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Administration of the recombinant analog of the pancreatic amyloid amylin, Pramlintide, has shown therapeutic benefits in aging and Alzheimer's disease (AD) models, both on cognition and amyloid-β (Aβ) pathology. However, the neuroprotective mechanisms underlying the benefits of Pramlintide remain unclear. Given the early and critical role of oxidative stress in AD pathogenesis and the known reactive oxygen species (ROS) modulating function of amyloids, we sought to determine whether Pramlintide's neuroprotective effects involve regulation of oxidative stress mechanisms. To address this, we treated APP/PS1 transgenic mice with Pramlintide for 3 months, starting at 5.5 months prior to widespread AD pathology onset, and measured cognition (Morris Water Maze), AD pathology, and oxidative stress-related markers and enzymes in vivo. In vitro, we determined the ability of Pramlintide to modulate H2O2-induced oxidative stress levels. Our data show that Pramlintide improved cognitive function, altered amyloid-processing enzymes, reduced plaque burden in the hippocampus, and regulated endogenous antioxidant enzymes (MnSOD and GPx1) and the stress marker HO-1 in a location specific manner. In vitro, Pramlintide treatment in neuronal models reduced H2O2-induced endogenous ROS production and lipid peroxidation in a dose-dependent manner. Together, these results indicate that Pramlintide's benefits on cognitive function and pathology may involve antioxidant-like properties of this compound.

MDMA is one of the most used drugs by adolescents and its consumption has been associated with many psychobiological problems, among them psychomotor problems. Moreover, some authors described that early exposure to MDMA may render the dopaminergic neurons more vulnerable to the effects of future neurotoxic insults. Alzheimer disease (AD) is the main cause of dementia in the elderly and a percentage of the patients have predisposition to suffer nigrostriatal alterations, developing extrapyramidal signs. Nigrostriatal dysfunction in the brain of aged APPswe/PS1dE9 (APP/PS1), a mouse model of familiar AD (FAD), has also been described. The aim of the present study was to investigate the consequences of adolescent exposure to MDMA in APP/PS1 mice, on nigrostriatal function on early adulthood. We used a MDMA schedule simulating weekend binge abuse of this substance. Our MDMA schedule produced a genotype-independent decrease in dopaminergic neurons in the substantia nigra that remained at least 3months. Shortly after the injury, wild-type animals showed a decrease in the locomotor activity and apparent DA depletion in striatum, however in the APP/PS1 mice neither the locomotor activity nor the DA levels were modified, but a reduction in dopamine transporter (DAT) expression and a higher levels of oxidative stress were observed. We found that these disturbances are age-related characteristics that this APP/PS1 mice develops spontaneously much later. Therefore, MDMA administration seems to anticipate the striatal dopaminergic dysfunction in this FAD model. The most important outcome lies in a potentiation, by MDMA, of the amyloid beta deposition in the striatum.

Addictive drugs produce forms of structural plasticity in the nucleus accumbens and prefrontal cortex. The aim of this study was to investigate the impact of chronic MDMA exposure on pyramidal neurons in the CA1 region of hippocampus and drug-related spatial learning and memory changes.Adolescent rats were exposed to saline or MDMA in a regime that mimicked chronic administration. One week later, when acquisition or reference memory was evaluated in a standard Morris water maze (MWM), no differences were obtained between groups. However, MDMA-exposed animals performed better when the MWM was implemented under more difficult conditions. Animals of MDMA group were less anxious and were more prepared to take risks, as in the open field test they ventured more frequently into the central area. We have demonstrated that MDMA caused an increase in brain-derived neurotrophic factor (BDNF) expression. When spine density was evaluated, MDMA-treated rats presented a reduced density when compared with saline, but overall, training increased the total number of spines, concluding that in MDMA-group, training prevented a reduction in spine density or induced its recovery.This study provides support for the conclusion that binge administration of MDMA, known to be associated to neurotoxic damage of hippocampal serotonergic terminals, increases BDNF expression and stimulates synaptic plasticity when associated with training. In these conditions, adolescent rats perform better in a more difficult water maze task under restricted conditions of learning and memory. The effect on this task could be modulated by other behavioural changes provoked by MDMA.

