Inflammation plays an important role in Alzheimer's disease (AD) and other neurodegenerative disorders. Although chronic inflammation in later stages of AD is well described, little is known about the inflammatory processes in preclinical or early stages of the disease prior to plaque deposition. In this study, we report that the inflammatory mediator S100A8 is increased with aging in the mouse brain. It is observed as extracellular aggregates, which do not correspond to corpora amylacea. S100A8 aggregation is enhanced in the hippocampi of two different mouse models for amyloid-ß (Aß) overproduction (Tg2576 and TgAPParctic mice). S100A8 aggregates are seen prior the formation of Aß plaques and do not colocalize. In vitro treatment of glial cells from primary cultures with Aß42 resulted in an increased production of S100A8. In parallel, treatment of a neuronal cell line with recombinant S100A8 protein resulted in enhanced Aß42 and decreased Aß40 production. Our results suggest that important inflammatory processes are occurring prior to Aß deposition and the existence of a positive feedback between S100A8 and Aß productions. The possible relevance of aging- or AD-dependent formation of S100A8 aggregates in the hippocampus thus affecting learning and memory processes is discussed.

Quercetin has been identified as a promising compound with a neuroprotective potential against age-related neurodegenerative diseases such as Alzheimer's disease (AD). Nevertheless, the clinical application of quercetin is hampered by its low oral bioavailability. The aim of this work was to evaluate the capability of nanoencapsulated quercetin in zein nanoparticles (NPQ), that significantly improves the oral absorption and bioavailability of the flavonoid, as potential oral treatment for AD. For this purpose, SAMP8 mice were orally treated for two months with either NPQ (25mg/kg every 48h) or a solution of quercetin (Q; 25mg/kg daily). NPQ displayed a size of 260nm and a payload of about 70¿g/mg. For Q, no significant effects were observed in animals. On the contrary, the oral administration of NPQ improved the cognition and memory impairments characteristics of SAMP8 mice. These observations appeared to be related with a decreased expression of the hippocampal astrocyte marker GFAP. Furthermore, significant levels of quercetin were quantified in the brain of mice treated with nanoparticles. These findings highlight the potential of zein nanoparticles to promote the oral absorption of quercetin as well as the therapeutic potential of this flavonoid in AD pathogenesis.

Quercetin has been identified as a promising compound with a neuroprotective potential against age-related neurodegenerative diseases such as Alzheimer's disease (AD). Nevertheless, the clinical application of quercetin is hampered by its low oral bioavailability. The aim of this work was to evaluate the capability of nanoencapsulated quercetin in zein nanoparticles (NPQ), that significantly improves the oral absorption and bioavailability of the flavonoid, as potential oral treatment for AD. For this purpose, SAMP8 mice were orally treated for two months with either NPQ (25mg/kg every 48h) or a solution of quercetin (Q; 25mg/kg daily). NPQ displayed a size of 260nm and a payload of about 70¿g/mg. For Q, no significant effects were observed in animals. On the contrary, the oral administration of NPQ improved the cognition and memory impairments characteristics of SAMP8 mice. These observations appeared to be related with a decreased expression of the hippocampal astrocyte marker GFAP. Furthermore, significant levels of quercetin were quantified in the brain of mice treated with nanoparticles. These findings highlight the potential of zein nanoparticles to promote the oral absorption of quercetin as well as the therapeutic potential of this flavonoid in AD pathogenesis.

Growing evidence suggests that changes in histone acetylation in specific sites of the chromatin modulate neuronal plasticity and contribute to antidepressant-like action. Sirtuin 2 (SIRT2) is a class III NAD+-dependent histone deacetylase involved in transcriptional repression of genes regulating synaptic plasticity. Importantly, a key role for the glutamate system in prefrontal cortex (PFC) synaptic plasticity changes induced by antidepressants has been suggested. Here, we asked whether SIRT2 could be a pharmacological target for depression therapy. The compound 2-{3-(3-fluorophenethyloxy)phenylamino}benzamide (33i), a selective SIRT2 inhibitor in vitro, was studied in mice (C57Bl6). Firstly, the inhibitory effect of subchronic 33i (5-15 mg/kg, 10 days) on SIRT2 activity in the PFC was evaluated. Moreover, the effect of SIRT2 inhibition on the expression of synaptic plasticity markers linked to glutamate neurotransmission (VGLUT1, synaptophysin, mGluR4, GluA1, GluN2B, GluN2A) and on serotonin levels was studied. Further, neurochemical and behavioral effects of chronic (5 weeks) 33i (15 mg/kg) on the chronic mild stress (CMS) model were analyzed. Subchronic 33i inhibited SIRT2, increased GluN2A, GluN2B and serotonin levels in the PFC. Moreover, chronic 33i reverted CMS-induced anhedonia and social avoidance.

