Elena Gatta

17A, a novel non-coding RNA, regulates GABA B alternative splicing and signaling in response to inflammatory stimuli and in Alzheimer disease

Alternative splicing is a central component of human brain complexity; nonetheless, its regulatory mechanisms are still largely unclear. In this work, we describe a novel non-coding (nc) RNA (named 17A) RNA polymerase (pol) III-dependent embedded in the human G-protein-coupled receptor 51 gene (GPR51, GABA B2 receptor). The stable expression of 17A in SHSY5Y neuroblastoma cells induces the synthesis of an alternative splicing isoform that abolish GABA B2 intracellular signaling (i.e., inhibition of cAMP accumulation and activation of K(+) channels). Indeed, 17A is expressed in human brain, and we report that it is upregulated in cerebral tissues derived from Alzheimer disease patients. We demonstrate that 17A expression in neuroblastoma cells enhances the secretion of amyloid β peptide (Aβ) and the Aβ x-42/Αβ x-40 peptide ratio and that its synthesis is induced in response to inflammatory stimuli. These data correlate, for the first time, the activity of a novel pol III-dependent ncRNA to alternative splicing events and, possibly, to neurodegeneration induced by abnormal GABA B function. We anticipate that further analysis of pol III-dependent regulation of alternative splicing will disclose novel regulatory pathways associated to brain physiology and/or pathology.

An Alu-like RNA promotes cell differentiation and reduces malignancy of human neuroblastoma cells

Neuroblastoma (NB) is a pediatric cancer characterized by remarkable cell heterogeneity within the tumor nodules. Here, we demonstrate that the synthesis of a pol III‐transcribed noncoding (nc) RNA (NDM29) strongly restricts NB development by promoting cell differentiation, a drop of malignancy processes, and a dramatic reduction of the tumor initiating cell (TIC) fraction in the NB cell population. Notably, the overexpression of NDM29 also confers to malignant NB cells an unpredicted susceptibility to the effects of antiblastic drugs used in NB therapy. Altogether, these results suggest the induction of NDM29 expression as possible treatment to increase cancer cells vulnerability to therapeutics and the measure of its synthesis in NB explants as prognostic factor of this cancer type.—Castelnuovo, M., Massone, S., Tasso, R., Fiorino, G., Gatti, M., Robello, M., Gatta, E., Berger, A., Strub, K., Florio, T., Dieci, G., Cancedda, R., Pagano, A. An Alu‐like RNA promotes cell differentiation and reduces malignancy of human neuroblastoma cells. FASEB J. 24, 4033–4046 (2010). www.fasebj.org

RNA polymerase III drives alternative splicing of the potassium channel–interacting protein contributing to brain complexity and neurodegeneration

An RNA polymerase III–transcribed noncoding RNA promotes alternative splicing of KCNIP4, altering amyloid precursor protein processing and contributing to neurodegeneration.

Neurounina-1, a Novel Compound That Increases Na+/Ca2+ Exchanger Activity, Effectively Protects against Stroke Damage

Previous studies have demonstrated that the knockdown or knockout of the three Na+/Ca2+ exchanger (NCX) isoforms, NCX1, NCX2, and NCX3, worsens ischemic brain damage. This suggests that the activation of these antiporters exerts a neuroprotective action against stroke damage. However, drugs able to increase the activity of NCXs are not yet available. We have here succeeded in synthesizing a new compound, named neurounina-1 (7-nitro-5-phenyl-1-(pyrrolidin-1-ylmethyl)-1H-benzo[e][1,4]diazepin-2(3H)-one), provided with an high lipophilicity index and able to increase NCX activity. Ca2+ radiotracer, Fura-2 microfluorimetry, and patch-clamp techniques revealed that neurounina-1 stimulated NCX1 and NCX2 activities with an EC50 in the picomolar to low nanomolar range, whereas it did not affect NCX3 activity. Furthermore, by using chimera strategy and site-directed mutagenesis, three specific molecular determinants of NCX1 responsible for neurounina-1 activity were identified in the α-repeats. Interestingly, NCX3 became responsive to neurounina-1 when both α-repeats were replaced with the corresponding regions of NCX1. In vitro studies showed that 10 nM neurounina-1 reduced cell death of primary cortical neurons exposed to oxygen-glucose deprivation followed by reoxygenation. Moreover, in vitro, neurounina-1 also reduced γ-aminobutyric acid (GABA) release, enhanced GABAA currents, and inhibited both glutamate release and N-methyl-d-aspartate receptors. More important, neurounina-1 proved to have a wide therapeutic window in vivo. Indeed, when administered at doses of 0.003 to 30 μg/kg i.p., it was able to reduce the infarct volume of mice subjected to transient middle cerebral artery occlusion even up to 3 to 5 hours after stroke onset. Collectively, the present study shows that neurounina-1 exerts a remarkable neuroprotective effect during stroke and increases NCX1 and NCX2 activities.