The senescence-accelerated mouse (SAM) is a useful animal model to study aging or age-associated disorder. In the present study, we have used a multidisciplinary approach to the characterization of changes that occur in aging and in the modelling of brain aging. The SAMP8 mouse at 5 months of age exhibited an increase in gliosis and molecular oxidative damage. Likewise, we found that superoxide dismutase activity decreased compared with age-matched SAMR1 while there were no differences in activity of catalase and glutathione reductase. These results indicate that the decrease of superoxide dismutase may be involved in the increase of oxidative stress in brain of SAMP8 at younger stages. This suggestion is supported by an increase in the expression of alpha-synuclein together with phosphorylated tau protein, which is concurrent with the decline of that antioxidant enzyme. Alpha-synuclein aggregates are invariably associated with tau pathologies and our results demonstrate that alpha-synuclein accumulation is a potent inducer of tau pathologies not only in neurodegenerative diseases but also in normal aging. These results also imply that SAMP8 are exposed to elevated levels of oxidative stress from an early age, and that could be a very important cause of the senescence-related impairments and degeneration in the brain seen in this strain.

The pro-oxidative effect of methamphetamine (METH) in dopamine terminals was studied in rat striatal synaptosomes. Flow cytometry analysis showed increased production of reactive oxygen species (ROS) in METH-treated synaptosomes, without reduction in the density of dopamine transporters. In synaptosomes from dopamine (DA)-depleted animals, METH did not induce ROS production. Reserpine, in vitro, completely inhibited METH-induced ROS production. These results point to endogenous DA as the main source of ROS induced by METH. Antioxidants and inhibitors of neuronal nitric oxide synthase and protein kinase C (PKC) prevented the METH-induced oxidative effect. EGTA and the specific antagonist methyllycaconitine (MLA, 50 microM) prevented METH-induced ROS production, thus implicating calcium and alpha7 nicotinic receptors in such effect. Higher concentrations of MLA (>100 microM) showed nonspecific antioxidant effect. Preincubation of synaptosomes with METH (1 microM) for 30 min reduced [(3)H]DA uptake by 0%. The METH effect was attenuated by MLA and EGTA and potentiated by nicotine, indicating that activation of alpha(7) nicotinic receptors and Ca(2+) entry are necessary and take place before DAT inhibition. From these findings, it can be postulated that, in our model, METH induces DA release from synaptic vesicles to the cytosol. Simultaneously, METH activates alpha(7) nicotinic receptors, probably inducing depolarization and an increase in intrasynaptosomal Ca(2+). This would lead to DAT inhibition and NOS and PKC activation, initiating oxidation of cytosolic DA.

The consequences of the neurotoxic insult induced by 3,4-methylenedioxymethamphetamine (MDMA, an amphetamine derivative with specific action on the serotonergic system) were compared with those of methamphetamine (a derivative with specific action on dopaminergic system) in rats. Both drugs induced a very similar loss of body weight, especially evident 24 h after treatment. Their hyperthermic profile was also very similar and was dependent on ambient temperature, corroborating the thermo-dysregulatory effect of both substances. Methamphetamine (four injections of 10 mg kg(-1) s.c. at 2-h intervals) induced the loss of dopaminergic (35%) but not of serotonergic, terminals in the rat striatum and, simultaneously, a significant increase in striatal peripheral-type benzodiazepine receptor density, pointing to a glial reaction. Evidence for this drug-induced astrogliosis was the increased heat shock protein 27 (HSP27) expression in striatum, cortex and hippocampus. MDMA (20 mg kg(-1) s.c. b.i.d. for 4 days) induced a similar dopaminergic lesion in the striatum 3 days post-treatment, which reversed 4 days later. An important neurotoxic effect on serotonergic terminals was also observed in the cortex, striatum and hippocampus 3 days post-treatment, which partially reversed 4 days later in the striatum and hippocampus. No microglial activation was noticeable at either 3 or 7 days after MDMA treatment. This lack of effect on microglial cells was assessed by [(3)H]PK 11195 binding and OX-6 immunostaining, which were unchanged in the striatum and cortex after MDMA treatment. A non-significant tendency to increase was noted in the hippocampus 3 days after MDMA treatment. Furthermore, in MDMA-treated rats, neither HSP27 expression nor an increase in HSP27 immunoreactivity were detected. This result, together with the lack of increase in glial fibrilliary acidic protein (GFAP) immunoreactivity, indicate no astroglial activation at either 3 or 7 days post-treatment. Without microglial activation, an inflammatory process would not accompany the lesion induced by MDMA. The differences in glial activation between methamphetamine and MDMA observed in the present study could have implications for the prognosis of the injury induced by these drugs.

