Neurodegenerative diseases share many pathological pathways like abnormal protein aggregation, mi... more Neurodegenerative diseases share many pathological pathways like abnormal protein aggregation, mitochondrial dysfunction, extensive oxidative stress and neuroinflammation. Cells have an intrinsic mechanism of protection, the Nrf2 transcriptional factor, known as the master regulator of redox homeostasis. Based on the common features of these diseases we have designed a multi-target hybrid structure derived from melatonin and ethyl cinnamate. The obtained derivatives were Nrf2 inducers and potent-free radical scavengers. These new compounds showed a very interesting neuroprotective profile in several in vitro models of oxidative stress, Alzheimer's disease and brain ischemia. We have designed a new hybrid structure with complementary activities. We have identified compound 5h as an interesting Nrf2 inducer, very potent antioxidant and neuroprotectant.
CERTIFICAN, que Don FRANCISCO JAVIER EGEA MÁIQUEZ ha realizado bajo su dirección el presente trab... more CERTIFICAN, que Don FRANCISCO JAVIER EGEA MÁIQUEZ ha realizado bajo su dirección el presente trabajo: "Rutas de señalización celular activadas por receptores nicotínicos y su implicación en la neuroprotección y el dolor", como Tesis para alcanzar el grado de Doctor por la Universidad Autónoma de Madrid.
The mutated form of the Ca(2+) channel CALHM1 (Ca(2+) homeostasis modulator 1), P86L-CALHM1, has ... more The mutated form of the Ca(2+) channel CALHM1 (Ca(2+) homeostasis modulator 1), P86L-CALHM1, has been correlated with early onset of Alzheimer's disease (AD). P86L-CALHM1 increases production of amyloid beta (Aβ) upon extracellular Ca(2+) removal and its subsequent addback. The aim of this study was to investigate the effect of the overexpression of CALHM1 and P86L-CALHM, upon Aβ treatment, on the following: (i) the intracellular Ca(2+) signal pathway; (ii) cell survival proteins ERK1/2 and Ca(2+) /cAMP response element binding (CREB); and (iii) cell vulnerability after treatment with Aβ. Using aequorins to measure the effect of nuclear Ca(2+) concentrations ([Ca(2+) ]n ) and cytosolic Ca(2+) concentrations ([Ca(2+) ]c ) on Ca(2+) entry conditions, we observed that baseline [Ca(2+) ]n was higher in CALHM1 and P86L-CALHM1 cells than in control cells. Moreover, exposure to Aβ affected [Ca(2+) ]c levels in HeLa cells overexpressing CALHM1 and P86L-CALHM1 compared with control cells...
Nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout the central nervous ... more Nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout the central nervous system, being expressed in neurons and non-neuronal cells, where they participate in a variety of physiological responses like memory, learning, locomotion, attention, among others. We will focus on the α7 nAChR subtype, which has been implicated in neuroprotection, synaptic plasticity and neuronal survival, and is considered as a potential therapeutic target for several neurological diseases. Oxidative stress and neuroinflammation are currently considered as two of the most important pathological mechanisms common in neurodegenerative diseases such as Alzheimer, Parkinson or Huntington diseases. In this review, we will first analysed the distribution and expression of nAChR in mammalian brain. Then, we focused on the function of the α7 nAChR subtype in neuronal and non-neuronal cells and its role in immune responses (cholinergic anti-inflammatory pathway). Finally, we will revise the anti-inflammatory pathway promoted via α7 nAChR activation that is related to recruitment and activation of Jak2/STAT3 pathway, which on the one hand inhibits NF-κB nuclear translocation, and on the other hand, activates the master regulator of oxidative stress Nrf2/HO-1. This review provides a profound insight into the role of the α7 nAChR subtype in microglia and point out to microglial α7/HO-1 pathway as an anti-inflammatory therapeutic target.
Subchronic oxidative stress and inflammation are being increasingly implicated in the pathogenesi... more Subchronic oxidative stress and inflammation are being increasingly implicated in the pathogenesis of numerous diseases, such as Alzheimeŕs or Parkinsońs disease. This study was designed to evaluate the potential protective role of α7 nicotinic receptor activation in an in vitro model of neurodegeneration based on subchronic oxidative stress. Rat organotypic hippocampal cultures (OHCs) were exposed for 4 days to low concentration of lipopolysaccharide (LPS) and the complex III mitochondrial blocker, antimycin-A. Antimycin-A (0.1μM) and lipopolysaccharide (1 ng/ml) caused low neurotoxicity on their own, measured as propidium iodide fluorescence in CA1 and CA3 regions. However, their combination (LPS/AA) caused a greater detrimental effect, in addition to mitochondrial depolarization, overproduction of reactive oxygen species (ROS) and Nox4 overexpression. Antimycin-A per se increased ROS and mitochondrial depolarization, although these effects were significantly higher when combined ...
