Biosafety assessment of conducting nanostructured materials by using co-cultures of neurons and astrocytes.

Neural electrode implants are made mostly of noble materials. We have synthesized a nanostructured material combining the good electrochemical properties of iridium oxide (IrOx) and carbon-nanotubes (CNT) and the properties of poly(3,4-ethylenedioxythiophene) (PEDOT). IrOx-CNT-PEDOT charge storage capacity was lower than that of IrOx and IrOx-CNT, but higher than that of other PEDOT-containing hybrids and Pt. Cyclic voltammetry, SEM, XPS and micro-Raman spectroscopy suggest that PEDOT encapsulates IrOx and CNT. In our search for a cell culture platform that could optimize modelling the in vivo environment, we determined cell viability, neuron and astrocyte functionality and the response of astrocytes to an inflammatory insult by using primary cultures of neurons, of astrocytes and co-cultures of both. The materials tested (based on IrOx, CNT and PEDOT, as well as Pt as a reference) allowed adhesion and proliferation of astrocytes and full compatibility for neurons grown in co-cultures. Functionality assays show that uptake of glutamate in neuron-astrocyte co-culture was significantly higher than the sum of the uptake in astrocytes and neurons. In co-cultures on IrOx, IrOx-CNT and IrOx-CNT-PEDOT, glutamate was released by a depolarizing stimulus and induced a significant increase in intracellular calcium, supporting the expression of functional NMDA/glutamate receptors. LPS-induced inflammatory response in astrocytes showed a decreased response in NOS2 and COX2 mRNA expression for IrOx-CNT-PEDOT. Results indicate that neuron-astrocyte co-cultures are a reliable model for assessing the biocompatibility and safety of nanostructured materials, evidencing also that hybrid IrOx-CNT-PEDOT nanocomposite materials may offer larger resistance to inflammatory insults.

Clarified Açaí (Euterpe oleracea) Juice as an Anticonvulsant Agent: In Vitro Mechanistic Study of GABAergic Targets.

Seizures affect about 50 million people around the world. Approximately 30% of seizures are refractory to the current pharmacological arsenal, so, the pursuit of new therapeutic alternatives is essential. Clarified Euterpe oleracea (EO) juice showed anticonvulsant properties similar to diazepam in an in vivo model with pentylenetetrazol, a GABAA receptor blocker. This study investigated the effects of EO on the main GABAergic targets for anticonvulsant drugs, analyzing the effect on the GABA receptor's benzodiazepine and picrotoxinin binding sites and the GABA uptake. Primary cultures of cortical neurons and astrocytes were treated with EO (0-25%) for up to 90 min. [3H]Flunitrazepam and [3H]TBOB binding, [3H]GABA uptake, cell viability, and morphology were assayed. Nonlethal concentrations of EO increased agonist binding and decreased antagonist binding in cortical neurons. Low concentrations significantly inhibited GABA uptake, especially in astrocytes, suggesting an accumulation of endogenous GABA in the synaptic cleft. The results demonstrate, for the first time, that EO can improve GABAergic neurotransmission via interactions with GABAA receptor and modulation of GABA uptake. Understanding these molecular mechanisms will help in the treatment of seizures and epilepsy, especially in developing countries where geographic isolation and low purchasing power are the main barriers to access to adequate treatment.

Ghrelin Causes a Decline in GABA Release by Reducing Fatty Acid Oxidation in Cortex.

Lipid metabolism, specifically fatty acid oxidation (FAO) mediated by carnitine palmitoyltransferase (CPT) 1A, has been described to be an important actor of ghrelin action in hypothalamus. However, it is not known whether CPT1A and FAO mediate the effect of ghrelin on the cortex. Here, we show that ghrelin produces a differential effect on CPT1 activity and γ-aminobutyric acid (GABA) metabolism in the hypothalamus and cortex of mice. In the hypothalamus, ghrelin enhances CPT1A activity while GABA transaminase (GABAT) activity, a key enzyme in GABA shunt metabolism, is unaltered. However, in cortex CPT1A activity and GABAT activity are reduced after ghrelin treatment. Furthermore, in primary cortical neurons, ghrelin reduces GABA release through a CPT1A reduction. By using CPT1A floxed mice, we have observed that genetic ablation of CPT1A recapitulates the effect of ghrelin on GABA release in cortical neurons, inducing reductions in mitochondrial oxygen consumption, cell content of citrate and α-ketoglutarate, and GABA shunt enzyme activity. Taken together, these observations indicate that ghrelin-induced changes in CPT1A activity modulate mitochondrial function, yielding changes in GABA metabolism. This evidence suggests that the action of ghrelin on GABA release is region specific within the brain, providing a basis for differential effects of ghrelin in the central nervous system.

