Amyloid Oligomer Neurotoxicity, Calcium Dysregulation, and Lipid Rafts

Amyloid proteins constitute a chemically heterogeneous group of proteins, which share some biophysical and biological characteristics, the principal of which are the high propensity to acquire an incorrect folding and the tendency to aggregate. A number of diseases are associated with misfolding and aggregation of proteins, although only in some of them—most notably Alzheimer's disease (AD) and transmissible spongiform encephalopathies (TSEs)—a pathogenetic link with misfolded proteins is now widely recognized. Lipid rafts (LRs) have been involved in the pathophysiology of diseases associated with protein misfolding at several levels, including aggregation of misfolded proteins, amyloidogenic processing, and neurotoxicity. Among the pathogenic misfolded proteins, the AD-related protein amyloid β (Aβ) is by far the most studied protein, and a large body of evidence has been gathered on the role played by LRs in Aβ pathogenicity. However, significant amount of data has also been collected for several other amyloid proteins, so that their ability to interact with LRs can be considered an additional, shared feature characterizing the amyloid protein family. In this paper, we will review the evidence on the role of LRs in the neurotoxicity of huntingtin, α-synuclein, prion protein, and calcitonin

TNFα expressed on the surface of microparticles modulates endothelial cell fate in rheumatoid arthritis

Background: Rheumatoid arthritis (RA) is associated with a high prevalence of atherosclerosis. Recently increased levels of microparticles (MPs) have been reported in patients with RA. MPs could represent a link between autoimmunity and endothelial dysfunction by expressing tumor necrosis factor alpha (TNFα), a key cytokine involved in the pathogenesis of RA, altering endothelial apoptosis and autophagy. The aim of this study was to investigate TNFα expression on MPs and its relationship with endothelial cell fate. Methods: MPs were purified from peripheral blood from 20 healthy controls (HC) and from 20 patients with RA, before (time (T)0) and after (T4) 4-month treatment with etanercept (ETA). Surface expression of TNFα was performed by flow cytometry analysis. EA.hy926 cells, an immortalized endothelial cell line, were treated with RA-MPs purified at T0 and at T4 and also, with RA-MPs in vitro treated with ETA. Apoptosis and autophagy were then evaluated. Results: RA-MPs purified at T0 expressed TNFα on their surface and this expression significantly decreased at T4. Moreover, at T0 RA-MPs, significantly increased both apoptosis and autophagy levels on endothelial cells, in a dose-dependent manner. RA-MPs did not significantly change these parameters after 4 months of in vivo treatment with ETA. Conclusions: Our data demonstrate that MPs isolated from patients with RA exert a pathological effect on endothelial cells by TNFα expressed on their surface. In vivo and in vitro treatment with ETA modulates this effect, suggesting anti-TNF therapy protects against endothelial damage in patients with RA

Role of Electrostatic Interactions in Calcitonin Prefibrillar Oligomer-Induced Amyloid Neurotoxicity and Protective Effect of Neuraminidase

Salmon calcitonin is a good model for studying amyloid behavior and neurotoxicity. Its slow aggregation rate allows the purification of low molecular weight prefibrillar oligomers, which are the most toxic species. It has been proposed that these species may cause amyloid pore formation in neuronal membranes through contact with negatively charged sialic acid residues of the ganglioside GM1. In particular, it has been proposed that an electrostatic interaction may be responsible for the initial contact between prefibrillar oligomers and GM1 contained in lipid rafts. Based on this evidence, the aim of our work was to investigate whether the neurotoxic action induced by calcitonin prefibrillar oligomers could be counteracted by treatment with neuraminidase, an enzyme that removes sialic acid residues from gangliosides. Therefore, we studied cell viability in HT22 cell lines and evaluated the effects on synaptic transmission and long-term potentiation by in vitro extracellular recordings in mouse hippocampal slices. Our results showed that treatment with neuraminidase alters the surface charges of lipid rafts, preventing interaction between the calcitonin prefibrillar oligomers and GM1, and suggesting that the enzyme, depending on the concentration used, may have a partial or total protective action in terms of cell survival and modulation of synaptic transmission

The Slowly Aggregating Salmon Calcitonin: A Useful Tool for the Study of the Amyloid Oligomers Structure and Activity

Amyloid proteins of different aminoacidic composition share the tendency to misfold and aggregate in a similar way, following common aggregation steps. The process includes the formation of dimers, trimers, and low molecular weight prefibrillar oligomers, characterized by the typical morphology of globules less than 10 nm diameter. The globules spontaneously form linear or annular structures and, eventually, mature fibers. The rate of this process depends on characteristics intrinsic to the different proteins and to environmental conditions (i.e., pH, ionic strength, solvent composition, temperature). In the case of neurodegenerative diseases, it is now generally agreed that the pathogenic aggregates are not the mature fibrils, but the intermediate, soluble oligomers. However, the molecular mechanism by which these oligomers trigger neuronal damage is still unclear. In particular, it is not clear if there is a peculiar structure at the basis of the neurotoxic effect and how this structure interacts with neurons. This review will focus on the results we obtained using salmon Calcitonin, an amyloid protein characterized by a very slow aggregation rate, which allowed us to closely monitor the aggregation process. We used it as a tool to investigate the characteristics of amyloid oligomers formation and their interactions with neuronal cells. Our results indicate that small globules of about 6 nm could be the responsible for the neurotoxic effects. Moreover, our data suggest that the rich content in lipid rafts of neuronal cell plasma membrane may render neurons particularly vulnerable to the amyloid protein toxic effect