Stopping the ischemic cascade by targeting its components is a potential strategy for acute ische... more Stopping the ischemic cascade by targeting its components is a potential strategy for acute ischemic stroke treatment. During ischemia and especially over reperfusion, oxidative stress plays a major role in causing neuronal cell death. Melatonin has been previously reported to provide neuroprotective effects in in vivo models of stroke by a mechanism that implicates melatonin receptors. In this context, this study was planned to test the potential neuroprotective effects of the novel melatonin MT1/MT2 receptor agonist, Neu-P11, against brain ischemia in in vitro and in vivo models, and to elucidate its underlying mechanism of action. Neu-P11 proved to be a good antioxidant, to protect against glutamate-induced excitotoxicity and oxygen and glucose deprivation in hippocampal slices, and to reduce infarct volume in an in vivo stroke model. Regarding its mechanism of action, the protective effect of Neu-P11 was reverted by luzindole (melatonin receptor antagonist), AG490 (JAK2 inhibito...
Melatonin is a neurohormone whose levels are significantly reduced or absent in Alzheimer's disea... more Melatonin is a neurohormone whose levels are significantly reduced or absent in Alzheimer's disease (AD) patients. In these patients, acetylcholinesterase inhibitors (AChEI) are the major drug class used for their treatment; however, they present unwanted cholinergic side effects and have provided limited efficacy in clinic. Because combination therapy is being extensively used to treat different pathological diseases such as cancer or acquired immune deficiency syndrome, we posed this study to evaluate if melatonin in combination with an AChEI, galantamine, could provide beneficial properties in a novel in vitro model of AD. Thus, we subjected organotypic hippocampal cultures (OHCs) to subtoxic concentrations of βamyloid (0.5 μM βA) plus okadaic acid (1 nM OA), for 4 days. This treatment increased by 95 % cell death, which was mainly apoptotic as shown by positive TUNEL staining. In addition, the combination of βA/OA increased Thioflavin S aggregates, hyperphosphorylation of Tau, oxidative stress (increased DCFDA fluorescence), and neuroinflammation (increased IL-1β and TNFα). Under these experimental conditions, melatonin (1-1000 nM) and galantamine (10-1000 nM), coincubated with the toxic stimuli, caused a concentrationdependent neuroprotection; maximal neuroprotective effect was achieved at 1 μM of melatonin and galantamine. Most effective was the finding that combination of sub-effective concentrations of melatonin (1 nM) and galantamine (10 nM) provided a synergic anti-apoptotic effect and reduction of most of the AD-related pathological hallmarks observed in the βA/OA model. Therefore, we suggest that supplementation of melatonin in combination with lower doses of AChEIs could be an interesting strategy for AD patients.
We have studied if curcumin can protect glial cells under an oxidative stress and inflammatory en... more We have studied if curcumin can protect glial cells under an oxidative stress and inflammatory environment which is known to be deleterious in neurodegeneration. Primary rat glial cultures exposed to the combination of an oxidative (rotenone/oligomycin A) and a pro-inflammatory LPS stimuli (RO/LPS) reduced by 50% glial viability. Under these experimental conditions, curcumin afforded significant glial protection and reduction of reactive oxygen species; these effects were blocked by the HO-1 inhibitor tin protoporphyrin-IX (SnPP). These findings correlate with the observation that curcumin induced the antioxidative protein HO-1. Most interesting was the observation that the glial protective effects related to HO-1 induction were microglial specific as shown in glial cultures from LysM(Cre) Hmox(∆/∆) mice where curcumin lost its protective effect. Under LPS conditions, curcumin reduced the microglial pro-inflammatory markers iNOS and TNF, but increased the anti-inflammatory cytokine IL4. Analysis of the microglial phenotype showed that curcumin favored a ramified morphology towards a microglial alternative activated state against LPS insult also by a HO-1 dependent mechanism. The curry constituent curcumin protects glial cells and promotes a microglial anti-inflammatory phenotype by a mechanism that implicates HO-1 induction; these effects may have impact in brain protection under oxidative and inflammatory conditions. This article is protected by copyright. All rights reserved.
Agmatine, an endogenous neuromodulator, is a potential candidate to constitute an adjuvant/monoth... more Agmatine, an endogenous neuromodulator, is a potential candidate to constitute an adjuvant/monotherapy for the management of depression. A recent study by our group demonstrated that agmatine induces Nrf2 and protects against corticosterone effects in a hippocampal neuronal cell line. The present study is an extension of this previous study by assessing the antidepressant-like effect of agmatine in an animal model of depression induced by corticosterone in mice. Swiss mice were treated simultaneously with agmatine or imipramine at a dose of 0.1 mg/kg/day (p.o.) and corticosterone for 21 days and the daily administrations of experimental drugs were given immediately prior to corticosterone (20 mg/kg/day, p.o.) administrations. Wild-type C57BL/6 mice (Nrf2 (+/+)) and Nrf2 KO (Nrf2 (−/−)) were treated during 21 days with agmatine (0.1 mg/kg/day, p.o.) or vehicle. Twenty-four hours after the last treatments, the behavioral tests and biochemical assays were performed. Agmatine treatment for 21 days was able to abolish the corticosterone-induced depressive-like behavior and the alterations in the immunocontent of mature BDNF and synaptotagmin I, and in the serotonin and glutamate levels. Agmatine also abolished the corticosterone-induced changes in the morphology of astrocytes and microglia in CA1 region of hippocampus. In addition, agmatine treatment in control mice increased noradrenaline, serotonin, and dopamine levels, CREB phosphorylation, mature BDNF and synaptotagmin I immunocontents, and reduced pro-BDNF immunocontent in the hippocampus. Agmatine's ability to produce an antidepressant-like effect was abolished in Nrf2 (−/−) mice. The present results reinforce the participation of Nrf2 in the antidepressant-like effect produced by agmatine and expand literature data concerning its mechanisms of action.
