Andrés Jiménez

Evaluation of potential pro‐survival pathways regulated by melatonin in a murine senescence model

Abstract:  We examined the effect of melatonin on pro‐survival processes in three groups of mice. Untreated senescence‐accelerated mice (SAMP8), melatonin‐treated SAMP8 and untreated senescence‐accelerated resistant mice (SAMR1) of 10 months old were studied. Melatonin (10 mg/kg) or vehicle (ethanol at 0.066%) was supplied in the drinking water from the end of the first month until the end of the ninth month of life. Differences in the Akt/Erk1‐2 pathway and downstream targets were examined and no significant changes were observed, except for β‐catenin. However, sirtuin 1 expression was significantly lower in SAMP8 than in SAMR1. In addition, acetylated p53 and NFκB expression were lower in SAMP8 than in SAMR1. These changes were prevented by melatonin. Moreover, the concentration/expression of α‐secretase was lower and that of amyloid β aggregates (Aβ) was higher in untreated SAMP8 than in SAMR1. Likewise, the levels of Bid were higher, whereas Bcl‐2XL levels were lower in SAMP8 than in SAMR1. Melatonin reduced all these changes. We conclude that melatonin improves pro‐survival signals and reduces pro‐death signals in age‐related impairments of neural processes.

Kainic acid-induced apoptosis in cerebellar granule neurons: an attempt at cell cycle re-entry

This study was undertaken to investigate whether kainic acid (KA) may regulate the expression of several proteins which plays an important role in cell-cycle progression in cerebellar granule neurons (CGNs). KA induced decrease in MTT values in a concentration dependent way. Flow cytometric analysis showed that KA was able to induce 30% apoptosis in CGNs. Apoptotic nuclear condensation were detected 24 h of exposure to KA (200 μM). An associated marked increase in DNA synthesis, measured by BrdU incorporation, was observed. Western blot analysis showed that KA induced an increase in the expression of Cdk2, cyclin E and E2F-1. It is proposed that, in post-mitotic cells like CGNs, re-entry cell cycle could be responsible for the apoptotic effect of KA.

Microgliosis and down-regulation of adenosine transporter induced by methamphetamine in rats

Chronic administration of methamphetamine to rats induces neurotoxicity characterized by a loss of striatal dopaminergic terminals and reactive gliosis. Subcutaneous administration of methamphetamine in a scheduled procedure of four doses (10 mg/kg) at 2 h interval also induces a significant increase in the peripheral-type benzodiazepine receptor (PBR) density. This increase is maximum (76%) at 72 h post-treatment in the striatum and disappears at 7 days, suggesting that microglia may have a predominant role in necrosis-phagocytosis of neuronal debris rather than acting in a restorative manner. Microgliosis is not restricted to the striatum since it is also evident in cerebellum (75.4% of PBR increase) and hippocampus (37.2% of PBR increase). In the areas with high density of adenosine transporter, the microgliosis phenomenon correlates well with a decrease of this nucleoside transporter (about 39%). Although the microgliosis and the decrease in adenosine transporter could be parallel and not related events, we can speculate that when microglia are activated, a down-regulation of adenosine transporter occurs, playing a role in tissue homeostasis. With the same dosing schedule, methamphetamine induces HSP72 expression in both cytoplasmic and nuclear fractions of the striatum, cerebellum and hippocampus. This expression is also evident in the cerebral cortex, where adenosine transporter population did not show any variation.

Evidence in favour of a role for peripheral-type benzodiazepine receptor ligands in amplification of neuronal apoptosis.

