for 1 h produced an elevation of ROS, as determined by increased levels of 2,7-dichlorofluorescein (DCF). This rapid elevation of ROS was followed by an increase in lipid peroxidation, and both the increase in DCF fluorescence and in lipid peroxidation were significantly blocked by pretreatment with EUK-134. STS treatment for 3-6 h increased cytochrome c release from mitochondria into the cytosol, an effect also blocked by pretreatment with EUK-134. These results indicate that intracellular oxidative stress and mitochondrial dysfunction are critically involved in STS-induced neurotoxicity. However, there are additional cellular responses to STS, which are insensitive to treatment with radical scavengers that also contribute to its neurotoxicity.

Toxic effects of MPP(+) and MPTP in PC12 cells independent of reactive oxygen species formation.

MPTP is a toxin presumed to damage dopamine-secreting neurons by an oxygen free radical-mediated mechanism. Two steps in MPTP metabolism are the primary candidates for oxygen free radical generation: (a) MPTP oxidation to MPP(+) by a monoamine oxidase and (b) NADH dehydrogenase inhibition by MPP(+). In order to test the idea that MPTP toxicity is mediated by oxygen free radicals, we assessed lipid peroxidation and the effects of antioxidants in dopaminergic PC12 cells treated with MPTP or MPP(+). For comparison purposes, we also examined the effects of the pro-oxidant tert-butyl-hydroperoxide (TBHP) and of the dopaminergic toxin 6-hydroxydopamine (6-OHDA) in PC12 cells. MPTP and MPP(+), unlike TBHP, failed to induce lipid peroxidation in PC12 cells after a 4-h exposure. All toxins tested (MPTP, MPP(+), TBHP and 6-OHDA) caused a dose-dependent decrease in [(3)H]dopamine ((3)H-DA) uptake in PC12 cultures. The hydroperoxide scavengers glutathione and N-acetyl-cysteine and the superoxide and peroxide scavenger EUK-134 protected PC12 cells from TBHP- and 6-OHDA-induced decrease in (3)H-DA uptake. However, no protection by these antioxidants at various concentrations and time regimens was observed against MPTP- or MPP(+)-induced decreases in (3)H-DA uptake in PC12 cells. In addition, incubation of PC12 cells with the energy-rich substrate, NADH, attenuated MPP(+)-induced decrease in (3)H-DA uptake. These results suggest that MPTP-induced toxicity in dopaminergic PC12 cell cultures, does not involve oxygen free radical production, but rather may be caused by impairment in energy metabolism.

Proteolysis of glutamate receptor-interacting protein by calpain in rat brain: implications for synaptic plasticity.

Activation of the calcium-dependent protease calpain has been proposed to be a key step in synaptic plasticity in the hippocampus. However, the exact pathway through which calpain mediates or modulates changes in synaptic function remains to be clarified. Here we report that glutamate receptor-interacting protein (GRIP) is a substrate of calpain, as calpain-mediated GRIP degradation was demonstrated using three different approaches: (i) purified calpain I digestion of synaptic membranes, (ii) calcium treatment of frozen-thawed brain sections, and (iii) NMDA-stimulated organotypic hippocampal slice cultures. More importantly, calpain activation resulted in the disruption of GRIP binding to the GluR2 subunit of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors. Because GRIP has been proposed to function as an AMPA receptor-targeting and synaptic-stabilizing protein, as well as a synaptic-organizing molecule, calpain-mediated degradation of GRIP and disruption of AMPA receptor anchoring are likely to play important roles in the structural and functional reorganization accompanying synaptic modifications in long-term potentiation and long-term depression.

Synthetic superoxide dismutase/catalase mimetics reduce oxidative stress and prolong survival in a mouse amyotrophic lateral sclerosis model.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that causes motoneuron degeneration, paralysis and death. Mutations in Cu, Zn superoxide dismutase (SOD1) are one cause of this disease. It is widely suspected that increased reactive oxidative species (ROS) is involved in motoneuron degeneration but whether such an involvement plays a role in ALS progression in vivo is uncertain. We treated mice expressing human mutant SOD1 G93A with EUK-8 and EUK-134, two synthetic SOD/catalase mimetics that have shown efficacy in several animal models of human diseases. These treatments reduced levels of oxidative stress and prolonged survival. The results suggest that oxidative stress plays an active role in ALS and illustrate the potential for treatment strategies aimed specifically against ROS.