Creatine is the substrate for creatine kinase in the synthesis of phosphocreatine (PCr). This ene... more Creatine is the substrate for creatine kinase in the synthesis of phosphocreatine (PCr). This energetic system is endowed of antioxidant and neuroprotective properties and plays a pivotal role in brain energy homeostasis. The purpose of this study was to investigate the neuroprotective effect of creatine and PCr against 6-hydroxydopamine (6-OHDA)-induced mitochondrial dysfunction and cell death in rat striatal slices, used as an in vitro Parkinson's model. The possible involvement of the signaling pathway mediated by phosphatidylinositol-3 kinase (PI3K), protein kinase B (Akt), and glycogen synthase kinase-3b (GSK3b) was also evaluated. Exposure of striatal slices to 6-OHDA caused a significant disruption of the cellular homeostasis measured as 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction, lactate dehydrogenase release, and tyrosine hydroxylase levels. 6-OHDA exposure increased the levels of reactive oxygen species and thiobarbituric acid reactive substances production and decreased mitochondrial membrane potential in rat striatal slices. Furthermore, 6-OHDA decreased the phosphorylation of Akt (Serine 473 ) and GSK3b (Serine 9 ). Coincubation with 6-OHDA and creatine or PCr reduced the effects of 6-OHDA toxicity. The protective effect afforded by creatine or PCr against 6-OHDA-induced toxicity was reversed by the PI3K inhibitor LY294002. In conclusion, creatine and PCr minimize oxidative stress in striatum to afford neuroprotection of dopaminergic neurons.
Hyperactivation of the hypothalamic-pituitaryadrenal axis is a common finding in major depression... more Hyperactivation of the hypothalamic-pituitaryadrenal axis is a common finding in major depression; this may lead to increased levels of cortisol, which are known to cause oxidative stress imbalance and apoptotic neuronal cell death, particularly in the hippocampus, a key region implicated in mood regulation. Agmatine, an endogenous metabolite of L-arginine, has been proposed for the treatment of major depression. Corticosterone induced apoptotic cell death and increased ROS production in cultured hippocampal neuronal cells, effects that were abolished in a concentration-and timedependent manner by agmatine. Interestingly, the combination of sub-effective concentrations of agmatine with fluoxetine or imipramine afforded synergic protection. The neuroprotective effect of agmatine was abolished by yohimbine (α 2 -adrenoceptor antagonist), ketanserin (5-HT 2A receptor antagonist), LY294002 (PI3K inhibitor), PD98059 (MEK1/2 inhibitor), SnPP (HO-1 inhibitor), and cycloheximide (protein synthesis inhibitor). Agmatine increased Akt and ERK phosphorylation and induced the transcription factor Nrf2 and the proteins HO-1 and GCLc; induction of these proteins was prevented by yohimbine, ketanserin, LY294002, and PD98059. In conclusion, agmatine affords neuroprotection against corticosterone effects by a mechanism that implicates Nrf2 induction via α 2 -adrenergic and 5-HT 2A receptors, Akt and ERK pathways, and HO-1 and GCLc expression.
Positive allosteric modulation of a7 isoform of nicotinic acetylcholine receptors (a7-nAChRs) is ... more Positive allosteric modulation of a7 isoform of nicotinic acetylcholine receptors (a7-nAChRs) is emerging as a promising therapeutic approach for central nervous system disorders such as schizophrenia or Alzheimer's disease. However, its effect on Ca 2+ signaling and cell viability remains controversial. This study focuses on how the type II positive allosteric modulator (PAM II) PNU120596 affects intracellular Ca 2+ signaling and cell viability. We used human SH-SY5Y neuroblastoma cells overexpressing a7-nAChRs (a7-SH) and their control (C-SH). We monitored cytoplasmic and endoplasmic reticulum (ER) Ca 2+ with Fura-2 and the genetically encoded cameleon targeting the ER, respectively. Nicotinic inward currents were measured using patch-clamp techniques. Viability was assessed using methylthiazolyl blue tetrazolium bromide or propidium iodide staining. We observed that in the presence of a nicotinic agonist, PNU120596 (i) reduced viability of a7-SH but not of C-SH cells; (ii) significantly increased inward nicotinic currents and cytosolic Ca 2+ concentration; (iii) released Ca 2+ from the ER by a Ca 2+ -induced Ca 2+ release mechanism only in a7-SH cells; (iv) was cytotoxic in rat organotypic hippocampal slice cultures; and, lastly, all these effects were prevented by selective blockade of a7-nAChRs, ryanodine receptors, or IP 3 receptors. In conclusion, positive allosteric modulation of a7-nAChRs with the PAM II PNU120596 can lead to dysregulation of ER Ca 2+ , overloading of intracellular Ca 2+ , and neuronal cell death.
