Lukas H. Margaritis

Cell-produced alpha-synuclein is secreted in a calcium-dependent manner by exosomes and impacts neuronal survival.

α-Synuclein is central in Parkinsons disease pathogenesis. Although initially α-synuclein was considered a purely intracellular protein, recent data suggest that it can be detected in the plasma and CSF of humans and in the culture media of neuronal cells. To address a role of secreted α-synuclein in neuronal homeostasis, we have generated wild-type α-synuclein and β-galactosidase inducible SH-SY5Y cells. Soluble oligomeric and monomeric species of α-synuclein are readily detected in the conditioned media (CM) of these cells at concentrations similar to those observed in human CSF. We have found that, in this model, α-synuclein is secreted by externalized vesicles in a calcium-dependent manner. Electron microscopy and liquid chromatography–mass spectrometry proteomic analysis demonstrate that these vesicles have the characteristic hallmarks of exosomes, secreted intraluminar vesicles of multivesicular bodies. Application of CM containing secreted α-synuclein causes cell death of recipient neuronal cells, which can be reversed after α-synuclein immunodepletion from the CM. High- and low-molecular-weight α-synuclein species, isolated from this CM, significantly decrease cell viability. Importantly, treatment of the CM with oligomer-interfering compounds before application rescues the recipient neuronal cells from the observed toxicity. Our results show for the first time that cell-produced α-synuclein is secreted via an exosomal, calcium-dependent mechanism and suggest that α-synuclein secretion serves to amplify and propagate Parkinsons disease-related pathology.

Mechanism for action of electromagnetic fields on cells

A biophysical model for the action of oscillating electric fields on cells, presented by us before [Biochem. Biophys. Res. Commun. 272(3) (2000) 634-640], is extended now to include oscillating magnetic fields as well, extended to include the most active biological conditions, and also to explain why pulsed electromagnetic fields can be more active biologically than continuous ones. According to the present theory, the low frequency fields are the most bioactive ones. The basic mechanism is the forced-vibration of all the free ions on the surface of a cells plasma membrane, caused by an external oscillating field. We have shown that this coherent vibration of electric charge is able to irregularly gate electrosensitive channels on the plasma membrane and thus cause disruption of the cells electrochemical balance and function [Biochem. Biophys. Res. Commun. 272(3) (2000) 634-640]. It seems that this simple idea can be easily extended now and looks very likely to be able to give a realistic basis for the explanation of a wide range of electromagnetic field bioeffects.

RBC‐derived vesicles during storage: ultrastructure, protein composition, oxidation, and signaling components

BACKGROUND: Red cells (RBCs) lose membrane in vivo, under certain conditions in vitro, and during the ex vivo storage of whole blood, by releasing vesicles. The vesiculation of the RBCs is a part of the storage lesion. The protein composition of the vesicles generated during storage of banked RBCs has not been studied in detail.

Progressive oxidation of cytoskeletal proteins and accumulation of denatured hemoglobin in stored red cells

Red blood cell (RBC) membrane proteins undergo progressive pathological alterations during storage. In conditions of increased cellular stress, the cytoskeleton also sustains certain modifications. The hemoglobin (Hb) content and oxidative status of the RBC cytoskeletons as a function of the storage period remain unclear. The possible Hb content and oxidative alterations occurring in the cytoskeletons in the course of storage were monitored in six units, by means of electrophoresis, immunoblotting and protein carbonylation assays. A proportion of the ghost‐bound Hb consists of non‐reducible crosslinkings of probably oxidized(denatured Hb or hemichromes.The defective Hb‐membrane association was strongly affected by the prolonged storage. A progressive accumulation of Hb monomers, multimers and high molecular weight aggregates to corresponding cytoskeletons were also evident. The oxidative index of the cytoskeletal proteins was found increased, signalizing oxidative modifications in spectrin and possibly other cytoskeletal proteins. The reported data corroborate the evidence for oxidative damage in membrane proteins with emphasis to the cytoskeletal components. They partially address the pathophysiological mechanisms underlying the RBC storage lesion, add some new insight in the field of RBC storage as a hemoglobin‐ and cytoskeleton‐associated pathology and suggest the possible use of antioxidants in the units intended for transfusion.

Stage-specific apoptotic patterns during Drosophila oogenesis

In the present study we demonstrate the existence of two apoptotic patterns in Drosophila nurse cells during oogenesis. One is developmentally regulated and normally occurs at stage 12 and the other is stage-specific and is sporadically observed at stages 7 and 8 of abnormally developed follicles. The apoptotic manifestation of the first pattern begins at stage 11 and is marked by a perinuclear rearrangement of the actin cytoskeleton and the development of extensive lobes and engulfments of the nurse cell nuclei located proximal to the oocyte. Consequently, at late stage 12 (12C), half of the nurse cell nuclei exhibit condensed chromatin, while at late stage 13 all the nuclei have fragmented DNA, as it is clearly shown by TUNEL assay. Finally, the apoptotic vesicles that are formed during stage 13, are phagocytosed by the neighboring follicle cells and at stage 14 the nurse cell nuclear remnants can be easily detected within the adjacent follicle cell phagosomes. In the second sporadic apoptotic pattern, all the nurse cell nuclei are highly condensed with fragmented DNA, accompanied by a completely disorganized actin cytoskeleton. When we induced apoptosis in Drosophila follicles through an etoposide and staurosporine in vitro treatment, we observed a similar pattern of stage-specific cell death at stages 7 and 8. These observations suggest a possible protective mechanism throughout Drosophila oogenesis that results in apoptosis of abnormal, damaged or spontaneously mutated follicles before they reach maturity.