Role of electrophoretic mobility of protein on its retention by an ultrafiltration membrane. Comparison to chromatography mechanisms.

Lysozyme and lactoferrin, two globular proteins, were first studied separately in order to elaborate a strategy for the improvement of their separation by ultrafiltration (UF) with zirconia-based membranes of different charge sign and pore radius. The electrophoretic mobility (mu) at fixed pH and variable ionic strength was used for the characterisation of both proteins and zirconia particles, similar to the active layer of the membrane during the UF run. Specific adsorption of phosphate ions was shown for both proteins resulting in new isoelectric points. The occurrence of electrostatic exclusion mechanism in addition to the molecular sieving in UF of charged solutes was shown for: * Low molecular weight solute: multivalent citrate at pH 6 was specifically adsorbed on zirconia and its transmission through the membrane (defined as the ratio of the concentration in the permeate to that of the feed solution) was reduced in the range 0.001-0.01 mol l(-1) of citrate concentration * Proteins: their transmissions increase when the ionic strength increases (ion-exchange is not the relevant mechanism because transmission is irrespective of the initial charge of the membrane compared with the protein charge). A model based on convection, diffusion and electrophoretic migration mechanisms (CDE model) was proposed to take into account this behaviour. The CDE model predicts the possible existence of a depleted sub-layer of the charged protein in the concentration polarisation layer, located in the close vicinity of the membrane surface. A strategy for the separation of two proteins in mixed solution was proposed by varying both the physico-chemical environment in the feed solution (pH, ionic strength, chemical nature of the electrolyte) and the membrane pore radius. Maximum selectivity was obtained when the target protein (to be transmitted in the permeate side) is close to being uncharged due to specific adsorption of electrolyte ions. Ultrafiltration selectivity is enhanced with membrane of large pore radius, which provides high transmission of the target protein and efficient electrostatic exclusion of the solute to be retained in the retentate side. This UF approach corresponds roughly to the separation of one uncharged and one charged protein from a mixed solution by size exclusion chromatography of the uncharged protein combined with electrostatic exclusion of the charged protein due to packing of similar charge.

Specific adsorption of phosphate ions on proteins evidenced by capillary electrophoresis and reversed-phase high-performance liquid chromatography.

Specific adsorption of phosphate ions at pH=7.0 was studied on different proteins, either counter-ions of phosphate (lysozyme, lactoferrin) or co-ion of phosphate (alpha-lactalbumin). The theoretical electrophoretic mobility of globular proteins lysozyme and alpha-lactalbumin (apo and holo (+1 calcium per molecule) forms) was compared with those measured by capillary electrophoresis in phosphate at pH 7.0, versus the ionic strength (I) in the range 0-0.775 mol L(-1). The specific adsorption of phosphate ions was evidenced by difference. From the experimental charge number (Z(eff)) of protein in phosphate medium, a phosphate content per protein molecule was determined at pH=7.0. * For lactoferrin (pI=8-9), the electrophoretic mobility (mu) was constant and negative, highlighting a charge reversal due to phosphate adsorption. * For alpha-lactalbumin (holo form) experimental mu was roughly constant and more negative than predicted. Z(eff) increased continuously from -4 to -11 in the ionic strength range from 0.005 to 0.775 mol l(-1), respectively. Accordingly, one to six phosphates were bound per molecule, respectively. * For lysozyme, experimental electrophoretic mobility was positive but lower than predicted. Z(eff) was only discrete values +5 for I in the range 0.001-0.020 mol l(-1) and about +3 in the range 0.050-0.500 mol l(-1), whereas the theoretical Z value was +7 at pH = 7.0. Lysozyme bounds one phosphate at low ionic strength and about two-three at higher ionic strength. Reversed-phase HPLC confirms that adsorption of phosphate is different for the three proteins.