Neurodegenerative diseases are a major problem afflicting aging populations; however, there are n... more Neurodegenerative diseases are a major problem afflicting aging populations; however, there are no effective treatments to stop their progression. Oxidative stress and neuroinflammation are common factors in their pathogenesis. Nrf2 is the master regulator to control oxidative stress and melatonin is an endogenous hormone with antioxidative properties that reduces its levels with aging. We have designed a new compound that combines melatonin with Nrf2 induction properties with the idea of achieving improved neuroprotective properties. Compound ITH12674 is a hybrid of melatonin and sulforaphane designed to exert a dual drug-prodrug mechanism of action. We have obtained the proposed hybrid in a single step; to test its neuroprotective properties we have used different in vitro models of oxidative stress related to neurodegenerative diseases and brain ischemia. ITH12674 shows an interesting neuroprotective profile, improving that of melatonin and sulforaphane; ITH12674 exhibits (i) con...
Benzothiazepine CGP37157 is widely used as tool to explore the role of mitochondria in cell Ca 2+... more Benzothiazepine CGP37157 is widely used as tool to explore the role of mitochondria in cell Ca 2+ handling, by its blocking effect of the mitochondria Na + /Ca 2+ exchanger. Recently, CGP37157 has shown to exhibit neuroprotective properties. In the trend to improve its neuroprotection profile, we have synthesized ITH12505, an isosteric analogue having a methyl instead of chlorine at C2′ of the phenyl ring. ITH12505 has exerted neuroprotective properties similar to CGP37157 in chromaffin cells and hippocampal slices stressed with veratridine. Also, both compounds afforded neuroprotection in hippocampal slices stressed with glutamate. However, while ITH12505 elicited protection in SH-SY5Y cells stressed with oligomycin A/rotenone, CGP37157 was ineffective. In hippocampal slices subjected to oxygen/glucose deprivation plus reoxygenation, ITH12505 offered protection at 3−30 μM, while CGP37157 only protected at 30 μM. Both compounds caused blockade of Ca 2+ channels in high K + -depolarized SH-SY5Y cells. An in vitro experiment for assaying central nervous system penetration (PAMPA-BBB; parallel artificial membrane permeability assay for blood-brain barrier) revealed that both compounds could cross the blood−brain barrier, thus reaching their biological targets in the central nervous system. In conclusion, by causing a mild isosteric replacement in the benzothiazepine CGP37157, we have obtained ITH12505, with improved neuroprotective properties. These findings may inspire the design and synthesis of new benzothiazepines targeting mitochondrial Na + /Ca 2+ exchanger and L-type voltage-dependent Ca 2+ channels, having antioxidant properties.
We have investigated the protective effects of ITH91/IQM157, a hybrid of 13 melatonin and N,N-dib... more We have investigated the protective effects of ITH91/IQM157, a hybrid of 13 melatonin and N,N-dibenzyl(N-methyl)amine, in an in vitro model of Alzheimer's disease 14 (AD)-like pathology that combines amyloid beta (Aβ) and tau hyperphosphorylation 15 induced by okadaic acid (OA), in the human neuroblastoma cell line SH-SY5Y. 16 Combination of subtoxic concentrations of Aβ and OA caused a significant toxicity of 17 40% cell death, which mainly was apoptotic; this effect was accompanied by retraction of 18 the cells' prolongations and accumulation of thioflavin-S stained protein aggregates. In 19 this toxicity model, ITH91/IQM157 (1−1000 nM) reduced cell death measured as MTT 20 reduction; at 100 nM, it prevented apoptosis, retraction of prolongations, and Aβ 21 aggregates. The protective actions of ITH91/IQM157 were blocked by mecamylamine, 22 luzindol, chelerythrine, PD98059, LY294002, and SnPP. We show that the combination of melatonin with a fragment endowed 23 with AChE inhibition in a unique chemical structure, ITH91/IQM157, can reduce neuronal cell death induced by Aβ and OA by 24 a signaling pathway that implicates both nicotinic and melatonin receptors, PKC, Akt, ERK1/2, and induction of hemoxygenase-25
The guanidine-like compound creatine exerts bioenergetic, antiexcitotoxic, antioxidant and neurop... more The guanidine-like compound creatine exerts bioenergetic, antiexcitotoxic, antioxidant and neuroprotective properties; however, the intracellular mechanisms responsible for these effects are still not well established. The purpose of this study was to investigate the protective effect of creatine against 6-hydroxydopamine (6-OHDA)-induced cell death in neuroblastoma SH-SY5Y cells and the possible intracellular signaling pathways involved in such effect.
