Generic interaction of pre-fibrillar amyloid aggregates with NMDA and AMPA receptors results in free Ca increase in primary neuronal cells
ABSTRACT 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+).
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- "Coverslips were transferred to a recording chamber mounted onto a Nikon Eclipse TE300 inverted microscope. Cells were continuously perfused with the appropriate solution and visualized using 1009 objective in oil (N.A. 1.3) (Pellistri et al. 2008). Fluorescence was detected using a Hamamatsu digital CCD camera with a 450–490-nm excitation filter, a 505-nm dichroic mirror, and a 520-nm emission filter (Nikon Italia, Florence, Italy). "
ABSTRACT: 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 β-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.Neurotoxicity Research 05/2013; 23(4):301-314. DOI:10.1007/s12640-012-9340-9 · 3.15 Impact Factor
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ABSTRACT: alpha-Synuclein (ASN), a small presynaptic protein that is abundant in the brain, is implicated in the pathogenesis of neurodegenerative disorders including Parkinson's and Alzheimer's disease. The central domain of alpha-synuclein, the non-amyloid beta component of the Alzheimer's disease amyloid (NAC) is probably responsible for its toxicity. However, the molecular mechanism of alpha-synuclein action remains largely elusive. The present study examined the effect of alpha-synuclein and the NAC peptide on nitric oxide synthase (NOS) activity in rat brain cortical and hippocampal slices using a radiochemical technique. Moreover, nitrite levels in brain slices incubated in the presence of alpha-synuclein were measured using the Griess reaction. ASN and the NAC stimulated NOS activity by about 70% and 40%, respectively. beta-Synuclein, a homologous protein of ASN that lacks the NAC domain, had no effect on NOS activity. Under the same experimental conditions, alpha-synuclein increased nitrite levels by 27%. alpha-Synuclein and the NAC affected the activity of constitutive neuronal isoform of NOS, but had no impact on the endothelial or inducible NOS isoforms. The effect of alpha-synuclein and the NAC peptide on NOS activity was inhibited by MK-801 and APV, antagonists of the NMDA receptor. These results indicate that the NMDA receptor plays an important role in alpha-synuclein-evoked nitric oxide synthesis. We suggest that nitric oxide liberated by the over-activated neuronal isoform of NOS could react with superoxide to form peroxynitrite, which modulates the function of a variety of biomolecules including proteins, lipids, and DNA.Pharmacological reports: PR 11/2009; 61(6):1078-85. DOI:10.1016/S1734-1140(09)70170-7 · 2.17 Impact Factor
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ABSTRACT: The whole set of the so-called »conformational« disorders, among them systemic amyloidoses, various demen-tias and other neurodegenerative diseases such as Parkinson's, Alzheimer's and amyotropic lateral sclerosis, may have similar molecular backgrounds: changes in protein conformation and aggregation, which lead to toxic amyloid oligomers and fibrils. The so called aggresomes in eukaryotes (equivalent to inclusion bodies in prokaryotes), located at the centriole by the nucleus and composed of aggregated proteins, are believed to sequester the toxic material. They eventually get cleared from the cell by autophagy. When the cell defense system fails due to continuous production of a mutated protein or to other damage to the cell, such as oxidative stress or protein modification as part of normal aging, familial or sporadic neurodegenerative diseases develop. Initially – for years -they are silent with no or mild symptoms. It could well be that aggregates represent a response to some other trigger or even a means of defense. However, the inherited cases with muta-tions leading to increased aggregation suggest the opposite to be the case. Evidence has accumulated that the soluble oli-gomers of amyloidogenic proteins are themselves cytotoxic and trigger a cascade of detrimental events in the cell, as summarized in the "amyloid cascade hypothesis". Among other plausible hypotheses for the mechanism of toxicity is the "channel hypothesis", which states that the soluble oligomers interact with cell membranes, causing influx of Ca 2+ ions, which is an early sign of pathology and contributes to uncontrolled neurotransmission. Another factor are metal ions, such as Zn 2+ , Cu 2+ , Fe 3+ , Al 3+ , etc., leading to the "metal hypothesis". The delicate balance of metal ions in the brain is impor-tant to prevent oxidative stress, which can itself modify proteins and make them aggregation-prone. The advances in mo-lecular and cellular studies will hopefully lead to novel therapies and eventually to a cure.