Norberto Aguirre

Sildenafil restores cognitive function without affecting β-amyloid burden in a mouse model of Alzheimer's disease.

BACKGROUND AND PURPOSE Inhibitors of phosphodiesterase 5 (PDE5) affect signalling pathways by elevating cGMP, which is a second messenger involved in processes of neuroplasticity. In the present study, the effects of the PDE5 inhibitor, sildenafil, on the pathological features of Alzheimers disease and on memory‐related behaviour were investigated.

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.

MDMA ('Ecstasy') enhances 5-HT1A receptor density and 8-OH-DPAT-induced hypothermia: blockade by drugs preventing 5-hydroxytryptamine depletion.

One week after a single administration of 3,4-methylenedioxymethamphetamine (MDMA HCI, 30 mg/kg i.p.), 5-HT1A receptor density was significantly increased by approximately 25-30% in the frontal cortex and hypothalamus of rats. The increased density correlated with the potentiation of the hypothermic response to the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 1 mg/kg s.c.). Hypothalamic 5-HT7 receptors, which also bind 8-OH-DPAT, were not changed, however, by MDMA. Fluoxetine (5 mg/kg s.c.), ketanserin (5 mg/kg s.c.) or haloperidol (2 mg/kg i.p.), given 15 min prior to MDMA, prevented the depletion of 5-hydroxytryptamine (5-HT) induced by MDMA and also blocked the effects of this neurotoxin on 5-HT1A receptor density and on 8-OH-DPAT-induced hypothermia. The protection afforded by drugs against 5-HT loss did not correlate, however, with the antagonism of the acute hyperthermic effect of MDMA. The present results indicate that drugs able to prevent or to attenuate MDMA-induced 5-HT loss also prevent the changes in 5-HT1A receptor density as well as the enhanced hypothermic response to the 5-HT1A receptor agonist 8-OH-DPAT in MDMA-treated rats.

Differential regulation by methylenedioxymethamphetamine of 5-hydroxytryptamine1A receptor density and mRNA expression in rat hippocampus, frontal cortex, and brainstem: the role of corticosteroids.

Abstract: The present study examined the effects of repeated administration to rats of 3,4‐methylenedioxymethamphetamine (MDMA, “Ecstasy”) on 5‐hydroxytryptamine1A (5‐HT1A) receptor density and mRNA expression in the hippocampus, frontal cortex, and brainstem. As expected, 7 days after subacute MDMA administration (20 mg/kg i.p. twice daily for 4 consecutive days) 5‐HT content was markedly reduced (−70%) in the hippocampus and the frontal cortex. 5‐HT1A receptor density was increased in the frontal cortex by 23% and decreased in the hippocampus and the brainstem by 25%. These changes correlated with an enhanced or diminished 5‐HT1A receptor mRNA expression in the three regions studied. To examine the influence of corticosteroids on these changes, adrenalectomized (ADX) rats received the same dosage regimen as above. Adrenalectomy by itself did not modify 5‐HT content in the brain regions examined and increased 5‐HT1A receptor density in the hippocampus (+20%) but produced no change in the frontal cortex and brainstem. Adrenalectomy also prevented MDMA‐induced changes in receptor number in the hippocampus and brainstem but not in the frontal cortex. Dexamethasone (1 mg/kg/day i.p.) administered for 7 consecutive days reversed the effects of adrenalectomy in the hippocampus but not in the frontal cortex. In the brainstem, MDMA no longer reduced 5‐HT1A receptor number in ADX rats, but a significant reduction was restored when ADX animals received the glucocorticoid treatment. The present data show that MDMA may affect 5‐HT1A receptors in a regionally dependent manner, notably through a drug effect on corticosterone release, which attenuates 5‐HT1A receptor gene transcription selectively in the hippocampus.

