Enzymatic hydrolysis of cellulose nanoplatelets as a source of sugars with the concomitant production of cellulose nanofibrils.

Cellulose, the most abundant biopolymer on earth, is produced at different ratios by all land plants. Since the morphology and crystallinity of cellulose are key factors involved in its enzymatic hydrolysis, in the present work, we tackled the study of the effects of such variables on the nanocellulose conversion into glucose. Cellulase from Trichoderma sp at 37 °C was used to produce glucose, the best results were found for the cellulose nanoplatelets (S-CNP) after 60 h of hydrolysis, which afforded a conversion of 47% to glucose, in contrast to 15% for the non-purified sample (W-CP) and 22% for microcrystalline cellulose (MCC20) used as control. The X-ray diffractogram recorded on the samples showed an initial crystallinity index of 45%, 54% and 72% for W-CNP, S-CNP and MCC20, respectively. Also, we showed that after 24 h of hydrolysis, long cellulose nanofibrils (∅ ≈ 30 nm) were found as a residue.

Sildenafil Decreases BACE1 and Cathepsin B Levels and Reduces APP Amyloidogenic Processing in the SAMP8 Mouse.

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.

3,4-methylenedioxymethamphetamine induces gene expression changes in rats related to serotonergic and dopaminergic systems, but not to neurotoxicity.

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 α-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.

Implication of JNK pathway on tau pathology and cognitive decline in a senescence-accelerated mouse model.

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 (7.5mg/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 (0.2mg/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.

Inhibition of calpain-regulated p35/cdk5 plays a central role in sildenafil-induced protection against chemical hypoxia produced by malonate.

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 (1.5µmol/2µL), 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.

Propranolol restores cognitive deficits and improves amyloid and Tau pathologies in a senescence-accelerated mouse model.

Ageing is associated with a deterioration of cognitive performance and with increased risk of neurodegenerative disorders. Hypertension is the most-prevalent modifiable risk factor for cardiovascular morbidity and mortality worldwide, and clinical data suggest that hypertension is a risk factor for Alzheimer's disease (AD). In the present study we tested whether propranolol, a ß-receptor antagonist commonly used as antihypertensive drug, could ameliorate the cognitive impairments and increases in AD-related markers shown by the senescence-accelerated mouse prone-8 (SAMP8). Propranolol administration (5 mg/kg for 3 weeks) to 6-month-old SAMP8 mice attenuated cognitive memory impairments shown by these mice in the novel object recognition test. In the hippocampus of SAMP8 mice it has been found increases in Aß(42) levels, the principal constituent of amyloid plaques observed in AD, accompanied by both an increased expression of the cleaving enzyme BACE1 and a decreased expression of the degrading enzyme IDE. All these effects were reversed by propranolol treatment. Tau hyperphosphorylation (PHF-1 epitope) shown by SAMP8 mice at this age was also decreased in the hippocampus of propranolol-treated mice, an effect probably related to a decrease in JNK1 expression. Interestingly, propranolol also phosphorylated Akt in SAMP8 mice, which was associated with an increase of glycogen synthase kinase-3ß phosphorylation, contributing therefore to the reductions in Tau hyperphosphorylation. Synaptic pathology in SAMP8 mice, as shown by decreases in synaptophysin and BDNF, was also counteracted by propranolol treatment. Overall, propranolol might be beneficial in age-related brain dysfunction and could be an emerging candidate for the treatment of other neurodegenerative diseases. This article is part of a Special Issue entitled 'Cognitive Enhancers'.

Propranolol reduces cognitive deficits, amyloid ß levels, tau phosphorylation and insulin resistance in response to chronic corticosterone administration.

Chronic exposure to glucocorticoids might result not only in insulin resistance or cognitive deficits, but it is also considered as a risk factor for pathologies such as Alzheimer's disease. Propranolol is a ß-adrenergic antagonist commonly used in the treatment of hypertension or acute anxiety. The effects of propranolol (5 mg/kg) have been tested in a model of chronic corticosterone administration (100 µg/ml, 4 wk) in drinking water. Corticosterone administration led to cognitive impairment in the novel object recognition test that was reversed by propranolol. Increased levels of Aß in the hippocampus of corticosterone-treated mice were counteracted by propranolol treatment, purportedly through an increased IDE expression. Chronic corticosterone treatment induced responses characteristic of insulin resistance, as increased peripheral insulin levels, decreased activation of the insulin receptor (pIR) and decreased associated intracellular pathways (pAkt). These effects might be related to a decreased c-Jun N terminal kinase 1 expression. Again, propranolol was able to counteract all corticosterone-induced effects. One of the main kinases involved in tau phosphorylation, glycogen synthase kinase 3ß (GSK3ß), which is inactivated by phosphorylation by pAkt, was found to be decreased after corticosterone and increased after propranolol treatment. Concomitant changes in pTau expression were found. Overall, these data further strengthen the potential of propranolol as a therapeutic agent for pathologies associated with the interaction glucocorticoids-insulin resistance and the development of relevant cellular processes for Alzheimer's disease.