The mitochondrial peripheral benzodiazepine receptor (PBR) is involved in a functional structure designated as the mitochondrial permeability transition (MPT) pore, which controls apoptosis. PBR expression in nervous system has been reported in glial and immune cells. We now show expression of both PBR mRNA and protein, and the appearance of binding of a synthetic ligand fluo-FGIN-1-27 in mitochondria of rat cerebellar granule cells (CGCs). Additionally, the effect of PBR ligands on colchicine-induced apoptosis was investigated. Colchicine-induced neurotoxicity in CGCs was measured at 24 h. We show that, in vitro, PBR ligands 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195), 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4- benzodiazepin-2-one (Ro5-4864) and diazepam (25– 50 μM) enhanced apoptosis induced by colchicine, as demonstrated by viability experiments, flow cytometry and nuclear chromatin condensation. Enhancement of colchicine-induced apoptosis was characterized by an increase in mitochondrial release of cytochrome c and AIF proteins and an enhanced activation of caspase-3, suggesting mitochondrion dependent mechanism that is involved in apoptotic process. Our results indicate that exposure of neural cells to PBR ligands generates an amplification of apoptotic process induced by colchicine and that the MPT pore may be involved in this process.

Neuroprotective action of flavopiridol, a cyclin-dependent kinase inhibitor, in colchicine-induced apoptosis

Flavopiridol was developed as a drug for cancer therapy due to its ability to inhibit cell cycle progression by targeting cyclin-dependent kinases (CDKs). In this study, we show that flavopiridol may also have a neuroprotective action. We show that at therapeutic dosage (or at micromolar range), flavopiridol almost completely prevents colchicine-induced apoptosis in cerebellar granule neurones. In agreement with this, flavopiridol inhibits both the release of cyt c and the activation of caspase-3 induced in response to colchicine treatment. We demonstrate that in this cellular model for neurotoxicity, neither re-entry in the cell cycle nor activation of stress-activated protein kinases, such as c-Jun N-terminal kinase (JNK) or p38 MAP kinase, is involved. In contrast, we show that colchicine-induced apoptosis correlates with a substantial increase in the expression of cdk5 and Par-4, which is efficiently prevented by flavopiridol. Accordingly, a cdk5 inhibitor such as roscovitine, but not a cdk4 inhibitor such as 3-ATA, was also able to protect neurons from apoptosis as well as prevent accumulation of cdk5 and Par-4 in response to colchicine. Our data suggest a potential therapeutic use of flavopiridol in disorders of the central nervous system in which cytoskeleton alteration mediated by cdk5 activation and Par-4 expression has been demonstrated, such as Alzheimers disease.

Kainic acid-induced neuronal cell death in cerebellar granule cells is not prevented by caspase inhibitors.

We examined the role of non‐NMDA receptors in kainic acid (KA)‐induced apoptosis in cultures of rat cerebellar granule cells (CGCs). KA (1 – 500 μM) induced cell death in a concentration‐dependent manner, which was prevented by NBQX and GYKI 52466, non‐NMDA receptor antagonists. Moreover, AMPA blocked KA‐induced excitotoxicity, through desensitization of AMPA receptors. Similarly, KA raised the intracellular calcium concentration of CGCs, which was inhibited by NBQX and GYKI 52466. Again, AMPA (100 μM) abolished the KA (100 μM)‐induced increase in intracellular calcium concentration. KA‐induced cell death in CGCs had apoptotic features, which were determined morphologically, by DNA fragmentation, and by expression of the prostate apoptosis response‐4 protein (Par‐4). KA (500 μM) slightly (18%) increased caspase‐3 activity, which was strongly enhanced by colchicine (1 μM), an apoptotic stimulus. However, neither Z‐VAD.fmk, a pan‐caspase inhibitor, nor the more specific caspase‐3 inhibitor, Ac‐DEVD‐CHO, prevented KA‐induced cell death or apoptosis. In contrast, both drugs inhibited colchicine‐induced apoptosis. The calpain inhibitor ALLN had no effect on KA or colchicine‐induced neurotoxicity. Our findings indicate that colchicine‐induced apoptosis in CGCs is mediated by caspase‐3 activation, unlike KA‐induced apoptosis.

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.