Some histone deacetylase (HDACs) enzymes have been proposed as epigenetic targets involved in the pathophysiology of depression and antidepressant-like action. Among them, we have recently identified SIRT2, a class III NAD(+)-dependent HDAC, as being oppositely regulated by stress and antidepressants. Moreover, SIRT2 inhibition has shown antianhedonic-like action in the chronic mild stress model of depression. Here we have extended the study using an alternative model of depression based in a genetic manipulation of glutamate function. Specifically, mice heterozygous for the vesicular glutamate transporter 1 (VGLUT1 + / -) were used. Firstly, mRNA expression of the different members of the HDAC superfamily in the prefrontal cortex (PFC) of VGLUT1 + / - mice and WT littermates were studied by RT-PCR. Secondly, the effect of repeated treatment with the selective SIRT2 inhibitor 33i and the antidepressant imipramine on anhedonic behaviour of VGLUT1 + / - mice was studied by weekly monitoring of sucrose intake. Further, the interaction of 33i towards specific monoaminergic targets such as serotonin or noradrenaline transporters as well as the monoaminooxidase enzyme was studied. The mRNA occurance of the different members of HDAC superfamily was not altered in the PFC of VGLUT1 + / - mice. While repeated imipramine showed an anti-anhedonic action in both VGLUT1 + / - and WT, the selective SIRT2 inhibitor 33i fully reversed anhedonia of VGLUT1 + / -. Further, 33i showed no interaction with the above mentioned monoaminergic molecular targets. These results confirm that SIRT2 inhibition is able to reverse anhedonia in different animal models and highlight the need to further investigate the role of SIRT2 inhibitors as new antidepressant agents.

Several studies support the relation between leptin and Alzheimer's disease (AD). We show that leptin levels in CSF are unchanged as subjects progress to AD. However, in AD hippocampus, leptin signalling was decreased and leptin localization was shifted, being more abundant in reactive astrocytes and less in neurons. Similar translocation of leptin was found in brains from Tg2576 and apoE4 mice. Moreover, an enhancement of leptin receptors was found in hippocampus of young Tg2576 mice and in primary astrocytes and neurons treated with A(1-42). In contrast, old Tg2576 mice showed decreased leptin receptors levels. Similar findings to those seen in Tg2576 mice were found in apoE4, but not in apoE3 mice. These results suggest that leptin levels are intact, but leptin signalling is impaired in AD. Thus, A accumulation and apoE4 genotype result in a transient enhancement of leptin signalling that might lead to a leptin resistance state over time.

Many studies suggest that the prefrontal cortex (PFC) is a target limbic region for stress response because a dysfunction here is linked to anhedonia, a decrease in reactivity to rewards, and to anxiety. It is suggested that stress-induced persistent molecular changes in this brain region could bring some light on the mechanisms perpetuating depressive episodes. In order to address this issue, here we have studied the long-term PFC gene expression pattern and behavioral effects induced by a chronic mild stress (CMS) model and antidepressant treatment in mice. CMS was applied to mice for six weeks and imipramine (10 mg/kg, i.p.) or saline treatment was administered for five weeks starting from the third week of CMS. Mice were sacrificed one month after CMS and following two weeks after the discontinuation of drug treatment and the PFC was dissected and prepared for gene (mRNA) and protein expression studies. Using the same experimental design, a separate group of mice was tested for anhedonia, recognition memory, social interaction and anxiety. CMS induced a long-term altered gene expression profile in the PFC that was partially reverted by imipramine. Specifically, the circadian rhythm signaling pathway and functions such as gene expression, cell proliferation, survival and apoptosis as well as neurological and psychiatric disorders were affected. Of these, some changes of the circadian rhythm pathway (Hdac5, Per1, and Per2) were validated by RT-PCR and western-blot. Moreover, CMS induced long-lasting anhedonia that was reverted by imipramine treatment. Impaired memory, decreased social interaction and anxiety behavior were also induced by chronic stress. We have identified in the PFC molecular targets oppositely regulated by CMS and imipramine that could be relevant for chronic depression and antidepressant action. Among these, a possible candidate for further investigation could be the circadian rhythm pathway.

