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Calcitonin native prefibrillar oligomers but not monomers induce membrane damage that triggers NMDA-mediated Ca²⁺-influx, LTP impairment and neurotoxicity.

ABSTRACT: Amyloid protein misfolding results in a self-assembling aggregation process, characterized by the formation of typical aggregates. The attention is focused on pre-fibrillar oligomers (PFOs), formed in the early stages and supposed to be neurotoxic. PFOs structure may change due to their instability and different experimental protocols. Consequently, it is difficult to ascertain which aggregation species are actually neurotoxic. We used salmon Calcitonin (sCT) as an amyloid model whose slow aggregation rate allowed to prepare stable samples without photochemical cross-linking. Intracellular Ca²⁺ rise plays a fundamental role in amyloid protein-induced neurodegerations. Two paradigms have been explored: (i) the "membrane permeabilization" due to the formation of amyloid pores or other types of membrane damage; (ii) "receptor-mediated" modulation of Ca²⁺ channels. In the present paper, we tested the effects of native sCT PFOs- with respect to Monomer-enriched solutions in neurons characterized by an increasing degree of differentiation, in terms of -Ca²⁺-influx, cellular viability, -Long-Term Potentiation impairment, Post-Synaptic Densities and synaptophysin expression. Results indicated that PFOs-, but not Monomer-enriched solutions, induced abnormal -Ca²⁺-influx, which could only in part be ascribed to NMDAR activation. Thus, we propose an innovative neurotoxicity mechanism for amyloid proteins where "membrane permeabilization" and "receptor-mediated" paradigms coexist.

SUBMITTER: Belfiore M

PROVIDER: S-EPMC6435710 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Calcitonin native prefibrillar oligomers but not monomers induce membrane damage that triggers NMDA-mediated Ca<sup>2+</sup>-influx, LTP impairment and neurotoxicity.

Belfiore Marcello M Cariati Ida I Matteucci Andrea A Gaddini Lucia L Macchia Gianfranco G Fioravanti Raoul R Frank Claudio C Tancredi Virginia V D'Arcangelo Giovanna G Diociaiuti Marco M

Scientific reports 20190326 1

Amyloid protein misfolding results in a self-assembling aggregation process, characterized by the formation of typical aggregates. The attention is focused on pre-fibrillar oligomers (PFOs), formed in the early stages and supposed to be neurotoxic. PFOs structure may change due to their instability and different experimental protocols. Consequently, it is difficult to ascertain which aggregation species are actually neurotoxic. We used salmon Calcitonin (sCT) as an amyloid model whose slow aggre ...[more]

PMID: 30914688

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Project description:Transthyretin (TTR) is a protein linked to a number of different amyloid diseases including senile systemic amyloidosis and familial amyloidotic polyneuropathy. The transient nature of oligomeric intermediates of misfolded TTR that later mature into fibrillar aggregates makes them hard to study, and methods to study these species are sparse. In this work we explore a novel pathway for generation of prefibrillar aggregates of TTR, which provides important insight into TTR misfolding. Prefibrillar amyloidogenic oligomers and protofibrils of misfolded TTR were generated in vitro through induction of the molten globule type A-state from acid unfolded TTR through the addition of NaCl. The aggregation process produced fairly monodisperse oligomers (300-500 kD) within 2 h that matured after 20 h into larger spherical clusters (30-50 nm in diameter) and protofibrils as shown by transmission electron microscopy. Further maturation of the aggregates showed shrinkage of the spheres as the fibrils grew in length, suggesting a conformational change of the spheres into more rigid structures. The structural and physicochemical characteristics of the aggregates were investigated using fluorescence, circular dichroism, chemical cross-linking, and transmission electron microscopy. The fluorescent dyes 1-anilinonaphthalene-8-sulfonate (ANS), 4-4-bis-1-phenylamino-8-naphthalene sulfonate (Bis-ANS), 4-(dicyanovinyl)-julolidine (DCVJ), and thioflavin T (ThT) were employed in both static and kinetic assays to characterize these oligomeric and protofibrillar states using both steady-state and time-resolved fluorescence techniques. DCVJ, a molecular rotor, was employed for the first time for studies of an amyloidogenic process and is shown useful for detection of the early steps of the oligomerization process. DCVJ bound to the early prefibrillar oligomers (300-500 kD) with an apparent dissociation constant of 1.6 muM, which was slightly better than for ThT (6.8 muM). Time-resolved fluorescence anisotropy decay of ANS was shown to be a useful tool for giving further structural and kinetic information of the oligomeric aggregates. ThT dramatically increases its fluorescence quantum yield when bound to amyloid fibrils; however, the mechanism behind this property is unknown. Data from this work suggest that unbound ThT is also intrinsically quenched and functions similarly to a molecular rotor, which in combination with its environmental dependence provides a blue shift to the characteristic 482 nm wavelength when bound to amyloid fibrils.

Dataset Information

Calcitonin native prefibrillar oligomers but not monomers induce membrane damage that triggers NMDA-mediated Ca²⁺-influx, LTP impairment and neurotoxicity.

Publications

Calcitonin native prefibrillar oligomers but not monomers induce membrane damage that triggers NMDA-mediated Ca<sup>2+</sup>-influx, LTP impairment and neurotoxicity.

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Dataset Information

Calcitonin native prefibrillar oligomers but not monomers induce membrane damage that triggers NMDA-mediated Ca2+-influx, LTP impairment and neurotoxicity.

Publications

Calcitonin native prefibrillar oligomers but not monomers induce membrane damage that triggers NMDA-mediated Ca<sup>2+</sup>-influx, LTP impairment and neurotoxicity.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Calcitonin native prefibrillar oligomers but not monomers induce membrane damage that triggers NMDA-mediated Ca²⁺-influx, LTP impairment and neurotoxicity.