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.

The Interaction between Amyloid Prefibrillar Oligomers of Salmon Calcitonin and a Lipid-Raft Model: Molecular Mechanisms Leading to Membrane Damage, Ca2+-Influx and Neurotoxicity

“…In particular, partially structured and mainly disordered PFOs resulted to be able to damage the lipid bilayer by the formation of "amyloid-pores" and this was made possible by Chol that rendered fluid membranes. This is in good agreement with the biological results we recently published about Ca 2+ -influx induced damage in hippocampal primary neurons [20] and allows us to speculate about the molecular mechanisms at the basis of neurotoxicity. Structural results gave interesting information about the molecular mechanisms.…”

“…Notably, in our previous paper concerning neurotoxicity in hippocampal neurons treated by sCT-PFOs we showed that, under physiological conditions, other cellular mechanisms were involved [20]. Nevertheless, in the same study we showed that the Ca 2+ -influx due to membrane permeabilization was the triggering factor that induced NMDA-mediated neurotoxicity and this is in good agreement with the results obtained now with both HT22-DIFF cells and liposomes.…”

“…Interestingly, since 2003 Glabe et al proposed that a common core of pathologic pathways exists for amyloid-associated diseases, based on the cellular membrane permeabilization and subsequent abnormal Ca 2+ -influx, independent from the primary sequence of the involved protein [8,[18][19][20]. Lashuel and Lansbury summarized the existing supportive circumstantial evidence about the hypothesis that amyloid diseases were caused by protein aggregates that mimic bacterial pore-forming toxins, which in general form well-ordered oligomeric membrane-spanning pores characterized by β-sheet structure [21].…”

“…Thus, to investigate this outcome we performed biophysical studies on the interaction of sCT with model membranes mimicking the presence of "lipid-rafts" confirming the GM1 pivotal role [47]. Recently, we fractionated the sCT aggregates and demonstrated that PFOs was the species able to induce neurotoxicity in primary hippocampal neurons and mouse brain slices [5,20].…”

Effects of disulfide bond and cholesterol derivatives on human calcitonin amyloid formation

“…Furthermore, it has been reported that electrostatic interactions between sCT and GM1 (a ganglioside component of cellular membranes) which are stabilized by cholesterol cause spherical amyloid aggregation to occur on the surface of liposomes . In addition, hCT is capable to form channels within the membrane and cause ion leakage, and this hCT‐induced membrane pore formation is enhanced by the presence of cholesterol . Therefore, cholesterol may play a crucial role in modulating calcitonin amyloid formation and cellular toxicity in vivo .…”