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Ruthenium–bipyridinetriphenylphosphine–GABA (RuBi–GABA) is a caged compound that allows studying the neuronal transmission in a specific region of a neuron. The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is bound to a caged group that blocks the interaction of the neurotransmitter with its receptor site. Following linear—one-photon (1P)—and non-linear—multi-photon—absorption of light, the covalent bond of the caged molecule is broken, and GABA is released. Such a controlled release in time and space allows investigating the interaction with its receptor in four dimensions (X,Y,Z,t). Taking advantage of this strategy, we succeeded in addressing the modulation of GABAA in rat cerebellar neurons by coupling the photoactivation process, by confocal or two-photon excitation microscopy, with the electrophysiological technique of the patch-clamp in the whole-cell configuration. Key parameters have been comprehensively investigated and correlated in a temporally and spatially confined way, namely: photoactivation laser power, time of exposure, and distance of the uncaging point from the cell of interest along the X, Y, Z spatial coordinates. The goal of studying specific biological events as a function of controlled physical parameters has been achieved.

The antisecretory factor is an endogenous protein found in all mammalian tissues investigated so far. It acts by counteracting intestinal hypersecretion and various forms of inflammation, but the detailed mechanism of antisecretory factor (AF) action is unknown. We tested neuronal GABAA receptors by means of AF-16, a potent AF peptide derived from amino acids 36–51 from the NH2 part of AF. Cultured rat cerebellar granule cells were used, and the effects on the GABA-mediated chloride currents were determined by whole-cell patch clamp. Both the neurotransmitter GABA and AF-16 were added by perfusion of the experimental system. A 3-min AF-16 preincubation was more efficacious than 30 s in significantly elevating the rapidly desensitizing GABA-activated chloride current. No effect was found on the tonic, slowly desensitizing current. The GABA-activated current increase by AF-16 demonstrated a low k of 41 pM with a maximal increase of 37% persisting for some minutes after AF washout, independent from GABA concentration. This indicates an effect on the maximal stimulation (E%Max) excluding an altered affinity between GABA and its receptor. An immunocytochemical fluorescence approach with anti γ2 subunit antibodies demonstrated an increased expression of GABAA receptors. Thus, both the electrophysiological and the immunofluorescence approach indicate an increased appearance of GABAA receptors on the neuronal membrane. The rationale of the experiments was to test the effect of AF on a defined neuronal population of GABAA receptors. The implications of the results on the impact of AF on the enteric nervous system or on brain function are discussed.

We tested the efficacy of novel cyclooxygenase 2 (COX-2) inhibitors in counteracting glia-driven neuroinflammation induced by the amyloidogenic prion protein fragment PrP90-231 or lipopolysaccharide (LPS). In search for molecules with higher efficacy than celecoxib, we focused our study on its 2,3-diaryl-1,3-thiazolidin-4-one analogues. As experimental models, we used the immortalized microglial cell line N9, rat purified microglial primary cultures, and mixed cultures of astrocytes and microglia. Microglia activation in response to PrP90-231 or LPS was characterized by growth arrest, morphology changes and the production of reactive oxygen species (ROS). Moreover, PrP90-231 treatment caused the overexpression of the inducible nitric oxide synthase (iNOS) and COX-2, with the consequent nitric oxide (NO), and prostaglandin E2 (PGE2) accumulation. These effects were challenged by different celecoxib analogues, among which Q22 (3-[4-(sulfamoyl)phenyl]-2-(4-tolyl)thiazolidin-4-one) inhibited microglia activation more efficiently than celecoxib, lowering both iNOS and COX-2 activity and reducing ROS release. During neurodegenerative diseases, neuroinflammation induced by amyloidogenic peptides causes the activation of both astrocytes and microglia with these cell populations mutually regulating each other. Thus the effects of PrP90-231 and LPS were also studied on mixed glial cultures containing astrocytes and microglia. PrP90-231 treatment elicited different responses in the co-cultures induced astrocyte proliferation and microglia growth arrest, resulting in a differential ability to release proinflammatory molecules with the production of NO and ROS mainly attributable on microglia, while COX-2 expression was induced also in astrocytes. Q22 effects on both NO and PGE2 secretion were more significant in the mixed glial cultures than in purified microglia, demonstrating Q22 ability to revert the functional interaction between astrocytes and microglia. These results demonstrate that Q22 is a powerful drug able to revert glial neuroinflammatory responses and might represent a lead to explore the chemical space around celecoxib frameworks to design even more effective agents, paving the way to novel approaches to contrast the neuroinflammation-dependent toxicity.