Neurodegenerative diseases share many pathological pathways like abnormal protein aggregation, mi... more Neurodegenerative diseases share many pathological pathways like abnormal protein aggregation, mitochondrial dysfunction, extensive oxidative stress and neuroinflammation. Cells have an intrinsic mechanism of protection, the Nrf2 transcriptional factor, known as the master regulator of redox homeostasis. Based on the common features of these diseases we have designed a multi-target hybrid structure derived from melatonin and ethyl cinnamate. The obtained derivatives were Nrf2 inducers and potent-free radical scavengers. These new compounds showed a very interesting neuroprotective profile in several in vitro models of oxidative stress, Alzheimer's disease and brain ischemia. We have designed a new hybrid structure with complementary activities. We have identified compound 5h as an interesting Nrf2 inducer, very potent antioxidant and neuroprotectant.
CERTIFICAN, que Don FRANCISCO JAVIER EGEA MÁIQUEZ ha realizado bajo su dirección el presente trab... more CERTIFICAN, que Don FRANCISCO JAVIER EGEA MÁIQUEZ ha realizado bajo su dirección el presente trabajo: "Rutas de señalización celular activadas por receptores nicotínicos y su implicación en la neuroprotección y el dolor", como Tesis para alcanzar el grado de Doctor por la Universidad Autónoma de Madrid.
The mutated form of the Ca(2+) channel CALHM1 (Ca(2+) homeostasis modulator 1), P86L-CALHM1, has ... more The mutated form of the Ca(2+) channel CALHM1 (Ca(2+) homeostasis modulator 1), P86L-CALHM1, has been correlated with early onset of Alzheimer's disease (AD). P86L-CALHM1 increases production of amyloid beta (Aβ) upon extracellular Ca(2+) removal and its subsequent addback. The aim of this study was to investigate the effect of the overexpression of CALHM1 and P86L-CALHM, upon Aβ treatment, on the following: (i) the intracellular Ca(2+) signal pathway; (ii) cell survival proteins ERK1/2 and Ca(2+) /cAMP response element binding (CREB); and (iii) cell vulnerability after treatment with Aβ. Using aequorins to measure the effect of nuclear Ca(2+) concentrations ([Ca(2+) ]n ) and cytosolic Ca(2+) concentrations ([Ca(2+) ]c ) on Ca(2+) entry conditions, we observed that baseline [Ca(2+) ]n was higher in CALHM1 and P86L-CALHM1 cells than in control cells. Moreover, exposure to Aβ affected [Ca(2+) ]c levels in HeLa cells overexpressing CALHM1 and P86L-CALHM1 compared with control cells...
Nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout the central nervous ... more Nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout the central nervous system, being expressed in neurons and non-neuronal cells, where they participate in a variety of physiological responses like memory, learning, locomotion, attention, among others. We will focus on the α7 nAChR subtype, which has been implicated in neuroprotection, synaptic plasticity and neuronal survival, and is considered as a potential therapeutic target for several neurological diseases. Oxidative stress and neuroinflammation are currently considered as two of the most important pathological mechanisms common in neurodegenerative diseases such as Alzheimer, Parkinson or Huntington diseases. In this review, we will first analysed the distribution and expression of nAChR in mammalian brain. Then, we focused on the function of the α7 nAChR subtype in neuronal and non-neuronal cells and its role in immune responses (cholinergic anti-inflammatory pathway). Finally, we will revise the anti-inflammatory pathway promoted via α7 nAChR activation that is related to recruitment and activation of Jak2/STAT3 pathway, which on the one hand inhibits NF-κB nuclear translocation, and on the other hand, activates the master regulator of oxidative stress Nrf2/HO-1. This review provides a profound insight into the role of the α7 nAChR subtype in microglia and point out to microglial α7/HO-1 pathway as an anti-inflammatory therapeutic target.
Subchronic oxidative stress and inflammation are being increasingly implicated in the pathogenesi... more Subchronic oxidative stress and inflammation are being increasingly implicated in the pathogenesis of numerous diseases, such as Alzheimeŕs or Parkinsońs disease. This study was designed to evaluate the potential protective role of α7 nicotinic receptor activation in an in vitro model of neurodegeneration based on subchronic oxidative stress. Rat organotypic hippocampal cultures (OHCs) were exposed for 4 days to low concentration of lipopolysaccharide (LPS) and the complex III mitochondrial blocker, antimycin-A. Antimycin-A (0.1μM) and lipopolysaccharide (1 ng/ml) caused low neurotoxicity on their own, measured as propidium iodide fluorescence in CA1 and CA3 regions. However, their combination (LPS/AA) caused a greater detrimental effect, in addition to mitochondrial depolarization, overproduction of reactive oxygen species (ROS) and Nox4 overexpression. Antimycin-A per se increased ROS and mitochondrial depolarization, although these effects were significantly higher when combined ...
