Marc Yeste-Velasco

Comparative analysis of the effects of resveratrol in two apoptotic models: Inhibition of complex I and potassium deprivation in cerebellar neurons

The mechanism involved in neuronal apoptosis is largely unknown. Studies performed on neuronal cell cultures provide information about the pathways which orchestrate the process of neuronal loss and potential drugs for the treatment of neurological disorders. In the present study we select resveratrol, a natural antioxidant, as a potential drug for the treatment of neurodegenerative diseases. We evaluate the neuroprotective effects of resveratrol in two apoptotic models in rat cerebellar granule neurons (CGNs): the inhibition of mitochondrial complex I using 1-methyl-4-phenylpyridinium (MPP(+)) (an in vitro model of Parkinsons disease) and serum potassium withdrawal. We study the role of the mammalian silent information regulator 2 (SIRT1) in the process of neuroprotection mediated by resveratrol. Because recent studies have demonstrated that SIRT1 is involved in cell survival and has antiaging properties, we also measured changes in the expression of this protein after the addition of these two apoptotic stimuli. MPP(+)--induced loss of cell viability and apoptosis in CGNs was prevented by the addition of RESV (1 microM to 100 microM). However, the neuroprotective effects were not mediated by the activation of SIRT1, since sirtinol-an inhibitor of this enzyme--did not attenuate them. Furthermore MPP(+) decreases the protein expression of SIRT1. RESV did not prevent serum potassium withdrawal-induced apoptosis although it did completely attenuate oxidative stress production by these apoptotic stimuli. Furthermore, serum potassium withdrawal increases the expression of SIRT1. Our results indicate that the antiapoptotic effects of RESV in MPP(+) are independent of the stimulation of SIRT1 and depend on its antioxidant properties. Furthermore, because SIRT1 is involved in neuronal survival depending on the apoptotic stimuli, changes in the expression of SIRT1 could be involved in the regulation of the apoptotic route.

Modulation of SIRT1 expression in different neurodegenerative models and human pathologies

We examined the expression of SIRT1 in several experimental paradigms of human pathologies. We used a neuroblastoma cell line (B65), neuronal primary cultures (hippocampus and cerebellar granule cells) and in vivo approaches in rat and senescence murine models (SAM). Cell cultures and rats were treated with several well-know neurotoxins, i.e. rotenone, MPP(+), kainate and 3-nitropropionic acid. Subsequently, SIRT1 expression was compared in these different paradigms of neurotoxicity. The pattern of expression of SIRT1 in proliferating cell cultures (B65) was different to that in quiescent cell cultures. In the murine model of senescence (senescence-accelerated mice prone, SAMP8), SIRT1 expression progressively decreased, while in the control strain (senescence-accelerated mice resistant, SAMR1) it increased. Finally, we studied human samples of Parkinsons disease (PD), dementia with Lewy bodies (DLB) and Huntingtons diseases (HD). SIRT1 expression decreased dramatically in HD, but there were no significant changes in Parkinson-related illnesses. In conclusion, SIRT1 expression may be a good sensor of toxic neuronal processes.

The antiproliferative activity of melatonin in B65 rat dopaminergic neuroblastoma cells is related to the downregulation of cell cycle-related genes

Abstract:  A potential application of melatonin is its ability to rescue many cell types from cell death, because of its antioxidant properties. Likewise, recent studies suggest that melatonin may also be used as an anti‐tumor drug, due to its anti‐proliferative properties in tumor cells when administered at physiologic or pharmacologic doses. In the present study, we investigated the mechanisms involved in the apoptosis induced by acute exposure to melatonin and roscovitine in the rat dopaminergic neuroblastoma B65 cell line. Cell growth studies revealed that, at 24 hr of treatment, roscovitine blocked cell growth and induced apoptosis whereas melatonin delayed cell growth and induced a slight increase in the number of apoptotic nuclei. Melatonin also increased the percentage of cells in the G1‐phase of the cell cycle, whereas roscovitine blocked cells in the G2/M‐phase. Both compounds significantly downregulated the transcriptional activity of cdk4, while melatonin also downregulated cdk2 and cyclin D1. Taken together, our data show that melatonin at millimolar concentrations inhibits dopaminergic B65 proliferation, induces cell apoptosis, and modulates cell cycle progression by inhibiting the transcriptional activity of cyclins and cdks related to the progression of the G1‐phase.