Changes in histone acetylation could contribute to the pathogenesis of depression and antidepressant therapy. Using the chronic social defeat stress (CSDS) model of depression and different antidepressant treatments we studied the regulation of histone deacetylases (Hdac's) and synaptic plasticity markers in the prefrontal cortex (PFC). Further, functional implication of identified Hdac's in brain plasticity was explored. Mice were exposed to CSDS (10 days) followed by saline or imipramine (4 weeks). PFC Hdac's mRNA abundance was studied and compared to human's. Further, protein expression of acetylated histones (AcH3 and AcH4), neuroplasticity markers (CREB and pro-BDNF) and selected Hdac's were analyzed. Moreover, other antidepressants (fluoxetine and reboxetine) and selective HDAC inhibitors were studied. CSDS increased Hdac5 and Sirt2 mRNA whereas repeated imipramine did the opposite. Accordingly, stress and imipramine induced opposite changes on AcH3, AcH4 and CREB expression. At protein level, CSDS upregulated nuclear fraction of Hdac5 and repeated imipramine and reboxetine increased its phosphorylated form (p-Hdac5), mainly located in the cytoplasm. Moreover, Sirt2 was downregulated by all monoaminergic antidepressants. Further, repeated treatment with the class IIa Hdac inhibitor MC1568 and the Sirt2 inhibitor 33i for three weeks increased synaptic plasticity in the prefrontal cortex. Our results suggest that Hdac5 and Sirt2 upregulation could constitute stable stress-induced neuronal adaptations. Noteworthy, the SIRT2 upregulation in depressed patients supports the interest of this target for therapeutic intervention. On the other hand, cytoplasmic Hdac5 export and Sirt2 downregulation induced by monoaminergic antidepressants could contribute to the well-known beneficial effects of antidepressants on brain plasticity.

Methylenedioxymethamphetamine (MDMA) causes a persistent loss of dopaminergic cell bodies in the substantia nigra of mice. Current evidence indicates that MDMA-induced neurotoxicity is mediated by oxidative stress probably due to the inhibition of mitochondrial complex I activity. In this study we investigated the contribution of dopamine (DA) to such effects. For this, we modulated the dopaminergic system of mice at the synthesis, uptake or metabolism levels. Striatal mitochondrial complex I activity was decreased 1h after MDMA; an effect not observed in the striatum of DA depleted mice or in the hippocampus, a dopamine spare region. The DA precursor, L-dopa, caused a significant reduction of mitochondrial complex I activity by itself and exacerbated the dopaminergic deficits when combined with systemic MDMA. By contrast, no damage was observed when L-dopa was combined with intrastriatal injections of MDMA. On the other hand, dopamine uptake blockade using GBR 12909, inhibited both, the acute inhibition of complex I activity and the long-term dopaminergic toxicity caused by MDMA. Moreover, the inhibition of DA metabolism with the monoamine oxidase (MAO) inhibitor, pargyline, afforded a significant protection against MDMA-induced complex I inhibition and neurotoxicity. Taken together, these findings point to the formation of hydrogen peroxide subsequent to DA metabolism by MAO, rather than a direct DA-mediated mitochondrial complex I inhibition, and the contribution of a peripheral metabolite of MDMA, as the key steps in the chain of biochemical events leading to DA neurotoxicity caused by MDMA in mice.