The anticholinergic drug orphenadrine is used in the treatment of Parkinson's disease. In this study we evaluate the neuroprotective effects of orphenadrine on excitotoxicity in vivo and in vitro. Orphenadrine prevented the mitochondrial and the cytoplasmic membrane potential decrease evoked by NMDA (100 microM) in rat dissociated cerebellar granule cells showing an IC50 value of 11.6 +/- 4.7 microM (mean +/- SEM, n = 5) and 13.5 +/- 2.3 microM (n = 3), respectively. Orphenadrine was able to protect cerebellar granule cell cultures from glutamate-induced neurotoxicity. Kainic acid (KA, 10 mg/kg)-induced excitotoxicity was evaluated in vivo using the microglial marker peripheral-type benzodiazepine receptor (PBR) and heat shock protein 72 (HSP72) expression in the hippocampus. The Bmax of PBR for control tissues was 589.1 +/- 40.0 fmol/mg protein (n = 4), increasing to 1692.5 +/- 51.6 fmol/mg protein (n = 5) after the KA treatment. Pretreatment with orphenadrine (10 mg/kg) blocked the KA-induced increase in PBR density. As expected, KA-administration induced the expression of HSP72 that was blocked in the orphenadrine + KA-treated rats. We demonstrate that orphenadrine, interacting at the NMDA receptor, is able to prevent the neurotoxicity mediated by activation at glutamate ionotropic receptors.

Previous work from our laboratory has demonstrated the presence of high-affinity binding sites for [3H]nitrobenzylthioinosine ([3H]NBTI), a marker of adenosine uptake systems, in the mitochondrial fraction of rat testis. Here, we characterize this system functionally through [3H]adenosine uptake assays. This system (K(m)=2+/-1.3 microM; V(max)=86.2+/-15.5 pmol/mg protein/min) was found to be saturable, non sodium-dependent and sensitive to temperature, pH and osmolarity. [3H]Adenosine incorporation was potently inhibited by hydroxynitrobenzylthioguanosine (HNBTG, IC(50)=3 nM) although NBTI inhibited this uptake weakly (IC(50)=72. 7+/-37.1 microM). Dilazep>dipyridamole>/=hexobendine inhibited [3H]adenosine incorporation at low micromolar concentrations. The nucleosides inosine and uridine were weak inhibitors of this system. The adenosine receptor ligands N(6)-phenylisopropyladenosine (PIA) and 2-chloroadenosine inhibited the uptake only at micromolar concentrations. Neither 5'-(N-ethylcarboxamido)-adenosine (NECA) nor theophylline inhibited adenosine uptake by more than 60% but the mitochodrial benzodiazepine receptor ligands 4'-chloro-diazepam (Ro 5-4864) and 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl) isoquinoline carboxamide (PK 11195) were able to inhibit it. The lack of inhibition by the blockers of the mitochondrial adenine-nucleotide carrier, atractyloside and alpha, beta-methylene-ATP, indicates that [3H]adenosine uptake occurs via a transporter other than this carrier. All these results support the existence of an equilibrative adenosine transport system, which might mediate the passage of adenosine formed in the mitochondria to the cytoplasm.

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.

The effect of kainic acid (KA) on mitochondrial membrane potential (MMP) and reactive-oxygen species (ROS) production was studied in dissociated cerebellar granule cells from rat pups. KA induced a maximum increase of 361%+/-35% in ROS production. The lazaroid compound U-83836E (at concentrations ranging from 10(-9) to 5x10(-6) M) completely inhibited this increase, with an IC50 value of 3.02+/-1.08x10(-7) M. KA also decreased the mitochondrial membrane potential (MMP), with a maximum decrease of about 30%. Absence of Na+ in the incubation medium did not significantly alter the effect of KA on MMP. As expected, the AMPA/kainate receptor antagonist NBQX inhibited the effects of KA on MMP with an IC50 value of 1.1+/-0.8 microM. However, the lazaroid U-83836E, indomethacin, nor-dihydroguaiaretic acid and L-nitroarginine all failed to inhibit the KA-induced decrease in the MMP. Finally, to assess the neuroprotective effect of U-83836E on KA-induced neurotoxicity in vivo, the increase in the peripheral-type benzodiazepine receptor density in rat hippocampus was measured. Treatment with KA increased the Bmax to 1341+/-192 fmol mg(-1). When U-83836E was coadministered with KA, the Bmax was reduced to 765+/-122 fmol mg(-1), which was not significantly different from the Bmax obtained from untreated rats (Bmax: 518+/-33 fmol mg(-1)). We conclude that treatment with the lazaroid U-83836E might be a suitable therapeutic strategy in neurodegenerative disorders.

