Epigallocatechin-3-gallate and related phenol compounds redirect the amyloidogenic aggregation pathway of ataxin-3 towards non-toxic aggregates and prevent toxicity in neural cells and Caenorhabditis elegans animal model.

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.

Different Molecular Mechanisms Mediate Direct or Glia-Dependent Prion Protein Fragment 90-231 Neurotoxic Effects in Cerebellar Granule Neurons.

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.

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-ß isoform (AßpE3-42) as one of the most neurotoxic peptides involved in the onset of Alzheimer's disease. Furthermore, AßpE3-42 co-oligomerized with excess of Aß1-42, produces oligomers and aggregates that are structurally distinct and far more cytotoxic than those made from Aß1-42 alone. Here, we investigate quantitatively the influence of AßpE3-42 on biophysical properties and biological activity of Aß1-42. We tested different ratios of AßpE3-42/Aß1-42 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% AßpE3-42, induces the highest disruption of intracellular calcium homeostasis and the highest neuronal toxicity. These data correlate to an high content of relaxed antiparallel ß-sheet structure and the coexistence of a population of big spheroidal aggregates together with short fibrils. Our experiments provide also evidence that AßpE3-42 causes template-induced misfolding of Aß1-42 at ratios below 33%. This means that there exists a critical concentration required to have seeding on Aß1-42 aggregation, above this threshold, the seed effect is not possible anymore and AßpE3-42 controls the total aggregation kinetics.

Study of the Interaction of 1,4- and 1,5-Benzodiazepines with GABAA Receptors of Rat Cerebellum Granule Cells in Culture.

The effects of a classical 1,4-benzodiazepine agonist, such as diazepam, its catabolite N-desmethyl-diazepam (nordiazepam), and 1,5-benzodiazepines such as clobazam and RL 214 (a triazolobenzodiazepine previously synthesized in our labs) were evaluated on native GABAA receptors of cerebellar granule cells in culture. The parameter studied was the increase of GABA-activated chloride currents caused by these substances. The contributions of α6 ß2/3 γ2 and α1 α6 ß2/3 γ2 receptor subtypes to the increase of GABA-activated chloride current were investigated by comparing the effects of such substances in the presence vs. the absence of furosemide. Furosemide is in fact able to block such receptors. It was found that the percent enhancement of peak GABA-activated current doubled for diazepam, clobazam, and RL 214. However, it did not change for N-desmethyl-diazepam. These results indicate that diazepam, clobazam, and RL 214 interact exclusively with α1 ß2/3 γ2 receptors, while N-desmethyl-diazepam seems to interact with not only α1- but also α6-containing receptors.

GABAA Receptors of Cerebellar Granule Cells in Culture: Interaction with Benzodiazepines.

GABAA receptor mediated inhibition plays an important role in modulating the input/output dynamics of cerebellum. A characteristic of cerebellar GABAA receptors is the presence in cerebellar granule cells of subunits such as α6 and δ which give insensitivity to classical benzodiazepines. In fact, cerebellar GABAA receptors have generally been considered a poor model for testing drugs which potentially are active at the benzodiazepine site. In this overview we show how rat cerebellar granule cells in culture may be a useful model for studying new benzodiazepine site agonists. This is based on the pharmacological separation of diazepam-sensitive α1 ß2/3 γ2 receptors from those which are diazepam-insensitive and contain the α6 subunit. This is achieved by utilizing furosemide/Zn2+ which block α6 containing and incomplete receptors.

Different ataxin-3 amyloid aggregates induce intracellular Ca(2+) 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.

Excitotoxicity through NMDA receptors mediates cerebellar granule neuron apoptosis induced by prion protein 90-231 fragment.

