Daniel Alvira

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.

Inhibition of the cdk5/p25 fragment formation may explain the antiapoptotic effects of melatonin in an experimental model of Parkinson's disease

Abstract:  In this study, the effects of melatonin on MPP+‐treated cerebellar granule neurons (CGNs) in culture were investigated. Results showed that MPP+ treatment significantly decreased cell viability and increased the apoptotic cell population at 24 and 48 hr. Calpain and caspase‐3 activation was also determined, with results showing a strong increase in calpain (74%) and caspase 3 activity (70%), as measured by α‐spectrin cleavage and fluorometric and colorimetric analysis, respectively. There are several studies suggesting that the activation of the cdk5/p35 pathway at its cleavage to cdk5/p25 may play a role in neuronal cell death in neurodegenerative diseases. Moreover, these studies indicate that this cleavage is mediated by calpains, and that MPP+ prompted an increase in cdk5 expression, as well as the cleavage of p35–p25, in a time‐dependent manner. 1 mm Melatonin not only reduced the neurotoxic effects of MPP+ on cell viability, but also prevented apoptosis mediated by this Parkinsonian toxin in CGNs. 1 mm Melatonin reduced cdk5 expression, as well as the cleavage of p35–p25. These data indicate that melatonin possesses some neuro‐protective properties against MPP+‐induced apoptosis. Moreover, these data suggest that the calpain/cdk5 signaling cascade has a potential role in the MPP+‐mediated apoptotic process in CGNs.

Inhibition of cyclin-dependent kinases is neuroprotective in 1-methyl-4-phenylpyridinium-induced apoptosis in neurons

The biochemical pathways involved in neuronal cell death in Parkinsons disease are not completely characterized. Mitochondrial dysfunction, specifically alteration of the mitochondrial complex I, is the primary target of the parkinsonian neurotoxin 1-methyl-4-phenylpyridinium (MPP+) induced apoptosis in neurons. In the present study, we examine the role of caspase-dependent and -independent routes in MPP+-induced apoptosis in rat cerebellar granule neurons (CGNs). We show a distinct increase in the expression of the cell cycle proteins cyclin D, cyclin E, cdk2, cdk4 and the transcription factor E2F-1 following a MPP+ treatment of CGNs. Flavopiridol (FLAV), a broad inhibitor of cyclin-dependent kinases (CDKs), attenuated the neurotoxic effects of MPP+ and significantly attenuates apoptosis mediated by MPP+ 200 microM. Likewise, the antioxidant vitamin E (vit E) increases neuronal cell viability and attenuates apoptosis induced by MPP+. Moreover, the expression levels of cyclin D and E2F-1 induced by this parkinsonian neurotoxin were also attenuated by vit E. Since, the broad-spectrum caspase inhibitor zVAD-fmk did not attenuate MPP+-induced apoptosis in CGNs, our data provide a caspase-independent mechanism mediated by neuronal reentry in the cell cycle and increased expression of the pro-apoptotic transcription factor E2F-1. Our results also suggest a potential role of oxidative stress in neuronal reentry in the cell cycle mediated by MPP+. Finally, our data further support the therapeutic potential of flavopiridol, for the treatment of Parkinsons disease.

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.

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.

Activation of ataxia telangiectasia muted under experimental models and human Parkinson’s disease

In the present study we demonstrated that neurotoxin MPP+-induced DNA damage is followed by ataxia telangiectasia muted (ATM) activation either in cerebellar granule cells (CGC) or in B65 cell line. In CGC, the selective ATM inhibitor KU-55933 showed neuroprotective effects against MPP+-induced neuronal cell loss and apoptosis, lending support to the key role of ATM in experimental models of Parkinson’s disease. Likewise, we showed that knockdown of ATM levels in neuroblastoma B65 cells using an ATM-specific siRNA attenuates the phosphorylation of retinoblastoma protein without affecting other cell-cycle proteins involved in the G0/G1 cell-cycle phase. Moreover, we demonstrated DNA damage, in human brain samples of PD patients. These findings support a model in which MPP+ leads to ATM activation with a subsequent DNA damage response and activation of pRb. Therefore, this study demonstrates a new link between DNA damage by MPP+ and cell-cycle re-entry through retinoblastoma protein phosphorylation.

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.