The protein ataxin-3 (ATX3) triggers an amyloid-related neurodegenerative disease when its polyglutamine stretch is expanded beyond a critical threshold. We formerly demonstrated that the polyphenol epigallocatechin-3-gallate (EGCG) could redirect amyloid aggregation of a full-length, expanded ATX3 (ATX3-Q55) towards non-toxic, soluble, SDS-resistant aggregates. Here, we have characterized other related phenol compounds, although smaller in size, i.e. (-)-epigallocatechin gallate (EGC), and gallic acid (GA). We analysed the aggregation pattern of ATX3-Q55 and of the N-terminal globular Josephin domain (JD) by assessing the time course of the soluble protein, as well its structural features by FTIR and AFM, in the presence and the absence of the mentioned compounds. All of them redirected the aggregation pattern towards soluble, SDS-resistant aggregates. They also prevented the appearance of ordered side-chain hydrogen bonding in ATX3-Q55, which is the hallmark of polyQ-related amyloids. Molecular docking analyses on the JD highlighted three interacting regions, including the central, aggregation-prone one. All three compounds bound to each of them, although with different patterns. This might account for their capability to prevent amyloidogenesis. Saturation transfer difference NMR experiments also confirmed EGCG and EGC binding to monomeric JD. ATX3-Q55 pre-incubation with any of the three compounds prevented its calcium-influx-mediated cytotoxicity towards neural cells. Finally, all the phenols significantly reduced toxicity in a transgenic Caenorhabditis elegans strain expressing an expanded ATX3. Overall, our results show that the three polyphenols act in a substantially similar manner. GA, however, might be more suitable for antiamyloid treatments due to its simpler structure and higher chemical stability.

Glia over-stimulation associates with amyloid deposition contributing to the progression of central nervous system neurodegenerative disorders. Here we analyze the molecular mechanisms mediating microglia-dependent neurotoxicity induced by prion protein (PrP)90-231, an amyloidogenic polypeptide corresponding to the protease-resistant portion of the pathological prion protein scrapie (PrPSc). PrP90-231 neurotoxicity is enhanced by the presence of microglia within neuronal culture, and associated to a rapid neuronal [Ca++] i increase. Indeed, while in "pure" cerebellar granule neuron cultures, PrP90-231 causes a delayed intracellular Ca++ entry mediated by the activation of NMDA receptors; when neuron and glia are co-cultured, a transient increase of [Ca++] i occurs within seconds after treatment in both granule neurons and glial cells, then followed by a delayed and sustained [Ca++] i raise, associated with the induction of the expression of inducible nitric oxide synthase and phagocytic NADPH oxidase. [Ca++] i fast increase in neurons is dependent on the activation of multiple pathways since it is not only inhibited by the blockade of voltage-gated channel activity and NMDA receptors but also prevented by the inhibition of nitric oxide and PGE2 release from glial cells. Thus, Ca++ homeostasis alteration, directly induced by PrP90-231 in cerebellar granule cells, requires the activation of NMDA receptors, but is greatly enhanced by soluble molecules released by activated glia. In glia-enriched cerebellar granule cultures, the activation of inducible nitric oxide (iNOS) and NADPH oxidase represents the main mechanism of toxicity since their pharmacological inhibition prevented PrP90-231 neurotoxicity, whereas NMDA blockade by D(-)-2-amino-5-phosphonopentanoic acid is ineffective; conversely, in pure cerebellar granule cultures, NMDA blockade but not iNOS inhibition strongly reduced PrP90-231 neurotoxicity. These data indicate that amyloidogenic peptides induce neurotoxic signals via both direct neuron interaction and glia activation through different mechanisms responsible of calcium homeostasis disruption in neurons and potentiating each other: the activation of excitotoxic pathways via NMDA receptors and the release of radical species that establish an oxidative milieu.