Prion diseases recognize, as a unique molecular trait, the misfolding of CNS-enriched prion protein (PrP(C)) into an aberrant isoform (PrP(Sc)). In this work, we characterize the in vitro toxicity of amino-terminally truncated recombinant PrP fragment (amino acids 90-231, PrP90-231), on rat cerebellar granule neurons (CGN), focusing on glutamatergic receptor activation and Ca(2+) homeostasis impairment. This recombinant fragment assumes a toxic conformation (PrP90-231(TOX)) after controlled thermal denaturation (1 h at 53 °C) acquiring structural characteristics identified in PrP(Sc) (enrichment in ß-structures, increased hydrophobicity, partial resistance to proteinase K, and aggregation in amyloid fibrils). By annexin-V binding assay, and evaluation of the percentage of fragmented and condensed nuclei, we show that treatment with PrP90-231(TOX), used in pre-fibrillar aggregation state, induces CGN apoptosis. This effect was associated with a delayed, but sustained elevation of [Ca(2+)]i. Both CGN apoptosis and [Ca(2+)]i increase were not observed using PrP90-231 in PrP(C)-like conformation. PrP90-231(TOX) effects were significantly reduced in the presence of ionotropic glutamate receptor antagonists. In particular, CGN apoptosis and [Ca(2+)]i increase were largely reduced, although not fully abolished, by pre-treatment with the NMDA antagonists APV and memantine, while the AMPA antagonist CNQX produced a lower, although still significant, effect. In conclusion, we report that CGN apoptosis induced by PrP90-231(TOX) correlates with a sustained elevation of [Ca(2+)]i mediated by the activation of NMDA and AMPA receptors.

Nonspecific interaction of prefibrillar amyloid aggregates with glutamatergic receptors results in Ca2+ increase in primary neuronal cells.

It is widely reported that the Ca(2+) increase following nonspecific cell membrane permeabilization is among the earliest biochemical modifications in cells exposed to toxic amyloid aggregates. However, more recently receptors with Ca(2+) channel activity such as alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl D-aspartate (NMDA), ryanodine, and inositol 1,4,5-trisphosphate receptors have been proposed as mediators of the Ca(2+) increase in neuronal cells challenged with beta-amyloid peptides. We previously showed that prefibrillar aggregates of proteins not associated with amyloid diseases are toxic to exposed cells similarly to comparable aggregates of disease-associated proteins. In particular, prefibrillar aggregates of the prokaryotic HypF-N were shown to be toxic to different cultured cell lines by eliciting Ca(2+) and reactive oxygen species increases. This study was aimed at assessing whether NMDA and AMPA receptor activations could be considered a generic feature of cell interaction with amyloid aggregates rather than a specific effect of some aggregated protein. Therefore, we investigated whether NMDA and AMPA receptors were involved in the Ca(2+) increase following exposure of rat cerebellar granule cells to HypF-N prefibrillar aggregates. We found that the intracellular Ca(2+) increase was associated with the early activation of NMDA and AMPA receptors, although some nonspecific membrane permeabilization was also observed at longer times of exposure. This result matched a significant co-localization of the aggregates with both receptors on the plasma membrane. Our data support the possibility that glutamatergic channels are generic sites of interaction with the cell membrane of prefibrillar aggregates of different peptides and proteins as well as the key structures responsible for the resulting early membrane permeabilization to Ca(2+).

ERK1/2 and p38 MAP kinases control prion protein fragment 90-231-induced astrocyte proliferation and microglia activation.

Astrogliosis and microglial activation are a common feature during prion diseases, causing the release of chemoattractant and proinflammatory factors as well as reactive free radicals, involved in neuronal degeneration. The recombinant protease-resistant domain of the prion protein (PrP90-231) displays in vitro neurotoxic properties when refolded in a beta-sheet-rich conformer. Here, we report that PrP90-231 induces the secretion of several cytokines, chemokines, and nitric oxide (NO) release, in both type I astrocytes and microglial cells. PrP90-231 elicited in both cell types the activation of ERK1/2 MAP kinase that displays, in astrocytes, a rapid kinetics and a proliferative response. Conversely, in microglia, PrP90-231-dependent MAP kinase activation was delayed and long lasting, inducing functional activation and growth arrest. In microglial cells, NO release, dependent on the expression of the inducible NO synthase (iNOS), and the secretion of the chemokine CCL5 were Ca(2+) dependent and under the control of the MAP kinases ERK1/2 and p38: ERK1/2 inhibition, using PD98059, reduced iNOS expression, while p38 blockade by PD169316 inhibited CCL5 release. In summary, we demonstrate that glial cells are activated by extracellular misfolded PrP90-231 resulting in a proliferative/secretive response of astrocytes and functional activation of microglia, both dependent on MAP kinase activation. In particular, in microglia, PrP90-231 activated a complex signalling cascade involved in the regulation of NO and chemokine release. These data argue in favor of a causal role for misfolded prion protein in sustaining glial activation and, possibly, glia-mediated neuronal death.

A quantum-dot nanocrystal study of GABAA receptor subunits in living cerebellar granule cells in culture.