The NMDA receptor subunit GluN3A protects against 3-nitroproprionic-induced striatal lesions via inhibition of calpain activation.

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 GluN1, 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 II/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.

Sildenafil ameliorates cognitive deficits and tau pathology in a senescence-accelerated mouse model.

Aging is associated with a deterioration of cognitive performance and with increased risk of neurodegenerative disorders. In the present study we tested whether the specific phosphodiesterase 5 inhibitor sildenafil could ameliorate the age-dependent cognitive impairments shown by the senescence-accelerated mouse prone-8 (SAMP8). Sildenafil administration (7.5 mg/kg for 4 weeks) to 5-month-old SAMP8 mice attenuated spatial learning and memory impairments shown by these mice in the Morris Water Maze. Tau hyperphosphorylation (AT8 but not PHF-1 epitope) shown by SAMP8 mice at this age was also decreased in the hippocampus of sildenafil-treated mice, an effect probably related to a decrease in cyclin-dependent kinase 5 protein expression and activity (p25/p35 ratio). Interestingly, sildenafil also phosphorylated Akt, which was associated with an increase of glycogen synthase kinase-3ß phosphorylation, providing a plausible explanation for the reductions in tau hyperphosphorylation (AT8 and PHF-1 epitopes) and attenuation of cognitive deficits shown by 9-month-old SAMP8 mice. Overall, sildenafil might be beneficial in age-related brain dysfunction and could be an emerging candidate for the treatment of other neurodegenerative diseases.

Long-lasting neuroprotective effect of sildenafil against 3,4-methylenedioxymethamphetamine- induced 5-hydroxytryptamine deficits in the rat brain.

Sildenafil, given shortly before 3,4-methylenedioxymethamphetamine (MDMA), affords protection against 5-hydroxytryptamine (5-HT) depletions caused by this amphetamine derivative by an acute preconditioning-like mechanism. Because acute and delayed preconditionings do not share the same mechanisms, we investigated whether sildenafil would also protect the 5-HT system of the rat if given 24 hr before MDMA. For this, MDMA (3 × 5 mg/kg i.p., every 2 hr) was administered to rats previously treated with sildenafil (8 mg/kg p.o.). One week later, 5-HT content and 5-HT transporter density were measured in the striatum, frontal cortex, and hippocampus of the rats. Our findings indicate that sildenafil afforded significant protection against MDMA-induced 5-HT deficits without altering the acute hyperthermic response to MDMA or its metabolic disposition. Sildenafil promoted ERK1/2 activation an effect that was paralleled by an increase in MnSOD expression that persisted 24 hr later. In addition, superoxide and superoxide-derived oxidants, shown by ethidium fluorescence, increased after the last MDMA injection, an effect that was prevented by sildenafil pretreatment. Similarly, MDMA increased nitrotyrosine concentration in the hippocampus, an effect not shown by sildenafil-pretreated rats. In conclusion, our data demonstrate that sildenafil produces a significant, long-lasting neuroprotective effect against MDMA-induced 5-HT deficits. This effect is apparently mediated by an increased expression of MnSOD and a subsequent reduced susceptibility to the oxidative stress caused by MDMA.

Minocycline exerts uncoupling and inhibiting effects on mitochondrial respiration through adenine nucleotide translocase inhibition.

The present study was aimed to provide a better understanding of the mitochondria-targeted actions of minocycline (MC), a second-generation tetracycline which has cytoprotective effects. Although the specific mechanisms underlying its activity remained elusive, considerable amounts of data indicated mitochondria as the primary pharmacological target of MC. Previous reports have shown that MC affects the oxygen-uptake rate by isolated mitochondria in different respiratory states. Here, we report on the effect of MC, in the range 50-200µM, on mitochondrial respiration. State 3 respiration titration with carboxyatractyloside revealed that MC inhibits the adenine nucleotide translocase. Furthermore, we analyze MC channel-forming capacity in the lipid membrane bilayer. Our results confirmed the crucial role of Δψ and showed a dependence on Ca(2+) for MC to have an effect on mitochondria. Our data also indicated that outer and inner mitochondrial membranes contribute differently to this effect, involving the presence of Δψ (the inner membrane) and VDAC (the outer membrane). Data from three isosmotic media indicate that MC does not increase the permeability of the inner membrane to protons or potassium. In addition, by using mitoplasts and ruthenium red, we showed that Ca(2+) uptake is not involved in the MC effect, suggesting involvement of VDAC in the MC interaction with the outer membrane. Our data contribute to unravel the mechanisms behind the mitochondria-targeted activity of the cytoprotective drug MC.