Activation of Akt by lithium: pro-survival pathways in aging.

The effects of lithium on senescence were investigated using the senescence-accelerated mouse prone 8 (SAMP8) mice and cultures of aging cerebellar granule cells. Our in vitro findings, using cerebellar granule neurons, demonstrate that lithium (1-10mM) exerts neuroprotective effects in young cultures (7-8 days) against LY294002-induced Akt inhibition. Furthermore, lithium (10mM) inhibits GSK-3beta activity by upregulating p-GSK-3beta (ser-9) and increases p-FOXO1 (Ser256) suggesting an effective anti-apoptotic effect. Our data also showed that lithium in aged cultures exerts anti-apoptotic effects via Akt activation and consequent inhibition of downstream targets regulated by this enzyme. Finally, the administration of lithium to senescence-accelerated mice (SAMP8) and senescence-accelerated resistant mice (SAMR1) at 3 months of age also caused increased Akt activity and p-FoxO-1. These results demonstrate the effectiveness of lithium in preventing age-related neural loss and the potential therapeutic applications of lithium in treatment/prevention of neurological disease.

Neuroprotection by c-Jun NH2-terminal kinase inhibitor SP600125 against potassium deprivation-induced apoptosis involves the Akt pathway and inhibition of cell cycle reentry.

Increasing evidence implicates the c-Jun NH(2)-terminal kinase (JNK) pathway in the regulation of apoptosis in neurodegenerative diseases. In this study, we examined the neuroprotective effect of SP600125, a selective JNK inhibitor, in cerebellar granule cells (CGNs) deprived of serum and potassium (S/K withdrawal). S/K withdrawal-induced apoptosis occurs via activation of multiple pro-apoptotic pathways, including re-entry into the cell cycle, activation of glycogen synthase kinase-3 beta (GSK-3beta), cyclin-dependent kinase 5 (cdk5/p35) breakdown, formation of cdk5/p25 and JNK activation. Here we demonstrate that SP600125 is able to inhibit all these pro-apoptotic pathways via the inhibition of JNK. Further, we found that JNK inhibition maintains the phosphorylation/activation of Akt after S/K withdrawal. For further confirmation of this result, we studied several targets downstream of Akt including GSK-3beta, p-FOXO1, p-CREB and p35. In addition, the specific PI3K/Akt inhibitor LY294002 greatly diminished the antiapoptotic effects of SP600125 upon S/K withdrawal, confirming that Akt is involved in the neuroprotection achieved by SP600125. These results suggest that the maintenance of the PI3-kinase/Akt pathway by inhibition of JNK contributes to the prevention of apoptosis in rat cerebellar granule neurons mediated by S/K withdrawal. Furthermore, we propose that JNK may regulate the cell cycle re-entry by a novel mechanism that involves Akt, GSK-3beta and Rb phosphorylation.

Glycogen synthase kinase-3 is involved in the regulation of the cell cycle in cerebellar granule cells.

