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

AbstractThis 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 Ca2+ levels and the abnormal Ca2+ 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 Ca2+ responses. However, only the pre-fibrillar aggregates of expanded ATX3 (the only variant which forms bundles of mature fibrils) triggered a characteristic Ca2+ 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

GABA A Receptors of Cerebellar Granule 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 a6 and d 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 a1 b2/3 c2 receptors from those which are diazepam-insensitive and contain the a6 subunit. This is achieved by utilizing furosemide/Zn2? which block a6 containing and incomplete receptor

Study of the Interaction of 1,4- and 1,5-Benzodiazepines with GABA<inf>A</inf> 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 GABA(A) 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 alpha 6 beta 2/3 gamma 2 and alpha 1 alpha 6 beta 2/3 gamma 2 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 alpha 1 beta 2/3 gamma 2 receptors, while N-desmethyl-diazepam seems to interact with not only alpha 1-but also alpha 6-containing receptors

Different Molecular Mechanisms Mediate Direct or Glia-Dependent Prion Protein Fragment 90\u2013231 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\u2013231, an amyloidogenic polypeptide corresponding to the protease-resistant portion of the pathological prion protein scrapie (PrPSc). PrP90\u2013231 neurotoxicity is enhanced by the presence of microglia within neuronal culture, and associated to a rapid neuronal [Ca++]i increase. Indeed, while in \u201cpure\u201d cerebellar granule neuron cultures, PrP90\u2013231 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\u2013231 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\u2013231 neurotoxicity, whereas NMDA blockade by d( 12)-2-amino-5-phosphonopentanoic acid is ineffective; conversely, in pure cerebellar granule cultures, NMDA blockade but not iNOS inhibition strongly reduced PrP90\u2013231 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

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 \ub0C) acquiring structural characteristics identified in PrP(Sc) (enrichment in \u3b2-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

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-\u3b2 isoform (A\u3b2pE3-42) as one of the most neurotoxic peptides involved in the onset of Alzheimer's disease. Furthermore, A\u3b2pE3-42 co-oligomerized with excess of A\u3b21-42, produces oligomers and aggregates that are structurally distinct and far more cytotoxic than those made from A\u3b21-42 alone. Here, we investigate quantitatively the influence of A\u3b2pE3-42 on biophysical properties and biological activity of A\u3b21-42. We tested different ratios of A\u3b2pE3-42/A\u3b21-42 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\u3b2pE3-42, induces the highest disruption of intracellular calcium homeostasis and the highest neuronal toxicity. These data correlate to an high content of relaxed antiparallel \u3b2-sheet structure and the coexistence of a population of big spheroidal aggregates together with short fibrils. Our experiments provide also evidence that A\u3b2pE3-42 causes template-induced misfolding of A\u3b21-42 at ratios below 33%. This means that there exists a critical concentration required to have seeding on A\u3b21-42 aggregation, above this threshold, the seed effect is not possible anymore and A\u3b2pE3-42 controls the total aggregation kinetics

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

Nonspecific Interaction of Prefibrillar Amyloid Aggregates with Glutamatergic Receptors Results in Ca2+ Increase in Primary Neuronal Cells*

It is widely reported that the Ca2+ increase following nonspecific cell membrane permeabilization is among the earliest biochemical modifications in cells exposed to toxic amyloid aggregates. However, more recently receptors with Ca2+ channel activity such as α-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 Ca2+ increase in neuronal cells challenged with β-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 Ca2+ 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 Ca2+ increase following exposure of rat cerebellar granule cells to HypF-N prefibrillar aggregates. We found that the intracellular Ca2+ 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 Ca2+