Inês M. Araújo

Adenosine A2A receptor antagonists exert motor and neuroprotective effects by distinct cellular mechanisms

To investigate whether the motor and neuroprotective effects of adenosine A2A receptor (A2AR) antagonists are mediated by distinct cell types in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) model of Parkinsons disease.

Nitric oxide stimulates the proliferation of neural stem cells bypassing the epidermal growth factor receptor.

Nitric oxide (NO) was described to inhibit the proliferation of neural stem cells. Some evidence suggests that NO, under certain conditions, can also promote cell proliferation, although the mechanisms responsible for a potential proliferative effect of NO in neural stem cells have remained unaddressed. In this work, we investigated and characterized the proliferative effect of NO in cell cultures obtained from the mouse subventricular zone. We found that the NO donor NOC‐18 (10 μM) increased cell proliferation, whereas higher concentrations (100 μM) inhibited cell proliferation. Increased cell proliferation was detected rapidly following exposure to NO and was prevented by blocking the mitogen‐activated kinase (MAPK) pathway, independently of the epidermal growth factor (EGF) receptor. Downstream of the EGF receptor, NO activated p21Ras and the MAPK pathway, resulting in a decrease in the nuclear presence of the cyclin‐dependent kinase inhibitor 1, p27KIP1, allowing for cell cycle progression. Furthermore, in a mouse model that shows increased proliferation of neural stem cells in the hippocampus following seizure injury, we observed that the absence of inducible nitric oxide synthase (iNOS−/− mice) prevented the increase in cell proliferation observed following seizures in wild‐type mice, showing that NO from iNOS origin is important for increased cell proliferation following a brain insult. Overall, we show that NO is able to stimulate the proliferation of neural stem cells bypassing the EGF receptor and promoting cell division. Moreover, under pathophysiological conditions in vivo, NO from iNOS origin also promotes proliferation in the hippocampus. STEM CELLS 2010;28:1219–1230

Neurotoxicity induced by antiepileptic drugs in cultured hippocampal neurons: a comparative study between carbamazepine, oxcarbazepine, and two new putative antiepileptic drugs, BIA 2-024 and BIA 2-093.

Summary:  Purpose: Newly designed antiepileptic drugs (AEDs) are being evaluated for their efficacy in preventing seizures and for their toxic profiles. We investigated and compared the toxic effects of two dibenz[b,f]azepine derivatives with anticonvulsant activity, 10,11‐dihydro‐10‐hydroxyimino‐5H‐dibenz[b,f]azepine‐5‐carboxamide (BIA2‐024) and (S)‐(‐)‐10‐acetoxy‐10,11‐dihydro‐5H‐dibenz[b,f] azepine‐5‐carboxamide (BIA2‐093), with the structurally related compounds carbamazepine (CBZ) and oxcarbazepine (OXC), both in current use for the treatment of epilepsy.

Neurotoxic/neuroprotective profile of carbamazepine, oxcarbazepine and two new putative antiepileptic drugs, BIA 2-093 and BIA 2-024

We investigated and compared the toxicity profile, as well as possible neuroprotective effects, of some antiepileptic drugs in cultured rat hippocampal neurons. We used two novel carbamazepine derivatives, (S)-(-)-10-acetoxy-10,11-dihydro-5H-dibenz[b, f]azepine-5-carboxamide (BIA 2-093) and 10, 11-dihydro-10-hydroxyimino-5H-dibenz[b,f]azepine-5-carboxamide (BIA 2-024), and compared their effects with the established compounds carbamazepine and oxcarbazepine. The assessment of neuronal injury was made by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay, as well as by analysing morphology and nuclear chromatin condensation (propidium iodide staining), after hippocampal neurons were exposed to the drugs for 24 h. The putative antiepileptic drugs, BIA 2-093 or BIA 2-024 (at 300 microM), only slightly decreased MTT reduction, whereas carbamazepine or oxcarbazepine were much more toxic at lower concentrations. Treatment with the antiepileptic drugs caused nuclear chromatin condensation in some neurons, which is characteristic of apoptosis, and increased the activity of caspase-3-like enzymes, mainly in neurons treated with carbamazepine and oxcarbazepine. The toxic effect caused by carbamazepine was not mediated by N-methyl-D-aspartate (NMDA) or by alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptors. Moreover, the antiepileptic drugs failed to protect hippocampal neurons from the toxicity caused by kainate, veratridine, or ischaemia-like conditions.