The senescence-accelerated mouse-prone 8 (SAMP8), used as a model of aging, displays many established pathological features of Alzheimer's disease. Cognitive impairments and increased levels of hyperphosphorylated tau are found in the hippocampus of SAMP8 mice along with an increased ß-secretase activity and amyloid-ß (Aß) depositions that increase in number and extent with age. Based on a previous study from our laboratory showing an amelioration of cognitive impairments and tau pathology by sildenafil, in this study we tested whether this drug could also modulate the amyloid precursor protein amyloidogenic processing in this mouse model. Our results show that the protein levels of the ß-secretases ß-site amyloid precursor protein cleaving enzyme 1 and cathepsin B are higher in the hippocampus of 9-month-old SAMP8 mice than those of age-matched senescence-resistant-1. Sildenafil (7.5mg/kg for 4 weeks) attenuated learning and memory impairments shown by SAMP8 mice in the passive avoidance test. The increased expression of ß-site amyloid precursor protein cleaving enzyme 1 was also reduced by sildenafil, an effect paralleled to decreases in the activities of two ß-site amyloid precursor protein cleaving enzyme 1 modulators, calpain and cyclin-dependent kinase 5 protein. Interestingly, sildenafil enhanced both Akt and glycogen synthase kinase-3ß (ser9) phosphorylation, which could be mediating the reduction in cathepsin B levels found in the hippocampus of sildenafil-treated SAMP8 mice. Sildenafil-induced reduction in ß-site amyloid precursor protein cleaving enzyme 1 and cathepsin B expression in SAMP8 mice was associated with a decrease in hippocampal Aß42 levels which, in turn, could mediate the parallel decline in glial fibrillary acidic protein expression observed in these animals. These findings highlight the therapeutic potential of sildenafil in Alzheimer's disease pathogenesis.

It is believed that glucocorticoids control the proliferation of neural progenitor cells, and this process is highly involved in mood disorders and cognitive processes. Using the maternal separation model of chronic neonatal stress, it has been found that stress induced depressive-like behavior, cognitive deficits and a decrease in proliferation in the subventricular zone (SVZ). Venlafaxine reversed all deleterious effects of chronic stress by modulating HPA activity. These outcomes suggest modulation of stress-mediated glucocorticoid secretion as a target for the treatment of mood disorders and neurodegenerative processes.

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is an amphetamine derivative widely abused by young adults. Although many studies have reported that relatively high doses of MDMA deplete serotonin (5-HT) content and decrease the availability of serotonin transporters (5-HTT), limited evidence is available as to the adaptive mechanisms taking place in gene expression levels in the brain following a dosing regimen of MDMA comparable to human consumption. In order to further clarify this issue, we used quantitative PCR to study the long-term changes induced by acute administration of MDMA (5 mg/kg × 3) in the expression of genes related to serotonergic and dopaminergic systems, as well as those related to cellular toxicity in the cortex, hippocampus, striatum, and brain stem of rats. Seven days after MDMA administration, we found a significantly lower expression of the 5-HTT (Slc6a4) and the vesicular monoamine transporter (Slc18a2) genes in the brain stem area. In the hippocampus, monoamine oxidase B (Maob) and tryptophan hydroxylase 2 (Tph2) gene expressions were increased. In the striatum, tyrosine hydroxylase (Th) expression was decreased, and a lower expression of ¿-synuclein (Snca) was observed in the cortex. In contrast, no significant changes were observed in the genes considered to be biomarkers of toxicity including the glial fibrillary acidic protein (Gfap) and the heat-shock 70 kD protein 1A (Hspa1a) in any of the structures assayed. These results suggest that MDMA promotes adaptive changes in genes related to serotonergic and dopaminergic functionality, but not in genes related to neurotoxicity.

Aging is the primary risk factor for Alzheimer's disease (AD), and it is known that inflammation is associated with both aging and AD. To resolve inflammation, biosynthesis of the specialized pro-resolving mediators (SPMs) is enhanced in a programmed and active manner. We investigated the effect of age on resolution by analyzing hippocampal tissue from 2- and 9-month-old senescence-accelerated mouse prone 8 (SAMP8), as well as age-matched senescence-accelerated mouse resistant 1 (SAMR1). Pro-inflammatory markers increased upon age in SAMP8 mice and were also higher than those in age-matched SAMR1 mice. However, neither SPMs nor their receptors were enhanced upon age in SAMP8 mice compared to age-matched SAMR1 mice. Analysis of SPM biosynthetic enzymes revealed elevated levels of leukocyte type 12-lipoxygenase (L12-LOX) and decreased 5-LOX levels upon age in SAMR1 mice, whereas they remained unchanged in SAMP8 mice. Moreover, we found partial co-localization of L12-LOX and amyloid beta (Aß) staining, as well as correlation between L12-LOX and phosphorylated tau levels in SAMP8, but not SAMR1 mice. Thus, we conclude that the resolution response in SAMP8 mice is insufficient to counteract the increased inflammation with age, and this may have a role in the development of AD-like pathologies.