Activation of microglia is a central event in the atypical inflammatory response occurring during prion encephalopathies. We report that the prion protein fragment encompassing amino acids 90-231 (PrP90-231), a model of the neurotoxic activity of the pathogenic prion protein (PrP(Sc)), causes activation of both primary microglia cultures and N9 microglial cells in vitro. This effect was characterized by cell proliferation arrest and induction of a secretory phenotype, releasing prostaglandin E2 (PGE2) and nitric oxide (NO). Conditioned medium from PrP90-231-treated microglia induced in vitro cytotoxicity of A1 mesencephalic neurons, supporting the notion that soluble mediators released by activated microglia contributes to the neurodegeneration during prion diseases. The neuroinflammatory role of COX activity, and its potential targeting for anti-prion therapies, was tested measuring the effects of ketoprofen and celecoxib (preferential inhibitors of COX1 and COX2, respectively) on PrP90-231-induced microglial activation. Celecoxib, but not ketoprofen significantly reverted the growth arrest as well as NO and PGE2 secretion induced by PrP90-231, indicating that PrP90-231 pro-inflammatory response in microglia is mainly dependent on COX2 activation. Taken together, these data outline the importance of microglia in the neurotoxicity occurring during prion diseases and highlight the potentiality of COX2-selective inhibitors to revert microglia as adjunctive pharmacological approach to contrast the neuroinflammation-dependent neurotoxicity.

Herein, we tested in a model of generalized reflex epilepsy in mice different 1,4-benzodiazepines and 1,5-benzodiazepines with agonistic activity at the GABAA receptor population contributing to the peak component of the chloride current elicited by GABA in cerebellar granule cells (CGCs) in culture. The substances have all higher lipophilia than clobazam, an antiepileptic drug well known and used in human therapy. This ensures that they all can pass relatively easily the blood-brain barrier (BBB). The benzodiazepines were administered intraperitoneally (i.p.) and tested for their activity against sound-induced tonic and clonic seizures in a genetic model of experimental epilepsy, the DBA/2 mouse. Our data demonstrates an interesting inverse correlation between the ED50s and the efficacy (E %) of the drugs in increasing the peak chloride current elicited by GABA in cerebellar granule cells in culture. There is indication of the existence of a threshold of E % above which the increase of ED50 with increasing E % becomes linear. This is statistically significant for the clonic phase, whereas it is at the limit of significance for the tonic one. A possible interpretation of these results is that in this epilepsy model, projections from the cerebellum exert a convulsion prevention activity.

A wide consensus based on robust experimental evidence indicates pyroglutamylated amyloid-beta isoform (A beta pE3-42) as one of the most neurotoxic peptides involved in the onset of Alzheimer's disease. Furthermore, A beta pE3-42 co-oligomerized with excess of A beta 1-42, produces oligomers and aggregates that are structurally distinct and far more cytotoxic than those made from A beta 1-42 alone. Here, we investigate quantitatively the influence of A beta pE3-42 on biophysical properties and biological activity of A beta 1-42. We tested different ratios of A beta pE3-42/A beta 1-42 mixtures finding a correlation between the biological activity and the structural conformation and morphology of the analyzed mixtures. We find that a mixture containing 5% A beta pE3-42, induces the highest disruption of intracellular calcium homeostasis and the highest neuronal toxicity. These data correlate to an high content of relaxed antiparallel beta-sheet structure and the coexistence of a population of big spheroidal aggregates together with short fibrils. Our experiments provide also evidence that A beta pE3-42 causes template-induced misfolding of A beta 1-42 at ratios below 33%. This means that there exists a critical concentration required to have seeding on A beta 1-42 aggregation, above this threshold, the seed effect is not possible anymore and A beta pE3-42 controls the total aggregation kinetics. (C) 2016 Elsevier Ltd. All rights reserved.