Evaluation of transcriptional activity of caspase-3 gene as a marker of acute neurotoxicity in rat cerebellar granular cells.

Caspase-3 is a key protein involved in the classical apoptosis mechanism in neurons, as in many other cells types. In the present research, we describe the transcriptional activity of caspase-3 gene as a marker of acute toxicity in a primary culture model of rat cerebellar granule neurons (CGNs). CGNs were incubated for 16h in complete medium containing the chemicals at three concentrations (10, 100microM and 1mM). A total of 48 different compounds were tested. Gene transcriptional activity was determined by low-density array assays, and by single Taqman caspase-3 assays. Results from the PCR arrays showed that the caspase-3 gene was up-regulated when CGNs were exposed to neurotoxic chemicals. Significative correlations were found between the transcriptional activity of caspase-3 and the activity of some other genes related to apoptosis, cell-cycle and ROS detoxification. In our experiments, acute exposure of CGNs to well-documented pro-apoptotic xenobiotics modulated significantly caspase-3 gene expression, whereas chemicals not related to apoptosis did not modify caspase-3 gene expression. In conclusion, acute exposure of CGNs to neurotoxic compounds modulates the transcriptional activity of genes involved in the classical apoptotic pathway, oxidative stress and cell-cycle control. Transcriptional activity of caspase-3 correlates significantly with these changes and it could be a good indicator of acute neurotoxicity.

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.

GSK-3 beta inhibition and prevention of mitochondrial apoptosis inducing factor release are not involved in the antioxidant properties of SB-415286.

The antioxidant effects of lithium and SB-415286, two glycogen synthase kinase-3 beta (GSK-3 beta) inhibitors, were studied in cerebellar granule neurons by measuring changes in 2, 7-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescence. GSK-3 beta inhibitors inhibit apoptosis mediated by serum and potassium withdrawal (S/K withdrawal) and GSK-3 beta activation, as measured by beta-catenin degradation. Furthermore, as both drugs prevent mitochondrial apoptosis inducing factor (AIF) release, these data indicate that GSK-3 beta inhibitors prevent caspase-independent apoptosis in cerebellar granule neurons induced by S/K withdrawal. While the most specific GSK-3 beta inhibitor, SB-415286, demonstrated antioxidant effects, Li+ 10 mM did not. These results indicate that lithium 10 mM and SB-415286 20 microM exert anti-apoptotic effects in cases of S/K withdrawal mediated by GSK-3 beta inhibition. However, these antioxidant properties are independent of GSK-3 beta inhibition and prevention of mitochondrial AIF release.

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). H(2)O(2) 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 H(2)O(2) (1mM). 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 H(2)O(2) damage. Furthermore, inhibition of GSK-3 activity was not involved in this protective effect.

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

Cytoskeletal alteration is a key factor in neurodegenerative processes like Alzheimer's or Parkinson's 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.

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 alpha-spectrin. Flavopiridol (1 microM) 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 microM) 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-3beta 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.

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

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. 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. PD150606 and PD151746 prevented apoptosis mediated by S/K withdrawal through inhibition of calpain. Furthermore, PD151746 was able to inhibit caspase-3 activity. After S/K withdrawal, we observed an increase in cdk5/p25 formation and MEF2 phosphorylation that was prevented by 40 microM PD150606 and PD151746. This indicates that calpain inhibition may be an upstream molecular target that prevents neuronal apoptosis in vitro. 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.

p21(WAF1/Cip1) is not involved in kainic acid-induced apoptosis in murine cerebellar granule cells.

Kainic acid (KA) treatment induced neuronal death and apoptosis in murine cerebellar granule cells (CGNs) cultures from both wild-type and knockout p21(-/-) mice. There was not statistically significant difference in the percentage of neuronal apoptosis among strains. KA-induced neurotoxicity was prevented in the presence of NBQX (20 microM) and GYKI 52446 (20 microM), but not by z-VAD-fmk, suggesting that caspases are not involved in the apoptotic process. Data suggest that p21(WAF/Cip) was unable to modulate KA-induced apoptosis in murine CGNs.

Cyclosporin A enhances colchicine-induced apoptosis in rat cerebellar granule neurons.

1. Cyclosporin A (CsA, 1-50 microM), an immunosuppressive drug with known neurotoxic effects, did not decrease the viability of primary cultures of rat cerebellar granule neurons (CGN) or induce apoptotic features. However, CsA specifically enhanced the cytotoxicity and apoptosis induced by colchicine (1 microM). 2. Flavopiridol, an inhibitor of cyclin-dependent kinases (CDKs), prevented the neurotoxic effects of colchicine plus CsA. At 0.1-5 microM, it also showed antiapoptotic effects, as revealed by propidium iodide staining, flow cytometry and counting of cell nuclei. 3. Roscovitine (25-50 microM), a selective cdk1, 2 and 5 inhibitor, showed an antiapoptotic effect against colchicine- and colchicine plus CsA-induced apoptosis. 4. CsA increased the expression of cdk5 and cdk5/p25 mediated by colchicine, a CDK involved in neuronal apoptosis. After treatment of CGN with colchicine plus CsA, the changes in the p25/p35 ratio pointed to cdk5 activation. 5. Immunohistochemical results showed a nuclear localization of cdk5 after neurotoxic treatment, which was prevented by cdk inhibitors. Thus, we propose a new mechanism of modulation of CsA neurotoxicity mediated by cdk5.

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.