The senescence accelerated mouse-prone 8 (SAMP8) strain of mice is an experimental model of accelerated senescence that also shares several pathological features with Alzheimer's disease. Among them, cognitive impairments and abnormal hyperphosphorylation of tau are ameliorated by the phosphodiesterase 5 inhibitor sildenafil, possibly through the modulation of Cdk5/p25 and Akt/GSK-3ß pathways. Here we studied the implication of protein phosphatase 2A (PP2A) and c-Jun N-terminal kinase (JNK) in the therapeutic effects of sildenafil. Results demonstrated that there were no differences in hippocampal PP2A protein levels or activity (measured by its inactive isoform phopho-PP2A Y307) when we compared 6-month old SAMP8 mice and age-matched control, SAMR1 mice, treated with saline or sildenafil (7.5 mg/kg i.p. for 4 weeks). However, this same treatment of sildenafil, that had been shown to reverse the cognitive impairment and tau hyperphosphorylation in this animal model, also reversed the increased levels of activated JNK (p-JNK) found in the hippocampus of SAMP8 mice. Moreover, the administration of the JNK inhibitor, D-JNKI-1 (0.2 mg/kg i.p. for 3 weeks) also ameliorated the cognitive deficits shown by SAMP8 mice in the Morris water maze and decreased hippocampal levels of phospho-c-Jun(Ser73). When phosphorylated tau (AT8 epitope) was analyzed a significant reduction was observed in the hippocampus of D-JNKI-1 treated SAMP8 mice, providing a plausible explanation for the attenuation of cognitive decline shown by these animals. These findings suggest the involvement of the JNK pathway on tau pathology and cognitive deficits shown by 6-month old SAMP8 mice. They also point to the modulation of this kinase to be among the mechanisms responsible for the beneficial effects shown by sildenafil

Phosphodiesterase 5 (PDE5) inhibitors have recently been reported to exert beneficial effects against ischemia-reperfusion injury in several organs but their neuroprotective effects in brain stroke models are scarce. The present study was undertaken to assess the effects of sildenafil against cell death caused by intrastriatal injection of malonate, an inhibitor of succinate dehydrogenase; which produces both energy depletion and lesions similar to those seen in cerebral ischemia. Our data demonstrate that sildenafil (1.5mg/kg by mouth (p.o.)), given 30min before malonate (1.5¿mol/2¿L), significantly decreased the lesion volume caused by this toxin. This protective effect can be probably related to the inhibition of excitotoxic pathways. Thus, malonate induced the activation of the calcium-dependent protease, calpain and the cyclin-dependent kinase 5, cdk5; which resulted in the hyperphosphorylation of tau and the cleavage of the protective transcription factor, myocyte enhancer factor 2, MEF2. All these effects were also significantly reduced by sildenafil pre-treatment, suggesting that sildenafil protects against malonate-induced cell death through the regulation of the calpain/p25/cdk5 signaling pathway. Similar findings were obtained using inhibitors of calpain or cdk5, further supporting our contention. Sildenafil also increased MEF2 phosphorylation and Bcl-2/Bax and Bcl-xL/Bax ratios, effects that might as well contribute to prevent cell death. Finally, sildenafil neuroprotection was extended not only to rat hippocampal slices subjected to oxygen and glucose deprivation when added at the time of reoxygenation, but also, in vivo when administered after malonate injection. Thus, the therapeutic window for sildenafil against malonate-induced hypoxia was set at 3h.

We previously described a heterozygous mouse model overexpressing human HA-tagged 24S- hydroxylase (CYP46A1) utilizing a ubiquitous expression vector. In this study, we generated homozygotes of these mice with circulating levels of 24OH 30-60% higher than the heterozygotes. Female homozygous CYP46A1 transgenic mice, aged 15 months, showed an improvement in spatial memory in the Morris water maze test as compared to the wild type mice. The levels of N-Methyl-D-Aspartate receptor 1, phosphorylated-N-Methyl-D-Aspartate receptor 2A, postsynaptic density 95, synapsin-1 and synapthophysin were significantly increased in the hippocampus of the CYP46A1 transgenic mice as compared to the controls. The levels of lanosterol in the brain of the CYP46A1 transgenic mice were significantly increased, consistent with a higher synthesis of cholesterol. Our results are discussed in relation to the hypothesis that the flux in the mevalonate pathway in the brain is of importance in cognitive functions.

