Nonspecific Interaction of Prefibrillar Amyloid Aggregates with Glutamatergic Receptors Results in Ca2+ Increase in Primary Neuronal Cells
Department of Physics, University of Genoa, Via Dodecaneso, 33, I-16146 Genoa, Italy.Journal of Biological Chemistry (Impact Factor: 4.57). 09/2008; 283(44):29950-60. DOI: 10.1074/jbc.M803992200
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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- "First, it forms fibrils that are morphologically, tinctorially and structurally similar to those found in amyloid deposition diseases (Chiti et al., 2001;Relini et al., 2004). Second, HypF-N forms pre-fibrillar oligomers that are toxic to cells in culture and in animal models (Bucciantini et al., 2002Bucciantini et al., , 2004Cecchi et al., 2005;Baglioni et al., 2006;Pellistri et al., 2008). Furthermore, HypF-N pre-fibrillar oligomers have similar pathogenic effects as Aβ oligomers on cells at the biochemical, molecular, electrophysiological and animal level (Bucciantini et al., 2004;Tatini et al., 2013). "
ABSTRACT: Living systems protect themselves from aberrant proteins by a network of chaperones. We have tested in vitro the effects of different concentrations, ranging from 0 to 16 μM, of two molecular chaperones, namely αB-crystallin and clusterin, and an engineered monomeric variant of transthyretin (M-TTR), on the morphology and cytotoxicity of preformed toxic oligomers of HypF-N, which represent a useful model of misfolded protein aggregates. Using atomic force microscopy imaging and static light scattering analysis, all were found to bind HypF-N oligomers and increase the size of the aggregates, to an extent that correlates with chaperone concentration. SDS-PAGE profiles have shown that the large aggregates were predominantly composed of the HypF-N protein. ANS fluorescence measurements show that the chaperone-induced clustering of HypF-N oligomers does not change the overall solvent exposure of hydrophobic residues on the surface of the oligomers. αB-crystallin, clusterin and M-TTR can diminish the cytotoxic effects of the HypF-N oligomers at all chaperone concentration, as demonstrated by MTT reduction and Ca
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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.
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- "The aggregation process of Ub closely matches, in terms of size and morphology of intermediate species, those described for α-syn incubated with CaII ions  or undergoing oxidation induced by FeIII ions in dithiothreitol . In both studies, α-syn was shown to form spherical and annular oligomers that might represent potentially toxic protofibrils , . The intermediate annular species, common to several neurodegenerative disorders, appear to be the most cytotoxic in vivo by forming pore-like structures which cause membrane permeabilization and disruption of ion homeostasis . "
ABSTRACT: Neurodegenerative disorders share common features comprising aggregation of misfolded proteins, failure of the ubiquitin-proteasome system, and increased levels of metal ions in the brain. Protein aggregates within affected cells often contain ubiquitin, however no report has focused on the aggregation propensity of this protein. Recently it was shown that copper, differently from zinc, nickel, aluminum, or cadmium, compromises ubiquitin stability and binds to the N-terminus with 0.1 micromolar affinity. This paper addresses the role of copper upon ubiquitin aggregation. In water, incubation with Cu(II) leads to formation of spherical particles that can progress from dimers to larger conglomerates. These spherical oligomers are SDS-resistant and are destroyed upon Cu(II) chelation or reduction to Cu(I). In water/trifluoroethanol (80:20, v/v), a mimic of the local decrease in dielectric constant experienced in proximity to a membrane surface, ubiquitin incubation with Cu(II) causes time-dependent changes in circular dichroism and Fourier-transform infrared spectra, indicative of increasing beta-sheet content. Analysis by atomic force and transmission electron microscopy reveals, in the given order, formation of spherical particles consistent with the size of early oligomers detected by gel electrophoresis, clustering of these particles in straight and curved chains, formation of ring structures, growth of trigonal branches from the rings, coalescence of the trigonal branched structures in a network. Notably, none of these ubiquitin aggregates was positive to tests for amyloid and Cu(II) chelation or reduction produced aggregate disassembly. The early formed Cu(II)-stabilized spherical oligomers, when reconstituted in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes and in POPC planar bilayers, form annular and pore-like structures, respectively, which are common to several neurodegenerative disorders including Parkinson's, Alzheimer's, amyotrophic lateral sclerosis, and prion diseases, and have been proposed to be the primary toxic species. Susceptibility to aggregation of ubiquitin, as it emerges from the present study, may represent a potential risk factor for disease onset or progression while cells attempt to tag and process toxic substrates.