IrOx-carbon nanotube hybrids: a nanostructured material for electrodes with increased charge capacity in neural systems.

Nanostructured iridium oxide-carbon nanotube hybrids (IrOx-CNT) deposited as thin films by dynamic electrochemical methods are suggested as novel materials for neural electrodes. Single-walled carbon nanotubes (SWCNT) serve as scaffolds for growing the oxide, yielding a tridimensional structure with improved physical, chemical and electrical properties, in addition to high biocompatibility. In biological environments, SWCNT encapsulation by IrOx makes more resistant electrodes and prevents the nanotube release to the media, preventing cellular toxicity. Chemical, electrochemical, structural and surface characterization of the hybrids has been accomplished. The high performance of the material in electrochemical measurements and the significant increase in cathodal charge storage capacity obtained for the hybrid in comparison with bare IrOx represent a significant advance in electric field application in biosystems, while its cyclability is also an order of magnitude greater than pure IrOx. Moreover, experiments using in vitro neuronal cultures suggest high biocompatibility for IrOx-CNT coatings and full functionality of neurons, validating this material for use in neural electrodes.

ATP and noradrenaline activate CREB in astrocytes via noncanonical Ca(2+) and cyclic AMP independent pathways.

In neurons, it is well established that CREB contributes to learning and memory by orchestrating the translation of experience into the activity-dependent (i.e., driven by neurotransmitters) transcription of plasticity-related genes. The activity-dependent CREB-triggered transcription requires the concerted action of cyclic AMP/protein kinase A and Ca(2+) /calcineurin via the CREB-regulated transcription co-activator (CRTC). It is not known, however, whether a comparable molecular sequence occurs in astrocytes, despite the unquestionable contribution of these cells to brain plasticity. Here we sought to determine whether and how ATP and noradrenaline cause CREB-dependent transcription in rat cortical astrocyte cultures. Both transmitters induced CREB phosphorylation (Western Blots), CREB-dependent transcription (CRE-luciferase reporter assays), and the transcription of Bdnf, a canonical regulator of synaptic plasticity (quantitative RT-PCR). We indentified a Ca(2+) and diacylglycerol-independent protein kinase C at the uppermost position of the cascade leading to CREB-dependent transcription. Notably, CREB-dependent transcription was partially dependent on ERK1/2 and CRTC, but independent of cyclic AMP/protein kinase A or Ca(2+) /calcineurin. We conclude that ATP and noradrenaline activate CREB-dependent transcription in cortical astrocytes via an atypical protein kinase C. It is of relevance that the signaling involved be starkly different to the one described in neurons since there is no convergence of Ca(2+) and cyclic AMP-dependent pathways on CRTC, which, moreover, exerts a modulatory rather than a central role. Our data thus point to the existence of an alternative, non-neuronal, glia-based role of CREB in plasticity.

JNK/ERK/FAK mediate promigratory actions of basic fibroblast growth factor in astrocytes via CCL2 and COX2.

While the role of cytokines in causing pro- and anti-inflammatory cascades in the brain and that of chemokines in promoting chemotaxis is well recognized, the immunomodulatory actions of neurotrophins released during brain injury remains largely undetermined. This knowledge gap affects basic fibroblast growth factor (FGF2), which in the brain is mainly produced by astrocytes and characteristically upregulated in reactive astrocytes. The goal of this study was to characterize the inflammatory actions of FGF2 in astrocytes using primary cultures. We report that FGF2 induced the upregulation of monocyte chemoattractant protein (CCL2) and cyclo-oxygenase type 2 (COX2), and the inhibition of lipopolysaccharide-elicited ICAM1 upregulation. The effects of FGF2 were: (i) dependent on gene transcription as revealed by the concomitant regulation of CCL2 or ICAM1 mRNAs; (ii) mediated by the FGF2 receptor type 2; (iii) dependent on ERK, JNK and FAK, and (iv) NF-κB-independent. FGF2 also caused accelerated wound closure dependent on CCL2, COX2, ERK, JNK and FAK in a scratch assay. We conclude that the signaling network triggered by FGF2 in astrocytes converged into accelerating directed motion. It follows that astrocyte migration to injury sites may be a key factor in the repair mechanisms orchestrated by FGF2.