BACKGROUND AND PURPOSE: PD5 inhibitors have recently been reported to exert beneficial effects against ischaemia-reperfusion injury in several organs. However, there are few studies regarding their neuroprotective effects in brain ischaemia. The present study was designed to assess the effects of sildenafil against chemical hypoxia induced by malonate. Intrastriatal injection of malonate produces energy depletion and striatal lesions similar to that seen in cerebral ischaemia through mechanisms that involve generation of reactive oxygen species (ROS). EXPERIMENTAL APPROACH: Volume lesion was analysed by cytochrome oxidase histochemistry. Generation of reactive species was determined by in situ visualization of superoxide production and nitrotyrosine measurement. Protein levels were determined by Western blot after subcellular fractionation. KEY RESULTS: Sildenafil, given 30 min before malonate, significantly decreased the lesion volume in the rat. This protective effect cannot be attributed to any effect on ROS production but to the inhibition of downstream pathways. Thus, malonate induced the activation of apoptosis signal-regulating kinase-1 (ASK1) and two MAPK kinases, MKK3/6 and MKK7, which lead to an increased phosphorylation of JNK and p38 MAPK, effects that were blocked by sildenafil. Selective inhibitors of p38 and JNK (SB203580 or SP600125, respectively) were used in combination with malonate in order to evaluate the plausible implication of these pathways in the p

The presence of the E4 allele of apolipoprotein E (apoE) is the strongest known genetic risk factor for sporadic Alzheimer's disease (AD). Other risk factors for developing AD have been identified, including lifestyle such as dietary habits. The present study was designed to explore the impact of the interaction between variant human apoE isoforms and a high carbohydrate diet (HCD) on mechanisms behind learning and memory retention. As an investigative model, we compared young apoE3 and apoE4 target replacement mice fed on a HCD for 6 months. Our results indicate that HCD compromises memory processes in apoE4 mice. ApoE4 mice on HCD showed decreased activity-regulated cytoskeletal-associated protein (Arc) and brain derived neurotrophic factor (BDNF) levels, as well as decreased BDNF signaling in the hippocampus. In contrast, apoE3 mice were resistant to the deleterious effects of HCD on both behavior and memory-related proteins. Our results support the hypothesis that already in mid-life, genetic, and environmental risk factors act together on the mechanisms behind cognitive impairment.

Excitotoxicity due to excessive activation of glutamate receptors is a primary mediator of cell death in acute and chronic neurological disorders, and NMDA-type glutamate receptors (NMDARs) are thought to be involved. NMDARs assemble from heteromeric combinations of GluNl, GluN2 and GluN3 subunits, yielding a variety of receptor subtypes that differ in biophysical properties, signaling, and synaptic targeting. Inclusion of inhibitory GluN3 subunits reduces Ca2+ influx via NMDAR channels and alters their synaptic targeting, thus modifying the two hallmarks of NMDARs that are critical for their roles on neuronal death and survival. Here we evaluated the neuroprotective potential of GluN3A subunits by analyzing the susceptibility to striatal excitotoxic damage of transgenic mice overexpressing GluN3A. We found that mild GluN3A overexpression protected susceptible striatal neurons from lesions induced by the neurotoxin 3-nitropropionic acid (3-NP), an inhibitor of mitochondrial complex 11/succinate dehydrogenase. GluN3A-mediated neuroprotection was dose-dependent and correlated with the levels of transgenic GluN3A expressed by two different mice strains. Neuroprotection was associated with a potent reduction of the activation of calpain, a Ca2+-dependent protease, which was measured as a decrease in 3-NP-induced fodrin and STEP cleavage in GluN3A transgenic mice relative to controls. We further show that transgenic GluN3A subunits incorporate into extrasynaptic compartments in mouse striatum, suggesting that reductions of toxic calpain activation might be linked to inhibition by GluN3A of pathological extrasynaptic NMDAR activity.