Quantum dots (QDs) are semiconductor nanocrystals emerging as a new class of fluorescent labels with large brightness, multi color fluorescence emission and resistance against photobleaching. Here we have used QDs as biological markers in an immunofluorescence approach. In this work GABA(A )receptors of rat cerebellar granule cells have been studied and in particular we have visualized the beta(2/3) and delta subunits in live cells. The results obtained were compared to those gathered with conventional probes. The images of the delta subunit in living cells appear to correspond to those expected for a subunit part of GABA(A )receptors mediating tonic inhibition in the granules cell bodies.

Two-photon analysis of lead accumulation in rat cerebellar granule neurons.

Lead (Pb2+) is a common pollutant and potent central neurotoxin. We have studied its pathways of permeation by two-photon fluorescence microscopy in rat cerebellar granule neurons loaded with the fluorescent dye indo-1. Pb2+ binds indo-1 with high affinity acting as a quencher. Its permeation through the neuronal membrane was indicated by a decrease of the fluorescence emission, which occurred even in resting condition. In the presence of 20 microM Pb2+, uptake reached a plateau level (approximately 45% of initial fluorescence) in 4 min and was partially antagonized by 25 microM lanthanum. Subsequent addition of a membrane permeant ionophore caused a further (>70%) quenching of the dye, suggesting that previous saturation was due to inactivation of the transport system. Intracellular Pb2+ concentrations were evaluated from the fluorescence intensity and this estimate indicated that the concentration of free Pb2+ sufficient to inactivate the transport system is close to 50 pM.

Different chloride electrochemical gradients across the plasma membrane in subcellular compartments of rat cerebellum granules.

The effects of GABA on intracellular Ca2+ have been studied in neonatal rat cerebellum granule cells (CGC) in culture by Oregon Green and two-photon excitation fluorescence microscopy. This technique allowed the study of [Ca2+]i both in cell bodies and neurites. Working with a perfusion chloride concentration corresponding to the average extracellular level, we found that GABA induced an increase in [Ca2+]i in the cell bodies in many of the cells studied with a maximum at day 4 in vitro. This effect disappeared after day 6. However, no increase in [Ca2+]i was ever found in neurites at standard [Cl-]e. On the other hand, an increase of [Ca2+]i was found also in neurites when [Cl-]e was close to zero. The [Ca2+]i increases were blocked by both bicuculline methiodide and nimodipine. The results indicate the presence of an outward directed electrochemical gradient for chloride in the cell bodies which results in depolarization by GABA via GABA(A) receptor activation. Calcium ion influx ensues due to activation of voltage-gated calcium channels (VGCC). This phenomenon may mediate the well-known trophic effect of GABA on these cells at this developmental stage, via an action of [Ca2+]i on the transcriptional activity of the nucleus. No calcium accumulation takes place in neurites due to either no or a reverse (hyperpolarizing) electrochemical gradient for chloride ions. Such a circumstance in later developmental stages may be of importance for the phasic component of GABA-mediated inhibition.

Two-photon imaging of calcium accumulation in rat cerebellar granule cells.

Topical accumulation of calcium ions in neurites and cell bodies of rat cerebellar granule cells was studied by two-photon microscopy in neurons loaded with the Ca-sensitive fluorescent indicator Oregon Green 488 Bapta. High potassium caused a rapid surge of internal calcium ([Ca2+]i) in the cell body, followed by a plateau. In neurites, [Ca2+]i reached a peak and then decreased back to the control level. In contrast, in neurons stimulated by NMDA, [Ca2+]i reached a steady level and remained constant as long as the agonist was present in the bath, either in the cell bodies or in neurites. In the latter, the response to NMDA treatment was smaller and heterogeneous, and [Ca2+]i increased in certain segments of the neurite, but not in others.

The combined disruption of microfilaments and microtubules affects the distribution and function of GABAA receptors in rat cerebellum granule cells in culture.

The role of the microfilaments and microtubules cytoskeleton in the stability of the subcellular distribution and function of GABAA receptors has been studied in rat cerebellar granule cells in culture. The disruption of either the microfilaments or the microtubules structures did not result in detectable changes in the receptors distribution, as assessed by immunocytochemistry, or in their function, as assessed by the whole-cell patch-clamp approach. A distinct disruption of both the subcellular distribution and the function of the GABAA receptors was found only if both microfilaments and microtubules were destroyed. The results suggest that, in the short term, the plasma membrane localization/stabilization and function of these receptors in granule cells are largely independent from microfilaments and microtubules individually, although they obviously depend on the presence of an organized cellular framework.