Staging anti-inflammatory therapy in Alzheimer's disease.

The use of non-steroidal anti-inflammatory drugs (NSAIDs) in Alzheimer's disease (AD) is controversial because conclusions from numerous epidemiological studies reporting delayed onset of AD in NSAID users have not been corroborated in clinical trials. The purpose of this personal view is to revise the case for NSAIDs in AD therapeutics in light of: (i) the last report from the only primary prevention trial in AD, ADAPT, which, although incomplete, points to significant protection in long-term naproxen users, and (ii) the recently proposed dynamic model of AD evolution. The model contends that there is a clinical silent phase in AD that can last up to 20 years, the duration depending on life style habits, genetic factors, or cognitive reserve. The failure of many purported disease-modifying drugs in AD clinical trials is forcing the view that treatments will only be efficacious if administered pre-clinically. Here we will argue that NSAIDs failed in clinical trials because they are disease-modifying drugs, and they should be administered in early stages of the disease. A complete prevention trial in cognitively normal individuals is thus called for. Further, the shift of anti-inflammatory treatment to early stages uncovers a knowledge void about the targets of NSAIDs in asymptomatic individuals. AD researchers have mostly relied on post-mortem analysis of Aß plaque-laden brains from demented patients or animal models, thus drawing conclusions about AD pathogenesis based on late symptoms. We will discuss evidence in support that defective, not excessive, inflammation underlies AD pathogenesis, that NSAIDs are multifunctional drugs acting on inflammatory and non-inflammatory targets, and that astrocytes and microglia may play differing roles in disease progression, with an emphasis of ApoEε4 as a key, undervalued target of NSAIDs. According to a meta-analysis of epidemiological data, NSAIDs afford an average protection of 58%. If this figure is true, and translated into patient numbers, NSAID treatment may revive as a worth pursuing strategy to significantly reduce the socio-economical burden imposed by AD.

Secretase-independent and RhoGTPase/PAK/ERK-dependent regulation of cytoskeleton dynamics in astrocytes by NSAIDs and derivatives.

Profens like ibuprofen, R-flurbiprofen, or CHF5074 are being considered for the treatment of Alzheimer's disease because epidemiological data indicates that non-steroidal anti-inflammatory drugs are protective against neurodegeneration. Rho-GTPases are small G proteins, including RhoA, Cdc42, and Rac1, which control cytoskeleton dynamics. Because ibuprofen promotes axon growth via RhoA in neurons, we examined whether profens modulate astrocyte plasticity via Rho-GTPases. We report that ibuprofen (100-500 µM), R-flurbiprofen (100-500 µM), and CHF5074 (10-30 µM) caused a concentration-dependent stellation of astrocytes in primary cultures, associated with the reorganization of GFAP and actin filaments. The stellation was independent of COX2, α-, ß- or γ-secretase as judged by the lack of effect of inhibitors of these enzymes. RhoA, PAK, and Cdc42, but not Rac1, accounted for the profen-mediated stellation, as concluded from the joint analyses of activities and reversal experiments with adenoviral or pharmacological manipulations. Ibuprofen accelerated migration in a scratch-wound assay, while R-flurbiprofen had no effect and CHF5074 caused deceleration. Cell polarity regulation by Cdc42 and ERK1/2 may underlie the paradoxical effects of profens on migration. We conclude that profens regulate cytoskeleton dynamics in astrocytes via Rho-GTPases, PAK, and ERK1/2. Since migration is a hallmark of astrocyte response during inflammation we propose that, in addition to (or instead of) lowering amyloid-ß42 via secretases, ibuprofen and its derivatives may prevent Alzheimer's disease instead of AD by modulating astrocyte reactivity through Rho-GTPase/PAK/ERK-dependent signaling.