Stopping the ischemic cascade by targeting its components is a potential strategy for acute ische... more Stopping the ischemic cascade by targeting its components is a potential strategy for acute ischemic stroke treatment. During ischemia and especially over reperfusion, oxidative stress plays a major role in causing neuronal cell death. Melatonin has been previously reported to provide neuroprotective effects in in vivo models of stroke by a mechanism that implicates melatonin receptors. In this context, this study was planned to test the potential neuroprotective effects of the novel melatonin MT1/MT2 receptor agonist, Neu-P11, against brain ischemia in in vitro and in vivo models, and to elucidate its underlying mechanism of action. Neu-P11 proved to be a good antioxidant, to protect against glutamate-induced excitotoxicity and oxygen and glucose deprivation in hippocampal slices, and to reduce infarct volume in an in vivo stroke model. Regarding its mechanism of action, the protective effect of Neu-P11 was reverted by luzindole (melatonin receptor antagonist), AG490 (JAK2 inhibito...
Melatonin is a neurohormone whose levels are significantly reduced or absent in Alzheimer's disea... more Melatonin is a neurohormone whose levels are significantly reduced or absent in Alzheimer's disease (AD) patients. In these patients, acetylcholinesterase inhibitors (AChEI) are the major drug class used for their treatment; however, they present unwanted cholinergic side effects and have provided limited efficacy in clinic. Because combination therapy is being extensively used to treat different pathological diseases such as cancer or acquired immune deficiency syndrome, we posed this study to evaluate if melatonin in combination with an AChEI, galantamine, could provide beneficial properties in a novel in vitro model of AD. Thus, we subjected organotypic hippocampal cultures (OHCs) to subtoxic concentrations of βamyloid (0.5 μM βA) plus okadaic acid (1 nM OA), for 4 days. This treatment increased by 95 % cell death, which was mainly apoptotic as shown by positive TUNEL staining. In addition, the combination of βA/OA increased Thioflavin S aggregates, hyperphosphorylation of Tau, oxidative stress (increased DCFDA fluorescence), and neuroinflammation (increased IL-1β and TNFα). Under these experimental conditions, melatonin (1-1000 nM) and galantamine (10-1000 nM), coincubated with the toxic stimuli, caused a concentrationdependent neuroprotection; maximal neuroprotective effect was achieved at 1 μM of melatonin and galantamine. Most effective was the finding that combination of sub-effective concentrations of melatonin (1 nM) and galantamine (10 nM) provided a synergic anti-apoptotic effect and reduction of most of the AD-related pathological hallmarks observed in the βA/OA model. Therefore, we suggest that supplementation of melatonin in combination with lower doses of AChEIs could be an interesting strategy for AD patients.
We have studied if curcumin can protect glial cells under an oxidative stress and inflammatory en... more We have studied if curcumin can protect glial cells under an oxidative stress and inflammatory environment which is known to be deleterious in neurodegeneration. Primary rat glial cultures exposed to the combination of an oxidative (rotenone/oligomycin A) and a pro-inflammatory LPS stimuli (RO/LPS) reduced by 50% glial viability. Under these experimental conditions, curcumin afforded significant glial protection and reduction of reactive oxygen species; these effects were blocked by the HO-1 inhibitor tin protoporphyrin-IX (SnPP). These findings correlate with the observation that curcumin induced the antioxidative protein HO-1. Most interesting was the observation that the glial protective effects related to HO-1 induction were microglial specific as shown in glial cultures from LysM(Cre) Hmox(∆/∆) mice where curcumin lost its protective effect. Under LPS conditions, curcumin reduced the microglial pro-inflammatory markers iNOS and TNF, but increased the anti-inflammatory cytokine IL4. Analysis of the microglial phenotype showed that curcumin favored a ramified morphology towards a microglial alternative activated state against LPS insult also by a HO-1 dependent mechanism. The curry constituent curcumin protects glial cells and promotes a microglial anti-inflammatory phenotype by a mechanism that implicates HO-1 induction; these effects may have impact in brain protection under oxidative and inflammatory conditions. This article is protected by copyright. All rights reserved.
Agmatine, an endogenous neuromodulator, is a potential candidate to constitute an adjuvant/monoth... more Agmatine, an endogenous neuromodulator, is a potential candidate to constitute an adjuvant/monotherapy for the management of depression. A recent study by our group demonstrated that agmatine induces Nrf2 and protects against corticosterone effects in a hippocampal neuronal cell line. The present study is an extension of this previous study by assessing the antidepressant-like effect of agmatine in an animal model of depression induced by corticosterone in mice. Swiss mice were treated simultaneously with agmatine or imipramine at a dose of 0.1 mg/kg/day (p.o.) and corticosterone for 21 days and the daily administrations of experimental drugs were given immediately prior to corticosterone (20 mg/kg/day, p.o.) administrations. Wild-type C57BL/6 mice (Nrf2 (+/+)) and Nrf2 KO (Nrf2 (−/−)) were treated during 21 days with agmatine (0.1 mg/kg/day, p.o.) or vehicle. Twenty-four hours after the last treatments, the behavioral tests and biochemical assays were performed. Agmatine treatment for 21 days was able to abolish the corticosterone-induced depressive-like behavior and the alterations in the immunocontent of mature BDNF and synaptotagmin I, and in the serotonin and glutamate levels. Agmatine also abolished the corticosterone-induced changes in the morphology of astrocytes and microglia in CA1 region of hippocampus. In addition, agmatine treatment in control mice increased noradrenaline, serotonin, and dopamine levels, CREB phosphorylation, mature BDNF and synaptotagmin I immunocontents, and reduced pro-BDNF immunocontent in the hippocampus. Agmatine's ability to produce an antidepressant-like effect was abolished in Nrf2 (−/−) mice. The present results reinforce the participation of Nrf2 in the antidepressant-like effect produced by agmatine and expand literature data concerning its mechanisms of action.