The aim of the present study was to investigate whether late pre-conditioning using 3-nitropropionic acid (3NP) prevents the 5-hydroxytryptamine (5-HT) deficits caused by the amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA) in the rat. For this purpose we administered 3NP 24 h before MDMA (3 x 5 mg/kg i.p., every 2 h) and rats were killed 7 days later. Pre-treatment of 3NP afforded complete protection against MDMA-induced 5-HT deficits independent of any effect on MDMA-induced hyperthermia or 5-HT transporter activity. To identify the transductional mechanisms responsible for the neuroprotective effect of 3NP, we first examined the involvement of nitric oxide (NO) by using selective inhibitors of all three nitric oxide synthase isoforms. Inhibition of endothelial and neuronal nitric oxide synthase, but not inducible nitric oxide synthase, reversed 3NP-induced pre-conditioning. The NO donor S-Nitroso-N-acetylpenicilamine mimicked 3NP effects further suggesting the involvement of NO in mediating 3NP protection. To investigate the involvement of NOS/soluble guanylate cyclase (sGC)/protein kinase G/mitochondrial ATP-sensitive potassium channels (mitoK(ATP)) signaling pathway we examined the effect of 5-hydroxydecanoate (5-HD), a selective mitoK(ATP) blocker, and 1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one, a potent inhibitor of sGC, on 3NP-induced tolerance. 5-hydroxydecanoate, but not 1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one, suppressed 3NP-mediated protection suggesting that mitoK(ATP) opening, but not NO-mediated activation of sGC, participates in the mechanism underlying tolerance to MDMA. Our data also showed that the protective effect of 3NP was abolished by cycloheximide, supporting the involvement of de novo protein synthesis. In conclusion, 3NP-induced delayed tolerance against 5-HT deficits caused by MDMA occurs via NO production.

Methadone is a widely used therapeutic opioid in narcotic addiction and neuropathic pain syndromes. Oncologists regularly use methadone as a long-lasting analgesic. Recently it has also been proposed as a promising agent in leukemia therapy, especially when conventional therapies are not effective. Nevertheless, numerous reports indicate a negative impact on human cognition with chronic exposure to opiates. Thus, clarification of methadone toxicity is required. In SH-SY5Y cells we found that high concentrations of methadone were required to induce cell death. Methadone-induced cell death seems to be related to necrotic processes rather than typical apoptosis. Cell cultures challenged with methadone presented alterations in mitochondrial outer membrane permeability. A mechanism that involves Bax translocation to the mitochondria was observed, accompanied with cytochrome c release. Furthermore, no participation of known protein regulators of apoptosis such as Bcl-XL and p53 was observed. Interestingly, methadone-induced cell death took place by a caspases-independent pathway; perhaps due to its ability to induce a drastic depletion in cellular ATP levels. Therefore, we studied the effect of methadone on isolated rat liver mitochondria. We observed that methadone caused mitochondrial uncoupling, coinciding with the ionophoric properties of methadone, but did not cause swelling of the organelles. Overall, the effects observed for cells in the presence of supratherapeutic doses of methadone may result from a ¿bioenergetic crisis.¿ A decreased level of cellular energy may predispose cells to necrotic-like cell death.

BACKGROUND AND PURPOSE: 3,4-methylenedioxymethamphetamine (MDMA) causes a persistent loss of dopaminergic cell bodies in the substantia nigra of mice. Current evidence indicates that such neurotoxicity is due to oxidative stress but the source of free radicals remains unknown. Inhibition of mitochondrial electron transport chain complexes by MDMA was assessed as a possible source. EXPERIMENTAL APPROACH: Activities of mitochondrial complexes after MDMA were evaluated spectrophotometrically. In situ visualization of superoxide production in the striatum was assessed by ethidium fluorescence and striatal dopamine levels were determined by HPLC as an index of dopaminergic toxicity. KEY RESULTS: 3,4-methylenedioxymethamphetamine decreased mitochondrial complex I activity in the striatum of mice, an effect accompanied by an increased production of superoxide radicals and the inhibition of endogenous aconitase. alpha-Lipoic acid prevented superoxide generation and long-term toxicity independent of any effect on complex I inhibition. These effects of alpha-lipoic acid were also associated with a significant increase of striatal glutathione levels. The relevance of glutathione was supported by reducing striatal glutathione content with L-buthionine-(S,R)-sulfoximine, which exacerbated MDMA-induced dopamine deficits, effects suppressed by alpha-lipoic acid. The nitric oxide synthase inhibitor, N(G)-nitro-L-arginine, partially prevented MDMA-induced dopamine depletions, an effect reversed by L-arginine but not D-arginine. Finally, a direct relationship between mitochondrial complex I inhibition and long-term dopamine depletions was found in animals treated with MDMA in combination with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. CONCLUSIONS AND IMPLICATIONS: Inhibition of mitochondrial complex I following MDMA could be the source of free radicals responsible for oxidative stress and the consequent neurotoxicity of this drug in mice.