Implication of cyclin-dependent kinase 5 in the neuroprotective properties of lithium

Although numerous studies have demonstrated a neuroprotective and anti-apoptotic role of lithium in neuronal cell cultures, the precise mechanism by which this occurs, remains to be elucidated. In this study, we evaluated the lithium-mediated neuroprotection against colchicine-induced apoptosis in cultured cerebellar granule neurons. Previously, it has been demonstrated that colchicine mediates apoptosis in cerebellar granule neurons through cytoskeletal alteration and activation of an intrinsic pro-apoptotic pathway. Recently we also demonstrated a potential role of cyclin-dependent kinase 5 (cdk5) in this pathway. Here we report that colchicine induces dephosphorylation in Ser-9 and phosphorylation in Tyr-216, and thus activation, of glycogen synthase kinase-3beta in cerebellar granule neurons, and that this modification is inhibited by the presence of 5 mM lithium. However, the selective glycogen synthase kinase-3beta inhibitors SB-415286 and SB-216763 were unable to prevent colchicine-induced apoptosis in these cells, suggesting that the anti-apoptotic activity of lithium is not mediated by glycogen synthase kinase-3beta under these conditions. On the other hand, 5 mM lithium prevented the colchicine-induced increase in cdk5 expression and breakdown of cdk5/p35 to cdk5/p25. In addition, we show that up-regulation of cdk5/p25 is unrelated to inhibition of the activity of myocyte enhancer factor 2, a pro-survival transcription factor. These data suggest a previously undescribed neuroprotective mechanism of lithium associated with the modulation of cdk5/p35 or cdk5/p25 expression.

Antiapoptotic effects of roscovitine in cerebellar granule cells deprived of serum and potassium: a cell cycle-related mechanism

Neuronal apoptosis may be partly due to inappropriate control of the cell cycle. We used serum deprivation as stimulus and reduced potassium from 25 to 5mM (S/K deprivation), which induces apoptosis in cerebellar granule neurons (CGNs), to evaluate the direct correlation between re-entry in the cell cycle and apoptosis. Roscovitine (10 microM), an antitumoral drug that inhibits cyclin-dependent kinase 1 (cdk1), cdk2 and cdk5, showed a significant neuroprotective effect on CGNs deprived of S/K. S/K deprivation induced the expression of cell cycle proteins such as cyclin E, cyclin A, cdk2, cdk4 and E2F-1. It also caused CGNs to enter the S phase of the cell cycle, measured by a significant incorporation of BrdU (30% increase over control cells), which was reduced in the presence of roscovitine (10 microM). On the other hand, roscovitine modified the expression of cytochrome c (Cyt c), Bcl-2 and Bax, which are involved in the apoptotic intrinsic pathway induced by S/K deprivation. We suggest that the antiapoptotic effects of roscovitine on CGNs are due to its anti-proliferative efficacy and to an action on the mitochondrial apoptotic mechanism.

A new patient with dicarboxylic aciduria suggestive of medium-chain acyl-CoA dehydrogenase deficiency presenting as Reye's syndrome

A new patient with medium-chain dicarboxylic aciduria and suberyl glycinuria during an attack of acute illness is reported. When, inadvertently he was given medium-chain triglycerides for 2 days, the excretion of abnormal metabolites of medium-chain fatty acids increased and hepatomegaly became more pronounced. During remission a low excretion of the metabolites were observed. After 16 h of fasting hypoglycaemia was accompanied by an increase of urinary dicarboxylic acids and ψ-hydroxyacids similar to that found on admission. Interestingly this urinary organic acid pattern persisted 8 h after intravenous administration of glucose. In a blood sample obtained after 16 h of fasting there was hypoketonaemia and increased levels of total free fatty acids, octanoic, decanoic andcis-4-decenoic acids. These biochemical data suggest the existence of a deficiency at the level of medium-chain acyl-CoA dehydrogenase.