Intracellular Clusterin Inhibits Mitochondrial Apoptosis by Suppressing p53-Activating Stress Signals and Stabilizing the Cytosolic Ku70-Bax Protein Complex

Purpose: Secretory clusterin (sCLU)/apolipoprotein J is an extracellular chaperone that has been functionally implicated in DNA repair, cell cycle regulation, apoptotic cell death, and tumorigenesis. It exerts a prosurvival function against most therapeutic treatments for cancer and is currently an antisense target in clinical trials for tumor therapy. However, the molecular mechanisms underlying its function remained largely unknown. Experimental Design: The molecular effects of small interfering RNA-mediated sCLU depletion in nonstressed human cancer cells were examined by focusing entirely on the endogenously expressed sCLU protein molecules and combining molecular, biochemical, and microscopic approaches. Results: We report here that sCLU depletion in nonstressed human cancer cells signals stress that induces p53-dependent growth retardation and high rates of endogenous apoptosis. We discovered that increased apoptosis in sCLU-depleted cells correlates to altered ratios of proapoptotic to antiapoptotic Bcl-2 protein family members, is amplified by p53, and is executed by mitochondrial dysfunction. sCLU depletion-related stress signals originate from several sites, because sCLU is an integral component of not only the secretory pathway but also the nucleocytosolic continuum and mitochondria. In the cytoplasm, sCLU depletion disrupts the Ku70-Bax complex and triggers Bax activation and relocation to mitochondria. We show that sCLU binds and thereby stabilizes the Ku70-Bax protein complex serving as a cytosol retention factor for Bax. Conclusions: We suggest that elevated sCLU levels may enhance tumorigenesis by interfering with Bax proapoptotic activities and contribute to one of the major characteristics of cancer cells, that is, resistance to apoptosis.

Storage-dependent remodeling of the red blood cell membrane is associated with increased immunoglobulin G binding, lipid raft rearrangement, and caspase activation

BACKGROUND: The elucidation of the storage lesion is important for the improvement of red blood cell (RBC) storage. Ex vivo storage is also a model system for studying cell‐signaling events in the senescence and programmed cell death of RBCs. The membrane hosts critical steps in these mechanisms and undergoes widespread remodeling over the storage period.

Cell death during Drosophila melanogaster early oogenesis is mediated through autophagy.

Autophagy is a physiological and evolutionarily conserved process maintaining homeostatic functions, such as protein degradation and organelle turnover. Accumulating data provide evidence that autophagy also contributes to cell death under certain circumstances, but how this is achieved is not well known. Herein, we report that autophagy occurs during developmentally-induced cell death in the female germline, observed in the germarium and during middle developmental stages of oogenesis in Drosophila melanogaster. Degenerating germline cells exhibit caspase activation, chromatin condensation, DNA fragmentation and punctate staining of mCherry-DrAtg8a, a novel marker for monitoring autophagy in Drosophila. Genetic inhibition of autophagy, by removing atg1 or atg7 function, results in significant reduction of DNA fragmentation, suggesting that autophagy acts genetically upstream of DNA fragmentation in this tissue. This study provides new insights into the mechanisms that regulate cell death in vivo during development.

Red blood cell aging markers during storage in citrate‐phosphate‐dextrose–saline‐adenine‐glucose‐mannitol

BACKGROUND: It has been suggested that red blood cell (RBC) senescence is accelerated under blood bank conditions, although neither protein profile of RBC aging nor the impact of additive solutions on it have been studied in detail.

Effect of GSM 900-MHz Mobile Phone Radiation on the Reproductive Capacity of Drosophila melanogaster

Pulsed radio frequency, (RF), electromagnetic radiation from common GSM mobile phones, (Global System for Mobile Telecommunications) with a carrier frequency at 900 MHz, “modulated” by human voice, (speaking emission) decreases the reproductive capacity of the insect Drosophila melanogaster by 50%–60%, whereas the corresponding “nonmodulated” field (nonspeaking emission) decreases the reproductive capacity by 15%–20%. The insects were exposed to the near field of the mobile phone antenna for 6 min per day during the first 2–5 days of their adult lives. The GSM field is found to affect both females and males. Our results suggest that this field-radiation decreases the rate of cellular processes during gonad development in insects.