Methadone induces necrotic-like cell death in SH-SY5Y cells by an impairment of mitochondrial ATP synthesis.

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-X(L) 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.

Delayed pre-conditioning by 3-nitropropionic acid prevents 3,4-methylenedioxymetamphetamine-induced 5-HT deficits.

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.

Methylenedioxymethamphetamine inhibits mitochondrial complex I activity in mice: a possible mechanism underlying neurotoxicity.

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.

Sildenafil protects against 3-nitropropionic acid neurotoxicity through the modulation of calpain, CREB, and BDNF.

In this study we tested whether phosphodiesterase 5 (PDE5) inhibitors, sildenafil and vardenafil, would afford protection against 3-nitropropionic acid (3NP), which produces striatal lesions that closely mimic some of the neuropathological features of Huntington's Disease (HD). The neurotoxin was given over 5 days by constant systemic infusion using osmotic minipumps. Animals treated with PDE5 inhibitors (sildenafil or vardenafil) showed improved neurologic scores, reduced the loss of striatal DARPP-32 protein levels and lesion volumes, and decreased calpain activation produced by 3NP. This protective effect was independent of changes in 3NP-induced succinate dehydrogenase inhibition. Furthermore, striatal p-CREB levels along with the expression of BDNF were significantly increased in sildenafil-treated rats. In summary, PDE5 inhibitors protected against 3NP-induced striatal degeneration by reducing calpain activation and by promoting survival pathways. These data encourage further evaluation of PDE5 inhibitors in transgenic mouse models of HD.

Mitochondria and calcium flux as targets of neuroprotection caused by minocycline in cerebellar granule cells.

Minocycline, an antibiotic of the tetracycline family, has attracted considerable interest for its theoretical therapeutic applications in neurodegenerative diseases. However, the mechanism of action underlying its effect remains elusive. Here we have studied the effect of minocycline under excitotoxic conditions. Fluorescence and bioluminescence imaging studies in rat cerebellar granular neuron cultures using fura2/AM and mitochondria-targeted aequorin revealed that minocycline, at concentrations higher than those shown to block inflammation and inflammation-induced neuronal death, inhibited NMDA-induced cytosolic and mitochondrial rises in Ca(2+) concentrations in a reversible manner. Moreover, minocycline added in the course of NMDA stimulation decreased Ca(2+) intracellular levels, but not when induced by depolarization with a high K(+) medium. We also found that minocycline, at the same concentrations, partially depolarized mitochondria by about 5-30 mV, prevented mitochondrial Ca(2+) uptake under conditions of environmental stress, and abrogated NMDA-induced reactive oxygen species (ROS) formation. Consistently, minocycline also abrogates the rise in ROS induced by 75 microM Ca(2+) in isolated brain mitochondria. In search for the mechanism of mitochondrial depolarization, we found that minocycline markedly inhibited state 3 respiration of rat brain mitochondria, although distinctly increased oxygen uptake in state 4. Minocycline inhibited NADH-cytochrome c reductase and cytochrome c oxidase activities, whereas the activity of succinate-cytochrome c reductase was not modified, suggesting selective inhibition of complexes I and IV. Finally, minocycline affected activity of voltage-dependent anion channel (VDAC) as determined in the reconstituted system. Taken together, our results indicate that mitochondria are a critical factor in minocycline-mediated neuroprotection.

Phosphodiesterase 5 inhibitors prevent 3,4-methylenedioxymethamphetamine-induced 5-HT deficits in the rat.

Phosphodiesterase 5 (PDE5) inhibitors are often used in combination with club drugs such as 3,4-methylenedioxymethamphetamine (MDMA or ecstasy). We investigated the consequences of such combination in the serotonergic system of the rat. Oral administration of sildenafil citrate (1.5 or 8 mg/kg) increased brain cGMP levels and protected in a dose-dependent manner against 5-hydroxytryptamine depletions caused by MDMA (3 x 5 mg/kg, i.p., every 2 h) in the striatum, frontal cortex and hippocampus without altering the acute hyperthermic response to MDMA. Intrastriatal administration of the protein kinase G (PKG) inhibitor, KT5823 [(9S, 10R, 12R)-2,3,9,10,11,12-Hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid, methyl ester)], suppressed sildenafil-mediated protection. By contrast, the cell permeable cGMP analogue, 8-bromoguanosine cyclic 3',5'-monophosphate, mimicked sildenafil effects further suggesting the involvement of the PKG pathway in mediating sildenafil protection. Because mitochondrial ATP-sensitive K(+) channels are a target for PKG, we next administered the specific mitochondrial ATP-sensitive K(+) channel blocker, 5-hydroxydecanoic acid, 30 min before sildenafil. 5-hydroxydecanoic acid completely reversed the protection afforded by sildenafil, thereby implicating the involvement of mitochondrial ATP-sensitive K(+) channels. Sildenafil also increased Akt phosphorylation, and so the possible involvement of the Akt/endothelial nitric oxide synthase (eNOS)/sGC signalling pathway was analysed. Neither the phosphatidylinositol 3-kinase inhibitor, wortmannin, nor the selective eNOS inhibitor, L-N5-(1-iminoethyl)-L-ornithine dihydrochloride, reversed the protection afforded by sildenafil, suggesting that Akt/eNOS/sGC cascade does not participate in the protective mechanisms. Our data also show that the protective effect of sildenafil can be extended to vardenafil, another PDE5 inhibitor. In conclusion, sildenafil protects against MDMA-induced long-term reduction of indoles by a mechanism involving increased production of cGMP and subsequent activation of PKG and mitochondrial ATP-sensitive K(+) channel opening.

