IN ChAc RED CELLS THE ABNORMALLY ACTIVATED LYN AFFECTS ANKYRIN MULTIPROTEIN COMPLEXES AND IS INHIBITED BY DASATINIB

Chorea-acanthocytosis (ChAc) is a hereditary-neurodegenerative disorder, one of the neuroacanthocytosis syndromes (NA). One of the hallmarks of NA is the presence of circulating acanthocytes, generation of which is still under investigation. Recently, we reported increased Tyr-phosphorylation state of the red blood cell (RBC) membrane proteins from ChAc patients, related to abnormal activation of Lyn, an Src family kinase (SFKs) (Blood 118; 5652; 2011). In the context of international collaboration, we further characterized Lyn signaling pathway in RBC from ChAc patients. In ChAc RBCs, we found a weakness of ankyrin - based multiprotein complex bridging the membrane to the cytoskeleton, contributing to the generation of acanthocytes. We then evaluated the state of Lyn (active-inactive) in the cytoplasmic fraction from RBC of ChAc patients. In ChAc RBCs we found higher levels of Phospho- Lyn-396, corresponding to active Lyn, compared to controls. We then evaluated whether classical Lyn inhibitors such as PP2 or Dasatinib, a pharmacological Lyn inhibitor, might block Lyn in ChAc RBCs. We found that both PP2 (0.1\u3bcM) or Dasatinib (0.1 \u3bcM) were able to efficiently inhibit Lyn in both ChAc and healthy RBCs. These data suggest that in ChAc (i) the abnormal activation of Lyn affects RBC membrane mechanical stability weakening both multiprotein complexes, bridging the membrane to the cytoskeleton; (ii) Lyn activity is inhibited by either PP2 or Dasatinib, suggesting Lyn as possible new therapeutic target in ChAc

Evaluation of correct endogenous reactive oxygen species content for human sperm capacitation and involvement of the NADPH oxidase system

BACKGROUND Generation of controlled amounts of reactive oxygen species (ROS) and phosphorylation of protein tyrosine residues (Tyr) are two closely related changes involved in sperm capacitation. This study investigated the effect of altered endogenous ROS production on Tyr-phosphorylation (Tyr-P), acrosome reaction (AR) and cell viability during sperm capacitation. The possible origin of the altered ROS production was also evaluated by apocynin (APO) or oligomycin (Oligo) addition. METHODS A total of 63 samples of purified sperm were analysed for ROS production by enhanced chemiluminescence, Tyr-P pattern by immunocytochemistry, and AR and viability by fluorochrome fluorescein isothiocyanate (FITC)-labelled peanut (Arachis hypogaea) agglutinin and propidium iodide positivity, respectively. RESULTS Samples were divided into four categories depending on the ability of sperm to produce ROS, expressed as Relative Luminescence Units (RLU), in capacitating conditions: low ROS production (LRP), range about 0.0–0.05 RLU; normal (NRP), 0.05–0.1 RLU; high (HRP), 0.1–0.4 RLU; very high (VHRP) 0.4–2.0 RLU. In NRP sperm heads, capacitation induced Tyr-P in 87.9 ± 4.3%, and the AR occurred in 62.5 ± 5.4% of cells; in LRP, HRP and VHRP Tyr-P labelling rarely spread over the head, acrosome-reacted cells only accounted for a small number of sperm, and the non-viable cells (NVC) were increased. The addition of APO, but not Oligo, drastically decreased ROS production in analysed samples. CONCLUSIONS This study proposes the optimal threshold for endogenous ROS production for correct sperm viability and functioning, and indicates the direct involvement of APO-sensitive NADPH oxidase in ROS production