Different ataxin-3 amyloid aggregates induce intracellular Ca2+ deregulation by different mechanisms in cerebellar granule cells

This work aims at elucidating the relation between morphological and physicochemical properties of different ataxin-3 (ATX3) aggregates and their cytotoxicity. We investigated a non-pathological ATX3 form (ATX3Q24), a pathological expanded form (ATX3Q55), and an ATX3 variant truncated at residue 291 lacking the polyQ expansion (ATX3/291Δ). Solubility, morphology and hydrophobic exposure of oligomeric aggregates were characterized. Then we monitored the changes in the intracellular Ca(2+) levels and the abnormal Ca(2+) signaling resulting from aggregate interaction with cultured rat cerebellar granule cells. ATX3Q55, ATX3/291Δ and, to a lesser extent, ATX3Q24 oligomers displayed similar morphological and physicochemical features and induced qualitatively comparable time-dependent intracellular Ca(2+) responses. However, only the pre-fibrillar aggregates of expanded ATX3 (the only variant which forms bundles of mature fibrils) triggered a characteristic Ca(2+) response at a later stage that correlated with a larger hydrophobic exposure relative to the two other variants. Cell interaction with early oligomers involved glutamatergic receptors, voltage-gated channels and monosialotetrahexosylganglioside (GM1)-rich membrane domains, whereas cell interaction with more aged ATX3Q55 pre-fibrillar aggregates resulted in membrane disassembly by a mechanism involving only GM1-rich areas. Exposure to ATX3Q55 and ATX3/291Δ aggregates resulted in cell apoptosis, while ATX3Q24 was substantially innocuous. Our findings provide insight into the mechanisms of ATX3 aggregation, aggregate cytotoxicity and calcium level modifications in exposed cerebellar cells.

Celecoxib Inhibits Prion Protein 90-231-Mediated Pro-inflammatory Responses in Microglial Cells

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 (PrPSc), 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.

A critical concentration of N-terminal pyroglutamylated amyloid beta drives the misfolding of Ab1-42 into more toxic aggregates

A wide consensus based on robust experimental evidence indicates pyroglutamylated amyloid-β isoform (AβpE3-42) as one of the most neurotoxic peptides involved in the onset of Alzheimers disease. Furthermore, AβpE3-42 co-oligomerized with excess of Aβ1-42, produces oligomers and aggregates that are structurally distinct and far more cytotoxic than those made from Aβ1-42 alone. Here, we investigate quantitatively the influence of AβpE3-42 on biophysical properties and biological activity of Aβ1-42. We tested different ratios of AβpE3-42/Aβ1-42 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% AβpE3-42, induces the highest disruption of intracellular calcium homeostasis and the highest neuronal toxicity. These data correlate to an high content of relaxed antiparallel β-sheet structure and the coexistence of a population of big spheroidal aggregates together with short fibrils. Our experiments provide also evidence that AβpE3-42 causes template-induced misfolding of Aβ1-42 at ratios below 33%. This means that there exists a critical concentration required to have seeding on Aβ1-42 aggregation, above this threshold, the seed effect is not possible anymore and AβpE3-42 controls the total aggregation kinetics.

Novel celecoxib analogues inhibit glial production of prostaglandin E2, nitric oxide, and oxygen radicals reverting the neuroinflammatory responses induced by misfolded prion protein fragment 90-231 or lipopolysaccharide.

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.

New 1,5-benzodiazepine compounds: activity at native GABAA receptors

Various new 1,5-benzodiazepine compounds were synthesized and tested for their biological activity in terms of effects on GABA(A) receptors of rat cerebellar granules in culture. Their effects were compared to those of a 1,4-benzodiazepine agonist, flunitrazepam and the already known 1,5-benzodiazepine antiepileptic clobazam. The effects were evaluated for the two different GABA(A) receptor populations present in these neurons, one mediating phasic inhibition and the other one mediating tonic inhibition. Many such compounds display a profile of inverse agonist to both GABA(A) receptor populations. One of them presents a profile of full agonist at the component mediating phasic inhibition. Interestingly, substitution of just one oxygen atom in that compound with sulphur in a specific position of a morpholine ring resulted in a remarkable change of activity from full agonist to a probable inverse agonist. This indicates such a position as a proton accepting one for the ligand within the benzodiazepine binding pocket of the relevant GABA(A) receptors. In addition, that position appears to be critical for the pharmacological activity.

GABAA receptors of cerebellar granule cells in culture: explanation of overall insensitivity to ethanol

GABA-activated chloride currents were studied in cerebellar granule cells put in culture from neonatal rats. As previously described, 10 microM GABA perfusion of these cells recorded by whole cell patch-clamp elicits chloride currents displaying a peak and a steady-state component. The two components were studied in the presence of 1 mM furosemide, 1 microM Zn(2+) and a combination of the two in order to evaluate the contribution of the different types of GABA(A) receptors. Furosemide inhibits alpha(6) containing receptors whereas low levels of Zn(2+) specifically block incomplete GABA(A) receptors made up of alpha and beta subunits only. The results show that the peak component involves the following receptors: alpha(x) beta(y), 25%; alpha(1) beta(y) gamma(2), 45%; alpha(6) beta(y) gamma(2) plus alpha(1) alpha(6) beta(y) gamma(2), 30%. The steady state component is made up by alpha(x) beta(y), 38%; alpha(1) beta(y) delta, 62%. Ethanol at relatively high concentration, 100 mM, slows further down the desensitization of alpha(1) beta(y) delta receptors. The results indicate that the relative insensitivity to ethanol of GABA(A) receptors of neonatal cerebellar granule cells in culture is due to the absence of mature alpha(6) beta(y) delta receptors, a major receptor brand involved in tonic inhibition.