On the role of tyrosine and peripheral metabolism in 3,4-methylenedioxymethamphetamine-induced serotonin neurotoxicity in rats.

The mechanisms underlying 3,4-methylenedioxymethamphetamine (MDMA)-induced serotonergic (5-HT) toxicity remain unclear. It has been suggested that MDMA depletes 5-HT by increasing brain tyrosine levels, which via non-enzymatic hydroxylation leads to DA-derived free radical formation. Because this hypothesis assumes the pre-existence of hydroxyl radicals, we hypothesized that MDMA metabolism into pro-oxidant compounds is the limiting step in this process. Acute hyperthermia, plasma tyrosine levels and concentrations of MDMA and its main metabolites were higher after a toxic (15 mg/kg i.p.) vs. a non-toxic dose of MDMA (7.5mg/kg i.p.). The administration of a non-toxic dose of MDMA in combination with l-tyrosine (0.2 mmol/kg i.p.) produced a similar increase in serum tyrosine levels to those found after a toxic dose of MDMA; however, brain 5-HT content remained unchanged. The non-toxic dose of MDMA combined with a high dose of tyrosine (0.5 mmol/kg i.p.), caused long-term 5-HT depletions in rats treated at 21.5 degrees C but not in those treated at 15 degrees C, conditions known to decrease MDMA metabolism. Furthermore, striatal perfusion of MDMA (100 microM for 5h) combined with tyrosine (0.5 mmol/kg i.p.) in hyperthermic rats did not cause 5-HT depletions. By contrast, rats treated with the non-toxic dose of MDMA under heating conditions or combined with entacapone or acivicin, which interfere with MDMA metabolism or increase brain MDMA metabolite availability respectively, showed significant reductions of brain 5-HT content. Altogether, these data indicate that although tyrosine may contribute to MDMA-induced toxicity, MDMA metabolism appears to be the limiting step.

The relationship between core body temperature and 3,4-methylenedioxymethamphetamine metabolism in rats: implications for neurotoxicity.

RATIONALE: A close relationship appears to exist between 3,4-methylenedioxymethamphetamine (MDMA)-induced changes in core body temperature and long-term serotonin (5-HT) loss. OBJECTIVE: We investigated whether changes in core body temperature affect MDMA metabolism. MATERIALS AND METHODS: Male Wistar rats were treated with MDMA at ambient temperatures of 15, 21.5, or 30 degrees C to prevent or exacerbate MDMA-induced hyperthermia. Plasma concentrations of MDMA and its main metabolites were determined for 6 h. Seven days later, animals were killed and brain indole content was measured. RESULTS: The administration of MDMA at 15 degrees C blocked the hyperthermic response and long-term 5-HT depletion found in rats treated at 21.5 degrees C. At 15 degrees C, plasma concentrations of MDMA were significantly increased, whereas those of three of its main metabolites were reduced when compared to rats treated at 21.5 degrees C. By contrast, hyperthermia and indole deficits were exacerbated in rats treated at 30 degrees C. Noteworthy, plasma concentrations of MDMA metabolites were greatly enhanced in these animals. Instrastriatal perfusion of MDMA (100 microM for 5 h at 21 degrees C) did not potentiate the long-term depletion of 5-HT after systemic MDMA. Furthermore, interfering in MDMA metabolism using the catechol-O-methyltransferase inhibitor entacapone potentiated the neurotoxicity of MDMA, indicating that metabolites that are substrates for this enzyme may contribute to neurotoxicity. CONCLUSIONS: This is the first report showing a direct relationship between core body temperature and MDMA metabolism. This finding has implications on both the temperature dependence of the mechanism of MDMA neurotoxicity and human use, as hyperthermia is often associated with MDMA use in humans.