Creatine is the substrate for creatine kinase in the synthesis of phosphocreatine (PCr). This ene... more Creatine is the substrate for creatine kinase in the synthesis of phosphocreatine (PCr). This energetic system is endowed of antioxidant and neuroprotective properties and plays a pivotal role in brain energy homeostasis. The purpose of this study was to investigate the neuroprotective effect of creatine and PCr against 6-hydroxydopamine (6-OHDA)-induced mitochondrial dysfunction and cell death in rat striatal slices, used as an in vitro Parkinson's model. The possible involvement of the signaling pathway mediated by phosphatidylinositol-3 kinase (PI3K), protein kinase B (Akt), and glycogen synthase kinase-3b (GSK3b) was also evaluated. Exposure of striatal slices to 6-OHDA caused a significant disruption of the cellular homeostasis measured as 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction, lactate dehydrogenase release, and tyrosine hydroxylase levels. 6-OHDA exposure increased the levels of reactive oxygen species and thiobarbituric acid reactive substances production and decreased mitochondrial membrane potential in rat striatal slices. Furthermore, 6-OHDA decreased the phosphorylation of Akt (Serine 473 ) and GSK3b (Serine 9 ). Coincubation with 6-OHDA and creatine or PCr reduced the effects of 6-OHDA toxicity. The protective effect afforded by creatine or PCr against 6-OHDA-induced toxicity was reversed by the PI3K inhibitor LY294002. In conclusion, creatine and PCr minimize oxidative stress in striatum to afford neuroprotection of dopaminergic neurons.
Hyperactivation of the hypothalamic-pituitaryadrenal axis is a common finding in major depression... more Hyperactivation of the hypothalamic-pituitaryadrenal axis is a common finding in major depression; this may lead to increased levels of cortisol, which are known to cause oxidative stress imbalance and apoptotic neuronal cell death, particularly in the hippocampus, a key region implicated in mood regulation. Agmatine, an endogenous metabolite of L-arginine, has been proposed for the treatment of major depression. Corticosterone induced apoptotic cell death and increased ROS production in cultured hippocampal neuronal cells, effects that were abolished in a concentration-and timedependent manner by agmatine. Interestingly, the combination of sub-effective concentrations of agmatine with fluoxetine or imipramine afforded synergic protection. The neuroprotective effect of agmatine was abolished by yohimbine (α 2 -adrenoceptor antagonist), ketanserin (5-HT 2A receptor antagonist), LY294002 (PI3K inhibitor), PD98059 (MEK1/2 inhibitor), SnPP (HO-1 inhibitor), and cycloheximide (protein synthesis inhibitor). Agmatine increased Akt and ERK phosphorylation and induced the transcription factor Nrf2 and the proteins HO-1 and GCLc; induction of these proteins was prevented by yohimbine, ketanserin, LY294002, and PD98059. In conclusion, agmatine affords neuroprotection against corticosterone effects by a mechanism that implicates Nrf2 induction via α 2 -adrenergic and 5-HT 2A receptors, Akt and ERK pathways, and HO-1 and GCLc expression.
Positive allosteric modulation of a7 isoform of nicotinic acetylcholine receptors (a7-nAChRs) is ... more Positive allosteric modulation of a7 isoform of nicotinic acetylcholine receptors (a7-nAChRs) is emerging as a promising therapeutic approach for central nervous system disorders such as schizophrenia or Alzheimer's disease. However, its effect on Ca 2+ signaling and cell viability remains controversial. This study focuses on how the type II positive allosteric modulator (PAM II) PNU120596 affects intracellular Ca 2+ signaling and cell viability. We used human SH-SY5Y neuroblastoma cells overexpressing a7-nAChRs (a7-SH) and their control (C-SH). We monitored cytoplasmic and endoplasmic reticulum (ER) Ca 2+ with Fura-2 and the genetically encoded cameleon targeting the ER, respectively. Nicotinic inward currents were measured using patch-clamp techniques. Viability was assessed using methylthiazolyl blue tetrazolium bromide or propidium iodide staining. We observed that in the presence of a nicotinic agonist, PNU120596 (i) reduced viability of a7-SH but not of C-SH cells; (ii) significantly increased inward nicotinic currents and cytosolic Ca 2+ concentration; (iii) released Ca 2+ from the ER by a Ca 2+ -induced Ca 2+ release mechanism only in a7-SH cells; (iv) was cytotoxic in rat organotypic hippocampal slice cultures; and, lastly, all these effects were prevented by selective blockade of a7-nAChRs, ryanodine receptors, or IP 3 receptors. In conclusion, positive allosteric modulation of a7-nAChRs with the PAM II PNU120596 can lead to dysregulation of ER Ca 2+ , overloading of intracellular Ca 2+ , and neuronal cell death.