Specificity in S-Nitrosylation: A Short-Range Mechanism for NO Signaling?

SIGNIFICANCE Nitric oxide (NO) classical and less classical signaling mechanisms (through interaction with soluble guanylate cyclase and cytochrome c oxidase, respectively) operate through direct binding of NO to protein metal centers, and rely on diffusibility of the NO molecule. S-Nitrosylation, a covalent post-translational modification of protein cysteines, has emerged as a paradigm of nonclassical NO signaling. RECENT ADVANCES Several nonenzymatic mechanisms for S-nitrosylation formation and destruction have been described. Enzymatic mechanisms for transnitrosylation and denitrosylation have been also studied as regulators of the modification of specific subsets of proteins. The advancement of modification-specific proteomic methodologies has allowed progress in the study of diverse S-nitrosoproteomes, raising clues and questions about the parameters for determining the protein specificity of the modification. CRITICAL ISSUES We propose that S-nitrosylation is mainly a short-range mechanism of NO signaling, exerted in a relatively limited range of action around the NO sources, and tightly related to the very controlled regulation of subcellular localization of nitric oxide synthases. We review the nonenzymatic and enzymatic mechanisms that support this concept, as well as physiological examples of mammalian systems that illustrate well the precise compartmentalization of S-nitrosylation. FUTURE DIRECTIONS Individual and proteomic studies of protein S-nitrosylation-based signaling should take into account the subcellular localization in order to gain further insight into the functional role of this modification in (patho)physiological settings.

Changes in calcium dynamics following the reversal of the sodium-calcium exchanger have a key role in AMPA receptor-mediated neurodegeneration via calpain activation in hippocampal neurons

Proteolytic cleavage of the Na+/Ca2+ exchanger (NCX) by calpains impairs calcium homeostasis, leading to a delayed calcium overload and excitotoxic cell death. However, it is not known whether reversal of the exchanger contributes to activate calpains and trigger neuronal death. We investigated the role of the reversal of the NCX in Ca2+ dynamics, calpain activation and cell viability, in α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-stimulated hippocampal neurons. Selective overactivation of AMPA receptors caused the reversal of the NCX, which accounted for approximately 30% of the rise in intracellular free calcium concentration ([Ca2+]i). The NCX reverse-mode inhibitor, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R7943), partially inhibited the initial increase in [Ca2+]i, and prevented a delayed increase in [Ca2+]i. In parallel, overactivation of AMPA receptors strongly activated calpains and led to the proteolysis of NCX3. KB-R7943 prevented calpain activation, cleavage of NCX3 and was neuroprotective. Silencing of NCX3 reduced Ca2+ uptake, calpain activation and was neuroprotective. Our data show for the first time that NCX reversal is an early event following AMPA receptor stimulation and is linked to the activation of calpains. Since calpain activation subsequently inactivates NCX, causing a secondary Ca2+ entry, NCX may be viewed as a new suicide substrate operating in a Ca2+-dependent loop that triggers cell death and as a target for neuroprotection.

Neuropeptide Y stimulates retinal neural cell proliferation – involvement of nitric oxide

