Victor Rimbau

Protective effects of C-phycocyanin against kainic acid-induced neuronal damage in rat hippocampus.

The neuroprotective role of C-phycocyanin was examined in kainate-injured brains of rats. The effect of three different treatments with C-phycocyanin was studied. The incidence of neurobehavioral changes was significantly lower in animals receiving C-phycocyanin. These animals also gained significantly more weight than the animals only receiving kainic acid, whereas their weight gain did not differed significantly from controls. Equivalent results were found when the neuronal damage in the hippocampus was evaluated through changes in peripheral benzodiazepine receptors (microglial marker) and heat shock protein 27 kD expression (astroglial marker). Our results are consistent with the oxygen radical scavenging properties of C-phycocyanin described elsewhere. Our findings and the virtual lack of toxicity of C-phycocyanin suggest this drug could be used to treat oxidative stress-induced neuronal injury in neurodegenerative diseases, such as Alzheimers and Parkinsons.

Antiinflammatory activity of some extracts from plants used in the traditional medicine of North-African countries (II)

Aqueous, ethanol and chloroform extracts from Corrigiliola telephiifolia, Echinops spinosus, Kundmania sicula, Tamarindus indica and Zygophyllum gaetulum were evaluated for antiinflammatory properties in mice (ear oedema induced by arachidonic acid) and rats (subplantar oedema induced by carrageenan) after topical or i.p. administration, respectively. Our results showed that all the plants exhibit antiinflammatory activity, since at least one extract from each plant was active in one of the experimental models. Whereas all the extracts of Corrigiliola telephiifolia and Echinops spinosus were highly active on all the experimental models assayed (values of inflammation inhibition well above 50%), poorer activity profiles were recorded in Kundmania sicula, Tamarindus indica and Zygophyllum gaetulum. These results support the traditional uses for these plants but indicate that the active principles in the chloroform extracts are probably more active and/or are contained in larger concentrations than the principles in the polar extracts used in the traditional medicine of North‐African countries. Copyright

Inhibition of the cdk5/MEF2 pathway is involved in the antiapoptotic properties of calpain inhibitors in cerebellar neurons

1 Experimental data implicate calpain activation in the pathways involved in neuronal apoptosis. Indeed, calpain inhibitors confer neuroprotection in response to various neurotoxic stimuli. However, the pathways involved in calpain activation‐induced apoptosis are not well known. 2 We demonstrate that apoptosis (40%) induced by serum/potassium (S/K) withdrawal on cerebellar granule cells (CGNs) is inhibited by selective calpain inhibitors PD150606 (up to 15%) and PD151746 (up to 29%), but not PD145305 in CGNs. zVAD‐fmk, a broad spectrum inhibitor of caspases, attenuates apoptosis (up to 20%) mediated by S/K deprivation and protects against cell death, as measured by MTT ([3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium]) assay. 3 PD150606 and PD151746 prevented apoptosis mediated by S/K withdrawal through inhibition of calpain. Furthermore, PD151746 was able to inhibit caspase‐3 activity. 4 After S/K withdrawal, we observed an increase in cdk5/p25 formation and MEF2 phosphorylation that was prevented by 40 μM PD150606 and PD151746. This indicates that calpain inhibition may be an upstream molecular target that prevents neuronal apoptosis in vitro. 5 Taken together, these data suggest an apoptotic route in S/K withdrawal in CGNs mediated by calpain activation, cdk5/p25 formation and MEF2 inhibition. Calpain inhibitors may attenuate S/K withdrawal‐induced apoptosis and may provide a potential therapeutic target for drug treatment in a neurodegenerative process.

