Abstract
Multiple Sclerosis (MS) is a chronic disease of the central nervous system, the etiology of which, although not completely known, involves inflammation and autoimmunity. In the present study we aimed at identifying molecular markers of apoptosis, cellular stress and DNA damage in isolated peripheral blood mononuclear cells (PBMCs) of MS patients. The analysis was carried on 19 relapsing-remitting untreated MS patients and 13 healthy individuals. We investigated the emergency-driven synthesis of poly(ADP-ribose) (PAR), the expression level of the constitutive enzyme poly(ADP-ribose) polymerase-1 (PARP-1) and the DNA damage-induced phosphorylation of histone H2AX. PAR accumulation, PARP-1 and phosphorylated H2AX (γH2AX) were detected by immunofluorescence experiments on PBMCs isolated from 19 patients and 13 healthy volunteers. Our results show for the first time a net increased amount in PAR and γH2AX in MS patients compared to healthy individuals. Patients were further subdivided in three groups, according to the neuroimaging (MRI)-based classification of disease phase. Remarkably, we found a positive correlation between the level of γH2AX and MS aggressiveness. In addition, apoptosis in PBMCs was monitored by flow cytometry of both phosphatidylserine exposure (revealed by Annexin V-FITC labeling) and membrane permeability to propidium iodide. Our observations provide the evidence that the number of apoptotic cells was significantly higher in patients compared to healthy individuals, thus suggesting that apoptosis could affect MS lymphocyte function.
Highlights
The etiology of Multiple Sclerosis (MS) is not known and probably implies a multifactorial context
We focused on markers of DNA damage and cellular stress by analysing respectively DNA double strand break (DSB)-induced serine-139 phosphorylation of histone H2AX [6,7] and poly(ADP-ribose) accumulation, which is catalysed by poly(ADPribose) polymerases (PARPs) in response to cellular stress conditions [8]
We started a pilot study to search for peripheral biomarkers, based on the hypothesis that MS lymphocytes could have altered basal levels of stress and damage, which could possibly be linked to the stage of the disease
Summary
The etiology of Multiple Sclerosis (MS) is not known and probably implies a multifactorial context. Pathogenetic mechanisms of MS have been extensively investigated and imply loss of tolerance in the immune response [1,2] and inflammatory aggression towards oligodendrocytes in the myelin sheath as well as neurodegenerative contributions [3]. Oxidative stress that produces Reactive Oxygen Species (ROS) harmful for cells, proteins and DNA, has been claimed to be involved in MS at the target tissue, within the Central Nervous System (CNS) [4]. Similar effects may act at the inflammatory effector (i.e. lymphocyte) level, affecting the control of apoptosis, which has been involved in MS pathogenesis [5]. We evaluated the occurrence of apoptosis using flow cytometry. These investigations were conducted in peripheral blood mononuclear cells (PBMCs) from MS patients and control subjects, using samples collected on the same day. The association with the disease evolution and disease phase was explored assessing lesion load changes and presence of gadolinium (GD) enhancement in brain and spinal Magnetic Resonance Imaging (MRI)
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