Proteasome-mediated degradation of keratins 7, 8, 17 and 18 by mutant KLHL24 in a foetal keratinocyte model: Novel insight in congenital skin defects and fragility of epidermolysis bullosa simplex with cardiomyopathy

Epidermolysis bullosa simplex (EBS) with cardiomyopathy (EBS-KLHL24) is an EBS subtype caused by dominantly inherited, gain-of-function mutations in the gene encoding for the ubiquitin-ligase KLHL24, which addresses specific proteins to proteasomal degradation. EBS-KLHL24 patients are born with extensive denuded skin areas and skin fragility. Whilst skin fragility rapidly ameliorates, atrophy and scarring develop over time, accompanied by life-threatening cardiomyopathy. To date, pathogenetic mechanisms underlying such a unique disease phenotype are not fully characterized. The basal keratin 14 (K14) has been indicated as a KLHL24 substrate in keratinocytes. However, EBS-KLHL24 pathobiology cannot be determined by the mutation-enhanced disruption of K14 alone, as K14 is similarly expressed in foetal and postnatal epidermis and its protein levels are preserved both in vivo and in vitro disease models. In this study, we focused on foetal keratins as additional KLHL24 substrates. We showed that K7, K8, K17 and K18 protein levels are markedly reduced via proteasome degradation in normal foetal keratinocytes transduced with the mutant KLHL24 protein (Delta N28-KLHL24) as compared to control cells expressing the wild-type form. In addition, heat stress led to keratin network defects and decreased resilience in Delta N28-KLHL24 cells. The KLHL24-mediated degradation of foetal keratins could contribute to congenital skin defects in EBS-KLHL24. Furthermore, we observed that primary keratinocytes from EBS-KLHL24 patients undergo accelerated clonal conversion with reduced colony forming efficiency (CFE) and early replicative senescence. Finally, our findings pointed out a reduced CFE in Delta N28-KLHL24-transduced foetal keratinocytes as compared to controls, suggesting that mutant KLHL24 contributes to patients' keratinocyte clonogenicity impairment

Mu2e Crystal Calorimeter Readout Electronics: Design and Characterisation

The Mu2e experiment at Fermi National Accelerator Laboratory will search for the charged-lepton flavour-violating neutrinoless conversion of negative muons into electrons in the Coulomb field of an Al nucleus. The conversion electron with a monoenergetic 104.967 MeV signature will be identified by a complementary measurement carried out by a high-resolution tracker and an electromagnetic calorimeter, improving by four orders of magnitude the current single-event sensitivity. The calorimeter—composed of 1348 pure CsI crystals arranged in two annular disks—has a high granularity, 10% energy resolution and 500 ps timing resolution for 100 MeV electrons. The readout, based on large-area UV-extended SiPMs, features a fully custom readout chain, from the analogue front-end electronics to the digitisation boards. The readout electronics design was validated for operation in vacuum and under magnetic fields. An extensive radiation hardness certification campaign certified the FEE design for doses up to 100 krad and 1012 n1MeVeq/cm2 and for single-event effects. A final vertical slice test on the final readout chain was carried out with cosmic rays on a large-scale calorimeter prototype

The Mu2e Crystal Calorimeter: An Overview

The Mu2e experiment at Fermilab will search for the standard model-forbidden, charged lepton flavour-violating conversion of a negative muon into an electron in the field of an aluminium nucleus. The distinctive signal signature is represented by a mono-energetic electron with an energy near the muon's rest mass. The experiment aims to improve the current single-event sensitivity by four orders of magnitude by means of a high-intensity pulsed muon beam and a high-precision tracking system. The electromagnetic calorimeter complements the tracker by providing high rejection power in muon to electron identification and a seed for track reconstruction while working in vacuum in presence of a 1 T axial magnetic field and in a harsh radiation environment. For 100 MeV electrons, the calorimeter should achieve: (a) a time resolution better than 0.5 ns, (b) an energy resolution <10%, and (c) a position resolution of 1 cm. The calorimeter design consists of two disks, each loaded with 674 undoped CsI crystals read out by two large-area arrays of UV-extended SiPMs and custom analogue and digital electronics. We describe here the status of construction for all calorimeter components and the performance measurements conducted on the large-sized prototype with electron beams and minimum ionizing particles at a cosmic ray test stand. A discussion of the calorimeter's engineering aspects and the on-going assembly is also reported

Towards a Muon Collider

A muon collider would enable the big jump ahead in energy reach that is needed for a fruitful exploration of fundamental interactions. The challenges of producing muon collisions at high luminosity and 10 TeV centre of mass energy are being investigated by the recently-formed International Muon Collider Collaboration. This Review summarises the status and the recent advances on muon colliders design, physics and detector studies. The aim is to provide a global perspective of the field and to outline directions for future work.Comment: 118 pages, 103 figure

Towards a muon collider

A muon collider would enable the big jump ahead in energy reach that is needed for a fruitful exploration of fundamental interactions. The challenges of producing muon collisions at high luminosity and 10 TeV centre of mass energy are being investigated by the recently-formed International Muon Collider Collaboration. This Review summarises the status and the recent advances on muon colliders design, physics and detector studies. The aim is to provide a global perspective of the field and to outline directions for future work