Evaluation of pathways involved in pentachlorophenol-induced apoptosis in rat neurons

Pentachlorophenol (PCP) (C(6)HCl(5)O) is a synthetic toxic organochloride fungicide for humans which exhibit neurotoxic properties. In the present research, we describe the potential pathways implicated in PCP-induced apoptosis in an acute model of toxicity in rat cerebellar granule neurons (CGNs). In our experiments, acute exposure of CGNs to micromolar concentrations of PCP induced the transcriptional activity of genes related to the classical apoptosis pathway (caspase 3, caspase 8, Bad), oxidative stress and glutathione metabolism (glutathione peroxidase-1, catalase, glutathione-S-transferase-3 and superoxide dismutase-1), and mitogenic response (cyclin D1, cdk2, cdk4, cdkn2b). Results from Western blot also shown significative increases in the expression of cyclins D1, E and A and cdk4. The mitogenic response was also related to a significative increase in the phosphorylation of retinoblastoma protein (Rb). PCP would cause apoptosis up-regulating the transcriptional activity of p53 gene and also increasing their activation by phosphorylation, concomitant with a decrease in the sirtuin 1 content. In conclusion, acute exposure of CGNs to PCP induces the classical p53 apoptotic pathway, promotes the up-regulation of several genes related to oxidative stress and the over-expression of molecules involved in the cell cycle control.

3-amino thioacridone, a selective cyclin-dependent kinase 4 inhibitor, attenuates kainic acid-induced apoptosis in neurons

The mechanisms underlying selective neuronal cell death in kainic acid-mediated neurodegeneration are not fully understood. We have recently demonstrated that in cerebellar granule neurons, kainic acid induces the expression of proteins associated with cell-cycle progression. In the present study we show that 3-amino thioacridone (3-ATA), a selective cyclin-dependent kinase 4 inhibitor, attenuates kainic acid-induced apoptosis in cerebellar granule neurons. When neurons were pre-treated with 3-ATA 10 microM for 24 h, they were less susceptible to damage induced by kainic acid 500 microM, since the number of dead cells decreased significantly. In flow cytometry studies using propidium iodide staining, 3-ATA also reduced the ratio of apoptotic cells induced by kainic acid. Moreover, 3-ATA decreased the proportion of cells with a condensed nucleus from 55% to 22%. Our data suggest that the cell cycle pathway is involved in the mechanism of apoptosis mediated by kainic acid and that cyclin-dependent kinase 4 plays a prominent role in this process. 3-ATA may to prevent the apoptosis associated with neurodegenerative disorders without the over-activation of excitatory amino acid receptors.

Evaluation of acute antiapoptotic effects of Li+ in neuronal cell cultures

SummaryLi+ exerts protective effect against several neurotoxins in neuronal cell preparations. Here we examined the antiapoptotic effects of GSK3β in cerebellar granule neurons (CGNs) in the presence of several neurotoxins. Acute treatment with Li+ protected neurons against nocodazole and serum/potassium (S/K) deprivation, but were ineffective against kainic acid and MPP+. Li+ 5 mM also decreased caspase-3 activation induced by nocodazole and S/K deprivation as measured by Ac-DEVD-p-nitroaniline and the breakdown of α-spectrin. All the neurotoxins used in the present study activated GSK3β, evaluated with a specific antibody phospho-GSK-3β (Ser9) by Western-blot and immunocytochemistry and were always inhibited by Li+ 5 mM. Our results implicate Li+ in the regulation of apoptosis mediated by caspase activation (Type I). Furthermore inhibition of GSK3β by acute treatment with Li+ 5 mM is not an indicator of neuroprotection. The acute antiapoptotic function of Li+ is discussed in terms of its inhibition of Type I pathway, the intrinsic (mitochondrial) apoptotic pathway in cerebellar granule cells.

Inhibition of Multiple Pathways Accounts for the Antiapoptotic Effects of Flavopiridol on Potassium Withdrawal-Induced Apoptosis in Neurons