Recent studies have demonstrated that neuronal reentry in the cell cycle and specifically the expression of the transcription factor E2F-1, constitutes a pathway that may be involved in neuronal apoptosis after serum and potassium withdrawal. Other enzymes such as glycogen synthase kinase-3beta (GSK-3beta) are also involved in this apoptotic stimulus, and thus in the process of neuronal cell death. Primary cerebellar granule cells (CGNs) were used in this study to determine whether pharmacological inhibition of GSK-3beta is involved in neuronal modulation of the cell cycle, and specifically in the regulation of E2F-1 and retinoblastoma protein (Rb). CGNs showed a dramatic increase in GSK-3beta activity after 2h of serum and potassium deprivation. Immunoblot and activity assays revealed that lithium and SB415286 inhibit fully the activation of GSK-3beta and attenuate the expression of cyclin D, cyclin E, pRb phosphorylation and the transcription factor E2F-1. These data were confirmed using AR-014418, a selective GSK-3beta inhibitor that prevents the expression of cell-cycle proteins. Our data indicate that GSK-3beta inhibition regulates, in part, the cell cycle in CGNs by inhibiting Rb phosphorylation and thus inhibiting E2F-1 activity. However, the selective inhibition of GSK-3beta with AR-A014418 had not effect on cell viability or apoptosis mediated by S/K withdrawal. Furthermore, our results suggest that selective GSK-3beta inhibition is not sufficient to protect against apoptosis in this S/K withdrawal model, indicating that Li(+) and SB415286 neuroprotective effects are mediated by the inhibition of additional targets to GSK3beta. Therefore, there is a connection between cell cycle and GSK-3beta activation and that these, along with other mechanisms, are involved in the molecular paths leading to the apoptotic process of rat CGNs triggered by S/K withdrawal.

Neuroprotective effects of caffeine against complex I inhibition-induced apoptosis are mediated by inhibition of the Atm/p53/E2F-1 path in cerebellar granule neurons.

The aim of the present study was to evaluate the neuroprotective effects of caffeine, an inhibitor of ataxia telangiectasia mutated (ATM) enzyme and an antagonist of adenosine receptors, in two models of apoptosis in cerebellar granule neurons (CGNs): the inhibition of mitochondrial complex I by the neurotoxin MPP+ and serum and potassium deprivation. We used cerebellar granule neurons because of low glial contamination. Cell viability was measured by the MTT method, and apoptosis was evaluated by assessing DNA fragmentation with flow cytometry or quantification of nuclear condensation. Our data indicate that the neuroprotective effects of caffeine in the MPP+ model of apoptosis are mediated through activation of the ATM/p53 pathway. In addition, caffeine decreased the expression of cyclin D and the transcription factor E2F‐1, a regulator of apoptosis in neurons. Caffeine‐mediated neuroprotection was not mediated through blockade of adenosine receptors because DPCPX and CGS‐15943, two antagonists of these receptors, failed to attenuate apoptosis produced by MPP+ treatment. In addition, caffeine did not exert neuroprotective effects after serum and potassium withdrawal, a p53‐independent model of apoptosis. Taken together, our findings indicate that DNA damage/ATM activation is a key component of MPP+‐induced apoptosis in CGNs through activation of p53 and reentry into the cell cycle, specifically expression of the transcription factor E2F‐1.

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.

The p38MAPK signaling pathway regulates neuronal apoptosis through the phosphorylation of the retinoblastoma protein

We investigated the role of SB202190, a selective p38 mitogen-activated protein kinase (MAPK) inhibitor in cerebellar granule neurons (CGC) in response to serum potassium deprivation (S/K deprivation), an apoptotic stimulus. CGC apoptosis after S/K deprivation was shown to be mediated through cell cycle re-entry and the induction of transcription factor E2F-1. We found that SB 202190 (10muM) inhibits retinoblastoma protein (pRb) phosphorylation, in response to S/K deprivation. Moreover, the expression of cyclin E and E2F-1 were also significantly decreased. Interestingly, SB202190 did not affect or modulate the increase in the protein expression levels of cyclin D1. Similarly, p-Akt and p-GSK3 protein levels, measured after 12h S/K deprivation, did not appear to be regulated by SB 202190 (10muM). These data indicate that the neuroprotective effects of the p38 inhibitor were not mediated via Akt activation. In conclusion, these results suggest that p38MAPK converged with the cell cycle in S/K deprivation-induced apoptosis through pRb phosphorylation.

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.