Sigma (sigma) and phencyclidine (PCP) receptor ligands, apart from their main effects on sigma receptors and NMDA receptor-mediated neurotransmission, have been found to interact with catecholamine systems in several central and peripheral tissues. In the present study the binding profile of [3H]nisoxetine ([3H]NIS), a selective marker of the noradrenaline transporter, has been characterized in rat vas deferens membranes to further study its modulation by a number of characteristic sigma and PCP ligands. The binding of [3H]NIS was found to be of high affinity (Kd = 1.63 +/- 0.36 nM), saturable, sodium-dependent and to a single population of binding sites (nH = 1.003 +/- 0.017). The maximal binding capacity was 1,625 +/- 500 fmol/mg of protein. Kinetic experiments gave a k(+1) of 3.9 x 10(7) min(-1)M(-1) and a k(-1) of 0.005 min(-1). The [3H]NIS binding was totally inhibited, with IC50 values in the micromolar range, by all the sigma and PCP ligands tested, with the following order of potency: haloperidoldextromethorphandizocilpinedextrorphan(+)-3-PPPPCPtenocyclidine. This order correlates well with that described in other tissues using [3H]desmethylimipramine. The inhibition by all these compounds, except that of (+)-3-PPP, was competitive. These results suggest that sigma and PCP ligands bind, at low micromolar concentrations, to a site in the noradrenaline transporter that is labelled by [3H]NIS.

Kainic acid (KA) causes seizures and neuronal loss in the hippocampus. The present study investigated whether a recreational schedule of 3,4-methylenedioxymethamphetamine (MDMA) favours the development of a seizure state in a model of KA-induced epilepsy and potentiates the toxicity profile of KA (20 or 30 mg/kg). Adolescent male C57BL/6 mice received saline or MDMA t.i.d. (s.c. every 3 h), on 1 day a week, for 4 consecutive weeks. Twenty-four hours after the last MDMA exposure, the animals were injected with saline or KA (20 or 30 mg/kg). After this injection, we evaluated seizures, hippocampal neuronal cell death, microgliosis, astrogliosis, and calcium binding proteins. MDMA pretreatment, by itself, did not induce neuronal damage but increased seizure susceptibility in all KA treatments and potentiated the presence of Fluoro-Jade-positive cells in CA1. Furthermore, MDMA, like KA, significantly decreased parvalbumin levels in CA1 and dentate gyrus, where it potentiated the effects of KA. The amphetamine derivative also promoted a transient decrease in calbindin and calretinin levels, indicative of an abnormal neuronal discharge. In addition, treatment of cortical neurons with MDMA (1050 μM) for 6 or 48 h significantly increased basal Ca2 +, reduced basal Na+ levels and potentiated kainate response. These results indicate that MDMA potentiates KA-induced neurodegeneration and also increases KA seizure susceptibility. The mechanism proposed includes changes in Calcium Binding Proteins expression, probably due to the disruption of intracellular ionic homeostasis, or/and an indirect effect through glutamate release.

Inflammation and oxidative stress (OS) are key points in age progression. Both processes impact negatively in cognition and in brain functions. Resveratrol (RV) has been postulated as a potent antioxidant natural compound, with rejuvenating properties. Inducing a metabolic stress by high-fat (HF) diet in aged C56/BL6 (24 months) led to cognitive disturbances compared with control age mated and with young mice. These changes were prevented by RV. Molecular determinations demonstrated a significant increase in some inflammatory parameters (TNF-α, Cxcl10, IL-1, IL-6, and Ccl3) in old mice, but slight changes in OS machinery. RV mainly induced the recovery of the metabolically stressed animals. The study of key markers involved in senescence and rejuvenation (mitochondrial biogenesis and Sirt1-AMPK-PGC1-α) demonstrated that RV is also able to modulate the changes in these cellular metabolic pathways. Moreover, changes of epigenetic marks (methylation and acetylation) that are depending on OS were demonstrated. On the whole, results showed the importance of integrative role of different cellular mechanisms in the deleterious effects of age in cognition and the beneficial role of RV. The work presented in this study showed a wide range of processes modified in old age and by metabolic stress, weighting the importance of each one and the role of RV as a possible strategy for fighting against., This study was supported by Spanish MINECO (SAF-2016- 33307 and CSD2010-00045) and the European Regional Development Fund. CG-F and M-P belong to 2014SGR 525 and CS to 014SGR625 from AGAUR, Catalonia.

1. 1. The peripheral-type benzodiazepine receptor was characterized in rat prostate and seminal vesicle using [ 3 H]Ro 5-4864 as radioligand. 2. 2. The affinity of this radioligand for this receptor was higher in rat prostate ( K D = 4.36 ± 1.02 nM) than in seminal vesicle ( K D = 8.45 ± 1.34 nM). 3. 3. The density of binding sites obtained in these two tissues was B max = 4164 ± 873 fmol/mg in prostate and 5978 ± 1022 fmol/mg in seminal vesicle. 4. 4. The [ 3 H]Ro 5-4864 binding was inhibited non-competitively by atractyloside and α,β-methyleneATP, suggesting a modulation by the ADP/ATP mitochondrial carrier. 5. 5. Flutamide was able to displace bound [ 3 H]Ro 5-4864 with an IC 50 similar to protoporphyrin IX.