Serum and potassium (S/K) deprivation is a well-known apoptotic model in cerebellar granule neurons (CGNs), used to study the efficacy of potential neuroprotective drugs. The objective of this study was to determine the pathways involved in the neuroprotective role of flavopiridol, a pan-inhibitor of cyclin-dependent kinases (CDKs), upon S/K withdrawal-induced apoptosis in CGNs. Cell death in primary cultures of rat CGNs was accompanied by chromatin condensation and activation of caspases-3, -6, and -9. Caspase-3 activity was also evaluated by cleavage of 120-kDa α-spectrin. Flavopiridol (1 µM) prevented caspase activation and abolished apoptotic features mediated by S/K withdrawal. Re-entry in the cell cycle is also involved in apoptotic neuronal cell death. Flavopiridol (1 µM) inhibited DNA synthesis as measured by BrdU incorporation, thus enhancing proliferating cell nuclear antigen expression. Serum/potassium (S/K) deprivation induced apoptotic cell death mediated by the activation of several kinases such as glycogen synthase kinase-3β and CDK5, as well as the breakdown of p35 in the neurotoxic fragment p25; inactivation of myocyte enhancer factor-2 (MEF2) was also found. Pretreatment with flavopiridol prevented these biochemical and molecular alterations. Taken together, these findings suggest an apoptotic route in CGNs after S/K withdrawal mediated by the activation of several kinases involved in cell cycle deregulation and MEF2 inactivation. We propose that the antiapoptotic properties of flavopiridol are mediated through kinase pathway inhibition.

DNA low-density array analysis of colchicine neurotoxicity in rat cerebellar granular neurons

Cytoskeletal alteration is a key factor in neurodegenerative processes like Alzheimers or Parkinsons disease. Colchicine is a microtubule-disrupting agent that binds to tubuline, inhibiting microtubule assembly, and which triggers apoptosis. The present research describes the transcriptional activation of molecules related to alternative forms of apoptosis, in an acute colchicine model of apoptosis in rat cerebellar granule neurons (CGNs). Treatment with colchicine up-regulated significantly the activity of genes related to oxidative stress: glutathione peroxidase 1 and catalase; altered significantly genes related to cell cycle control (cyclin D1 and cyclin-dependent kinase 2), genes related to classical apoptosis pathway (caspase 3) and a neuronal cell-related gene (pentraxin 1). Colchicine treatment also down-regulated the gene expression of calpain 1. In conclusion, our experiments demonstrate that the cell damage caused by exposure to colchicine activates the classical apoptosis pathway, but also promotes the up-regulation of several genes related to oxidative stress and cell cycle control. Present data may help to a better understanding of the molecular mechanisms involved in cytoskeletal degradation-induced apoptosis in neurons.

Evaluation of neuronal cell death by laser scanning cytometry

We developed a method in which laser scanning cytometry (LSC) is applied to evaluate cell viability. Neuronal cell death induced by glutamic acid, serum potassium deprivation and 3-nitropropionic acid was studied in cerebellar granule cells by neutral red assay (NR) and LSC, using propidium iodide (PI) as fluorescent dye. PI labeled the nuclei of dead neurons and increased fluorescence was measured using a laser scanning cytometer. Similar levels of damage for each injury were detected by NR or LSC. The protocol presented here, provides a fast and sensitive assay for the analysis of neuronal viability using LSC, and can be used to study new neuroprotective drugs in neuronal cell cultures.

Neuroprotective effects of SB-415286 on hydrogen peroxide-induced cell death in B65 rat neuroblastoma cells and neurons

Glycogen synthase kinase‐3 (GSK‐3) is involved in the pathogenesis of several neurodegenerative diseases. In addition, as oxidative stress has been implicated in all neurodegenerative disorders, the inhibition of both pathways offers a potential strategy for preventing or delaying neurodegeneration. We examined the cytoprotective effects of lithium and SB‐415286, two inhibitors of GSK‐3, using a rat B65 cell line and also in cerebellar granule cells (CGN). H2O2 decreased the inactive form of GSK‐3 (phospho‐GSK‐3 at Ser9), as measured by immunoblot experiments involving an antibody against the inactive form of the enzyme. Moreover, lithium inhibited this effect. While SB‐415286 exerted a protective effect, lithium did not attenuate the toxic effects of H2O2 (1 mM). We then examined those mechanisms implicated in the protective effects of SB‐415286. When we analyzed reactive oxygen species (ROS) production using the fluorescent probe 2,7‐dichlorodihydrofluorescein diacetate in B65 cells, as well as in CGN, we found that SB‐415286 strongly reduced DCF fluorescence. Lithium, however, did not exhibit any antioxidant properties. We conclude that the GSK‐3 inhibitor SB‐415286 has antioxidant properties, which may explain the cytoprotective effects against H2O2 damage. Furthermore, inhibition of GSK‐3 activity was not involved in this protective effect.