Neuropeptide Y (NPY) is a 36 amino acid peptide widely present in the CNS, including the retina. Previous studies have demonstrated that NPY promotes cell proliferation of rat post‐natal hippocampal and olfactory epithelium precursor cells. The aim of this work was to investigate the role of NPY on cell proliferation of rat retinal neural cells. For this purpose, primary retinal cell cultures expressing NPY, and NPY Y1, Y2, Y4 and Y5 receptors [Álvaro et al., (2007) Neurochem. Int., 50, 757] were used. NPY (10–1000 nM) stimulated cell proliferation through the activation of NPY Y1, Y2 and Y5 receptors. NPY also increased the number of proliferating neuronal progenitor cells (BrdU+/nestin+ cells). The intracellular mechanisms coupled to NPY receptors activation that mediate the increase in cell proliferation were also investigated. The stimulatory effect of NPY on cell proliferation was reduced by l‐nitroarginine‐methyl‐esther (l‐NAME; 500 μM), a nitric oxide synthase inhibitor, 1H‐[1,2,4]oxadiazolo‐[4, 3‐a]quinoxalin‐1‐one (ODQ; 20 μM), a soluble guanylyl cyclase inhibitor or U0126 (1 μM), an inhibitor of the extracellular signal‐regulated kinase 1/2 (ERK 1/2). In conclusion, NPY stimulates retinal neural cell proliferation, and this effect is mediated through nitric oxide–cyclic GMP and ERK 1/2 pathways.

Calpain activation is involved in early caspase-independent neurodegeneration in the hippocampus following status epilepticus

Evidence for increased calpain activity has been described in the hippocampus of rodent models of temporal lobe epilepsy. However, it is not known whether calpains are involved in the cell death that accompanies seizures. In this work, we characterized calpain activation by examining the proteolysis of calpain substrates and in parallel we followed cell death in the hippocampus of epileptic rats. Male Wistar rats were injected with kainic acid (10 mg/kg) intraperitoneally and killed 24 h later, after development of grade 5 seizures. We observed a strong Fluoro‐Jade labeling in the CA1 and CA3 areas of the hippocampus in the rats that received kainic acid, when compared with saline‐treated rats. Immunohistochemistry and western blot analysis for the calpain‐derived breakdown products of spectrin showed evidence of increased calpain activity in the same regions of the hippocampus where cell death is observed. No evidence was found for caspase activation, in the same conditions. Treatment with the calpain inhibitor MDL 28170 significantly prevented the neurodegeneration observed in CA1. Taken together, our data suggest that early calpain activation, but not caspase activation, is involved in neurotoxicity in the hippocampus after status epilepticus.

Early calpain‐mediated proteolysis following AMPA receptor activation compromises neuronal survival in cultured hippocampal neurons

In this work, we investigated the involvement of calpains in the neurotoxicity induced by short‐term exposure to kainate (KA) in non‐desensitizing conditions of AMPA receptor activation (cyclothiazide present, CTZ), in cultured rat hippocampal neurons. The calpain inhibitor MDL28170 had a protective effect in cultures treated with KA plus CTZ (p < 0.01), preventing the decrease in MTT reduction caused by exposure to KA (p < 0.001). Caspase inhibition by ZVAD‐fmk was not neuroprotective against the toxic effect of KA. At 1 h after treatment, we could already observe significantly increased calpain activity, which was prevented by MDL 28170 and NBQX. Western blot analysis of calpain substrates, GluR1, neuronal nitric oxide synthase (nNOS) and nonerythroid spectrin (fodrin), showed a time‐dependent and MDL 28170‐sensitive proteolysis of these proteins. This effect was due to calpains, but not caspases, since ZVAD‐fmk was ineffective in preventing proteolytic events. Breakdown products of fodrin (BDPs) were detected as early as 15 min after exposure to KA. Overall, these results show early activation of calpains following activation of AMPA receptors as well as compromise of neuronal survival, likely due to proteolytic events that affect proteins involved in neuronal signaling.

Proteolysis of NR2B by calpain in the hippocampus of epileptic rats.

Overactivation of N-methyl-D-aspartate receptors is known to mediate excitotoxicity due to excessive entry of calcium, leading to the activation of several calcium-dependent enzymes. Calpains are calcium-activated proteases that appear to play a role in excitotoxic neuronal death. Several cellular proteins are substrates for these proteases, particularly the N-methyl-D-aspartate receptor. Recently, cleavage of NR2B subunits has been implicated in excitotoxic neurodegeneration in ischemia. In this work, we investigated the proteolysis by calpains of NR2B subunits of the N-methyl-D-aspartate receptor in the hippocampus of epileptic rats. Our results show that cleaved forms of NR2B subunits are formed after status epilepticus, in the same areas of the hippocampus where calpain activation was detected by immunohistochemical staining of calpain-specific spectrin breakdown products.