Administration of SCH 23390 into the Medial Prefrontal Cortex Blocks the Expression of MDMA-Induced Behavioral Sensitization in Rats: An Effect Mediated by 5-HT2C Receptor Stimulation and not by D1 Receptor Blockade

Akin to what has been reported for cocaine, systemic administration of the dopamine D1 receptor antagonist, SCH 23390 ((R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride), blocks the expression but not the induction of 3,4-methylenedioxymethamphetamine (MDMA)-induced behavioral sensitization. Since the medial prefrontal cortex (mPFC) appears to regulate the expression of sensitization to cocaine, this study examined whether microinjection of SCH 23390 into the mPFC would alter the expression of MDMA sensitization. Saline or MDMA was administered for 5 consecutive days. After 12 days of withdrawal, rats received a bilateral intra-mPFC microinjection of SCH 23390 or saline followed by an intraperitoneal (i.p.) challenge dose of MDMA. While SCH 23390 enhanced locomotion in MDMA-naïve rats, it completely suppressed the expression of sensitization in MDMA-pretreated animals. Since, SCH 23390 has a fairly good affinity for 5-HT2C receptors, we went further to study the role of mPFC D1 and 5-HT2C receptors in this, apparently, paradoxical effect shown by SCH 23390. Thus, the microinjection of both SKF 81297 (R-(+)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide) and MK 212 (6-chloro-2-(1-piperazinyl)pyrazine hydrochloride), a D1 and 5-HT2C receptor agonist, respectively, blocked MDMA sensitization. By contrast, the 5-HT2C receptor antagonist, RS 102221 (8-[5-(2,4-dimethoxy-5-(4-trifluoromethylphenylsulfonamido)phenyl-5-oxopentyl]-1,3,8-triazaspiro[4,5]decane-2,4-dione hydrochloride), had no effect in MDMA-naïve or MDMA-sensitized animals, but reversed the effects of SCH 23390 in MDMA-pretreated rats. These results demonstrate that suppression of MDMA-induced sensitization by SCH 23390 is mediated by 5-HT2C receptor stimulation in the mPFC and not by the blockade of mPFC D1 receptors. Furthermore, these data indicate that stimulation of 5-HT2C receptors by SCH 23390 is not a minor issue and should be considered when interpreting future data.

Involvement of mitochondrial potential and calcium buffering capacity in minocycline cytoprotective actions.

Minocycline, a semisynthetic derivative of tetracycline, displays beneficial activity in neuroprotective in models including, Parkinson disease, spinal cord injury, amyotrophic lateral sclerosis, Huntington disease and stroke. The mechanisms by which minocycline inhibits apoptosis remain poorly understood. In the present report we have investigated the effects of minocycline on mitochondria, due to their crucial role in apoptotic pathways. In mitochondria isolated suspensions, minocycline failed to block superoxide-induced swelling but was effective in blocking mitochondrial swelling induced by calcium. This latter effect might be mediated through dissipation of mitochondrial transmembrane potential and blockade of mitochondrial calcium uptake. Consistently, minocycline fails to protect SH-SY5Y cell cultures against reactive oxygen species-mediated cell death, including malonate and 6-hydroxydopamine treatments, but it is effective against staurosporine-induced cytotoxicity. The effects of this antibiotic on mitochondrial respiratory chain complex were also analyzed. Minocycline did not modify complex IV activity, and only at the higher concentration tested (100 microM) inhibited complex II/III activity. Other members of the minocycline antibiotic family like tetracycline failed to induce these mitochondrial effects.

Malonate induces cell death via mitochondrial potential collapse and delayed swelling through an ROS‐dependent pathway

1 Herein we study the effects of the mitochondrial complex II inhibitor malonate on its primary target, the mitochondrion. 2 Malonate induces mitochondrial potential collapse, mitochondrial swelling, cytochrome c (Cyt c) release and depletes glutathione (GSH) and nicotinamide adenine dinucleotide coenzyme (NAD(P)H) stores in brain‐isolated mitochondria. 3 Although, mitochondrial potential collapse was almost immediate after malonate addition, mitochondrial swelling was not evident before 15 min of drug presence. This latter effect was blocked by cyclosporin A (CSA), Ruthenium Red (RR), magnesium, catalase, GSH and vitamin E. 4 Malonate added to SH‐SY5Y cell cultures produced a marked loss of cell viability together with the release of Cyt c and depletion of GSH and NAD(P)H concentrations. All these effects were not apparent in SH‐SY5Y cells overexpressing Bcl‐xL. 5 When GSH concentrations were lowered with buthionine sulphoximine, cytoprotection afforded by Bcl‐xL overexpression was not evident anymore. 6 Taken together, all these data suggest that malonate causes a rapid mitochondrial potential collapse and reactive oxygen species production that overwhelms mitochondrial antioxidant capacity and leads to mitochondrial swelling. Further permeability transition pore opening and the subsequent release of proapoptotic factors such as Cyt c could therefore be, at least in part, responsible for malonate‐induced toxicity.

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 Huntingtons 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.

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 × 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.

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.