Neurodegenerative diseases are a major problem afflicting aging populations; however, there are n... more Neurodegenerative diseases are a major problem afflicting aging populations; however, there are no effective treatments to stop their progression. Oxidative stress and neuroinflammation are common factors in their pathogenesis. Nrf2 is the master regulator to control oxidative stress and melatonin is an endogenous hormone with antioxidative properties that reduces its levels with aging. We have designed a new compound that combines melatonin with Nrf2 induction properties with the idea of achieving improved neuroprotective properties. Compound ITH12674 is a hybrid of melatonin and sulforaphane designed to exert a dual drug-prodrug mechanism of action. We have obtained the proposed hybrid in a single step; to test its neuroprotective properties we have used different in vitro models of oxidative stress related to neurodegenerative diseases and brain ischemia. ITH12674 shows an interesting neuroprotective profile, improving that of melatonin and sulforaphane; ITH12674 exhibits (i) con...
Benzothiazepine CGP37157 is widely used as tool to explore the role of mitochondria in cell Ca 2+... more Benzothiazepine CGP37157 is widely used as tool to explore the role of mitochondria in cell Ca 2+ handling, by its blocking effect of the mitochondria Na + /Ca 2+ exchanger. Recently, CGP37157 has shown to exhibit neuroprotective properties. In the trend to improve its neuroprotection profile, we have synthesized ITH12505, an isosteric analogue having a methyl instead of chlorine at C2′ of the phenyl ring. ITH12505 has exerted neuroprotective properties similar to CGP37157 in chromaffin cells and hippocampal slices stressed with veratridine. Also, both compounds afforded neuroprotection in hippocampal slices stressed with glutamate. However, while ITH12505 elicited protection in SH-SY5Y cells stressed with oligomycin A/rotenone, CGP37157 was ineffective. In hippocampal slices subjected to oxygen/glucose deprivation plus reoxygenation, ITH12505 offered protection at 3−30 μM, while CGP37157 only protected at 30 μM. Both compounds caused blockade of Ca 2+ channels in high K + -depolarized SH-SY5Y cells. An in vitro experiment for assaying central nervous system penetration (PAMPA-BBB; parallel artificial membrane permeability assay for blood-brain barrier) revealed that both compounds could cross the blood−brain barrier, thus reaching their biological targets in the central nervous system. In conclusion, by causing a mild isosteric replacement in the benzothiazepine CGP37157, we have obtained ITH12505, with improved neuroprotective properties. These findings may inspire the design and synthesis of new benzothiazepines targeting mitochondrial Na + /Ca 2+ exchanger and L-type voltage-dependent Ca 2+ channels, having antioxidant properties.
We have investigated the protective effects of ITH91/IQM157, a hybrid of 13 melatonin and N,N-dib... more We have investigated the protective effects of ITH91/IQM157, a hybrid of 13 melatonin and N,N-dibenzyl(N-methyl)amine, in an in vitro model of Alzheimer's disease 14 (AD)-like pathology that combines amyloid beta (Aβ) and tau hyperphosphorylation 15 induced by okadaic acid (OA), in the human neuroblastoma cell line SH-SY5Y. 16 Combination of subtoxic concentrations of Aβ and OA caused a significant toxicity of 17 40% cell death, which mainly was apoptotic; this effect was accompanied by retraction of 18 the cells' prolongations and accumulation of thioflavin-S stained protein aggregates. In 19 this toxicity model, ITH91/IQM157 (1−1000 nM) reduced cell death measured as MTT 20 reduction; at 100 nM, it prevented apoptosis, retraction of prolongations, and Aβ 21 aggregates. The protective actions of ITH91/IQM157 were blocked by mecamylamine, 22 luzindol, chelerythrine, PD98059, LY294002, and SnPP. We show that the combination of melatonin with a fragment endowed 23 with AChE inhibition in a unique chemical structure, ITH91/IQM157, can reduce neuronal cell death induced by Aβ and OA by 24 a signaling pathway that implicates both nicotinic and melatonin receptors, PKC, Akt, ERK1/2, and induction of hemoxygenase-25
The guanidine-like compound creatine exerts bioenergetic, antiexcitotoxic, antioxidant and neurop... more The guanidine-like compound creatine exerts bioenergetic, antiexcitotoxic, antioxidant and neuroprotective properties; however, the intracellular mechanisms responsible for these effects are still not well established. The purpose of this study was to investigate the protective effect of creatine against 6-hydroxydopamine (6-OHDA)-induced cell death in neuroblastoma SH-SY5Y cells and the possible intracellular signaling pathways involved in such effect.
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Papers by Javier Egea